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ACKNOWLEDGMENTS
The author is sincerely grateful to Dr. Emil L. Smith for his
guidance and assistance. His interest and enthusiasm were valuable
stimuli during the course of this research.
The work of Mr. D. M. Brown in connection with the physical
studies on papain is deeply appreciated, and the author is equally
grateful to Miss Anne Stockell who carried out the amino acid analyses.
The author is greatly indebted to the American Canoer Society
for fellowships which permitted him to carry out this work.
ii
TABLE OF CONTENTS
" ..
. .. . . . .
Page
v LIST OF TABLES " 0 0 0
LIST OF ILLUSTRATIONS. o 0 . .
" vi
Chapter
I
II
INTRODUCTION .. . . . . 0
THE HISTORY OF PAPAIN. " (I: 0 . . . . . . . . .
The Activation of Papain
Discovery and Coenzyme Theory
The Development of the Sulfhydryl Concept
Reactions to the Sulfhydryl Concept
Other Activating Agents for Papain
Inhibitors of Papain
The Effect of pH on Papain Action
The Effect of Specific Anions on Papain Action
The Specificity of Papain
Synthetic Actions of Papain
The Purification of Papain
1
6
III EXPERIMENTAL METHODS " .. .. 0 0 0 0 0 31
The Determination of Protein Concentration
Measurement of Hydrolysis of Synthetic Substrates
Measurement of the Hydrolysis of Esters
Calculation of Specific Activity
Measurement of Proteolysis
Electrophoretic Studies
Sedimentation Studies
IV THE ISOLATION AND CRYSTALLIZATION OF PAPAIN AND
MERCURIPAPAIN. " 0 " " " 0
The Extraction of Proteolytic Activity
The Preparation of Crystalline Papain
Recrystallization
The Preparation of Mercuripapain
o 0 , 39
Elementary Analysis of Papain and Mercuripapain
Discussion
V THE ENZYMATIC PROPERTIES OF PAPAIN 0 " 0 0 0 " 0 " 52
The activation of Crystalline Papain
Effect of pH on Papain Activity
Inhibitors of Papain
Esterase Action of Papain
iii
The Specificity of Papain
Proteolysis ~ y Papain
Discussion
page
VI THE COMPOSITION OF CRYSTALLINE PAPAIN. 68
The Amino Acid Composition of Papain
Free Amino Groups and N-Terminal Sequence
Amino Acid'Sequences Containing Cysteine
Discussion
VII THE PHYSICAL PROPERTIES OF PAPAIN AND MERCURIPAPAIN 85
SUMMARY.
Electrophoretic Studies
Sedimentation Studies
Diffusion Studies
Molecular Weight
Properties of Mercuripapain
Nature of Mercuripapain Complex
Discussion
. . . . . . .
BIBLIOGRAPHY o
iv
. .

.106
. . o .110
LIST OF TABLES
Table page
I Enzymatic Activity of Mercuripapain. 45
II Elementary AnalTsis of Papain and Mercuripapa.in. 46
III Isolation of Cr,ystalline Papain from Dried Papaya
Latex. 47
IV Electrophoretic Components of Papaya Latex. o' 49
V Action of Cr.ystalline Papain on Synthetic Substrates 61
VI of by
Papain 62
VII Inhibition ot Papain by
VIII
IX
Acid 6:;
Compolition and Moleoular Weight of Papain

71
Ionio Groupe ot Papain

73
X DNP Amino Acids in Complete Hydrolysates of
DNP papain preparationl. 74
XI Cysteic Acid Peptides Isolated from Partial
XII
XIII
'XIV
HydrolTsates of Papain 82
GO Diffusion Constant of Crystalline Papain
Sedimentation of Mercuripapain at pH 8 0 0
93
98
Sedimentation of Papain at pH 8. 0
" 0
.100
v
LIST OF ILLUSTRATIONS
Figure page
1. The Standard Curve for the Bucher Protein
2.
4.
6.
7.
8.
10.
11.
Determination 0 .. .. .. .. 0 o . . . .
The Effect of pH on the Efficiency of Extraction
of Proteolytic Activity from Dried Papaya Latex
.. .

Crystals of Mercuripapain Photographed at 100
Times Enlargement .. 0 0 0 .0. 0
Electrophoretic Patterns of Clarified Crude
Extracts of Fresh Latex and Dried Latex from
Source 1. .. 0 0 .. o
Effect of pH on the Proteolytic Coefficient
for the Hydrolysis of Benzoyl-b-argininamide
by Papain in the Presence of 0.005 M. Cysteine
Effect of pH on the Activation of Crystalline
Papain as Measured by the Proteolytic Coefficient
for the Hydrolysis of Benzoyl-b-argininamide.
Effect of pH on the C
l
for Hydrolysis of Benzoyl-
1;argininamide by Crystalline Papain in the
Presence of 0.001 M Versene and 0.005 M Cysteine.
Action' of Papain on methyl ester
at the Various pH Values Indicated on the Curves.
Effect of pH on the Hydrolysis of Tosyl-b-arg-
.



inine and BAA 0 0
Effect of pH on the Hydrolysis of Four Synthetic
Substrates by Crystalline Papain in the presence
of 0.005 M Cysteine and 0.001 M Versene
.
The Digestion of Urea-Denatured Hemoglobin by
Crystalline Papain and Crystalline Tr,ypsin. .. .. .. ..
12 It Ionophoresis of Oxidized Partial Hydrolysates
of Papain .. .. .. .. (I .. .. 0 .. .. 0' .. 0 It .. .. 0
13. Two Dimensional Paper Chromatogram of Cysteic Acid
Peptides Isolated from Partial Hydrolysates of
Pa pain. .. 0' .. .. .. .. o 0 0 0 0 e 0 0 0 0
vi
32
40
44
49
54
55
58
59
64
65
7B
81
Figure page
14. Electrophoretic Mobility as a Function of
pH at 1.5
0
for Cr.ystalline Papain as Deter-
mined Univalent Buffers at an Ionic Strength
of 0.1 . . . . . . . . . . . . 86
15. Electrophoretic Patterns of Various Preparations

16. Sedimentation Patterns of Crystalline Papain
in Acetate Buffer at 0.2 Ionic Strength. 90
17. Sedimentation Constants (520 w) of CITstalline
Papain as a Function of Concentration. 91
18. Sedimentation of Mercuripapain at pH 4 in
0.1 Ionic Strength Acetate Buffer. 95
19. Electrophoretic Patterns of the Descending
Migration of Crystalline Mercuripapain

20. Sedimentation Constants ( ) of Mercuripapain
95
in 0.12 Ionic Strength Acetate Buffer Containing
0.02 M Cysteine and 0.OO2,MVersenELat pH,3.9 96
21. Sedimentation Patterns of Cr,ystalline
Mercuripapain at pH 8 at Four Different
Protein Concentrations 0 97
vii
CHAPTER I
INTRODUCTION
The tropical papaw or melon tree, Carica papaya, probably enjoys
greater popularity and usefulness than any other plant of the tropics.
It is said to have originated in Central America Or the West Indies and
to have been transported to the Philippines and the East Indies during
the seventeenth and eighteenth centuries by explorers and traders. To-
day, the papaya is found under oultivation in practically all of the
tr'opical countries of the world.
Carica papaya is a tall palm-like tree consisting of a single,
slim, erect stalk crowned by a cluster of broad flat leaves from two to
four feet in length. It grows rapidly in the tropios and requires only
ten to fourteen months from the germination of the seeds to the harvest
of the first ripe fruit. This fruit grows in clusters just below the
crown of the tree or hanging down a few feet from it. It resembles the
muskmelon in character and is green when immature and yellow to orange
when ripe.
Virtually every part of the papaya tree is put to some use by
the natives of the areas where it grows. ~ e ripe fruit is the uni-
versal breakfast food of the tropics and is served like a cantaloupe.
It is used in salads, fruit cocktails, papaya tarts, marmalades, and
sherbets. The juice is served as a drink or made into jelly. On the
other hand, the fully grown green fruit 'functions as a vegetable. It
-1-
-2-
is boiled, baked, or stewed like a summer squash. The seeds are l1Bee-r-
ated with vinegar and used for seasoning. The wood is too 50ft and
porous for use, but the tough sinewy bark is utilized by the natives
to make rope.
Tropical natives have a high regard for the medicinal properties
of the papaya. They use the roots for the preparation of a nerve tonic,
the seeds as a carminative, anthelminthic.and antip,yretic. The juice of
the ripe fruit is a common flavoring for other medicines. The milky
juice of the green fruit is used to treat almost all types of skin ail-
ments. A slice of the fruit rubbed on the skin is the native counter-
part of our modern soaps and lotions for improving the complexion. It
is interesting to note that the papaya aroused medical interest some two
centuries before the first chemical publications appeared. The Pharma-
copia of India (165) states that the anthelminthic properties of the
latex of the unripe fruit were demonstrated by Hernandez in the seven-
teenth century.
It is probably not unusual for a tropical plant to have several
culinary and medical uses, but the papaya has another unique and import-
and function in the everyday life of the tropical native. For centuries
various parts of the plant have been used to tenderize tough meat either
prior to, or during cooking. In practice, the meat is rubbed with a
slice of the green fruit, soaked and cooked in the juice, or wrapped
overnight in the leaves. The English naturalist Griffith Hughes (74)
expressed his amazement at this digestive property of papaya juice in
1750 when he commented:
This juice is of so penetrating a nature, that if the unripe
fruit when unpeeled is boiled with the toughest old salt meat, it
will soon make it soft and tender.
-3-
This digestive action is now known to be due to proteolytic en-
zymes which are present in various parts of the plant (11). The richest
source of these enzymes is the juice of the fully grown green fruit.
Just under the skin of these are fairly large latex vessels which, when
severed, exude a clear water-like fluid that rapidly becomes opaque on
exposure to the air. The modern method of obtaining this latex for com-
mercial use consists of making several scratches in the green
fruit while it still hang$ on the The latex is allowed to drip
off, or is scraped off into trays around the trunk of the tree. This
coagulated material is subsequentl.1 dried, powdered and sieved, and in
this form appears on the market a.s papain; a. name first applied by Wurtz
(175) in 18S0.
Papain was first imported into this country as a medicinal for
the treatment of chronic dyspepsia or indigestion late in the nineteenth
century. Kilmer (S6), in attempting to exPlain the lack of chronic in-
digestion in the tropical home, makes the following interesting comment
regarding this particular use:
Some of these people are great gluttons; they gorge themselves
until the skin on their distended stomachs is stretched to the ut-
most. It is certain that no human could digest the kind of
food and the enormous amounts they consume without the kindly aid
of the papaw fruit to assist digestion.
While some of the papain imported today is put to medical use,
the vast majority of it is consumed by various industries. Thus, papain
is \1sed in the packing industry for the tenderization of meat; in the
industry to prevent wool shrinkage; in the brewing industry to
prevent oxidation and chill hazes in beer; in the tanning industry to
bate skips and hides; and in the manufacture of chewing gum. The United
States imports about 500,000 pounds of crude papain annually. The mone-
tary value of this is estimated to be about two million dollars (149).
-4-
In addition to its commercial importance, papain has long been
of academic interest as a proteolytic enzyme. It was the first enzyme
upon which cyanide was demonstrated to have an activating effect. The
discover,r of this effect eventually led to the development of our pres-
ent ideas regarding sultpydryl enzymes. The experimental work directly
responsible for the hypothesis of reversible oxidation-reduction of
thiol groups on enzyme molecules was carried out on papain. Furthermore,
papain was the'first proteolytic enzyme to be tested against a synthetic
peptide substrate, in contrast to protein substrates. In the literature
prior to 1940 all of the preparations of papain that were used in 861en-
tific investigation were crude or partially purified extracts of fresh
or dried papaya latex. Although it was generally recognized that these
preparations might contain more than one proteolytic enzyme, this possi-
bility did not discourage investigation and the number of publications
on papain between 1920 and 1940 numbers well in the hundreds. The end
of this period marked a turning point in studies on papain because it
was at this time that the crystallization of two proteinases, papain and
chymopapain, from fresh papaya latex was reported. Following this, pap-
ain virtually disappeared from the biochemical literatUre. This is un-
derstandable since fresh papaya latex was expensive and scarce. Further-
more, it has not been generally possible to obtain crystalline papain
from the readily available dried latex by application of the published
procedure of Balls and Lineweaver (9). Therefore, published information
about the crystalline proteinases of Carica papaya is relatively scarce.
Like the cathepsins of animal origin, papain is regarded as an
intracellular proteinase. Its apparent sulfhydryl character and its a-
bility to synthesize peptide bonds under the proper conditions are prop-
erties also shared by the cathepsins. This similarity suggests that
-5- .
a study of crystalline papain could yield information of importance to
our understanding of the animal cathepsins, in addition to information
about the mechanism of proteolysis by sulfhydr,yl enzymes. The close re-
semblance between papain and certain of the cathepsins is particularly
worthy of attention when one considers the potential availability of
these enzymes. The concentration of papain in papaya latex is probably
several hundred fold greater than that of a cathepsin in its source ma-
terial. Therefore, it appeared to be theoretically possible to prepare
much larger quantities of papain than of a cathepsin. These reasons
were responsible for the initiation of the present study. The aim was
to develop a method whereby cr,ystalline papain could be prepared in good
yield from commercial dried papaya latex. This goal was achieved and it
is now possible to describe some of the physical, chemical and enzymatic
properties of cr,ystalline papain.
CHAPTER II
THE HISTORY OF PAPAIN
The medicinal properties of papaya latex were responsible for
much of the early interest in this material. Previous mention has been
made of the medical report regarding its anthelminthic value which ap-
peared during the seventeenth centur,y (165). Although two British nat-
uralists, Griffith Hughes (74) in 1750 and Patrick Browne (33) in 1756
described the use of papaya latex for tenderization of tough meat by
natives of the West Indies, no experimental work was published until
1873. At this time G. C. Roy (130) in India reported the digestive
action of papaya juice on animal and vegetable proteins. Roy's inter-
ests were undoubtedly medical because he recommended that papaya milk
be used to treat indigestion. Wittmack (173) in 1878 studied the di-
gestion of meat by papaya-latex, and the following year Peckolt (122)
made similar observations with a white odorless powder prepared from
fresh latex by alcohol precipitationo Peckolt named his product rtpa_
1
payotin". Both of these observers noted that their preparations caused
milk to clot. This action is now known to be inseparable from the pro-
teolytic action. It is interesting that these two reports appeared in
medioal literature.
The first well controlled scientific investigation of the active
IThe term "papay-otin" was used commercially by Merck and Coo to
describe their papain preparation until about 1940.
-6-
-7-
princi'p1e in papaya latex was carried out by-WUrtz and Bouchut (174) in
1879. These authors worked with fresh latex and noted that this mater-
ial could be separated into two fractions, the liquid latex, and the
pulp. The liquid latex when precipitated with 10 volumes of alcohol
yielded a white amorphous solid which digested fibrin in neutral, slightly
acid or slightly alkaline media. The digestion was observed to proceed
faster at 40
0
than at room temperature. Following removal of the liquid
latex, the pulp was washed with large quantities of water and the wash-
ings concentrated in yacuo. This material produced more extensive di-
gestion of fibrin than the liquid latex, but here it should be noted
that HeN was ueed as a preservative in the digestion mixtures. This
work was extended the following year by Wurtz (175, 176) and attention
was concentrated on the pulp since it was apparently the most active o
The treatment of the pulp washings was modified to include dialysis and
a lead acetate treatment prior to alcohol precipitation. The white amor-
phous powder obtained by this procedure was regarded as the pure active
ingredient
l
and was called "papain
t
'. Papain was characterized as an "al-
buminoid body" (i.e. a protein in modern terminology) but it did not fall
clearly into any known class of proteins according to the criteria of
that time. The nature of its digestive properties prompted WUrtz to
place papain in a class with tr,rpsin. Wurtz isolated crystals of leu-
cine from papain digests of fibrin. In these digests HeN had been added
as an antiseptic but it is stated that the rate of solution of fibrin in
the presence of papain was the same whether cyanide, boric acid or phenol
was the preservative. An attempt was made to gain some information about
IMoncorvo (113) in 1880 prepared a white amorphous powder from
papaya latex which he called t1caricintl. Martin (98) thought this might
also be the pure active ingredient. t
-8-
the mode of action of papain. A solution of papain was mixed with finely
.divided fibrin and allowed to incubate for 20 minutes. The liquid was then
filtered off and the undissolved fibrin subjected to prolonged washing with
cold watero It was noted that the last washing still contained digestive
activity. Furthermore, if the washed fibrin was resuspended in water and
incubated at 37
0
digestion still took place. Because of this, Wurtz rea-
soned that papain exerted its digestive action by becoming bound to the
fibrin.
The work of Wurtz and his collaborator was confirmed in ever,y re-
spect a few years later by Martin (98,99) who added the observation that
papain was active in neutral and alkaline media but inactive in acid media.
Martin found tyrosine in addition to leucine in his reaction mixtures af-
ter digestion of fibrin with papain.
The term "papain" applied by Wurtz to his purified material sub-
sequently came into general use and was applied to the crude latex as well
as to partially purified preparations. Until the actual crystallization
of a pure enzyme from papaya latex, "papain" was a relatively non-specific
term which was used to designate the proteolytic activity of papaya latexo
It will be used as such throughout this chapter 0
The Activation of Papain
Discovery and Coenzyme Theory.-- In the years following the work
of WUrtz and of Martin, interest in proteolytic enzymes became directed
towards the extent to which proteins were digested. In this respect pap-
ain became the center of a controversy. It will be recalled that both
WUrtz and Martin had demonstrated free amino acids in papain digests of
fibrin. Subsequent investigators were unable to duplicate thiso In
1892, Chittenden (36) reported that peptones were the major products af-
-9-
ter digestion of fibrin and raw or cooked m.eat with papain, and he could
demonstrate only minute amounts of leucine or tyrosine. Sharp (141) stud-
ied the digestion of egg and serum albumins and was unable to find even
peptones among the products. Dott (42) obtained similar results with egg
albumin. In 1898 .. Chittenden, Mendel and McDermott (37) repeated the
earlier work of Chittenden, confirmed the results, and concluded that
"even under the m.ost favorable conditions the formation of amino acids
or other crystalline products is extremely slight". Further confirma-
tion was provided by Mendel and Underhill(lll) who made extensive stud-
ies of papain digestion with a number of protein substrates. They could
demonstrate free amino acids among the digestion products from raw mus-
cle only and this they attributed to autolytic action. At this point
the controversy seemed settled, and it appeared that the original obser-
vations of WUrtz and of Martin were in error.
In the same year that Mendel and Underhill (111) completed their
thorough study, the English botanist, S. H. Vines (152) incidentally re-
ported positive tryptophan tests with chlorine water after digestion of
fibrin with papain. This, of course, immediately reopened the contro-
versy. Mendel (109), in a review article discussing the extent of di-
gestion of proteins by various proteolytic enzymes, openly criticised
the work of Vines on the grounds that no antiseptic had been used, and ,',:
therefore, the possibility of bacterial origin of the tryptophan had not
been excluded. Vines replied to Mendel in a series of papers (153, 154,
155) wherein he described experiments on papain digestion in the presence
of a number of antiseptics. He summarized his conclusions as follows:
It appears that neither chloroform nor sodium fluoride inhibits
the peptonizing action of papain, but that they both impede further
proteolysis with the formation of tryptophano----(The experiments)
strikinglY demonstrate the remarkable favorble effect of the pres-
ence of HeN upon the proteolytic activity.
-10-
This was the first demonstration of an acceleration of enzyme action by
cyanide; an effect, which today, is regarded as a general property of a
large group of enzymes known as sulfhydryl enzymes.
It appears that Vines regarded the effect of cyanide more as an
absence of inhibition than as an actual activation. Thus, he attributed
the low yields of amino acids obtained by Emmerling (45) from a six weeks
digestion of fibrin to a peculiarly unfavorable combination of conditions,
chief among which was the use of toluene as the antiseptic.
Following Vines' observations, reports in the literature still
continuedto deny the liberation of amino acids from proteins in signifi-
cant amounts after papain digestion (89). Abderhalden and Teruuchi (l)
reported that papain hydrolyzed but the yields of
amino acids were small even when relatively large amounts of enzyme
were used (1 gm. of papain per 1.5 gm. of peptide).
The next real contribution to the knowledge of papain was made
by Mendel and Blood (110) in 1910. These authors reviewed the litera-
ture up until that time with particular reference to the activating
effect of cyanide as reported by Vines. They pointed out that the pro-
longed digestion periods necessar.y in studies of proteolysis required
the use of an antiseptic to prevent bacterial contamination. In the
days of WUrtz and Martin, HeN was a common antiseptic but had fallen
into disuse in favor of "less harmf'ul
f1
SUbstances such as toluene,
thymol, chloroform, and sodium fluoride. Their study of the litera-
ture pointed to a direct correlation between the use of HeN as the an-
tiseptic in papain digestions and the yield of free amino acids after
digestion. Workers who had used other preservatives either failed to
isolate free amino acids or were able to do so after prolonged
digestion (up to ten months). The experimental work of Mendel and
-11-
Blood (110) unquestionably confirmed the work of Vines. Digestion of
proteins by papain was observed to be accelerated by HCN and free amino
acids w e ~ e easily demonstrated in these digests. This activating effect
was found to be shared by H
2
S but not by boric acid. Dialysis of the en-
zyme resulted in the loss of activity but re-addition of HeN restored the
activity. On the basis of these observations, Mendel and Blood concluded
that HCN functioned as a coenzyme, which imparted to papain the ability
to split peptones in addition to proteins.
Frankel (51) in 1917 made quantitative observations on papain hy-
drolysis of gelatin using the formol titration. He observed that cyan-
ide produced a two-fold increase in the specific activity of the enzyme.
He stated that HCN was not destroyed during the process of activation
since it could be completelY recovered at the end of the experiment.
This observation favored the coenzyme hypotheSiS, but Hellerman (67)
states that the experimental methods available to Frankel were prob-
ably inadequate to decide the question of cyanide recover,y. A few
years after the report by Frankel (51), Willstatter and GrassmannWf167)
published the results of an extensive study of papain. They noted that
,30 to 60 minutes was required for activation of papain by cyanide. In
relating enzyme concentration to rate of protein hydrolysis, they found
that the effect of cyanide corresponded essentially to an increase in
enzyme concentration. A later study from the same laboratory (168)
showed that papain could split gelatin but not peptone in the absence
of HeN, but when HeN or H
2
S was present both substances were rapidly
hydrolyzed. Cyanide activated to a greater extent than did H
2
S. These
findings led to the proposal by the authors that HCN or H
2
S acted to
broaden the specificity of papain and that the mode of action was simi-
lar to that of a coenzyme. Thus they wrote papain-HeN or papain-H
2
S.
-12-
The Deve1op!!nt of the Sulfhydryl Concept.-- The whole concept
of papain activation began to undergo drastic changes during the period
following the work of Wi11statter and his students. The change was ini-
tiated by the report of Ambros and Harteneck (5) in 1929 that latex fresh
from the papaya fruit itself was less amenable to the action of cyanide.
These studies demonstrated the presence of a dialyzable natural activator
in papaya latex which the authors named tlphytokinase
H
o
At the same time,
Waldschmidt-Leitz a1. (161) found that certain proteolytic enzymes in
animal tissues (cathepsins) closely resembled the papain-like plant pro-
teinases and also seemed to be associated with natural activators in the
tissue. Reduced glutathione was subsequently identified as such a natu-
ral activator for yeast proteinase by Grassmann a1.(63) and for liver
cathepsin by Waldschmidt-Leitz, Purr and Balls (162). However, it should
be noted that just prior to the identification of this natural activator,
Grassmann et a1 (61, 63) had demonstrated that cysteine and reduced glu-
tathione were as effective as HON or H
2
S in the activation of papain,
:yeast proteinase and lddney cathepsin. These observations were confirmed
later by Waldschmidt-Leitz and PUrr (160).
The addition of cysteine and glutathione to the list of activa-
tors of papain did not markedly alter the ideas concerning the action
of these substances. Grassmann (63) discussed the various possi"':'-
bi1ities and considered the usual coenzyme idea (e.g. papain-cysteine).
However, these authors also noted that all of the activators for papain
were excellent inhibitors for certain metal-catalyzed enzymatic reactions.
They mentioned the possibility that cyanide, H
2
S and cysteine might pro-
duce activation of papain simply by inhibitory heavy metals from
solution. Krebs (S7) had apparently come to the same conclusiono He car-
ried out studies showing the extreme sensitivity of papain to such metals
-13-
as copper, silver, gold, zinc, cadmium, and mercur,y. A long list of
other metals had no inhibitory effects. The inhibitory metals pro-
duced 50 per cent inhibition of gelatin Qydrolysis in concentrations
of the order of 10-5M This inhibition was found to be reversible by
cyanide, H
2
S or cysteine. Krebs pointed out that the heavy metal con-
tent of most protein preparations was sufficiently high to account for
a suppression of the major portion of papain activity. On these grounds,
he claimed that the activating agents functioned to remove heavy metals.
Furthermore, the activating effects of citrate and pyrophosphate were
explicable in the same manner. Waldschmidt-Leitz and Purr (159) criti-
cised the work of Krebs after experiments of their own wherein gelatin
was pretreated with cyanide prior to exposure to papain. Under these
conditions, papain was still activated by HeN. Thus, they claimed that
papain was activated by HeN even in the absence of heavy metals. Krebs
replied (8S) that the aforementioned authors had not taken into consid-
eration the nature of the metal impurities in gelatin of which zinc was
apparently the most prominent. He stated that zinc inhibition was not
reversible by cyanide; a phenomenon which had been demonstrated earlier
by Jacoby (81). An English investigator, Murray (117), provided the most
satisfactor,y follow-up to the work of Krebs. Murray confirmed Kreb
8
s ob-
servations about heavy metal inhibition of papain. He also found that
this inhibition was reversible, but in contrast to Krebs, his findings
demonstrated that citrate or pyrophosphate and cyanide were not inter-
changeable in their activating effects. These agents actually produced
additive stimulation of papain action on gelatin. The thorough experi-
ments of Murray justified his hypothesis that other factors, in addition
to heavy metal removal, were involved in papain activation.
-14-
It should be emphasized that cysteine and glutathione were ef-
fective activators of papain only in the reduced state. In this form
they each react with sodium in alkaline solution to give
a red color. The disulfide or oxidized form does not react., Maschmann
and Helmert (101) utilized this reaction to study papain activation.
They first demonstrated that papain was completelY inhibited by iodo-
acetic acid, a reagent known to react with thiol groups. This inhibi-
tion occurred even in the presence of cysteine. Papain-cysteine mix-
tures gave a positive nitroprusside test which did not decrease in in-
tensity after addition of iodoacetate. From this the authors concluded
that the inactivation of papain by iodoacetate was the result of a re-
action with the enzyme itself. This conclusion was not completely justi-
fied from their data, but later studies gave it some support. Solutions
of papain or liver cathepsin which gave a negative nitroprusside test
were observed to give a transient positive test atter activation with
cyanide. Maschmann and Helmert then postulated that the activation ot
papain and cathepsin resulted from the reduction of -5-5- groups to SH
groups. Finally, studied dialyzed cathepsin preparations and found
that HeN activation resulted in the development ot weakly positive nitro-
prusside reactions. However, their data did nqt permit them to make any
correlation between the strength of the nitroprusside test and the activ-
ity of the enzyme, and so they were unable to decide whether HeN or cyst-
eine activation reduced disulfide groups on the enzyme molecule or on
"impurities" in the enzyme preparations.
The publication of Maschmann and Helmert (101) was followed al-
most immediately by a report by Berein and Logemann (29) wherein it was
demonstrated that papain behaved in many respects like cysteine or cys-
tine. It was pointed out that all the known activators were capable of
-15-
reducing disulfide bonds. To the list of known aetivators they added
thioglycolic acid, thiolactic acid and sulfite. Like cysteine, active
papain apparently reacted with halogenated acetic acid derivatives (e.g.
iodoacetic acid). Cysteine could be reversibly oxidized with I2 or H
2
02
and papain when treated with these reagents was found to be inactivated.
The inactivation could be partially reversed by addition of the known ac-
tivating agents, all of which were capable of r.educing cystine. To fur-
ther sUbstantiate their idea that papain activation was the result of a
reduction reaction Bersin and Logemann set up a biological system (succ-
inic denydrogenase, succinate) and found that inactive papain was effect-
ively activated. In a later report, Bersin (26) produced more evidence
to support his ideas. Cystine is reduced to cysteine on exposure to ul-
traviolet light; similarly papain was activated on brief exposure to the
same radiation. On the basis of the known behavior of certain arsenic
compounds with cystine and cysteine, Bersin was able to correctly pre-
dict which compounds would activate papain and which would inhibit. Ber-
sin postulated that inactive papain existed as a disulfide (PaSSPa) and
on. reduction to PaSH it became active. It is interesting to note that
Hellerman, Perkins and Clark (69) had presented an identical hypothesis
in regard to cr,ystalline urease a very short time before the report of
Bersin and Logemann. Later, Hellerman and Perkins (68) extended their
observations to papain and agreed in all respects with Bersin and Loge-
mann. They demonstrated that papain was inactivated reversibly on ex-
posure to air or on oxidation with 1
2
- Furthermore, the enzyme was
strongly inhibited by univalent organic mercurials, such as phenyl-Hg-Cl,
compounds known to bind strongly to thiols. This inhibition was readily
reversed with cysteine.
It is now known that not only papain and urease, but a large
-16-
group of enzymes are activated by HeN, cysteine, etc. These enzymes
are generally referred to as sulfhydryl enzymes. The concept developed
by Bersin and Logemann, and by Hellerman, Perkins, and Clark is believed
to apply to this entire group (67; 27). The activation phenomenon is
commonly represented in the following schematic fashion:
2 En-SH
oxidation. En-S-S-En + 2 (H)
\
(active) reduction (inactive)
The potent inhibitor,y power of heavy metals on papain is a property
shared by most of the other enzymes, and the affinity of
the free thiol groups on the protein for these metals is
held to be responsible for this effect.
In this manner, papain figured prominently in the develop-
ment of a new and basic concept. A concept that has had far reach-
ing significance to biochemistry, but about which much remains to
be learned.
Reactions to the Sulfhydryl Concept.-- The experimental work
on which the concept of the thiol nature of papain was based had in-
volved a large variety of observations. Berein (28) later extended
his observations and was able to correlate positive nitroprusside
tests with enzyme activity. Several other investigators (57, 58, 125)
confirmed and added to the original observations on papain. In gen-
eral the theor,y was well accepted. Nevertheless, there were certain
findings which could not be readily explained by the thiol hypothesis
and this led to some criticism.
Maschmann and Helmert (102) objected to the sulfhydryl theory
on the grounds that iodoacetate inactivated papain even if added after
cysteine. Since they felt that iodoacetate inhibition was an oxidation
-17-
reaction similar to that with 1
2
, they eould not explain this phenom-
enon by the sulfhydr,yl theory. In a later paper these same authors
(107) again objected to the hypothesis because they had found that fer-
ricyanide activated papain. Bersin (28) answered this objection by
pointing out that ferricyanide would probably be reduced in the veronal
buffer used by Maschmann and Helmert and therefore, they were measuring
an effect of ferrocyanide.
The most valid objections to the thiol theor,y came from the lab-
oratories of Max Bergmann at the Rockefeller Institute. Bergmann and
his coworkers used protein substrates and espeeially synthetic substrates
such as hippur,ylamide and to study the action
of aqueous extracts of crude papaya latex. The initial observations of
Bergmann and Ross (19) appeared to demonstrate that their enzyme prepara-
tions contained not only a proteinase but also a polypeptidase. Although
they later abandoned this idea (14) some important findings were obtained.
It was noted that oxidation with I2 or H
2
02 was not reversible,
contrar,y to popular opinion (22). In addition, Bergmann and Fruton (14)
found that combinations of activators were more effective than a single
activating agent. The same authors (55) reported that activated papain,
when precipitated with isoprop,yl alcohol, was inactive when redissolved,
but could be reactivated fully with cyanide. In this paper it was also
reported that different activators produced degrees of activity
towards -benzoyl-b-argininamide than cyanide, but the latter was a more
effective activating agent when was
the substrate. On this basis they felt that the activator played a role
in determining the specificity of the enzyme and suggested that an enzyme-
activator complex existed. Greenberg and Winnick (64) found that cysteine-
activated papain lost half of its activity when precipitated by alcohol,
-18-
and if the precipitate was aerated a large irreversible loss of activity
took place. Later Winnick, Cone, and Greenberg (171) studied crystalline
ficin, a proteolytic enzyme from the fig which is very similar to papain,
and demonstrated that anaerobic dialysis did not inactivate cysteine-acti-
vated ficin. On the other hand aerobic dialysis produced 96 per cent in-
activation. Therefore, they expressed the opinion that papain, like ficin,
was highly susceptible to oxidation by air and that the alcohol precipita-
ted papain studied by Fruton and Bergmann (55) had been inactivated by
this mechanism. In addition, it was felt unnecessary to postulate an en-
zyme-activator complex since ficin remained active after removal of cyst-
eine by dialysis under anaerobic conditions.
Prior to the criticism of Winnick ~ 91, Bergmann's group made
other observations with dialyzed, partially purified preparations of pap-
ain (78, 79). They found that the hydrolysis of -benzoyl-1-argininamide
was a first order reaction. Cyanide or traces of cysteine, glutathione
or H
2
S would not cause acceleration of hydrolysis of this substrate. The
combination of HCN with one of the other agents resulted in activation of
the enzyme. Removal of the HeN in vacuo reversibly destroyed the activity.
When higher concentrations of H
2
S were used, it was found that this sub-
stance alone was capable of activating either dialyzed or undialyzed pap-
ain. In the case of the undialyzed enzyme, removal of the H
2
S in vacuo
did not bring about inactivation. Since the dialysate was known to con-
tain "natural activators", the authors reasoned that H
2
S was capable of
transforming potential activators into true activating agents while HeN
was not. These activation experiments were all carried out at pH 5. At
this pH, H
2
S will reduce oxidized glutathione but HeN will not
o
T h e r e f o r e ~
the authors expressed the opinion that the reversible oxidation-reduction
of thiol groups could not alone account for the reversible inactivation
-19-
and activation of papain by various agents. Their ideas regarding this
subject they expressed schematically as follows:
oxidation H
2
S
ct-papain ( (3 -papain ;) H
2
S-papain
(inactive) (inactive) (evacuation (active)
Using this hypothesis, it might be predicted that papain activity would
be dependent upon the nature of the activating agent or agents. This
was shown to be the case when precise kinetic studies were carried out
with partially purified papain (80). No satisfactory answer to the
postulates of Bergmann il.. was produced even when studies were carried
out with crystalline papain.
Several other investigators have either criticised the thiol the-
or,y or made postulations of their own. Seshagirarao and Giri (139, 140)
found that papaya latex, when treated with copper sulfate until it gave
a negative nitroprusside test, still retained 70 per cent of its activity
towards gelatin and peptone. It seems probable that the substrates in
this case were more effective copper-binding agents than papain. These
two authors suggested that a dienol group was the active center. Okumura
(120) postulated that an aldeq,de group was responsible for papain activ-
ity. Scott and Sandstrom (138) studied the activation of papain by a
series of aliphatic mercaptans and on the basis of their results suggested
that papain activation was a surface phenomenon. None of these theories
has received extensive support.
Other Activating Agents for Papain.-- Aside from the usual acti-
vators, HeN, H
2
S, cysteine and glutathione, a large number of other sub-
stances are now known to be capable of activating papain. Among these
are thioglycollic acid and thiolactic (29), thioglucose (19) and
thiosulfate (82). In addition, Maschmann and Helmert (103) found that
a whole series of aliphatic acids activated papain, but ex-
-20-
cept for thioglycollic acid, activation took place slowly. Thioacetic
acid (CH
3
COSH) was also found to be effective. Scott and Sandstrom (138)
activated papain effectively with thiophenol, benzylmercaptan and ethyl
through heptyl mercaptan. Thiophenol was found to be the best activator
in this group. More recently, Gawron and Cheslock (59) tested a series
of ar.ylamides of mercaptoacetic acid and found that all of these were as
effective as cysteine.
The ability of ascorbic acid to undergo reversible oxidation and
reduction led Maschmann and Helmert (103, 104, 105) to test the activat-
ing powers of this substance on papain. They found that ascorbate in-
hibited the enzyme, but a combination of ferrous iron and ascorbate was
as effective as cysteine in activating papain. Purr (126) confirmed this
observation but added that activation by ferrous iron and ascorbate occur-
red only in crude enzyme preparations. He was unable to demonstrate ac-
tivation in purified preparations and felt that this effect was an in-
direct one due to reduction of disulfide bonds on the impurities and sub-
sequent activation of papain by these thiol groups.
Inhibitors of Papain.-- All of the known inhibitors of papain
are either oxidizing agents or substances known to react with mercap-
tans readily. Iodoacetic acid and the heavy metals fall into the latter
class. The oxidation of papain with 12 of H
2
0
2
has been widely used to
inactivate the enzyme. This inactivation is only partly reversible.
Fischer and Bacq (46) found that certain war gases similar to Lewisite
were extremely potent inhibitors of papain presumably because of the
ability to react with thiols. Bersin and Logemann (29) found that qui-
none inactivated papain and Hoffmann-Ostenhof and Biach (70) extended
these observations to include a number of similar reagents
o
-21-
One interesting type of inhibition of papain activity has never
been adequately explained. It will be recalled that Bergmann and Ross
(19) felt that they had demonstrated two types of activity in crude pap-
ain preparations; proteinase and polypeptidase. They measured the first
by gelatin hydrolysis and the latter by the splitting of hippurylamide.
Phenylhydrazine was found to inhibit the hydrolysis of hippurylamide but
not the protein digestion. Phenylhydrazine inhibition was readily abol-
ished with benzaldehyde. Hydroxylmaine inhibited both actions of papain
especially the polypeptidase action (20). This two enzyme hypothesis
was later abandoned (14) but the action of phenylhydrazine remained un-
explained. It should be noted that phenylhydrazine was also found to
be capable of activating papain under certain conditions (14, 24). Schalee
!!.(135) studied the hydrolysis of egg white by papain and found that
hydrazine, semicarbazide, and hydroxylamine produced
about 30 per cent inPibition. One wonders whether the observations de-
scribed above actually represented inhibition. In the light of present
knowledge regarding hydroxylamine in particular, it appears likely that
transfer reactions resulting in the formation of hydroxamic acids or
phenylhydrazides could have obscured the changes which Bergmann's group
measured by titrating with alkali. In the study by Schales al.hydroly-
sis was measured by the decrease in turbidity of egg white suspensions.
Here it seems equally likely that the formation of insoluble products
resulting from transfer reactions could have prevented the usual de-
crease in turbidity observed in the absence of inhibitors.
The Effect of pH on Papain Action
The substrate for most of the early work with papain was fibrin
and it was generally agreed that this substance was digested most rapidly
-22-
in neutral media. Egg albumin digestion had been studied by Ripptoe
(128) and found to proceed best in slightly alkaline solutions. Simi-
lar findings were reported by Pratt (124) with regard to the milk
clotting action. One investigator, Graber (60), felt that papain di-
gestion was most rapid in acid solutions.
The first real pH-activity curve for papain was determined by
Frankel (51) in 1917. He found that the pH optimum for gelatin hydroly-
sis was at pH 5. Willstatter and Grassmann (167) confirmed this and
later (169) reported that the pH optimum for fibrin was at pH 7, and for
albumin peptone. near pH 5 . Greenberg and Winnick (65) found that de-
natured ovalbumin was hydrolyzed most rapidly at pH 7.5 and urea dena-
tured hemoglobin at pH 7 to 8. Lineweaver and Schwimmer (94) found that
crystalline papain split casein most rapidly at pH 6.5 to 7. It is
apparent that with protein substrates the optimum pH for papain hydroly-'
sis is dependent to a large extent upon the nature of the substrate it-
self.
With synthetic substrates pH 5 appears to be the optimal value.
The extensive speoificity studies of Bergmann all carried out
at this value. Hoover and Kokes (73), with partially purified papain,
found that a-benzoyl-&-argininamide was split best at pH 505, and hippur-
ylamide or at pH 5. Roche e Silva and And-
rade (129) found two pH optima for the hydrolysis
amide.
The Effect of Specific Anions on Papain Action
The accelerating effect of citrate and pyrophosphate on papain
action were attributed by Krebs (87) to complex formation with inhibi-
tory heavy metals. Murray's thorough investigations (117) demonstrated
-23-
that the pH optimum for gelatin hydrolysis actually varied according to
the buffer used. Thus, at a given pH hydrolysis was more rapid in ace-
tate than in phthalate and still more rapid in citrate. Maschmann (100)
and Maschmann and Helmert (106) made similar observations with regard to
acetate and citrate.
Strong emphasis was placed on the activating effect of citrate
by Fox and Pettinga (49). These authors noted that citrate ion markedly
accele rated the r,ate of synthe sis of acylamino acid anilides catalyzed
by papain. The yield of anilide was also increased in the presence of
citrate. Fox (48) later postulated a mechanism of protein synthesis
potentiated by citrate.
The Specificity of Papain
The liberation of free amino acids during the digestion of pro-
teins by papain which many of the early workers reported (155, 110) sug-
gests that papain could be expected to split peptide bonds involving a
wide variety of amino acids. Results of a quantitative nature were first
reported by Leipert and Hafner (91) who found that 47 per cent of the
total nitrogen of casein was liberated as amino nitrogen after twelve
days digestion with papain-HeN. More definitive results were obtained
by Calvery (35). This investigator studied the hydrolysis of crystal-
line egg albumin by papain, pepsin, and trypsin and found that after
papain digestion neither pepsin nor t ~ p s i n could bring about further
hydrolysis. On the other hand, digestion with pepsin or trypsin always
left room for further extensive action of papain. Bergmann and Niemann
(18) observed that 64 per cent of the peptide bonds of fibrin were split
by papain in a 20 day digestion.
The first use of synthetic substrates was made by Abderhalden
-24-
and Teruuchi (1) who found that papain hydrolyzed glycyl-1-ty-rosine very
slowly, if at all. Frankel (51) studied the of a group of di-
peptides of alanine and glycine and reported that activated papain was
incapable of splitting these. WillstUtter and Grassmann (167) came to
similar conclusions with glycylglycine and leucylglycine but found that
leucylglycylleucine was hydrolyzed at a significant rate. Glycyldiamino-
propionic acid and asparaginylhistidine were observed to be hydrolyzed
by papain by M1yanoki (112). In 1944 igren (2) studied the hydrolysis
of a number of synthetio di- and tripeptides by' HeN-activated aqueous ex-
tracts of papaya latex. He found that all the ':tripeptides were split and
a few of the dipeptides, e.g. valylglycine and alanylglycine.
The development by Bergmann and Zervas (21) of a practical method
for the synthesis of a wide variety of peptides and 'peptide derivatives
was followed by extensive studies of the specificity of papain. As each
new group of derivatives became available they were tested against papain.
Hippurylamide was one of the early synthetic substrates found to be rapidly'
split by papain (23). Later it was found that benzoyl-&-isoglutamine was
even more rapidly hydrolyzed (20), and finally Bergmann and Fruton (14)
demonstrated (BAA) appeared to be the most
sensitive substrate of all. In this sense papain appeared to resemble
trypsin in its specificity since BAA was also the best substrate for
this enzynie. However, after investigation of over 65 peptides and pep-
tide derivatives, it became apparent that the specificity of papain was
not nearly so limited as that of tr,rpsin (14, 16, 20, 23, 24, 25, 41).
The essential features of the specificity of papain have been reviewed
by Bergmann and Fruton (17). It was pointed out by these authors that
papain hydrolyzed peptides derived from a wide variety of amino acids,
and that it was capable of splitting substrates with or without free
-25-
alpha amino groups or alpha carboxyl groupsl. Papain was .found to ex-
hibit stereochemical specificity towards 1 forms since it was incapable
of splitting peptide bonds involving Q-amino acids.
All of the specificity work described in the literature was car-
ried out with crude or partially purified preparations of papain. The
fact that these preparations might contain more than one proteolytic en-
zyme was never overlooked by any of the investigators, Berg-
mann and his coworkers. This fact was a major deterrent to the conclu-
sive description of the specificity of papain, but as will be evident
later in the studies on cr,ystalline papain, much ot the intormation can
be applied to the pure enzyme. The first evidence in this regard was re-
ported by Balls and Lineweaver (8) who demonstrated the hydrolysis of hip...
pur,y1amide by crystalline papain.
Synthetic Actions of Papain
Since it was known that certain of the enzymes were
capable of synthesizing peptide bonds as well as hydrolyze them (166),
the development of the sulfhydryl hypothesis led to considerable specu-
lation with respect to papain and especially the cathepsins. The opin-
ion was expressed that the oxidation-reduction potential of the intra-
cellular spaces were a major factor controlling the synthesis and break-
down of protein (27). It was postulated that -5-S- forms of the eathep-
sins were synthetically active while the SH forms, arising from post-mor-
tern lowering of oxygen tension, were hydrolytically active. This was
offered as an explanation of post-mortem autolysis. Voegtlin .:t . YO!' ,
I
With regard to free carboxyl groups it should be noted that most
of the substrates with free carboxyl groups that were tested were tri- or
tetrapeptides. In these cases, splitting occurred one or more peptide
bonds removed from the bond adjacent to the free carboxyl.
-26-
(156, 157, 158) actually carried out experiments wherein they apparently
demonstrated that aeration of papain digest of casein resulted in an in-
crease in trichloracetic acid precipitable material. Strain and Linder-
strp'm-Lang (147) were unable to confirm this and later Linderstr,Jm-Lang
and Johansen (93) expressed the opinion that Voegtlin's observations re-
suIted from evaporation during vigorous aeration of the reaction mixtures.
A different approach was used by Bergmann and Fraenkel-Conrat (13)
in studying the synthesis of peptide bonds by papain. These authors in-
cubated carbobenzoxy-glycine and aniline with activated papain and found
the carbobenzoxyglycine anilide was synthesized. This reaction was sub-
ject to the same requirements as the hydrolytic reactions. Activating
agents were essential and the specificity requirements were no different

than for hydrolysis of peptide bonds by papain. Later, Behrens and Berg-
mann (12) extended this work and demonstrated that acetyl-1-phenylalanyl-
glyoine plus glyoine anilide yielded
anilide in the presence of activated papain. The same reactions have
been studied by Fox (49,50) and by Albertson (3). It should be em-
phasized that the products of these reactions were insoluble oompounds
that precipitated from the reaction mixtures almost as soon as they were
formed. Therefore, what was apparently an equilibrium reaction was per-
mitted to proceed to completion. While such reactions resnlt in the for-
mation of new peptide bonds, it is now recognized that, in reality, they
are transfer reactions. A peptide substrate when in combination with a
proteolytic enzyme, e.g. papain, can react with water resulting in hy-
drolysis; or it can react with other such as hydroxylamine to
form the corresponding hydroxamio acid. In a similar manner it can re-
aot with the free amino groups of another amino acid or peptide to form
a new peptide bond. For example, glycyl-b-phenylalaninamide reacts with
-27-
hydroxylamine in the presence of cathepsin C to yield
hine hydroxamic acid (84)0 Even more striking is the formation of an
octapeptide amide from glycyl-&-phenylalaninamide in the presence of
cathepsin C. This product is insoluble at pH 7.6 so that the apparent
pH optimum for the reaction is in this region (56). However, Durell and
Fruton (43) have shown that the same reaction proceeds at pH 5 also even
though the octapeptide is soluble at this pH. In the case of papain
Durell and Fruton (43) have demonstrated recently that transfer reactions
appear to be more likely to proceed than hydrolysis. Aecording to these
authors hydroxylamine is about 420 times more efficient in its reaction
with the papain-substrate complex than is water and this efficiency may
be even greater in reactions with other amino acids or peptides. In
contrast, the intestinal proteinase, tr,ypsin, is more efficient in the
hydrolytic reaction; a result which is not surprising in view of the
apparent purpose of this enzyme in the gastrointestinal tract.
The Purification of Papain
The question whether papaya latex contained more than one proteo-
lytic enzyme was first raised by Vines (155) who felt that the protein
splitting and peptone splitting actions were associated with different
entities. Willstatter, Grassmann, and Ambros (170) tried to separate
these components by salt fractionation and by adsorption on alumina but
were unable to do so and concluded that only one enzyme was involved.
The two enzyme hypothesis of Bergmann !1.has previously been discussed.
In general, it appears that most all investigators were of the opinion
that more than one proteolytic enzyme was present in papaya latex but
convincing proof was lacking.
The early attempts at purification were designed primarily to
-2S-
remove activating agents, but several workers achieved several fold puri-
,fication by their procedures (51, 52, 124). It is interesting to note
that Kilmer in 1901 (86) tried thirty different methods of purifying the
proteolytic ingredient in papaya latex. Some of these were quite dras-
tic in the modern sense, but some degree of purification was apparently
achieved. Two investigators used adsorption methods in attempts to
purify papain. and Grassmann (167) achieved a two-fold puri-
fication by adsorption of alumina, and Okumura (119) a lO-fold purifica-
tion by adsorption on takaamylase.
In 1937 Balls and Lineweaver (8) announced the cr,ystallization
of a proteolytic enzyme from fresh papaya latex. This enzyme was called
papain. It crystallized in tiny needles. The preparative procedure in-
volved first making a water extract of the fresh latex. Papain was pre-
cipitated from this by 0.4 saturation with ammonium sulfate. The precpi-
tate was redissolved and then could be reprecipitated by addition of e-
nough laCl to bring the concentration to 10 per cent. Finally the enzyme
was cr,ystallized after resuspending the precipitate from the 10 per cent
salt solution and adjusting the pH to 6.5. Papain was recr,ystallized by
redissolving the first cr.ystals and raising the salt concentration of the
solution to about 2 per cent. The striking decrease in the solubility of
the enzyme as it is separated from other proteins of the latex is apparent.
In fact, crystalline papain can be almost completely precipitated fram aque-
ous solution by 2 per cent NaCl. Papain was found to be readily soluble in
70 per cent ethanol. In this respect it could be classified as a prolamine.
Crystalline papain, like the crude enzyme, required the usual acti-
vating agent or agents for full activity. In the absence of cyanide it
gave a negative nitroprusside test. Iodoacetate inhibited the enzyme and

-29-
oxidation with H
2
0
2
caused inactivation. Papain was sensitive to oxida-
tion by air so that it was necessary to use cyanide, cysteine or a nitro-
gen atmosphere throughout the preparation to prevent irreversible inacti-
vation.
By osmotic pressure measurements, Balls and Lineweaver (9) con-
cluded that the molecular weight of papain was 27,000 ! 2000. The molec-
ular weight calculated from the diffusion constant was 38,200. The iso-
electric point was stated to be above pH 8.5.
Chemical analysis revealed that papain oontained 15.4 per cent
nitrogen, 1.2 per cent sulfur, and 3.7 per cent cystine. Van Slyke amino
-nitrogen determinations demonstrated the presence of approximately 8 free
amino groups per molecule. From the cystine content, and assuming the
molecular weight to be 30,000, Balls and Lineweaver (10) calculated that
crystalline papain contained about ten thio1 groups. Quantitative mea-
of iodoacetate inhibition showed that one molecular equiva-
lent was sufficient to completely inhibit papain action, and the authors
concluded that one sulfhydryl group was involved.
Lineweaver and Schwimmer (94) studied the stability of crystal-
line papain towards acid, heat and urea. They found that papain was
rapidly destroyed below pH 3.5 at 30
0
However, it was remarkably stable
towards heat at pH values near neutrality. For example, after 30 minutes
at 60
0
only about 10 per cent of the original activity was lost. Papain
was found to be stable for at least 18 hours in 9 M urea solution at
room temperature.
Balls and Lineweaver (9) attempted to prepare crystalline papain
from dried papaya latex by the same procedures used for fresh latex, but
were unsuccessful. Apparently many other investigators shared this ex-
-30-
perience, although only one published report is available (55). As a
result the data of Lineweaver and Balls (9, 10) constituted the
published observations on the cr,ystalline enzyme.
In the description of the preparation of'cr.ystalline papain,
the authors noted that considerable activity remained in a fraction
devoid of papain and not precipitable by 0.4 saturation with ammonium
sulfate. This they called "high salt fraction". In 1941, Jansen
and Balls (83) crystallized another proteolytic enzyme from this frac-
tion. This enzyme was called ffehymopapain". Its milk clotting activity
was equal to that of papain but its proteolytic activity was about one-
half as great. In several respects chymopapain had even more remark-
able properties than papain. It was crystallized in the form of broad
sabre-like needles at pH 2, and indeed, was found to be stable at this
pH for long periods of time at low temperatures. Chymopapain was con-
siderably more soluble than papain and was apparently present in much
greater quantities in the latex.
Thus, it was demonstrated that papaya latex contained
more than one proteolytic enzyme. Although the preparative procedures
for both enzymes have been adequately described, the scarcity and high
cost of fresh papaya latex have been a deterrent to duplication of the
work of Balls Because of this and the fact that commercial dried
papaya latex has not been useful as starting material, informa.tion con-
cerning crystalline papain is limited to the papers of Balls et al. and
- -
of Lineweaver and Schwimmer.
CHAPTER III
EXPERIMENTAL METHODS
In the course of this investigation certain techniques were em-
ployed routinelY. It is the purpose of this chapter to describe these
techniques. In some cases, special methods or alterations of the rou-
tine procedures were used. These will be described in connection with
the experiments where they were applied.
In general, all of the inorganic chemicals, e.g. those used for
the preparation of buffer solutions, were analytical reagent grade. All
enzymatic reactions were carried out in deionized water. This contained
less than one part per million of ionic material (expressed as NaCI) and
was prepared by passing distilled water through a mixed bed ion exchange
resin.
The Determination of-Protein Concentration
I
The determination of the specific activity of a given enzyme
preparation requires a knowledge of the amount of protein in the solu-
tion under study. Such knowledge is also necessary in physical studies
with the electrophoresis apparatus or the ultracentrifuge. The most
rapid and convenient methods for obtaining this information were found
to be the turbidimetric method of BUcher (34) and a refractive index
method.
The method of BUcher was employed in the preparative work and
~ 3 l -
-32-
in studies of enzymatic activity. The protein solution was diluted by
an approptiate amount and a 1 ml. aliquot was mixed with 0.5 ml. of a
9 per cent trichloracetic acid solution. After turbidity was fully de-
veloped (30 seconds) the optical density of this suspension was measured
at 420 mu in a spectrophotometer. Solutions of papain of accurately
kndwn nitrogen content were treated in the same manner to establish a
standard curve. These had been dialyzed prior to.ana1ysis
to insure the absence of non-protein nitrogen. From these data and a
knowledge of the nitrogen content and molecular weight or crystalline
papain, it was possible to construct standard curves sucrrthat protein
concentration in mg. protein N per ml., gm. per liter; or moles per
liter could be read directly. The most commonly used units were mg.
protein N. per ml. Fig. 1 is the standard curve for the BUcher method
in these units.
0.03
DE NSlTY
0.01
2 S 8 '0 '2
MG. N PER ML.X 10
2
Figure 1. The Standard Curve for the Bucher Protein Determina-
tion. Solutions of crystalline papain of accurately known nitrogen con-
tent were used to obtain these data. One ml. of each solution was mixed
with 0.5 ml. of 9 per cent trichloracetic acid and the optical density
read at 420 mu in a spectrophotometer.
-33-
A refractive index method was used to determine protein concen-
tration in most of the physical studies where knowledge of the relative
concentration is sufficient. The refractive indices of the protein so-
lution and a control solution of identical composition, except for the
absence of protein, were compared. The protein concentration was cal-
culated on the assumption that a 1 per cent solution produces a refrac-
tive index increment of 0.00184. This value is probablY within a few
per cent of the correct value as judged by Kjeldahl analyses.
Measurement of Hydrolysis of Synthetic Substrates
Assays of papain activity on synthetic peptides and peptide de-
rivatives were performed by the alcohol titration technique of Grassman
and Heyde (62). The reaction mixtures were prepared in 2.5 ml. volumetric
flasks. One ml. of a 0.125 M substrate solution or', 0.125 millimoles of
the solid substrate and 0.5 ml. of a 0.1 M buffer solution at the desired
pH were mixed in the reaction flask. If necessary, pH adjustments were
made at this time by addition of 1 N HCl or 1 I laOH. The enzyme solu-
tions were generally prepared in 0.05 M cysteine and O.QlO M Versena (eth-
ylenediaminetetraacetic acid dihydrate) at pH 5 to 7. Both the reaction
mixtures and the enzyme solutions were equilibrated at 39
0
in a constant
temperature bath prior to initiating the reaction. The reaction was
started by-adding 0.25 ml. of the enzyme solution to the reaction mix-
ture followed by immediate dilution to volume and mixing. Aliquots
(0.2 ml.) were removed and titrated for carboxyl groups immediately af-
ter mixing and at appropriate times thereafter.
The t i t r a t i o ~ s were carried out with approximately 0.01 N KOB in
90 per cent ethanol. This solution must be standardized each day of use
o
The end point of the titration was determined with thymolphthaleino The
':"34-
aliquot from the reaction mixture, plus two drops of indicator, was ti-
trated to a definite blue color. Following this, 1.8 ml. of absolute
ethanol was added and the titration was continued until a light blue
color developed. The difference between the titration value at a given
time and that at zero time was the increment of base from which the ex-
tent of hydrolysis was calculated.
After a sufficient number of samples had been titrated, the pH
of the remaining reaction mixture was determined at room temperature
with a Cambridge Instrument Co. glass electrode standardized against
0.05 M potassium acid phthalate taken to be pH 4.00.
The most sensitive substrate for papain appears to be
L-argininamide (BAA). Therefore, this substance was used as the refer-
ence standard in the preparative work. It should be noted that certain
other substrates studied were only sparingly soluble in the reaction med-
ium. In these cases enzymatic hydrolyses were run on the suspended ma-
terial, although it is obvious that the concentration of substrate may
be a limiting factor in determining the reaction rate. In most cases
the enzyme concentration is the rate limiting factor. When insoluble
substrates were studied great care was' taken to insure that this'material
was in a finelY divided state in the reaction mixture. This prevented
mechanical difficulties in removing aliquots for titration and promOted
more rapid solution of unhydrolyzed substrate as the reaction progressed.
Measurement of the Hydrolysis of Esters
The hydrolysis of a peptide bond liberates equal amounts of acid
and base so that large changes in pH are not ordinarily encountered in
such reactions. This is not true for esters. Therefore, if ester hy-
drolysis is to be studied at a known pH value some arrangement must be
-35-
made to prevent drastic pH changes as the reaction proceeds. Schwert et
al.(137) have described a procedure that accomplishes this very well.
Ester hydrolyses witn papain were carried out in a 10 ml. beaker
immersed in a constant temperature bath at 39. The outside electrodes
from a pH meter, a jet through which nitrogen could be bubbled to accom-
plish mixing, and a pipette were suspended in the beaker. The pipette
was a graduated 1 ml. pipette with the tip drqwn out to a fine capillary
and bent at right angles. A 1 ml. syringe was attached to the other end
of this pipette and held in a clamp. This arrangement facilitated the
addit.ion of small amounts of alkali during the' reaction. Substrate, di-
lute buffer, and water were placed in the beaker to make a total volume
of 4.5 ml. The stream of nitrogen was started and the solution was al-
lowed to come to bath temperature. At zero time, 0.5 ml. of the enzyme
solution was added and the pH of the reaction mixture was recorded. As
hydrolysis took place, the pH decreased. When this decrement amounted
to 0.2 of a pH unit, the time required for this drop to oecur was noted,
and approximately 0.1 rol. of 0.2 N NaOH was added under the surface of
the reaction mixture by means of t h ~ pipette described above. Again the
time required for the pH to fall to the desired value was recorded and
more NaOH was added. This procedure was repeated until hydrolysis was
complete. From the amount of alkali neutralized it was possible to cal-
culate the extent of hydrolysis. The time required for neutralization
of a given amount of base permitted the calculation of the rate of hydrol-
ysis.
With this method it is necessary to estimate the amount of alkali
consumed during the initial reaction period prior to the first addition
of base. This was d Olle by adding varying amounts of alkali during the ini-
-36-
tial periods of the reaction and recording the pH increases resulting.
With this information, a graph of ml. of base versus pH increment was
prepared. Then knowing the change in pH which occurred before the first
addition of alkali, it is possible to estimate from the graph the amount
of base neutralized during this
It should be pointed out that the volume of the reaction mixture
increases with each successive addition of alkali. This fact must be
taken into consideration when calculating specific activities.
Calculation of Specific Activity
Most of the reactions studied followed the kinetics of a first
order reaction. The first order rate constant, k
l
, was computed with
the following equation:
kl = l/t (log a/a-x)
where t is the time in minutes; a is the initial amount of substrate;
and x is the amount of substrate hydrol,-zed at time t. The specific
activity (01) is defined as k1 per mg. of protein N per ml. of reaction
mixture.
Measurement of Proteolysis
The digestion of urea-denatured hemoglobin was followed by the
method of Anson (6). A stock solution of substrate was prepared by mix-
ing seven parts of a 2 per cent solution of urea-denatured hemoglobin
and three parts of a 1.0 M buffer solution at the desired pH. Five ml.
of this solution was transferred to a 10 mI. volumetric flask and brought to
39
0
in a constant temperature bath. The enzyme solution, also at bath
temperature, was added followed by dilution to volume and mixing. Two
ml. aliquots were removed immediately and at appropriate times there-
-37':"
after. These were quickly mixed with 2 ml. of a 5 per cent trichloracetic
acid solution and incubated for 1 hour at 39
0
to coagulate the precipitate.
At the end of this period, the suspensions were filtered and the optical
density of the filtrates determined at 2S0 mu in a Beekman Model DU Spec-
trophotometer. Controls containing enzyme alone and substrate alone were
run in all cases. The optical density of the filtrates, when corrected
by subtraction of the optical density of the control filtrates, gives a
measure of the tyrosine and tr,yptophan liberated during proteolysis.
The pH of each reaction mixture was determined as previously described
at the end of the desired reaction period.
Electrophoretic Studies
These were made in a Tiselius apparatus equipped with the
schlieren scanning device described by Longsworth (95). All runs
were made at 1.5
0
in univalent buffers of ionic strength 0.1 and at
a known pH. The movement of the boundar,y was recorded on a photographic
plate at the beginning and at the end of each run. During each run the
current flow was measured in milliamperes several times and the average
value used for the calculation of mobilities. At the completion of the
run the specific conductance of the protein solution was determined in
a conductivity cell equipped with platinum electrodes 1 sq. cm. in area
and 1 cm. apart.
The electrophoretic mobility was calculated from the following
formula:
u = AU lit
where u is the mobility in sq. em. per volt per s e ~ o n d ; X is the number
of cm. which the boundary moved in time t; Ais the cross sectional area
of the electrophoresis cell in sq. cm.; A is the specific conductance of
-38-
the protein solution in reciprocal ohms; i is the electrical current in
amperes; and t is the time in seconds.
An enlarged tracing of the photographic record of the electro-
phoretic pattern was used to measure the distance the boundary had moved
from the origin. This distance was measured from the center of the peak
and was corrected for the degree of enlargement produced in making the
tracing.
Sedimentation. Studies
These observations were made in a Spinco Model E electrically
driven ultracentrifuge. This instrument is equipped with a Philpot-
Svenson type of optical system, which enables one to make a photographic
record of the boundary as it descends in the cell. All runs were made
at 59,780 r.p.m. and at room temperature. Position of the boundary at
a given time was determined from enlarged tracings of the photographic
record. When corrected for the degree of magnification produced in
making the tracing, the distance moved by the boundary represents the
distance from that portion of the cell nearest the center of rotation
of the ultracentrifuge rotor. The distance from the innermost part of
the ultracentrifuge cell to the axis of rotation is characteristic of
the centrifuge rotor. Sedimentation constants were calculated from
the following equation:
s = x / d ~ 2 x
where s is the sedimentation constant; x is the distance of the boundar.y
from the center of rotation in cm.;Wis the angular velocity in radians
per second; and t is the time in seconds (148). The sedimentation oon-
stant, s, is the sedimentation rate per unit field of force 0 All data
have been corrected to sedimentation values in Svedberg units (S ~ 1 X 10-
13
)
corresponding to water at 20
0
(s20,w).
CHAPTER IV
THE ISOLATION AND CRYSTALLIZATION OF PAPAIN AND MERCURIPAPAIN
It has been pointed out in an earlier chapter that the procedure
for the preparation of crystalline papain from fresh papaya latex (9)
was not successful when applied to commercial dried latex.
One reason for this failure appears to be that there is considerable
variation in dried latex from different sources. For example when pre-
parations were attempted with source material from three different sup-
pliers
l
, good yields or cr.ystalline papain were obtained from only two.
The third yielded a minute amount of cr.rstalline material, presumably
papain, but the specific activity was low.
Another factor of considerable importance is the method whereby
papain is extracted from the dried powder into aqueous solution.
The Extraction of Proteolytic Activity
The dried papaya latex as obtained from the supplier occurs in
two forms. One form is a ver,y fine powder, which is difficult to wet.
The second form consists of hard particles varying in diameter from
approximately 1 mm. to 5 mm. These particles are also difficult to
wet but in addition must be pulverized
2
for efficient extraction of
lrhe author is grateful to Dr. M. L. Tainter of the Sterling-
Winthrop Research Institute for generous gifts of dried papaya latex.
author is indebted to Dr. Alonza Johnson for informing us
of the importance of the grinding procedure in the extraction of papain
from dried latex.
-39-
-40-
proteolytic activity. Regardless of the nature of the starting material,
extraction of proteolytic activity from dried latex was most efficient
when a homogenization procedure was employed. Grinding with sand in a
mortar or blending for several minutes in a Waring Blendor are procedures
which have been used successfully.
For dried latex the pH permitting the most efficient extraction
is different from that described by Balls and Lineweaver (9) for fresh
latex. These authors recommended pH 6.5 to 7.0. In the case of dried
latex the greatest total activity and the highest specific activity of
the extract are observed when extraction is carried out at pH 5.7. The
effect of pH on the efficiency of extraction of proteolytic activity is
shown in Fig. 2.
>-
. ~ .
0,10
I-
0.5
-
".

> "'-
Ie .,
t- 0 .. 4
"
(.)
C
1
0.06
0.3

...J
I
<l 0.2
!
t-

,0 0.'
, l-
I
-2
Figure 2. The Effect of pH on the Efficiency of Extraction of
Proteolytic Activity from Dried Papaya Latex. One gm. samples of dried
papaya latex, 200 mg. of Celite, and 10 ml. of 0.04 M cysteine at the
desired pH were ground in a mortar with sand. Each solution was filtered
and the filtrate assayed against BAA in 0.05 M cysteine and 0.04 M cit-
rate, pH 5.5. Total activity is defined as the product of the specific
activity (01) and the total amount of protein N in each filtrate
o
The
pH of each filtrate was measured as described.
-41 ....
When the extraction of the proteolytic components of dried latex
is carried out as described, the original procedure of Balls and Line-
weaver (9) can be used almost without alteration for the preparation of
cr.ystalline papain from the commercial dried latex. This preparation
has now been repeated many times and by several individuals with little
variation in yield or specific activity.
The Preparation of CrYstalline Papain
The dried papaya latex (180 gm.), 100 gm. of Celite and 150 gm.
of washed sand are mixed in a mortar and ground thoroughly with 200 to
300 ml. of 0.04 M cysteine. This solution is made by dissolving 6.3
gm. of cysteine hydrochloride in 100 mI. of 0.054 N NaOH. This contains
an excess of alkali in order to bring the aqueous extract to pH 5.7.
The suspension is allowed to settle and the supernatant solution
is decanted. The extraction and grinding are repeated with another 300
ml. portion of the cysteine solution; this is decanted as before and the
mortar is washed with the cysteine solution until a total volume of one
liter has been used for extractions and washings. The resulting suspen-
sion is stirred well and filtered on a large BUchner funnel with mild
suction through a 0.5 cm. layer of Hyflo-Super-cel on Whatman No. 1 fil-
ter paper. The filtrate (Fraction 1) is opalescent, greenish yellow and
should be near pH 5.7.
If a Waring Blendor is used for the extraction it is of course
unnecessary to use sand. The homogenate resulting from this type of
treatment is much more difficult to filter and is best accomplished
with pressure filtration. A coarse filter pad (D-l) covered with a
thin layer of Hyflo-Super-cel has been used in a Hormann Filtero
Fraction 1 is adjusted to pH 90 0 with approximately 110 ml. of
-42-
N NaOH-which is added slowly with stirring. A fine gray precipitate
appears which is removed by centrifugation at 2600 r.p.m. for one hour.
The supernatant solution (Fraction 2) should be clear. The precipitate
consists of denatured protein which cannot be redissolved for assay.
Fraction 2 is brought to 0.4 saturation with solid ammonium sul-
fate (250 gm. per liter). A white precipitate appears and the suspension
is allowed to settle for one to two hours at 4
0
The precipitate (Frac-
tion 3) is then removed by centrifugation at 2500 r.p.m. for one hour
and the supernatant solution (Fraction 3a) is discarded. Fraction 3
is washed once with 400 to 500 ml. ot cold ammonium sulfate solution
(250 gm. per liter) and separated by centrifugation as before. Frac-
tion 3 is redissolved in 600 ml. of 0.02 M cysteine (pH 7 to 7.5) and
60 gm. of solid NaCl are added slowly. Papain is precipitated as a
fine white solid by this procedure. This suspension is also allowed
to stand for one hour at 4; it is then centrifuged in the cold for
one hour at 2500 r.p.m. and the supernatant solution (Fraction 4a) is
discarded.
The solid material from above (Fraction 4) is suspended in 400
ml. of 0.02 M cysteine at pH 6.5. A slight rise in pH generally occurs
when the protein is added to the cysteine solution, so that it is necess-
ary to adjust the suspension to pH 6.5. At room temperature the suspen-
sion develops a crystalline sheen in about 30 minutes; it is then kept
overnight at 4. The light crystals (Fraction 5) are removed by centri-
fugation at 2000 to 2500 r.p.m. for 4 to 5 hours. It is imperative that
this centrifugation be carried out in the cold, since some of the cr,ystal-
line material will redissolve if the temperature rises to 15
0
or higher.
More material will cr,ystallize from Fraction 5a but the overall increase
in yield is insignificant.
-43-
Recrystallization
This is performed as described by Balls and Lineweaver (9).
Fraction 5 is dissolved in the minimum amount of distilled water (pro-
tein concentration about 1 per cent) at room temperature and 10 ml. of
saturated sodium chloride solution per 300 ml. of protein solution are
added very slowly with stirring. When about 75 per cent of the salt
solution has been added, the enzj"De will begin to crystallize from the
solution at room temperature. The resulting suspension is allowed to
stand at 4
0
overnight and the cr,rstals are removed by centrifugation as
described for Fraotion 5.
Papain may also be recrystallized by solution in 70 per cent
ethanol and salting out with lithium sulfate or lithium chloride as
previously described by Balls and Lineweaver (9).
Crystals of papain prepared as described above appear as minute
needles if viewed under high magnification (400X). When stored as a
packed suspension at 4 no detectable loss of activity occurs for about
60 days. Lyophilization results in no loss of activity but acetone-
ether dried material is virtually insoluble and apparently inactive.
The Preparation of Mercuripapain
Further purification of papain has been accomplished by its
cr,ystaDization as a mercury derivative 0 It should be noted that War-
burg and Christian (164) o b t a ~ e d enolase as a cr,ystalline mercury deri-
vative, and Hughes (75) has taken advantage of the ability of thiol
groups to bind mercury in the isolation of mercaptalbumin as a mercury
complex. The apparent sulfhydryl nature of papain and its reported in-
hibition by certain of the heavy metals (.7), suggested that this enzyme
might readily form a mercury complex. This was found to be the case
o
-44-
Mercuripapain is prepared by dissolving twice recrystallized pa-
pain at room temperature in 70 per cent ethanol containing 0.001 M agC1
2

The protein concentration should be 1.5 to 2..0 per cent. If slightly
turbid, the solution is filtered and then stored in the cold. Within
24 hours, a white, obviously crystalline precipitate will form, and in
three to four days about 90 per cent of the activity will crystallize
from solution. These heav,r crystals are easily removed by centrifuga-
tion. They are insoluble in cold 70 per cent ethanol but may be easily
redissolved in water. Cr.ystals of mercuripapain are large: enough to be
seen with the naked eye. Indeed, in suspensions of mercuripapain which
were allowed to stand at 4
0
for several months, crystals one-half ineh
long have been observed. Under low power magnifieation, they appear as
long plates occurring singly or in sheaves (Fig. 3).
Figure 3. Crystals of Mercuripapain
Photographed at 100 Times Enlargement.
Mercuripapain can also be prepared by solution of the crystalline
enzyme in aqueous 0.001 M HgC1
2
, followed by addition of NaCl as described
above. The material thus obtained exhibits a cr,rstalline sheen in sus-
-45-
pension but crystals are not visible even when magnified 400 times.
Mercuripapain against BAA in 0.02 M Acetate, pH 5.5 is
inactive. In the presence of cysteine and a metal chelating agent such
as Versene full activity is restored (Table I). When dried by the usual
acetone-ether procedures, mercuripapain retains its cr,ystalline character,
can be redissolved in water easily and retains 80 to 90 per cent of the
enzymatic activity of the undried material (Table I).
TABLE I
ENZIMATIC ACTIVITY OF MERCURI PAPAIN
All assays were carried out as described with
as the substrate in 0.02 M acetate buffer at pH 5.5. I
Reactants ProteinN X 103 kl X 103
1
mg. per ml.
,,,H,\::
"
''\'
Mercuri papain ...
11.7 0 0
Mercuripapain + 0.001 M Versene 5.9 0 0
Mercuripapain + 0.005 M cysteine 5.9 0.75 0.13
Mercuripapain + 0.001 M Versene
5.9 6.9 + 0.005 M cysteine 1.19
Mercuripapa.in (acetone-ether
dried) + 0.001 M Versene +
0.005 M cysteine 8.2 8.2
__ 1.00
Elementary Analysis of Papain and Mercuripapain
Samples of three times recrystallized papain and mercuripapain
were prepared for analysis by washing with 90 per cent ethanol until
free of chloride, then three times each with acetone and ether, and air
dried. The sulfur content was determined by Dr. A. Elek by the method
of Elek and Hill(44). Nitrogen was determined in quadruplicate by a
-46-
micro-Kjeldahl method. Samples of two prepara.tions of m.ercuripapa.in were
each analyzed In triplicate for mercur,y by the method of Laug and ~ e l s o n
(90). All the controls and precautions recommended by these authors were
followed.
Table II summarizes the analytical data on papain and mercuripa-
pain. For purposes of comparison the analytical data obtained earlier
with cr,ystalline papain from fresh papaya latex are included (9, 38).
The sulfur content of one of our preparations was found to be identical
with that of Balls and Lineweaver's preparation. However, our value for
nitrogen is smaller than that of Close ~ !!.and higher than that of Balls
and Lineweaver.
TABLE II
ELEMENTARY ANALYSIS OF PAPAIN AND MERCURI PAPAIN
All values are corrected for moisture and ash.
Sample No. Nitrogen Sulfur
, per cent per cent
Data of Balls and Lineweaver (9 ) 15.5 1.2
Data of Clos e, Moore and Bigwo ad (38) 16.4
-
Papain 16.1 1.22
Mercuripapain I
-
-
Mercuripapain II
- -
Discussion
Mercury
per cent
-
-
-
0.49
0.44
The data recorded in Table III show typical results obtained with
preparations of the size described and illustrate the variations to be-ex-
pected in dried latex from different commercial sources. The most strik-
ing variations are apparent when Source 3 is the starting material.
-47-
TABLE III
ISOLATION OF CRYSTALLINE PAPAIN FROM DRIED PAPAYA LATEX
Representative runs giving yields and activities obtained with
180 gm. of dried latex from three different sources., The proteolytic
coefficients ' (C ) were determined with benzqrl-L-argininamide as sub ... ';',.'.
strate at 39
0
ana at pH 5.5 in the presence of 0.005 M cysteine and 0.001
M versene. Units of total activity are calculated by multiplying the 01
value by the total amount of protein nitrogen in the fraction.
Source 1.
Fraction Volume Total Protein
1
Total
Activitz
ml. gm. units
1 770 45 0.19 1300
2 760
43 0.23 1500
3 1.3 0 .30 580
.3at:
7.35 .36 0.25 1470
4a*
530 8 0.18 215
5 *
(orystals) 2.1 0.84 270
5a 400 3.8 0.12 72
6 (2 recr,ysta11izations) 1.2 1.06 190
Source 2.
1 725 41 0.18 1200
2 820 40 0.20 1390
.3
26 0.25 1080
.3a*
775
16.6 0.20 538
4
6.6 0.43 47.3
4a*
755
22 0.16 568
5
(crystals) 1.8 1.16 .329
5a* 448 4.8 0.28 217
Source 2.
1 820
.37 00 12 746
2 820
.35 0.09
545
.3
15 0.11 261
.3a'* 785 7.3 0.10 114
4
required 15 per cent NaC1
4a to produce precipitation
5
(?crysta1s) 0.48
'*
Fractions 4a and 5a are discarded. Fraction .3a may be saved for the
isolation of c ~ o p a p a i n o
-48-
Here, low specific activities are observed throughout the preparation and
an extremely small amount of crystalline product is obtained. The speci-
fic activity of this product (Fraction 5) is only about one-half that of
crystalline products from Sources 1 and 2.
The similar behavior of dried latex from Sources 1 and 2 is appar-
ent from the date in Table III. However, there is one difference that
should be mentioned. Fraction 5 from Source 1 has a specific activity
towards BAA of 0.84 and this increases to 1.06 after two recrystalliza-
tions. Fraction 5 from Source 2 exhibits a C value on the same sub-
1
strate of 1.16. In either case, further recrystallizations do not sig-
nificantly alter the specific activity. If Fraction 6 from Source 1 or
Fraction 5 from Source 2 are recrystallized as the mercury derivative,
then both products have the same specific activity towards BAA (01 = 1.20).
In addition, their electrophoretic and sedimentation behavior is identical.
It appears, therefore, that the same enzyme is obtained from both sources
but that certain steps in the procedure result in greater purification
with Source 2 than with Source I.
With Source 1 or 2 as the starting material, the overall increase
in specific activity from the first extract to the recr,ystallized enzyme
is approximately 6-fold. Since only 25 per cent of the crude latex is
soluble, the total increase in activity is less than 25-fold. As pre-
viously noted (9) for papain obtained from fresh latex, the overall puri-
fication is not high but the specific activity compares favorably with
that of the animal proteinases. In fact, papain has' a C value for BAA
1
of 1.20 as compared to a C
1
of about 0.04 for crystalline tr,ypsin on the
same substrate (71) at a lower temperature (25).
The extent of purification judged from activity measurements on
I'
-49-
BAA does not give a completely valid estimate since oath papain and chy-
mopapain act on this substrate. A better estimate may be obtained from
electrophoretic measurements. Figure 4 shows the electrophoretic patterns
8
4. Electrophoretic Patterns of Clarified Crude Extracts
of Fresh (A) Latex, and Dried Latex from Source 1 (B). The runs were
performed at pH 4.0 in acetate buffer at '0.1 ionic strength for 205 min-
utes (A), and 225 minutes (B). The peak marked p is that of papain.
TABLE IV
,
ELECTROPHORETIC COMPONENTS OF PAPAYA LATEX*
The measurements were made in 0.1 ionic strength acetate buffer at
pH 4.0. Mobilities (u) have been multiplied by 105 sq. em. per volt
d per secon
Compo 1 Camp. 2 Compo 3 Compo 4 Compo
5
\
(papain) (ehymo-
J)C3.p,ain)
.
4.0 6.9 8.6 9.6
Mobility to 5.5 to to to
4.3 7.2 8.9 9.7
percent percent percent percent percent
'Fresh" latex 7 14 65 1 13
UDried
lt
latex,
Fraction 1 6
9 53 7 25
"Dried" latex,
Fraction 6 99
-
- - --
*
The author is inde oted to Mr. D. M. Brown for these measurements 0
obtained on clarified fresh latex (A) and on Fraction 1 of the dried
latex from Source 1 (B). Estimates of mobility and area are given in
table IV. It is noteworthy that the two preparations very similar.
Component 1 which has been identified as papain itself from studies on
.... 50-
the crystalline, homogeneous protein represents 6 to 7 per cent of the
total migrating area; the purification from Fraction 1, is therefore,
about l6-fold. The overall yield of Fraction 6 (three times recrystal-
lized enzyme) from this source is 44 per cent of the papain present in
Fraction 1.
Referring back to Table III, it should be noted that Fraction 2
has a greater total activity than does Fraction 1. This phenomenon has
been observed repeatedly and is presumably due to the removal of inhib-
itors in the first steps of the preparation. In addition, when Source 1
is the starting material, the greater part of the proteolytic activity re-
mains in Fraction 3a. This is the -high salt fraction" referred to by
Jansen and Balls (93). In respect to this component, Source 2 behaves
a little differently but still a major portion of the proteolytic acti-
vity is in this fraction. Products high in nchymopapaintt have been pre-
pared from Fraction 3A and e1eetrophoretica11y contain more than 90 per
cent of Component 3 (Table IV). Such preparations are about one-fifth
as active as papain on BAA. Component 3 (Table IV) is probably not com-
posed of chymopapain completely since a lysozyme has been cr.ystallized
from dried papaya latex (144) which has the same mobility. Furthermore,
there is evidence to indicate that chymopapain not the only proteo-
lytic enzyme present in the "high salt fractionlt. At the time chymopa-
pain was crystallized, Jansen and Balls (93) noted that they could not
account for all of the proteolytic activity of papaya latex on the basis
of papain and chymopapain alone. Accordingly, they postulated that even
more proteolytic enzymes were present. Evidence from this laboratory is
in agreement with this postulate. When tested against various substrates,
Fraction 3a has been found to hydrolyze both BAA and N-aeety1-b-tyrosin-
amide. If this fraction is made 0.9 saturated with ammonium sulfate all
-51-
of the activity towards both substrates will be precipitated. On the other
hand, if the ammonium sulfate concentration of Fraction 3a is increased' to
0.5 per cent saturation the BAA activity alone is precipitated. The activ-
ity towards remains in solution and this solution
hydrolyzes BAA very slowly. Since "chymopapainrt has been obtained in a
relatively pure state and is quite active towards BAA it is unlikely that
much of the BAA activity in the "high salt fraction" is due to papain.
Furthermore, the lack of activity towards BAA in the fraction that splits
N-acetyl-1-tyrosinamide indicates the presence of a different enzyme.
Mercuripapain appears to be the purest form of the enzyme yet ob-
tained. Even though recr.ystallized papain appears homogeneous in the el-
ectrophoresis apparatus and monodisperse in the ultracentrifuge, an in-
crease in specific activity has invariably peen observed when this mater-
ial is converted to the mercury derivative. The remarkable stability of
mercuripapain makes it the most desirable fonn of the enzyme for study.
of the experimental work reported herein has been carried out with
this derivative.
The mercury content of mercuripapain indicates that 1 gm. atom
of mercur.y is combined with 43,500 gms. of protein. The molecular weight
of papain by sedimentation-diffusion measurements is 20,700. It thus ap-
pears, that within the precision of the analyses, there is a combining
ratio of 1 atom of mercury to two moles of papain. This will be dis-
cussed in more detail in connection with the physical studies on papain
and mercuripapain.
CHAPl'ER V
THE PROPERTIES OF PAPAIN
The various theories the nature of papain activation
by cyanide, sulfhydryl compounds and certain reducing agents have been
discussed in some detail in Chapter II. Although none of the studies
on which these theories were based were performed with highly purified
papain, almost all of the phenomena observed were encountered in this
investigation on cr,ystalline papain. For this reason it seems pertinent
to review briefly the various ideas about the mechanism of papain acti-
vation.
Bersin and Logemann (27) concluded that papain is active when
free sulfhydr,yl groups on the enzyme are in the reduced state, and in-
active when these sulfhydryl groups are oxidized to the disulfide form

Their conclusions were based on evidence showing the similarity between
the behavior of active papain and cysteine. Hellerman (67) later supported
this proposal by demonstrating that certain thiol reagents, such as
mercuribenzoate, inhibit papain action. This theory seems to have been the
most widely accepted sinee there were other enzynes (e.g. urease) that were
activated and inhibited in the same Nevertheless, this idea did
not completely explain all the facts and there were other proposals that
probably came closer in this regard. Notable among these was the theor.y
of Bergnann et ale (78, 79) that papain existed in an inactive form which,
when reduced, was capable of combining with the activator to form the act-
-52-
-53-
ive enzyme. This proposal was based on the observation that the activity
of papain depended to a considerable extent on the nature of the activat-
ing agent.
Prior to this, Krebs (S7) had reported that a number of heavy
metal ions inhibit the proteolytic activity of papain and suggested that
activation by cyanide and sulfide was due to the removal of metal ions
present as combined impurities in the enzyme preparations. Furthermore,
he proposed that citrate and pyrophosphate activated papain by the same
mechanism. Murray (117) noted that citrate and cyanide were not inter-
changeable in their effects on papain, but that their effects were addi-
tive. In view of these observations, he stated that removal of inhibi-
tor,r heavy metals could not be the sole mechanism of activation. Closely
related to these findings were the specific ion effects also noted by
Murray. His experiments demonstrated that at a given pH papain was more
active in acetate than in phthalate and still more active in citrate.
Fox and Pettinga (49) studied the synthesis of acyl amino acid anilides
catalyzed by papain, and also observed this activating effect of citrate.
The Activation of Crystalline Papain
Once crystalline papain had been prepared, it became essential to
establish the conditions wherein the enzyme was fully active. During the
development of the procedure for the isolation and crystallization of pa-
pain, the various fractions had been assayed at pH 5.5 in 0.02 M Citrate
buffers with cysteine alone as the activating agent. Using these condi-
tions, the of BAA was studied at various pH values ina var-
iety of buffer systems in order to establish the optimum pH for papain
action. When cysteine alone was the activating agent, it soon ap-
parent that the optimum pH was dependent on the nature of the buffer an-
-54-
ions. Thus at a given pH value, papain was considerably more active in
citrate than in acetate. Furthermore, at alkaline pH values no activity
was measurable in Tris but in phosphate
buffers at identical pH values the enzyme was very active. Typical ob-
servations with 4 different buffer systems are illustrated in Fig. 5. It
should be noted that the activity of paplin in each'mixture could well be
related to the ability of the anion to form complexes with metal ions.
Tris does not exist as an anion and is incapable of complex formation
with metal ions. These experiments clearly indicated that factors other
than reduction of a disulfide are involved and prompted a more thorough
.,,/
investigation of the requirements for full activation of papain.
to
PHOSPHAT{
\x
0.4
0.2
TRIS
7
Figure 5. Effect of pH on the Proteolytic Coefficient (01) for
the Hydrolysis of Benzoyl-&-argininamide by Papain in the Presence of
0.005 M. Cysteine. The buffers were present to 0.02 M.except for citrate
which was 0.04 M.
Activating agents which have been most commonly used are cysteine,
H
2
S and cyanide. All three reagents are capable of reducing disulfide
bonds and in addition form chelates or insoluble compounds with heavy met-
also To this group has been added Versene, an excellent metal-chelating
The results with these four -agents both alone and in various com-
-55-
binations, and in several buffer systems are shown in Fig. 6.
o.
~ s ,
o.
Figure 6. Effect of pH on the Activation of Crystalline Papain
as Measured by the Proteolytic Coefficient (Cl) for the Hydrolysis of
Benzoyl-1-argininamide. Activators were used singly and in various com-
binations. Qz! is cysteine and Y is Versene, all used at 0.005 M. The
buffers were used at 0.02 M concentration; acetate near pH 5, citrate at
pH 6, phosphate at pH 7, and Tris near pH 8. Cysteine plus Versene gave
the highest activity at all pH values and the C
l
values were independent
of the buffer used.
Since the buffer has a marked effect on the extent of activation,
the results are strictly comparable only at the same pH. Acetate was used
near pH 5, citrate at pH 6, phosphate at pH 7 and Tris near pH 8. The data
show that papain is not fully activated by anyone reagent. Cysteine alone
activates markedly in citrate and phosphate at pH 6 and 7 respectively, but
full activity is not attained unless Versene is also present. Cyanide ap-
pears to be capable of replacing cysteine at pH values above 7.0 but on
the acid side is less effective than cysteine; presumably this is due to
the weak dissociation of HeN at acid pH values. At all pH values the com-
bination of cysteine and Versene gave maximal specific activity_ It is
noteworthy that when Versene is present no specific ion effects are obser-
-56-
ved, despite the fact that Versene alone produced no activation.
These results confirm the suggestion of Krebs (87) that removal
of heavy metals is necessary for maximal activity of papain, but also
demonstrate that a reducing agent is required, presumably for the reduc-
tion of disulfide bonds to free thiol groups which are essential for ac-
tivity.
Effect of pH on Papain Activity
Fig. 7 shows a detailed study of the effect of pH on the rate of
hydrolysis of BAA by papain in the presence of cysteine and Versena. A
variety of buffers including Versene itself was used in establishing this
curve, and it is apparent that the specific ion effects shown in Fig. 4
are absent. Between pH 5.0 and 7.5 the specific activity of papain is at
a maximum.
1.0
o,e
Figure 7. Effect of pH on the Cl for Hydrolysis of Benzoyl-1-
argininamide by Crystalline Papain in the Presence of 0.001 M Versene
and 0.005 M cysteine. The buffers employed at 0.02 M were acetate (a),
Versene (v), citrate (e), phosphate (p), Veronal (vI), Tris (t) and
glycine (g).
Inhibitors of Papain
Sulhydryl reagents such as iodoacetamide and p-chloromercuriben-
-57-
benzoate have been shown to inhibit partially purified preparations of pa-
pain. Since these two agents are also capable of reacting with one of the
activators, i.e. cysteine, inhibition studies should be carried out in the
absence of activating agents. In this manner reactions with the enzyme it-
self can be investigated. With this in mind, crystalline papain was dis-
solved in a solution containing 0.05 M cysteine and 0.01 M Versene (pH 6)
and allowed to incubate for several minutes at 39. This solution was then
dialyzed against several changes of "air-free
n
deionized water at 4
0
for
two days. It was hoped that such treatment would provide an enzyme which
was active in the absence of activators. Assay of this enzyme preparation
with BAA at pH 5.5 in 0.02 M acetate failed to reveal any proteolytic ac-
tivity unless the usual activating agents were present. Thus it was not
possible to study the effects of iodoacetamide and p-chloromercuribenzoate
on papain itself. These agents will inhibit papain action when activators
are present, but one cannot r u l ~ out the possibility that the inhibitors
are acting to remove cysteine from the reaction mixture.
In contrast to tr,ypsin and chymotrypsin papain is not inhibited by
diisopropyl fluorophosphate, even when this substance is present at 5 tUnes
the molar concentration of the enzyme.
Esterase Action of Papain
It has been demonstrated that the three crystalline proteolytic en-
zymes of pancreas, trypsin, chymotrypsin, and carboxypeptidase, possess well
defined esterase activity on appropriate synthetic compounds which are
closely related in structure to substrates possessing amide or peptide
bonds. It has now been observed that ,cr,ystalline papain also has an es-
terase action.
The substrate was tosyl-1-arginine methyl ester at 0.025 M in the
-58-
presence of 0.001 M Versene and 0.005 M cysteine with various buffers at
0.01 M. Hydrolysis was followed at 39 by the titrimetric technique of
Schwert et ale (137). This procedure has been described in Chapter III.
The hydrolysis of the ester substrate follows the kinetics of a
first order reaction at the five pH values shown in Fig. B where 100 minus
per cent hydrolysis is plotted on a scale against time. The
initial enzyme concentration at pH 6.0 was 0.0088 mg. of protein N per
ml. and was 0.0195 mg. of protein N per ml. in the other experiments.
__

Figure B. Action of Papain on Tosyl-b-arginine Methyl Ester at
the Various pH Values Indicated on the Curves. The semilogarithmic plot
of 100 minus per cent hydrolysis against time shows that the kinetics are
first order. The upper ordinate applies to the data at pH 4. The enzyme
concentration at pH 6 was 0.0088 mg. of protein N per ml. and at the other
pH values was 0.0195 mg. of protein N per ml.
The pH activity curve for the esterase action is given in Fig. 9.
F'or comparison, the dash line shows the action on the amide substrate,
BAA. A much narrower optimum was found for the esterase action, but it
-59-
should be noted that the ester concentration was half that of the amide
(00 05 M). It is striking that the maximal 01 values for both types of
substrates are almost identical.
The main product of the esterase action was shown to be
arginine by isolation of this substance from the reaction mixture in 75
per cent of the theoretical yield; m.p., 257-259 (with decomposition);
for an authentic sample, m. p., 256-258
0
( with decomposition).
o.
r ______ -.1 __
I BAA,
5 6
pH
7
\
,
\
\
,
8
Figure 9. Effect of pH on the Hydrolysis of Tosyl-1-arginine
Methyl Ester (tsAME) and BAA (dash line). The data are taken from the
experiments in Fig. 8.
The Specificity of Papain
Much of the early work concerned with the digestion of protein
substrates by :papain made it clear that crude or partially purified pre ....
parations of papain produced fairly extensive hydrolysis of proteins o A
number of investigators observed that papain digestion a greater
-60-
proportion of free amino acids than did the animal proteinases, e.g.
trypsin 0 Such findings suggested that these papain preparations con-
tained either a mixture of proteolytic enzymes with different specifici-
ties or one enzyme with a broad hydrolytic action. Since papaya latex
is now known to contain more than one proteolytic enzyme it seemed
that the former was the most probable reason for the broad specificity.
This affinity of papain for peptide bonds involving wide variety of amino
acids is illustrated by the studies ot Sanger, Thompson and Tuppy (134)
on the hydrolysis of the oxidized A chain of insulin by crude papain.
The most definitive specificity studies, mainly by Bergmann and
Fruton (17), demonstrated that partially purified preparations of papain
attack many types of synthetic amide and peptide substrates. The state
of purity of some of their enzyme preparations and the methods used to
prepare these makes it likely that, at least in some of their experi-
ments, papain was the only enzyme present. However, this is not known
with certainty. The work with synthetic substrates clearly showed that
papain did exhibit a broad specificity in contrast to the more limited
types of compounds hydrolyzed by the animal proteinases. In view;of
this it was of interest to the specificity of crystalline pa-
pain.
With the substrates tested thus far, the cr,ystalline enzyme re-
sembles the cruder preparations. Table V gives the data for a represen-
tative selection of compounds. Thus far the most sensitive substrate
found is BAA, which is also a substrate for tr,ypsin. In addition typi-'
cal substrates for pepsin
peptidase (carbobenzoxyglycyl-1-tryptophan), chymotrypsin (acetyl-b.-t7M-
sinamide), and peptidases (1-leucylglycy1g1ycine and 1-leucinamide) are
-61-
TABLE V
ACTION OF CRYSTALLINE PAPAIN ON SYNTHETIC SUBSTRATES
The substrates were tested at 0.05 M in the presence of 0.005 K
cysteine and 0.001 M Versena at 39
0
at enzyme concentrations from
0.006 to 0.63 mg. of protein N per ml., the larger amounts being re-
quired for the least sensitive substrates. Crystalline mercuripapain
was used for these studies.
Substrate
a-Benzoyl-1-argininamide
acid diamide*


Hippurylami.de* .............................. .

tI
..........
"
..........
Carbobenzoxyglyeyl-L-pnenylalanine

Acetyl-L-tJrosinamide*
1-Leucylglycylglycine
1-Leucylg1yeine ............................. .
1-Leucinamide .............................. .
0.0
&-Histidinamide ........................... .
Carbobenzoxy-1;histidinamide
Carbobenzoxy-1;histidyl-1;leucinamide
pH
5.0-7.5
7.35
5.2
6.5
4.5-6.85
4.0
7.5
8.1
6.8
5.0
5.9
6.0
6.1
5.0
1.20
0.24
0.050/1
0.021
0.014
0.0036
0.0011
0.0009
0.0004
0.045#
0.003
0.004
0.0004
0.04
0.0211
0.01
0.11
0.013
* These compounds were only partially dissolved at the start of the test.
H The values given are extrapolated initial constants, since decreasing
values of 0
1
were observed.
-62-
hydrolyzed, as well as a number of others not susceptible to the puri-
fied enzymes of animal origin.
TABLE VI
HYDROLYSIS OF CARBOBENZOXY-1-ISOGLUTAMINE BY PAPAIN
Substrate concentration was 0.05 M in the presence of 0.005 M cyst-
eine and 0.001 M Versene. The enzyme concentration was 0.105 mg. pro-
tein N pel- ml.
Time
pH 4.45
pH 7.06
Hydrolysis
1
Hydrolysis
1
min. per cent per cent
15 19 0.046
30 32 0.043 13 0.016
45 35 0.032 18 0.015
60
37
0.026 23 0.015
75 41
0.023 28 0.015
While the present studies should be regarded as preliminar,y, some
interesting points can already be noted. In the hydrolysis of carboben-
(CGT) and carbobenzoxy-1-isoglutamine
the apparent C
l
values decrease progressively when studied at pH values
between 4 and 6; above this range the C
l
values are constant. An example
of this effect for CIG is given in Table VIo The decreasing constants
appear to be due to progressive inhibition by the liberated carbobenzoxy-
acid (GG). Indeed, when GG is added to the initial reaction
mixture, it produces strong inhibition of the hydrolysis of BAA and hip-
purylamide ((HA), as well as of eIG and carbobenzoxy-1-g1utamyl-1-tyrosine.
For the first two substrates, the inhibitor.y effect is very marked in
the region pH 3.9 to 4.5 (Table VII).
.. '
-63 ....
TABLE VII"
INHIBITION OF PAPAIN BY ACID
The substrate concentration was 0.05 M; that of the inhibitor was
0.02 M. The hippurlyamide was only partially dissolved at the begin-
ning of the experiment II Cysteine and Versene were also present.
Substrate Protein N pH Time Hydrolysis
mg. per. nU. min. per cent
BAA. 0 0 0 0.0053 3.9 75 23
BAA + CG.oo 0.0053 3.9 75 9
BAA 0 1/ 1/ 0.0053 5.0 75 48
BAA + CG ...... 0.0053 5.0 75 46
HA 0.11 4.5 240 49
HA + CG 0.11 4.5 240
14
HA 0 0.070 5.1 150
45
HA + CG 0.070 5.1 150 42
The effect of pH on the 'hydrolysis of several papain substrates
is shown in Fig. 10. It should be noted that when progressive inhibition
was found, as with CIG, extrapolated, initial values for 01 were used
o
The curve for hippurylamide resembles that for BAA, but the others are
obviously different. The curve for CIG suggests that progressive ioniza-
tion of the "Y'-carboxy-l group decreases the susceptibility of this sub-
strate. This is consonant with the effectiveness of the
glutamic acid as an inhibitor at pH 4 and its ineffectiveness near pH'7.
In addition, the much greater sensitivity of carbobenzoxy-1-glutamic acid
diamide as compared with CIG is in accord with this view. Fruton (54)
has shown that the diamide is hydrolyzed onlY at the a-amide linkage to
yield carbobenzoxy-L-glutamine.
-64-
4
PH
Figure]l Effect of pH on the Hydrolysis of Four Synthetic Sub-
strates by Crystalline Papain in the Presence of 0.005 M Cysteine and
0.001 M Versene. The substrates studied were carbobenzyoxy-L-leucina-
rnide (eLA), hippurylamide (HA), aeid-diamide
(CGDA) and (CIG)o
The marked effeot of pH on the hydrolysis of uncharged substrates
such as the diamide, and the ver,y different influence of pH on BAA are ex-
tremely strikingo From the information presently available, no definite
explanation of these findings is possible. However, it is noteworthy that
the two substrates tested that have a benzoyl radical substituted on the
a-amino group (BAA and HA) exhibit the same type of pH-activity curve o
The substrates with a carbobenzoxy group (eLA and CGDA) both exhibit the
same type of pH curve although it is different from that for BAA. Thus,
the influence of pH on the hydrolysis of a substrate may be related to
the type of substitution on the a-amino groupo
Proteolysis by Papain
A comparison of the action of papain and trypsin on a protein sub-
strate was made by studying the digestion of urea-denatured hemoglobin by
these two enzymes. The results are shown in Fig. 110 The experiment dem-
-65-
onstrates that crystalline papain digests this protein approximately three
times faster than crystalline trypsin. The optimum pH for digestion of de-
natured hemoglobin by crystalline papain extends from pH 7.0 to 80 5.
~ 0.2
CI)
z
IoU
o
~ 0.1
u
Ii:
o
10 '15 .2.0
MG. PROTEIN PER Ml. X 10+
Figure 11. The Digestion of Urea-Denatured Hemoglobin by Crys-
talline Papain (solid line) and Crystalline Trypsin (dash line). The
experiment with papain was carried out in 0.05 K phosphate buffer at
pH 7.0 in the presence of 0.005 M cysteine and 0.001 HVersene. Tryp-
sin was studied in 0.05 M phosphate buffer at pH 8.0. Aliquots of the
reaction mixtures were removed 10 minutes after starting the reaction.
These were mixed with 5 per cent trichloracetic acid to remove intact
protein. The filtrates were examined in a spectrophotometer at 280
muD Appropriate controls were run.,
Discussion
The fact that crystalline papain requires both a reducing agent
and a metal complexing agent for maximal activation suggests that a free
thiol group or groups is necessar.y for papain action. This is consist-
ent with the theory of Bersin and Logemann (27'i. In view of the marked
sensitivity of papain to heavy metals (Sf) it is not surprising that some
efficient means of removing these inhibitory substances is a requirement
for maximal activity. Undoubtedly these metals become tightly bound to
the enzyme during its collection and purification. The tightness of this
binding is illustrated by the fact that netther H
2
S, cysteine nor cyanidei'
-66-
all three excellent metal-complexing agents, can produce full activation
of papain, either when used alone or in combination. The observed speci-
fic ion effects on the activity of papain are also undoubtedly related to
the ability of these anions to form insoluble salts with heavy metals and
thus remove them from the protein. It may be noted in passing that m a ~
enzymes, e.g. urease, are now known in which sulfhydryl agents are essen-
tial for activity and for which specific ion effects have been reported.
It appears likely that such effects may be circumvented by the procedure
which has been adopted for papain, namely the addition of a metal che1a-
ting agent such as Versene as well as a reducing SUbstance.
It should be emphasized that the information available suggests
but does not prove that free thio1 groups are necessar.y for papain action.
For example, the lack of activity in the papain that had been activated
then dialyzed free of activators could be explained by the older coenzyme
theory. However, in the experiment described in this chapter it is possible
that the dialysis conditions were not rigidly anaerobic. If papain, like
ficin (171) is easily oxidized by exposure to air then this would result
in inactivity of the enzyme and it is not necessar.y to postulate the re-
moval of a coenzyme. Similarly, Bergmann et aL (79) interpreted the dif-
ferent levels of papain activity brought about by various activators as
indicating the presence of an enzyme-coenzyme complex as the active com-
ponent. Cysteine, H
2
S and cyanide were observed to produce'different
levels of activity with crystalline papain, but when one considers that
these three substances vary in their relative ability to reduce disulfide
bonds it appears that the results reported earlier in this chapter are
just as readily explained by the thio1 theory. The identical effects of
cyanide and cysteine above pH 7 supports this view especially since cyan-
-67-
ide is a good reducing agent for glutathione only above pH
70 0 (78, 79)0
Recent work has demonstrated that the thiol group has a pH in
the neighborhood of pI 8
0
5 in contrast to the older view that the pK
was near pH 10. If this is true for the thiol groups of papain that are
involved in the active site of the enzyme, then it is indeed interesting
that the falloff in the pH-activity curve between pH 7.5 and pH 10 ap-
pears to have a pI value of about 8.50
In view of the broad specificity of crystalline papain the exist-
ence of enzymatically active impurities in these preparations or the ex-
istence of more than one active center must be considered. The
and chemical homogeneity of crystalline papain to be discussed in later
chapters, renders it highly unlikely that more than one protein is in-
volved. However, the specific activity of the purified peptidases is
so much higher than that of the- -proteinases that it is virtually impossi-
ble to prove conclusively that some of the least sensitive substrates are
not being hydrolYzed by minute amounts of peptidase impurities in the pa-
pain preparations. The fact that carbobenzoxy-1-glutamic acid inhibits
the hydrolysis of all types of substrates studied is strong evidence that
only one active center is involvedo
CHAPTER VI
THE COMPOSITION OF CRYSTALLINE PAPAIN
Crystalline papain is available in amounts large enough to per-
mit a variety of physical and chemical studies .not commonly possible
with other enzymes. Such studies often require considerably more ma-
terial than enzymatic observations even though new techniques have greatly
increased the sensitivity of analytical procedures. The amounts of crys-
talline papain which were at hand permitted a detailed study of the amino
acid composition of this enzyme. In addition, the protein was further
characterized by determining the N-terminal amino acid sequence. Since
free thiol groups appear to be necessary for papain action it was of in-
terest to identify some of the peptide sequenQes involving cysteine in
the papain molecule.
The amino aeid composition of cr.ystalline papain was determined
by Miss Anne Stockell with some assistance from the author. The N-ter-
minal amino acid sequence was determined by Dr. E. o. P. Thompson. How ..
ever, this work was of such value in supplying evidence regarding the purity
of cr,ystalline papain and in establishing some of its fundamental character-
istics, that it is summarized herein.
The Amino Acid Composition of Papain
Until recently, the information about the amino acid composition
of papain has been fragmentar,y. A high tyrosine content had been demon-
strated in partially purified preparations by Fruton and Lavin (177).
-68-
-69-
Kassel and Brand (S5) had made studies of the sulfur-containing amino
acids and Balls and Linweaver (9) had determined the cystine content of
crystalline papain. A qualitative examination of a papain hydrolysate
on a two dimensional paper chromatogram was made by Blackburn (30) who
observed relatively strong spots of glycine, tyrosine, proline and lysine,
and weaker spots of other amino acids. The most recent study of the am-
ino acid composition of cr,ystalline papain was made by Close, Moore and
Bigwood (3S) with material prepared from fresh latex. For purposes of
comparison their data are included in the present summar,y.
The papain used for these studies was a twice recrystallized
preparation which was apparently homogeneous in the ultracentrifuge
and showed more than 98 per cent of a single component on electrophor-
esis. The material had been prepared for analysis as described for the
nitrogen and sulfur determinations. Samples were hydrolyzed for 20, 70
or 140 hours in sealed Pyrex glass tubes in the absence of air at 105
0
with approximately 500 volumes of 6 N HOI, three times redistilled in
glass. The hydrolysates of papain were quantitatively transferred to a
round-bottom flask and repeatedly concentrated in vacuo to remove excess
HCl. Finally, the hydrolysate was washed into a volumetric flask and made
up to volume. The amount of protein in the hydrolysates was determined by
duplicate micro-Kjeldahl analyses on suitablealiquots. T he nitrogen con-
tent of the preparations of anhydrous, ash-free papain was estimated to
be 16.1 per cent (cof. p. 46).
The amino acids in the hydrolysates of papain were separated by
chromatography on columns of Dowex-50 ion exchange resin by the method
of Moore and Stein (116), and estimated in the eluate fractions by the
photometric ninhydrin procedure of the same authors (115). Tryptophan
-70-
was determined separately by the method of Spies (146).
Table VIII summarizes the results of this study and includes the
data of Close, MOore and Bigwood (38). The data from this laborator.y have
been corrected for the loss of serine, threonine, aspartic acid and gluta-
mic acid during hydrolysis of the protein. The slow liberation of isoleu-
cine and valine resulting from the relative stability of peptide bonds in-
volving these amino acids has been taken into account. The yield of ~
moniaincreases during acid hydrolysis and appropriate corrections have
been applied for this. The details of this analysis have been described
by Smith, Stockell, and Kimmel (145). The data of Close, Moore and Big-
wood was arrived at from a single hydrolysis period of 20 hours. No cor-
rections f9r destruction or slow liberation of certain amino acids were
made by these authors.
In general, there is good agreement between the data from this
laboratory and that of Close et ale The discrepancies that do exist are
in most eases for those amino acids where oorrection factors must be used.
The most striking features of the amino acid composition of papain are the
high content of tyrosine and glycine, the presence of only one histidine
residue, and the complete absence of methionine.
The cystine content of papain has been determined by four differ-
ent laboratories and there is little agreement in these analyses
o
The
data from this laboratory was calculated from the sulfur content since
methionine is absent. When expressed on the basis of weight of free am-
ino acid (in contrast to weight of amino acid residue as in Table VIII)
the cystine content is 4.58 per cent. The data of Close et al. gives a
cystine content of 2014 per cent; the data of Balls and Lineweaver (9)
304 per cent; and the data of Kassel and Brand (85') 4 ~ 2 per cent. The
reasons for these wide variations are not readily apparent.
Amino
acid
Aspartic Acid
Threonine
Serine
Glutamic Acid
Proline
CIY"cine
Alanine
Valine
Isoleucine
Leucine
Tyrosine
Phenylalanine
Histidine
Lysine
Ammonia (NH
2
)
Arginine
Tryptophan
Cystinel
Total
-71-
TABLE VIII
COMPOSITION AND MOLECULAR WEIGHT OF PAPAIN
Gm. of amino acid
residue per 100 gm. Minimum. Calculated
protein molecular molecular
Close Smith weight weight
et al et al
(38) (145)
8.58 9 . 79 1176 19,992
3.12 3.30 3064 21,448
4.17 4.90 1778 19,558
9.96 10.91 1183 20,111
3.79 4.31 2253 20,277
6.33 6.39 893 20,539
3.73 4.49 1584 20,592
6.36 7.13 1390 20,850
4.88 5.22 2169 19,521
4.96 5.26 2152 19,.368
11.50 13.25 12.32 20,944
2 .38 2.82 5220 20,880
0.84 0.75 18293 18,293
4.49 4.97 2579 2e,632
-
1.51 1060 20,140
6.83 6.95 2247 20,223
4.02 4.27 4361 21,805
1.82 3.89 5249 20,996
98.60 (Average 20,343)
* This value is omitted from the total.
I Calculated from the sulfur content.
Assumed
number of
residues
17
7
,
11
17
9
23
13
\
15
9
9
17
4
1
8
19*
9
5
4
i
178
-72-
The recover,r of nitrogen in this amino acid analysis was 103.79
per cent. The weight recovery of amino acid residues was calculated to
be 98.60 per cent. These figures would indicate that the composition
of papain has been satisfactorily However, the high nitro-
gen recovery is somewhat puzzling since the quantity of protein in the
hydrolysates was estimated from the same nitrogen content (16.1 per cent)
as were the calculations of nitrogen recovery. In confirmation of the
results of Close et ale (38) no carbohydrate could be detected by the
carbazole method (7).
The molecular weight of papain calculated from the amino acid
composition is 20,343 which is in good agreement with the value of 20,700
found by sedimentation-diffusion measurements, and the value of 21,750
calculated from the mercury content of mercuripapain. The relatively
complete accounting of the weight of papain permits a calculation of the
partial specific volume (V) of this protein. Earlier studies have shown
that such calculations are in excellent agreement with direct measure-
ments (38, 108). These calculations were oarried out by using the values
for specific volumes of the amino acid residues given by Cohn and Edsall
(39) The amide groups were assigned equally to glutaminyl and aspara-
ginyl residues, although as will be pointed out later, it is likely that
few or no glutaminyl residues are present; the assignment of the amide
groups has no significant effect on V. The partial specific volume of
papain determined by this method is 0.724.
In Table IX the number of ionic groups of papain is given on the
assumption that a single terminal group is present which is
consistent with the single free a-amino group demonstrable by the DNP
method. The greater number of cationic groups is in accord with the
-73-
basic properties of this protein which has an isoelectric point near
TABLE IX
IONIC GROUPS OF PAPAII
Aspartic Acid 17 Arginine
9
Glutamic Acid 17 Lysine 8
Terminal Carboxyl 1 Terminal ct -Amino 1
35
Histidine
-L
. Amide Groups
=l2...
Total Cationic Groups 19
Total Anionic Groups 16
Free Amino Groups and I-Terminal Sequence
This work was performed by Dr. Eo O. P. Thompson on both crystal-
line papain and mercuripapa.in. The details of the study have been re-
ported elsewhere (151)0 The results are summarized here. The enzyme
preparations were prepared in the same manner as those for the amino
acid studies, and treated with I-fluoro-2,4-dinitrobenzene (FDNB) accord-
ing to the technique of Sanger (123) 0 The dinitrophenyl protein, (DNP
protein) was wa.shed three times successively with water, ethanol and
ether and air dried. The amount of protein in the DNP protein was es-
timated by determining the amide content of the two preparations. The
amide content of anhydrous, ash-free papain was found to be 1.6l per
cent as (NH2). This is slightly higher than the value arrived at in
the amino acid analyses.
Hydrolysis of the DNP protein was performed in 6 N Hel at 105
0
for 24 hours. For identification of the N-terminal sequence of amino
-74-
acids, partial hydrolyses were performed in 12 N Hel at 37 for 8 days.,
The hydrolysates in either case were extracted with ether'. The ether
phase was evaporated to dryness in vacno, then made up to an appropriate
volume in the desired solvent. The aqueous phase was made up to a given
volume with water. The DIP amino acids in each phase were isolated on
columns of buffered Celite 545 and determined speetrophotometrically.
TABLE I
DIP AMINO ACIDS II COMPLETE HIDROLYSATES
OF DNP PAPAIN PREPARATIONS
Preparation
Papain, recrystallized twice in
absence of cysteine, washed once
with 2 per cent NaCl
Mercuripapain I
Mercuripapain II
DIP animo acids
identified
Isoleucine
Aspartic ACiJ-
Serine
Glutamie Acid
Alanine
Valine
Glycine
Isoleucine
Aspartic ACi
J
Serine
Glutamic Acid
'-DNP Lysine
Isoleucine
Aspartic ACi
J
Serine
Glutamic A01
-DNP Lysine
Moles DIP amino
aeidper mole ot

0.80
0.1
Traces
0.84
0.07
707
0080
0.07
7.6
* These values are uncorrected for hydrolytic breakdown. The molecular
weight of papain is assumed to be 20,300.
Table X shows the of studies on various preparations of
papain and mercuripapain. The a-amino group of isoleucine and the e-amino
-75-
groups of lysine were the only free amino groups present in stoichio-
metric amounts. The small amounts of other DNPamino acids present in
the hydro1ysates could be indicative of contamination with other pro-
teins, but the possibility of slight splitting of peptide bonds during
coupling with FDNB in alkaline solution cannot be excluded. It is note-
worthy that these extraneous amino acids occur in greater abundance with
papain than with mercuripapain. This may be the result of autolysis of
papain. Autolysis is impossible with mercuripapain. In work with large
proteins with no N-termina1 reSidues, the presence of the amino acids ob-
served here in non-stoichiometric amounts has been reported (7, 32).
When corrected for the 10 per cent destruction during acid hydro-
lysis of the DNP protein, the yield of DNP-isoleucine is 0.83 to 0.87.
mole per mole of papain (Mol. Wt. 20,300). On the other hand, the mol-
ecular weight of papain calculated from the corrected yield of DNP iso-
leucine is 23,500. Within the precision of the method, this value is in
good agreement with values obtained by other means during this investi-
gation.
The uncorrected values for 6-DNP lysine in Table X give a mean
of 7.65 moles of lysine per mole of papain. This agrees with the ob-
servations made in the determination of the amino acid composition. How-
ever, a small amount of hydrolytic breakdown of -DNP lysine occurs in
the course of acid hydrolysis, and when this correction is applied the
lysine content amounts to 8.5 moles per mole of papain. The accuracy
of the DNP method is not sufficient to indicate whether the true value
is 8 or 9 moles. The determination of the correction factor for destruc-
tion of E -DNP 1ys ine is carried out under conditions slightly different
from those of the actual hydrolysis so that this may be responsible for
the discrepancy.
-76-
DNP isoleucine and two DNP peptides were isolated from the par-
tial hydrolysates of DNP papain. Both peptides contained DNP isoleucine
as the N-terminal residue. One was a dipeptide containing proline, thus
establishing the sequence isoleucylproline. The other peptide contained
both proline and glutamic acid. Partial hydrolysis of this tripeptide
permitted the identification of DNP isoleucylproline. Thus the sequence
of isoleucylprolylglutamic acid was established as the N-terminal amino
acid sequence of papain. These three amino acids were also shown to be
present in equimolecular amounts 1n both the dipeptide and the tripep-
tide.
Amino Acid Sequences Containing Cysteine
The apparent requirement of free thiol groups for papain activity
indicates that the enzymatically active center may involve a molecule of
cysteine. It was of interest to study some of the peptides containing
this amino acid which could be isolated from partial of pa-
pain. Since cysteine is not stable during acid hydrolysis, such studies
are more likely to be successful if thiol and disulfide groups are first
oxidized to the sulfonic acid, i.e., cysteic acid. This technique was
used by Sanger (131) with success in separating the A and B chains of
insulin. The strongly acidic character of cysteic acid and peptides
containing it greatly simplify the isolation of these compounds from
partial hydrolysates of proteins.
The papain used in these experiments was prepared in the same
manner as that used to determine the amino acid composition. The papain
(500 mg.) was hydrolyzed for 7 days in 12 N Hel at 37
0
The-hydrolysate
was concentrated in vacuo several times and finally treated with solid
AgO to remove traces of HCl. The precipitated Agel was removed by cen-
2
-77-
trifugation and the precipitate was washed once with water. The combined
supernatant solution and washings were concentrated and finally dried in
a desiccator under reduced pressure.
The dried hydrolysate was oxidized by solution in S ml. of anhy-
drous formic acid followed by addition of 0.5 ml. of H
2
0
2
After fif-
teen minutes at room temperature, the reaction mixture was diluted with
10 ml. of water and then concentrated under reduced pressure several times.
The final oxidized hydrolysate represented 500 mg. of protein in a volume
of about 2 ml.
An alternative procedure that gave greater yields of peptides con-
taining cysteic acid consisted of first oxidizing the protein and then
partially hydrolyzing it. The procedures used were exactly the same as
those previously described.
Two methods were used to isolate cysteic acid and cysteic acid pep-
tides from the partial hydrol1sates. Ionophoresis in an apparatus similar
to that described by Piynn and de Mayo (47) had the advantage of separating
free e.ysteic acid from the cysteic acid peptides but required considerable
amounts of time to collect sufficient material for further study. The
ionophoresis was carried out on a full sheet of Whatman 13 filter p a p e r . ~
This was folded at the center line and 0.2 mI. of the oxidized partial
hydrolysate was applied to the paper along this fold. The paper was sus-
pended on glass rods with the ends dipping in 1 N acetic acid, pH 2.),
and a potential difference of 400 volts D.C. was applied for four hours.
This operation was carried out in a covered glass aquarium at room tem-
perature. After completion of the ibnophoresis the sheet of filter paper
was dried in air and strips were cut from the center and both edges at
right angles to the center fold. These strips were sprayed with a solu-
-7'6-
tion of ninhydrin (0.1 per cent) in 95 per cent ethanol. The color was
allowed to develop at room temperature.
Five distinct bands appeared on the paper after staining with
ninhydrin. The results are illustrated in Fig. 12.
centimeters
t
origin
,
oentimeters
Figure 12. Ionophoresis of Oxidized Partial Hydrolysates of
Papain. The sample was applied along the center line (origin) of a
full sheet of Whatman #3 filter paper and subjeoted to a potential dif-
ference of 400 volts for four hours. The electrodes and the ends of
the paper were dipping in 1 N acetio acid pH 2.3. The bands were loc-
ated by spraying sample strips with 0.1 per cent ninhydrin in 95 per
cent ethanol and allowing the color to develop at room temperature.
This is a soale drawing showing the distance and the direction that
'each band moved.
The stained paper strips served as guides to locate the bands
on the unstained portions of the filter paper sheet. Once located,
these bands were cut out and the adsorbed material eluted with water
by the method of Sanger and Tuppy (132). Several ionophoretio runs
were made under identical conditions and the eluates from the indivi-
dual bands pooled.
To identif,y the amino acids in each band, samples of the pooled
eluates were hydrolyzed in sealed tubes in 6 N Hel for 24 hours at 37.
These hydrolysates were freed of excess Hel by repeated concentration
in vacuo and chromatographed on Whatman #4 filter paper with water-sat-
urated phenol: 0.3 per cent arrnnonia (31). 'Band 1 was found to contain
only free cysteic acid. Band 2 contained high concentrations of cysteic
acid, aspartic acid, glutamic acid, serine and glycine. Band 3 showed
only small amounts of cysteic acid and many other amino acids o Bands 4
-79-
and 5 contained no acid. Further investigation was carried out
on material from Band 2.
The second method for isolating the acid peptides was a
column chromatography procedure with the ion exchange resin Dowex-50
(4 per cent cross linked; mesh size 50 to 100). A column of this resin
(10 x 2.5 em.) was prepared and the resin was cycled several times by
passing through 2 M NaOM then 2 M Hel. Finally, the resin was left in
the hYdrogen and washed with water until the washings were neu-
tral. The partial was placed at the top of the column and
run through at a flow rate of about 1 ml. per minute. Under these con-
ditions cysteic acid and some peptides containing cysteic acid will not
be retarded by the resin. All of the other amino acids and peptides
will be tightly bound to the resin and will require elution at more
alkaline pH values. The eluate containing the cysteic acid peptides
was concentrated in vacuo until dr.y.
Band 2 from the ionophoresis and the cysteic acid fraction from
the column chromatographY were subjected to further fractionation by two
dimensional paper chromatography. Approximately one-fifth of each frac-
tion (in 0.05 ml.) was spotted on a full sheet of Whatman #3 filter pa-
per. The solvent for development in the first dimension was water-sat-
urated phenol: 0.3 per cent ammonia and for the second dimension was
n-butanol: acetic acid: water (4:1:5 v/v) (31). After completion of
development of the chromatogram, the paper was thoroughly dried and
sprayed with a solution of ninhydrin (0.025 per cent) in 95 per cent
ethanol. The color was allowed to develop at room temperature. When
the spots had appeared they were cut out and washed with acetone to
remove excess ninhydrin. The peptide was eluted from the paper by the
-80-
technique of Sanger and Tupp.y (132). A portion of each eluate was hy-
drolyzed in 6 N HCl in a sealed tube for 24 hours at 105
0
and the con-
stituent amino acids identified by paper chromatography with one of the
systems previously described. The remainder of the eluate was treated
with FDNB as described by Sanger and Tuppy (132). After completion of
the dinitrophenylation, excess FDNB was removed from the alkaline solu-
tion by extraction with ether. The aqueous phase was concentrated to
dryness several times, then redissolved in three times redistilled 6 N
HCl and hydrolyzed for 4 to 8 hours' at 105
0
The hydrolysates were ex-
tracted three times with ether to remove the DIP amino acids, and both
the ether and aqueous phases were freed of excess HOI by concentration
to dr,yness. The amino acids in the aqueous phase, including DNP cysteic
acid, were identified by paper chromatography as described previously.
DNP amino acids were identified by paper chromatography on Whatman 14
filter paper buffered at pH 6.0 with 0.1 M phthalate. The mobile phase
was tert-amyl alcohol saturated with 0.1 M phthalate buffer pH 600.
Figure 13 shows the approximate location of the peptide spots
on the two dimensional chromatogram. Fifteen distinct spots were read-
ily detected. Spot 5 which contained only free cysteic acid was not
present in the two dimensional of Band 2 from the iono-
phoresis. All of the spots except Spot 9 were found to contain cysteic
acid.
Table XI shows the amino acid composition of each spot, and the
DNP amino acid resulting when each peptide was treated with FDNB. In
the case of dipeptides it was possible to establish the amino acid se-
quence with this treatment. At least two tripeptides were identified
and in order to establish the amino acid sequence in these cases it was
-81-
necessary to hydrolyze the material partially and repeat the dinitro-
phenylation. Also included in Table XI is the probable amino acid se-
quence of some of the peptides containing cysteic acid.
<
~ +

CD a::(D
CD
CJ:)
ill
@
CID
CID
m
Figure 13. Two dimensional 'Paper Chromatogram of Cysteic Acid
Peptides Isolated from Partial Hydrolysates of Papain. The mixture of
peptides was flaeed on a full sheet of Whatman #3 filter paper at the
spot marked -.:. The solvent for the !irst dimension w a ~ water-saturated
with phenol: 0.3 per cent ammonia; the second dimension was developed
with n-butanol: acetic acid: water (4:1:5 v/v). The spots were located
by spraying with 0.025 per cent ninhydrin in 95 per cent ethanol.
From the data in Table 1I it is not possible to describe the com-
plete amino acid sequence of the tripeptides in Spots 8 and 15. The ma-
terial from Spot 8 was studied further by partial hydrolysis for 3 days
o
at 37. This hydrolysate was chromatographed on paper in the butanol:
acetic acid: water system and found to separate into three componentso
These components were eluted fram the paper and studied by the ~ N P method.
Spot
Number
1
2
3*
4
5*
6
7
8
9
10*
11

tl *
15
-82-
TABLE XI
CYSTEIC ACID PEPTIDES ISOLATED FROM PARTIAL
HYDROLYSATES OF PAPAIN
DNPAmino Acid Amino Acids Probable Sequence
Serine Cysteic Acid
Ser - CyS03H ?
#
Aspartic Acid Cysteic Acid Asp - C
Y
S0
3
H
Cysteic Acid
Aspartic Acid ?
Cysteic Acid Aspartic Acid
CyS03H - Asp
Cysteic Acid
Serine ?
Serine Cysteic Acid
Sar - CyS03H
Cysteic Acid Glycine
CyS03H - Gly
Cysteic Acid As part ic Acid CyS03H (Asp, Gly)
Glycine
Glutamic Acid
Cysteic Acid
Glycine
Valine C1steic Acid Val - CyS0
3
H
Cysteic Acid,
Serine, Glycine
Glutamic Acid
Glycine Cysteic Acid Gly (Pro, CySO H)
Proline
3
, This peptide is unquestionably different from the peptide in Spot 6
which also contains serine and cysteic acid, since the two spots are
widely separated on the two dimensional chromatogram. Its slow rate
of movement in both solvents suggests that it may contain two moles
of cysteic acid. '
* Not present in large enough quantities to study by the DNPmethod o
As a result, it was possible to identify the sequences and
glyeylaspartic acid. This established the amino acid sequence of the tri-
-83-
peptide from Spot 8 as cysteylglycylaspartic acid. There was not suffi-
cient material in Spot 15 to carry out similar studies but from the be-
havior of this tripeptide and its hydrolysates, certain inferences can
be made o Acid hydrolysates of material from Spot 15 invariably appeared
to contain much more cysteic acid than glycine or proline. The rapid
movement of this tripeptide in the pllenol:ammonia system practically
excludes the existence of more than one equivalent of cysteic acid in
the peptide. The low yield of glycine and proline could be explained
easily if the sequence glycy1proline was present because this dipeptide
will form the diketopiperazine during acid hydrolysis. The d1ketopip-
erazine will not stain with ninhydrin. Since the has glycine
as its residue, it is possible that the tripeptide in spot 15
has the sequence glycylprolylcysteic acid.
Discussion
The data from the amino acid analysis provide additional evi-
dence of the purity of crystalline papain. The relatively complete
accounting of the weight of the protein, the complete absence of meth-
ionine, the presence of only one histidine residue and the good agree-
ment between the individual molecular weight calculations can all be
cited as evidence that only one protein was being dealt with in these
studies. The finding of 1 N-terminal isoleucine residue per mole of pa-
pain also supports this conclusion 0 In fact, if the DNP amino acids in
papain hydrolysates are all assumed to arise from protein impurities in
the papain preparations, then the DNP studies indicate that the protein
is better than 99.9 per cent pure. This reasoning fails, of course, if
the protein impurities have the same N-terminal residue as papain.
The amino acid analysis and the end group determination indicate
- ~ -
that papain is a single peptide chain containing lS2 amino acid resi-
dues. This conclusion is arrived at on the basis that there are S cys-
teine residues in the molecule. It is not known how many of these are
present as cystine. More recent data from this laboratory suggests
that there may be only 6 cysteine residues in papain. This is being
investigated further.
The study of cysteic acid peptides from papain indicates that
at least four of the cysteine residues are accounted for. These sequen-
ces are serylcysteic acid, aspartylcysteic acid, valylcysteic acid and
glyeylprolylcysteic aCid
l
The peptide serylcysteic acid occurs in great
abundance in the partial hydrolysates of papain and it is possible that
this sequence occurs more than once. Furthermore, it is possible that
the sequence cysteylcysteic acid occurs as suggested from the observa-
tions on Spot 1 of the two dimensional chromatogram of cysteic acid
peptides. If these latter suggestions are correct, then six cysteine
residues are accounted for in peptide sequences.
lEven though the sequence of this tripeptide is not conclusively
established, the N-terminal glycine residue eliminates the possibility
that it contains sequences already identified.
CHAPTER VII
THE PHYSICAL PROPERTIES OF PAPAIN AND MERCURIPAPAIN
Electrophoretic studies ot cr,ystalline papain have been an im-
portant asset in establishing the purity of crystalline papain. In addi-
tion these studies have yielded considerable information about the nature
of the protein molecule. The ultracentrifuge has been of equal value par-
ticularly with regard to mercuripapain. The behavior of this substance
was of special interest because of its tendency to form polymers under
certain conditions. These physical studies have established the isoel-
ectric point of papain and have yielded excellent evidence regarding the
molecular weight.
Elegtrophoretic Studies
Crystalline papain was examined electrophoretically at pH values
ranging from 2.8 to 9.6. In order to avoid the possibility of autolysis
most of the runs were made in the absence of cysteine and Versene. In
some runs 0.005 M cysteine was present and this had no detectable effect
on the mobility of the protein. When both cysteine and Versene were
added to study the enzymatically active protein a mobility decrease at
pH 4.4 from a value of about + 4.6 X 10-
5
to + 4.1 X 10-
5
sq. cm. per
volt per second was observed.
The effect of pH on the mobility of crystalline papain is shown
in Fig. 14. The apparent isoelectric point is at pH 8.75 in good agree-
ment with the earlier findings which indicated an alkaline isoeleetrie
point near pH 9 (9, 127).
-85-
~
~ + 4
:!:::
o
~ T 2
E
o ot-------
'Q
)(
~ -2
-86-
- 4 - - + - - - - - - - ~ ________ __ __
Figure 14. Electrophoretic Mobility as a Function of pH for
Crystalline Papain. The runs were made at 1.5
0
in univalent buffers
at ionic strength 0.1. Ac is acetate, V is veronal, and G is glycine.
The most distinctive feature of the mobility of inactive papain
is the broad zone between pH 3.9 and 6.0 over which the mobility remains
relatively constant. If it is assumed that the form of the mobility curve
is similar to that of the titration curve which is the case for other pro-
teins (96), the character of this curve is of considerable interest in re-
lation to the amino acid composition of papain. This protein contains an
excess of three basic groups, which, at the isoelectric point, must be urt-
charged; these must be the single imidazole group of histidine, the ter-
minalQ"-amino group of isoleucine, and the equivalent of one t-amino group
of lysine
l
The change in mobility with pH indicates that the pK of the
imidazole group of histidine must be above 6 and probably lies in the
neighborhood of 6.S, as judged by the change in slope of the mobility
l.rhis view may have to be altered if free sulfhydryl groups on
the papain molec'ule have pK values near pH 8.5. If this is true then at
the isoelectric point the imidazole group of histidine would be uncharged
and the equivalent of two thiol groups would bear negative charges.
-87-
curve between pH 6.0 and the isoelectric point. Since papain has a con-
stant, maximal enzymatic activity on BAA over the region from pH 5.0 to
7.5, it is unlikely that the imidazole group which is titrated in this
region is directly concerned in the active catalytic center of the en-
zyme.
The constant mobility from pH 3.9 to 6.0 also demonstrates that
the sixteen ionizable carboxyl groups estimated to be present in papain
must have pK values considerably below 3.9, probably as low as 3.0 to
3.4. Cohn and Edsall (39) have estimated that the pK values of -car-
boxyl groups of aspartyl residues are between 3.0 and 4.7 and of""'-car-
boxyl groups of glutamyl residues to be near 4.4 from titration studies
on peptides of known structure. Since it is evident that the carboxyl
groups of papain must have relatively low pK values, it is possible that
the free carboxyl groups of aspartyl residues predominate in this pro-
tein and that there are few or no free carboxyl groups of glutaD.tVl resi-
dues; however, until the location of the amide groups of papain is de-
termined, this suggestion can only be regarded as tentative o
A comparison of papain with two other proteins of known compo-
sition, insulin and lysozyme, is of Sanger, Thompson and
Tuppy (134) have shown that in insulin all of the aspartic residues are
in the amide form. Hence, for one A chain and one B chain, there are
four II( -carboxyl groups of glutamyl residues, two terminal -carboxyl
groups and two histidine residues. In contrast to papain, the titra-
tion curve of insulin measured by Cohn, Ferry and Blanchard (39) shows a
steep slope at pH values below 6.0 in conformity with the relatively high
pK value of 4.4 to groups of residues o
Studies by Winsteiner and Abramson (172) of the electrophoretic mobility
of insulin crystals or of insulin adsorbed on quartz also show a steep
-88-
slope between pH 4 and 6.
Analyses of lysozyme indicate 1 histidine residue, 20 to 21 as-
partic residues, 5 to 6 glutamyl, and 18 amide groups per mole of protein
(53, 92, 114, 150). The electrophoretic mobility of lysozyme shows lit-
tle or no change" between pH 4 and 6, as judged by the studies of Longs-
worth et a1: (97) on whole egg white, or those of Alderton and coworkers
(4) on the crystalline enzyme. It would appear that most of the free
carboxyl groups of this protein have low pK values, like those of papain,
and probably belong to the predominating aspartyl groups; all or most of
the few residues of glutamic acid are probably in the amide for.m.
Papain is only sparingly soluble in the cold in the neighbor-
hood of its isoeleetric point and the electrophoretic studies in this
region had to be made at concentrations of less than 0.2 per cent. In
fact, papain in saturated solutions at room temperature between pH 7.5
and 9.5 will crystallize when solutions are chilled to the bath tempera-
ture of 1.5
0
of the electrophoresis apparatus. Because of the necessarily
low protein concentrations, electrophoretic runs above pH 7 were useful
for an estimate of mobility but not for a critical evaluation of homo-
geneity in this region.
Some of the electrophoretic patterns which were obtained are
shown in Fig. 15. It is apparent that crystalline papain is, in general,
monodisperse in the sense that it is free of grossly contaminating pro-
teins. The sample shown in Fig. 15, A contained less than 2 per cent of
a minor component which was not present in most of our preparationso
However, it should be noted that after periods of migration as long as
250 to 300 minutes, the patterns at pH 304 to 4.4 appeared slightly
broader than might be expected for a completely homogeneous substance.
The excessive spreading was not apparent in runs made for 100 to 150 mino
-89-
The run made at pH 208 was markedly hetergeneous; the mobility
value shown in Fig. 14 was that of the major component. It should be
noted that, at this pH value, papain is readily and irreversibly inacti-
vated
A
Figure 15. Electrophoretic Patterns of Cyrstalline Papain. A,
tracings of the electrophoretic patterns of twice recrystallized papain,
pH 3.9; protein concentration = 1.0 per cent in acetate buffer contain-
ing 0.001 M Versene and 0.02 M cysteine. Ascending and descending pat-
terns are shown after 275 minutes of migration. The minor peak evident
on the descending pattern represents less than 2 per cent of the total
protein and was absent in most of the preparations. B, shows the de-
scending pattern at 0.2 per cent protein concentration in Veronal buffer
at pH 7.5 after 150 minutes. C is the descending pattern after 150 min-
utes in acetate buffer at pH 6.0 at a protein concentration of 0.4 per
cent. Cysteine and Versene were omitted in runs Band C. The patterns
in Band C are at approximately twice the linear enlargement of those
in A.
The lack of complete homogeneity does not appear to reflect any
gross contamination of the cr,ystalline enzyme which appears to be rela-
tively free of other proteins by several criteria, e.g. amino acid com-
position, free amino groups studied by the dinitrophenyl method, and
sedimentation in the ultracentrifuge. It is more likely that the slight
electrical heterogeneity in the region of good stability may be due to
partial binding of ions by the basic protein or small variations in the
amide content.
-90-
Sedimentation Studies
Preparations of papain which had been crystallized two or three
times gave sedimentation patterns which appeared to be monodisperse; some
typical observations under conditions in the ultracen-
trifuge are shown in Fig. 16.


Figure 16. Sedimentation Patterns of Crystalline Papain in
Acetate Buffer at 0.1 Ionic Strength. A shows papain (2.0 per cent)
containing 0.02 M cysteine at pH 3.9. B is papain (1.25 per cent)
under the same conditions with cysteine omitted. C shows results
with papain (1.0 per cent) which had been oxidized with iodine at pH
6.0. For all the runs the exposures were at 16 minute intervals.
The absolute heights have no significance since the magnifications
were changed during the runs.
Most of the studies were performed at pH 3.9 to 4.0, since the
solubility of the protein in this region is much greater than it is
near the isoe1ectric point. Under these conditions, it was found that
there was a marked difference in behavior in the presence and absence
of cysteine. The sedimentation constants of several different prepara-
tions of crystalline papain obtained in fourteen runs in acetate buffer
(0.1 to 0.2 ionic strength) at a cysteine concentration of 0.02 Mare
shown in Fig. 17, 1. In four of these runs 0.002 M Versena was present
also, but this substance has no detectable effect on the sedimentation
constant. It is apparent from these results that there is no substan-
tial influence of protein concentration on s20,w. The average of all
-91 ....
these measurements is 2.42 0.04 S.

0'
(\J 2.
CI) 2.
. .
PROTEIN CONe. PER CENT
Figure 17. Sedimentation Conatanta (a ) of Crystalline Pa-
pain as a Function of Protein Concentration. 10Alows studies at pH
3.9 to 4.0 in acetate buffer (0.1 to 0.2 ionic strength) at a cysteine
concentration of 0.02 M , results with added Versene at 0.002 M;
0, without Versena. B shows results in acetate at pH 4.0 (0.1 ionic
strength) in the absence of cysteine (e) and for papain oxidized with
iodine (0). C gives measurements with 0.02 M cysteine at 0.1 ionic
strength in acetate at pH 5.4 (0) and in Veronal at pH 7.0 (.). For
B and C the straight lines have arbitrarily been drawn to an extrapol-
ated value of 2.42 S for s20 w at zero concentration; this is the av-
erage value found at pH 4 in'the presence of cysteine.
In Fig. 17, B are shown some results obtained under the same
conditions in the absence of cysteine, together with runs made on pa-
pain which was oxidized with iodine at slightly acid pH values to pro-
duce completely inactive material. It is evident that there is a pro-
nounced effect of protein on s20,w' although the extra-
polation leads to a value at zero concentration which is that found for
cysteine-papain within the precision of the measurements. It would ap-
pear from the few measurements in Fig. 17, B that crystalline papain
which, in the absence of cysteine and Versene is completely inactive,
behaves in the same manner as papain which has been oxidized with io-
dine.
Fig. 17, C shows a few measurements made at pH 5.4 and 7.0 in
the presence of cysteine (0.02 M). Here it is also clear that protein
concentration has a substantial effect on s20,w.
-92-
Previous studies on chymotrypsin (136, 142, 143) and trypsin (40)
have shown that these proteolytic enzymes tend to form mobile mixtures of
monomers and dimers under certain conditions. The behavior of cysteine-
papain at pH 4.5 to 7.0 and of the oxidized protein in the absence of cys-
teine at pH 4 demonstrates that papain also has a tendency to aggregate in
dimers and that the extent of dimerization is a function of protein con-
cent ration and pH. All of the measurements are in substantial agreement
that the papain monomer possesses a sedimentation oonstant of 2.42 So
Diffusion Studies
These measurements were made 1n the electrophoresis cell by the
procedure of Longsworth (96) from photographs taken by the schleiren
scanning method. The results were calculated by the height-area method
from the formula D = 2 / 4 ~ tH
2
, where A is the area under the ourve, H
the maximal height, t the time in seconds, and D the diffusion constant
in sq. cm, per seoond.
The studies were made at pH 3.9 in an acetate buffer containing
0.02 M cysteine of 0.12 ionic strength; these are the conditions under
which it was found, as described above, that papain exhibits completely
monomeric behavior in the ultracentrifuge. In fact, as a control for
each diffusion run an identical sample was tested in the ultracent:ri.i-
fuge and these measurements are given with the data of Fig. 17, A and
Fig. 20. The measurements were made at 1.5
0
and corrected for the di-
fference in viscosity and temperature in the manner described by Longs-
worth (96) to give values of D
20
,w' In each of three runs, the two halves
of the cell were used separately, giving duplicate determinations in'
each case. Six photographs were taken at intervals between 20 and 70
hours after the boundaries were established and the slopes were obtained
-93-
from plots of IIH against -It; satisfactory straight lines, calculated by
the method of least squares, were obtained in all cases. Areas were meas-
ured on tracings of projected enlargements of the individual schlieren
curves. The computed diffusion constants are given in Table XII. The av-
erage value for D
20
,w is 10,27 t 0.13 X 10-
7
sq. cm. per second.
T4BLE XI!
DIFFUSION CONSTANT OF CRYSTALLINE PAPAIN
The measurements made at pH 3.9 in acetate buffer of 0.12 ionic
strength containing 0.02 M cysteine.
Run No. Concent ra tion
D
20
,w X ,10
7
per cent sq. cm., per sec.
1 0.61 10.11, 10.34
2 1.OS 10.38, 10.10
3*
0.65 10.21, 10.49
I
AVf3rage 10.27 - 0.13
* Mercuripapain containing 0.02 M cysteine and 0.002 M Versene under
the same conditions as above.
Molecular" Weigl1t
The molecular weight (M) of the papain as determined
from sedimentation-diffusion measurements, has been computed from the
usual formula, (148) M = RTs/D{l - Vp), where V is the partial specific
volume, T is the absoiute temperature, R is the gas constant, and p is
the density of the water. V is 0.724, as calculated from the amino acid
composition of the protein. With s = 2.42 XlO-
13
and D = X 10-
7
the molecular weight is 20,712 or, within the precision of the data
20,700. The frictionai ratio (fifo) computed from the above data is
1.16, which is similar to that of ovalbundn and hemoglobin (148).
From the contour lines calculated by Cncley (121), the molecule is
-94-
about 40 per cent hydrated if assumed to be symmetrical. If the axial
ratio (a/b) is 2:1, the molecule may be hydrated about 26 per cent.
Properties of Mercuripapain
The isolation of a crystalline mercur,y complex of papain made
possible an investigation of the physical properties of this interesting
derivative. From the analytical results which indicate a combining ratio
of 1 mole of mereur,y to 2 of papain, it was anticipated that physical meas-
urements would reveal the presence of a dime ric complex, possibly similar
to that found by Hughes (76) for mercaptalbumin. This was not found to be
the case and mercuripapain represents a somewhat more complicated instance
of protein-metal interaction.
For these studies, papain which was recrystallized two or three
times was converted to the mercury derivative near neutrality by the method
previously given. Sedimentation studies have now shown that the character
of the combination with mercury is pH dependent. At pH 4 redissolved crys-
stalline mercuripapain shows the monomeric behavior described for papain
itself. Mercuripapain at 1.52 per cent in acetate buffer at 0.1 ionic
strength gave a monodisperse sedimentation curve (Fig. 18) which, in dup-
licate determinations, gave 8
20
,w values of 2.38 and 2.46 s. The first
determination was made as rapidly as possible after dissolving the protein
and the second after the solution was permitted to remain at 4
0
for 24 hours.
Papain crystallized three times (0.59 per cent) at pH 4.0 with 1 or
2 equivalents of mercur,y added, gave monodisperse sedimenting boundaries
with s values between 2.51 and 2.80 S (average = 2.62 S) when run im-
20,w
mediately or after standing from 1 to 4 days. These values are in the
range previously found for papain at pH 4 in the absence of cysteine at
the same protein concentration (Fig. 17, B).
-95-
Figure 18. Sedimentation of Mercuripapain (1.52 per cent) at
pH 4.0 in 0.1 Ionic Strength Acetate Buffer. The first exposure was
taken 45 minutes after attaining full speed, and the subsequent ones
at 16 minute intervals. The S20,w value of the monodisperse system
is identical with that of papain at this pH.
A
Figure 19. Electrophoretic Patterns ot the Descending Migration
of Cr,rstalline Mercuripapain. A, 0.1 ionic strength acetate buffer at
pH 3.9 at a protein concentration of 1.1 per cent; the photograph was
taken after 300 minutes. B, the same preparation under the same con-
ditions, but in the presence of 0.02 M cysteine and 0.002 M Versene at
a concentration of 1.5 per eent after migration for 250 minutes.
Although the sedimentation results at pH 4 do not indicate any
change in size, papain does remain combined with mercury at this pH as
shown by electrophoretic measurements. Fig. 19, A shows the descending
migration of mercuripapain after 300 minutes. The smaller and slower
peak, estimated to be approximately 35 per cent of the total area, had
a mobility ot + 4.7 X 10-
5
sq. cm. per volt per second which is essen-
tially identical with the value previously found for papain itself. The
larger peak Of higher mobility, + 5.5 I 10-
5
, has acquired a greater pos-
itive charge which suggests that it is papain combined with mercur,y. When
the same preparation of mercuripapain was tested in the presence of 0.02
M cysteine and 0.002 M Versena, it was found that the more rapidly migra-
-96-
ting peak had disappeared and the nearly monodisperse peak (Fig. 19, B)
had the mobility, + 4.1 X 10-
5
sq. cm. per volt per second of uncombined
papg.in under the same conditions. Mercuripapain treated with cysteine .
and Versene has given the highest proteolytic activity observed for pa-
pain. It is evident that cysteine and Versene together can remove bound
mercury from the protein.
2 . 6 1 - - - - - - ~ - - - - - - ~ - - - - - - ~ ~
2.4
S20,W Z.
u
o o Q
o
o
0.5 1.0 1,5
PROTE IN CONe.""' eyo
Figure 20. Sedimentation Constants (0) of Mercuripapa.in in
0.12 Ionic Strength Acetate Buffer Containing 0.02 M C,ysteine and
0.002 M Versene at pH 3.9. The values formercuripapain without added
cysteine and Versene are also shown (C).
The sedimentation beha.vior of mercuripapa.in at pH 4 in the pres-
ence of cysteine and Versene was identical with that of papain itself
under the same conditions and gave essentially the same average value,
The data are shown in Fig. 200 The diffusion

constant of mercuripapain in the presence of cysteine and Versene, al-
ready given in Table XII, is identical with that of papain.
The properties of mercuripapain at pH 8 are markedly different
from those at pH 4. The complex dissolves at pH 8 only after prolonged
standing at room temperature; this suggests a possible slow dissocia-
tion of strong bonds in the crystals. The sedimentation behavior of mer-
curipapain at the higher pH is strongly influenced by the protein con-
centration. Patterns obtained at four different protein concentrations
at pH 8.0 are shown in Fig. 210 It is evident that the amount of heavy
component diminishes with decreasing protein concentration. At 0.33 per
-97-
cent it is present only as a marked asymmetry on the main peak and rep-
resents less than three per cent of the total area; its sedimentation
constant cannot be estimated.
A
D
Figure 210 Sedimentation Patterns of C'ristalline Mercuripapain
at pH 8 at Four Different Protein Concentrations. The runs were made at
a total ionic strength of 0.1 in Tris buffer containing sodium chloride,
A is at 2.28 per cent, B at 1.3 per cent, C at 0.65 per cent, and D at
0.33 per cent. It is apparent that the amount of heavy component dimin-
ishes with protein concentration; at the lowest concentration, it is
present only as a slight asymmetry on the main component. All of the
tracings shown were made from photographs t a ~ e n between 31 and 41 min-
utes after reaching full speed (59,780 r.p.m.) ,
Table XIII gives for the two components the estimated sedimenta-
tion constants and relative concentrations. The sedimentation constant
of the light component is identical with that observed for the monomer
at acid pH values; the average s20,w for the nine values in Table XIII
is 2.43 t 0.13 s. The estimated maximal value for the heavy component
is 8.0 S.
There is no apparent time dependence of relative concentrations
or sedimentation constants of the two components as judged by a test at
a protein concentration of 2.28 per cent at which one run (No.8) was
made immediately after solution of the protein and the other (No.9)
after the preparation was allowed to remain at room temperature for an
additional 24 hours. However, it should be noted that before the first
run was made mercuripapain cr,ystals were suspended in the buffer for
about 90 minutes at room temperature
o
-98-
TABLE nIl
SEDIMENTATION OF PAPAIN AT pH 8
The runs were made in Tris-sodium chloride buffer at 0.1 ionic strength.
Component 1 Component 2
Protein
Run No. Concentration
Amount Amount s s
20,w

20,w
per cent per cent S per cent. S
1 0.33 97 2.49 3
*
2 0.44- 97 2.38 3
*
31 0.55 90 2.45 10
*
4
0.65 57 2.53 43 6.9
5# 1.09 33
2.26 67 8.0
6 1.30 27 2.54 73
8.0
7# 1.63 25 2.23 75
8.0
8 2.28 21 2.28
79 8.0
9
2.28 21 2.74 79
8.0
* The sedimentation oonstants of these beav,y materials cannot be meas-
ured ..
I This test was made after 24 hours in the same buffer oontaining 0.001
M Versena.
Runs 3, 5, and 7 (Table XIII), which were made in the presence of
0.001 M Versene, gave the same results as in the other studies. It is
evident that the chelating agent alone will not dissociate the heavy c o ~
plex, and it will not produce a regeneration of the enzymatic activity.
Estimations of the concentration given in Table XIII can be re-
garded only as approximations, since it is known that in mixtures of
proteins area measurements may deviate considerably from the true oon-
oentrations, generally in the direction of overestimating the concentra-
tion of the lighter component, as in the recent study of Harrington and
Schachman (66); however, it is evident that at high concentrations only
small amounts of monomer are present and that under the conditions of
crystallizing mercuripapain it must be the heavy form which is less sol-
I
uble and cr.ystallizes from solution.
-99-
Although the analysis of mercuripapain indicates a ratio of one
mole of mercury to 2 moles of papain, it is obvious that the heavy com-
penant possesses a sedimentation constant which is much larger than that
expected for a dimer. An approximate estimate of the size of this ma-
terial can be made on the basis that for spherical molecules the molec-
ular weight is proportional to s3/
2
From the available data, (8.0/
3/2
2.42) (20,7oo) = 124,000, or 6 times the size of the monomer. If,
however, the monomer and the heavier complex differ greatly in symmetry,
the calculated molecular weight may be greatly in error.
Studies of the activation behavior of papain have shown that, in
addition to a reducing 9ubstanJe such as cysteine, some chelating agent,
such as Versene, is essential for maximal activity. This has suggested
that crystalline papain contains heavy metal impurities. Papain was,
therefore, studied in the ultracentrifuge at pH 8 at which mercuripapain
shows the presence of large aggregates. Under these conditions, papain
contains heavy naterial as well as the monomer (Table XIV). It should be
noted that the heavy component has a smaller sedimentation constant (505
to 6.1 S) than does the heavy comppnent of mercuripapain (8
0
0S). Addi-
tion of 0.001 M Versene does have an effect in dissociating the heavy
complex of papain (Run 4, Table XIV). It is likely that the trace metal
impurity in crystalline papain is responsible for the aggregation into
heavy particles. This metal ion, whose nature is unknown, differs from
mercury insofar as it can be removed from the protein complex by the chela-
ting agent alone.
Nature of Merburipapain Complex
In order to attempt an explanation of the nature of mercuripa-
pain complex, it is necessary to recapitulate certain pertinent observa-
-100 ....
TABLE XIV
SEDIMENTATION OF PAPAIN AT pH g
Run NO
q
Protein Component 1 Component 2
Concentration
Amount s
20,w
Amount s
20,w
per cent per cent S per cent S
1 0.38 99
2.60 1
*
2 0.65 62 2.38 38 5q5
3
1.09 54 2.45 46 6.1
4# 1.09 90 2.87 10
*
'-
* The sedimentatiom constant cannot be estimated.
# After addition of 0.001 M Versena and 24 hours at room temperature.
tions on papain as well as mercuripapain. (1) Papain contains a atoms
1
of sulfur and no methionine (38). Since one or more sulhydr,yl groups
are essential for activity, these must be present in an even number,
that i s ~ at least two sulfhydryl groups per mole of protein. (2) Oxi-
dized papain is inactive but can be partially reactivated by the addi-
tion of cysteine. In addition, the oxidized form appears to be present
as a mixture of monomers and dimers, but, on dilution, the dimeric form
tends to disappear. This suggests that oxidized papain contains a mix-
ture of an intramolecular S-S form and a dimer linked by an S-S bridge:
f
apaif
+
S-s
-papain-S-S-papain-
An intermolecular S-S bridge could dissociate on dilution without any
energy change to form an intramolecular disulfide bond. (3) Mercuri-
papain which is cr,ystallized in the presence of excess mercury presumably
lTbe sulfur analyses indicate that papain contains 8 atoms of sul-
fur but more recent determinations of cysteine in this laboratory suggest
that only 6 residues of this amino acid are present in the protein. The
other two sulfur atoms possibly result from sulfate ion combined with the
basic protein. The discussion above requires only that an even number of
cysteine residues are present in papain.
-101-
represents the least soluble, stable form of the complex, and contains 1
mole of mercury for 2 moles of papain. At pH 4, the complex is present
only as monomers but is completely inactive. From the electorphoretic
studies, there are two types of monomers at this pH. It is likely that
these are HS-papain-S-HgCl and papain. Full reactivation is achieved with
I I
3-,5
Versene plus cysteine, and partial activation with cysteine alone. If
HS-papain-3H were present, the preparation should have 50 per cent of
the activity of active The electrophoretic data (Fig. 19, A)
indicate that somewhat more than half of the protein has a greater posi-
tive charge. The change in mobility of papain for an excess of three
positive charges is + 4.6 X 10-
5
sq. cm. per volt per second on proc-
eeding from the isoelectric point to pH 3.9. The mobility of the fast
component of mercuripapain is + 5.5 X 10-
5
sq. cm. per volt per second,
which is about the order of magnitude to be for a net ohange of
one positive charge at this pH. (4) At pH 8 the heavy complex (5 = 8)
of mercuripapain dissociates on dilution to the size of monomers. Hughes
(76) has previously noted that the mercaptalbumin dimer, which has the
form albumin-5-Hg-S-albumin, dissociates on dilution. It is, therefore,
likely that mercuripapain also contains linkages of this type.
On the basis of the above discussion, it is probable that the
heavy mercuripapain complex contains two types of intermolecular linkages,
papain-S-Hg-S-papain and papain-S-S-papain. The 5 = 8 unit nmst contain
an even number of papain units in order to fit the ratio of 1 mole of
Hg to 2 moles of papain. The approximate value of M, calculated above
from the sedimentation constants, suggests the presence of 6 moles of
papain in the complex. It should be reemphasized that the heavy complex
must be the least soluble type (strongest eyrsta! lattice forces), under
-102-
the conditions of cr,rstallization from 70 per cent ethanol, but many
other types of Hg-protein interactions must take place in such solu-
tions in the presence of excess mercury. The data given in Table XIII
indicate that the equilibrium between the heavy material (S = 8) and
the monomer is exceedingly sensitive to concentration. If 6 moles of
papain are combined in the polymer, this is to be expected, since for
the over-all equilibrium 6 moles of and K = (pa-
pain)6;{ papain)6. The amount of polymer formed would be a sixth power
function of the papain concentration. The available data are consist-
ent with a large exponential function but are not sufficiently precise

for an estimate of the value of the exponent. Increasing the protein
concentration from 0.44 to 0.65 per cent increases the amount of heavy
polymer from an estimated maximum of 3 to 43 per cent of the total sed-
imentating area.
In considering the nature of the heavy complex, there are a
number of possible formulations which will fit the known facts. One
of the simplest is a cyclic unit containing 6 moles of papain with al-
ternating disulfide and S-Hg-S bonds, but there is no direct evidence,
as yet, to support this or any 'other specific picture.
Discussion
From'the data presented, it is apparent that crystalline'papain
and mercuripapain exhibit in the ultracentrifuge the properties of es-
sentially homogeneous substances only under a limited and specified set
of conditions. Papain itself, which appears to be readily oxidizable
and reacts easily with heavy metals, exhibits a complex sedimentation
behavior in the oxidized state and at neutral pH values at which it
forms aggregrates with the heavy metal ion impurities. When cysteine
-103-
and Versene are present under stable reducing conditions at pH 4, papain
is a monomeric substance with a molecular weight near 20,700, a value
which is in excellent accord with analytical results on this protein.
Earlier estimates of the molecular weight of papain by Balls
and Lineweaver (9) were made mainly by osmotic pressure measurements
at pH 6.4. It is evident from these studies that papain at this pH
contains a mixture of monomers and dimers and that the average value
of 27,000 obtained in the earlier studies is consistent with such a
mixture. Balls and Lineweaver (9) also measured the diffusion constant
of their preparation of cr,rstalline papain. With the porous disc method,
they found D = 0.060 sq. em. per day at gOo This yields D
20
w = 9.9 X
,
10-
7
sq. cm. per second which is in good agreement with the value reported
here of D
20
,w = 10.3 X 10-
6
sq. cm. per second. It is evident that the
diffusion constant measured by the porous disc method is that of the more
rapidly diffusing monomers which migrate into an infinitely dilute buffer
solution.
It was hoped that the preparation and studies of mercuripapain
would throw some light on the number of sulfhydryl groups that are read-
ily available for reaction with heavY metals. The complex behavior of
mercuripapain renders it highly likely that there are at least two such
groups. However, the results do not allow any definite conclusions to
be reached. This problem was approached from another standpoint. Since
silver ion is univalent it was anticipated that a more concrete indica-
tion of the number of sulfhydryl groups could be arrived at by preparing
the silver derivative of papain. This was accomplished by adding 2 mole-
cular equivalents of AgN0
3
to a solution of papain i ~ 70 per cent ethanol.
This enzyme had been recrystallized with sodium acetate instead of sodium
chloride to eliminate contamination of the protein with insoluble AgC10
-104-
When the solution containing papain and AgN0
3
was stored in the cold over-
night rectangular plates, presumably of the silver derivative of papain,
crystallized from solution. In the presence of cysteine this material was
about 80 per cent as active as the starting protein and when examined in
the ultracentrifuge it showed no tendency to form dimers. Analyses were
not performed because reduction of Ag+ to metallic silver apparently oc-
curred during the reaction. This left the protein preparation a dark
brown color. It was not felt worth while to attempt analysis of this mat-
erial for silver until a preparation could be made that was free of such
impurities.
The evidence from a number of different types of studies leaves
little doubt that the state of purity of crystalline papain is well de-
fined. Enzymatic, analytical and physical studies all indicate that the
purity is high. Furthermore, the correlation of these studies enables
many characteristics of the enzyme to be described. Thus papain has a
molecular weight of about 20,500 and consists of a single peptide chain
of 182 (possibly 180) amino acid residues. Thiol groups appear to be
essential for enzymatic activity and it is likelY that two such groups
are free on the active enzyme. It is not known whether one or both are at
the active catalytic center of the enzyme. It should be reemphasized
that no conclusive proof exists to show how many sulfhydryl groups are
necessary for papain action. Such proof will require the preparation of
active papain which retains its activity in the absence of activating
agents. ,Then it will be possible to make quantitative estimates of the
number of free thiol groups on the active enzyme.
Perhaps the most important attribute of crystalline papain is
its availability in relatively large quantities in highly purified form.
This feature renders possible many studies and uses not ordinarily feas-
-105-
ible with other enzymeso For this reason papain will probably continue
to enjoy the academic popularity that it has had for many years
o
SUMMARY
Papain, a proteolytic enzyme found in the latex of the green
fruit of the tropical papaw, Carica papaya, has been known for many
years. It has a long and illustrious history and figured prominentlY
in the development of the concept of sulfhydryl enzymes. The latex of
the green fruit when dried has commercial uses because of its pro-
teolytic activity. Therefore, dried papaya latex is readilY available.
In the past it has not been possible to prepare or,ysta11ine papain with
dried latex as the starting material, but the crystalline enzyme had
been prepared from fresh papaya latex (9). This dissertation describes
a method whereby crystalline papain can be prepared in good yield from
certain sources of commercial dried papaya latex.
Crystalline papain was found to require both a metal-che1ating
agent (e.g. Versene) and a reduoing agent (e.g. oysteine) for maximal
activation. Specific ion effects on papain activity were noted in the
absence of Versene. The optimum pH for hydrolysis
ninamide by papain extends from pH 5.0 to 7.5. Like the crystalline
proteinases from pancreas, papain has esterase action on tosy1-L-arginine
methyl ester. The pH optimum for ester hydrolysis is similar to that for
the hydrolysis of the amide substrate. Papain is inhibited by iodoaceta-
mide and p-eh1oromerouribenzoate but not bydiisopropy1tluorophosphate
o
Crystalline papain has a broad specificity since it hydrolyzes a variety
of acyl and unsubstituted amino acid amides. The most sensitive substrate
yet found !mides of N-substituted aromatic
-106-
-107-
amino acids. are hydrolyzed ver,y slowly. Free dipeptides are resistant
but tripeptides are hydrolyzed slowly. is
split but strong product inhibition is observed. Carbobenzoxy-1-g1ut-
amic acid acts as an inhibitor for all types of substrates studied.
This indicates that the same active center on the enzyme is responsible
for the broad action of the enzyme. Cr,ystalline papain is about three
times as active hemoglobin as crystalline trypsin.
A cr,ystalline mercury derivative of papain has been prepared.
This derivative contains 1 gram atom of mercury per two moles of pa-
pain. The molecular weight of papain calculated from the mercury con-
tent of mercuriPapain is 21,700. Mercuripapain is inactive but is fully
activated in the presence of cysteine and Versene.
The amino acid composition of cr,ystalline papain has been deter-
mined. The protein has a righ content of tyrosine and glycine, 1 histi-
dine residue per mole and no methionine. The molecular weight calculated
from the amino acid composition is 20,3000 The N-terminal amino acid
sequence of papain is isoleueylprolylglutamic acid. There is one N-
terminal residue per 23,900 grams of papain indicating that the mole-
cule is a single peptide chain. On the basis of the amino acid compo-
sition this chain is 182 amino acid residues long. Since sulfhydr,yl
groups appear to be essential for papain action, some of the amino acid
sequences containing cysteine were identified. This was done with
formate-oxidized papain. The sequences identified were ser,ylcysteic acid,
aspartylcysteic acid, valylcysteic acid, and possibly glycylprolylcysteic
acid.
Physical studies with cr.ystalljne papain indicate that the mater-
ial is in a high state of purity. MOnodispersity in the ultracentrifuge
-108-
and the electrophoresis apparatus are observed only at pH 4. At this pH
the sedimentation constant (s20,w) is 2.42 Svedberg units; the diffusion
constant is 10.27 X 10-
7
sq. cm. per second. The partial specific vol-
ume (V) was calculated from the amino acid composition and was found to
by 0.724. These values were used to calculate the molecular weight and
yielded the figure 20,700.
Electrophoresis of papain indicates an isoe1ectric point at pH
S.75. The mobility curve has a broad zone between pH 3.9 and 6.0 over
which the mobility remains constant. Comparison of this curve with
that of insulin and ~ s o z y m e suggests that most of the free carboxyl
groups of papain and lysozyme are those of aspartyl residues and that
all, or nearly all, of the glutamyl residues are in the amide form.
Oxidized pap:iin exists in aqueous solution as a mixture of mon-
omeric and dimeric forms, The relative amounts being a function of the
protein concentration.
At pH S, mercuripapain contains a mixture of monomers and a
heavy aggregate apparently containing 6 moles of papain and 3 moles
of mercury. The relative amounts of the two forms are dependent upon
the protein concentration, the aggregate dissociating to monomers on
dilution. Ordinar,r papain appears to contain trace metal impurities
which also cause aggregation at pH 8.
All of the physical and chemical evidence accumulated indicate
that cr,ystalline papain is essentially homogeneous. There is no data
to suggest any gross contamination with protein impurities. Further-
more, these same studies all give values for the molecular weight that
are in good agreement with one another. Therefore, it is possible to
characterize papain as a protein consisting of 182 amino acid residues
arranged in a single peptide chain. This protein has a molecular weight
-109-
of about 20,500. It hydrolyzes many peptide bonds and one or more free
thiol groups on the protein molecule are necessary for this action.
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