THE JOURNAL OF BIOLOQICAL CHEMIWRY

Vol. 239, No. 7, July 1964

Printed in U.S.A.

Ficus Enzymes
I. SEPARATION OF THE PROTEOLYTIC ENZYMES OF FICUX CARICA AND
FICUS GLABRATA LATICES*

VALDEMIRO C. SGAnnIEnr,t SHASHIKANT M. GUPTE,$ DONALD E. KRAMER, AND JOHN R. WHITAKER

From the Department of Food Science and Technology, University of California, Davis, California

(Received for publication, July 5, 1963)

Ficin is the name given to the proteolytic enzyme activity of was also obtained from Enzyme Development Corporation, New
the latex of trees of the genus Ficus (1). While this proteolytic York (Lot No. 4540), and Paul Lewis Laboratories, Milwaukee,
activity has been utilized for centuries and Walti (2) crystal- Wisconsin. The gum, approximately 30% by weight in F.
lized a proteolytic enzyme from fig latex, little is known of the carica latices, was removed from the aqueous solution by cen-
physical, chemical, and enzymatic properties of this activity. trifugation at 100,000 x g for 20 minutes at 0’. The clear,

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The information available on ficin indicates that it closely re- straw-colored aqueous solutions, which contained between 10
sembles papain with regard to substrate specificity, esterase and 17.5% protein (biuret), were frozen and stored at -25’.
activity, transpeptidation reactions, and activation by reducing We calculate that approximately 90% of this protein in F. carica
agents (3). Bernhard and Gutfreund (4), Hammond and Gut- has proteolytic activity. Dried F. glabrata latex was obtained
freund (5), and Liener (6) have partially purified ficin and have from Merck and Company (Lot No. 50567, Sp. 709), Enzyme
studied some of its properties. Cohen (7) reported work on Development Corporation (“Special,” No. 273A), and Paul
some of the properties of crystallized ficin of undefined origin. Lewis Laboratories. Twice crystallized ficin (Lot No. 3110,
The presence of more than one proteolytic enzyme component prepared by the procedure of Walti (2)) was obtained from
in ficin is indicated by studies on substrate specificity and heat Nutritional Biochemicals Corporation, Cleveland, Ohio.
stability (8), pH-activity curves (9), and by separation pro- Reagents-Amberlite IRC-50 (XE-64, 200 to 400 mesh, Lot
cedures (3, 10, 11). No. 124), was from Rohm and Haas Company, Philadelphia,
The present work led to the development of a procedure for Pennsylvania. DEAE-cellulose’ was from Eastman Kodak
the complete and reproducible separation of the proteolytic Company, Rochester, New York. CM-cellulose was prepared
enzymes of ficin. It will be shown that the proteolytic enzymes in this laboratory from Whatman coarse grade, ashless cellulose
of Ficus glabrata and Ficus carica latices differ chromatographi- (carefully sieved dry to obtain loo-mesh size) by the method of
tally and electrophoretically and that there are both qualitative Peterson and Sober (12). It had a titratable carboxyl content
and quantitative differences in the proteolytic enzyme com- of 0.49 meq per g. Before use, it was washed with cysteine
ponents from different varieties of F. carica. (1.25 X 10m2 M) to remove all traces of monochloroacetic acid.
Sephadex G-100 (Lot No. To 1554, 140 to 400 mesh beads,
EXPERIMENTAL PROCEDURE water regain of 10 f 1) was from Pharmacia, Uppsala, Sweden.
Hydrolyzed starch (Lot No. 159) for gel electrophoresis was from
Materials
Connaught Medical Research Laboratories, Toronto, Canada.
La&es--Latices from varieties of F. carica L. were obtained Cellulose acetate strips were from Gelman Instrument Company,
by collection from the severed fruit stalks of green fruits grown Chelsea, Michigan.
in the variety plot at Fresno, California (maintained by the L-Cysteine hydrochloride, ninhydrin, hydrindantin, Al-ethyl-
California Citrus Research Center, Riverside, in cooperation maleimide, “Hammersten-quality” casein, and mercaptoethanol
with the United States Department of Agriculture), from the were from Nutritional Biochemicals Corporation, Cleveland,
University of California experimental plots, Davis and Winters, Ohio. Versene was from Eastman Organic Chemicals, Roches-
California, and from the orchard of Mr. Ed Scott of Planada, ter, New York. a-Benzoyl-L-argininamide hydrochloride mono-
California. After collection, the latices were immediately frozen hydrate was from Mann Research Laboratories, Inc., New York.
in Dry-Ice. Carefully preserved liquid latex of F. glabrata Ammonium sulfate and sodium chloride were reagent grade
Kunth was obtained from South America through the courtesy materials from J. T. Baker Chemical Company, Phillipsburg,
of Merck and Company, Rahway, New Jersey. Liquid latex New Jersey and Allied Chemical and Dye Corporation, New
York, New York, respectively. All other compounds were
* This work was supported in part by a research grant, GM-
05216, from the National Institutes of Health. Taken in part from reagent grade. Deionized water, obtained by passage through a
Master’s theses by V. C. Sgarbieri (M.S. Food Science, 1963), mixed bed resin (Barnstead bantam demineralizer), was used
S. M. Gupte (MS., Food Science, 1960), and D. E. Kramer (M.A., throughout.
Comparative Biochemistry, 1962), University of California, Davis.
t Present address, Instituto Agronomico, Campinas, S&o Paulo, 1 The abbreviations used are: DEAE-cellulose, diethylamino-
Brazil. ethyl cellulose; CM-cellulose, carboxymethyl cellulose; BAA, a-
$ Present address, B-3 Empress Mahal, Bombay, India. benzoyl-L-argininamide.

2170
 July 1964 V. C. Sgarbieri, S. M. Gupte, D. E. Kramer, and J. R. Whitaker 2171

Methods sorbed at 280 rnH and only 2 to 5y0 of the proteolytic enzyme
Protein Determination-Protein content was determined by activity. Much of this 280-rnp-absorbing material can be
either the biuret, Lowry, or spectrophotometric method (13). removed by prolonged dialysis but 30 to 50% of the protein is
Crystallized bovine serum albumin (Armour Laboratories) was precipitated and 30 to 60% of the proteolytic enzyme activity is
used as a standard protein for the first two methods. lost at pH 7.0. The peroxidase, amylase, and acid phosphatase
ElectrophoresisFor free boundary electrophoresis, the aque- activities present in latex were eliminated as these were not
ous latex solutions were diluted 5-fold with 0.05 M acetate-O.100 adsorbed. The enzyme-resin was immediately put on top of
M sodium chloride buffer, pH 5.0, and were dialyzed for 36 hours the column, and chromatography was performed at 4’. Frac-
at 2’ against this same buffer. After dialysis, the protein con- tions of 10 ml each were collected at the rate of 1.5 ml per minute.
tent of the latices was adjusted to 20.0 mg of protein (biuret) The ionic strength was changed in a stepwise manner by use of
per ml with the acetate buffer. Free boundary electrophoresis the appropriate concentration of sodium chloride in 0.01 M
was performed in a Perkin-Elmer model 38A Tiselius electropho- sodium phosphate buffer adjusted to pH 7.0. Absorbance at
resis apparatus. All runs were performed at 4’, 0.020 ampere, 280 rnp was determined on every fraction, and activity on casein
and 100 volts with a 2-ml cell assembly and silver-silver chloride was determined for every second or third fraction. All separa-
electrodes. Photographs of the ascending boundary were taken tions were performed at least in duplicate.
at four intervals by a modified Longsworth scanning method. Purijicatzon by Precipitation-Ammonium sulfate and ethanol
Conductivities of the solutions were determined with a Fisher fractionations were performed at controlled pH values and 0”.
conductivity cell with a Leeds-Northrup bridge. All elec- Ammonium sulfate-sodium chloride purification of Merck F.
trophoretic runs were done in duplicate. For removal of com- gZa&-ata dried latex was also carried out as described by Ham-

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ponents from the electrophoresis cell, Kadota latex (28.2 mg of mond and Gutfreund (5).
protein per ml) was run for 5400 seconds, and the boundaries Enzymatac ActititiesProteolytic activity was determined by
were carefully removed with a syringe. Starch gel electropho- the method of Kunitz (16) in the presence of 1% casein and
resis was performed horizontally as described by Smithies (14). 1.25 X 10e2 M each cysteine and Versene at pH 7.0 and 35.0’.
Zone electrophoresis on cellulose acetat’e was performed horizon- Relationship between change in absorbance and enzyme con-
tally on l.O- X 12.5-cm strips. centration was established over the range used. Specific activity
Chromatography-Amberlite IRC-50 (XE-64) was purified, and was expressed as the initial change in absorbance at 280 rnp per
columns were prepared by the method of Hirs (15). DEAE- minute per mg of enzyme protein.
cellulose columns (1.9 x 18 cm) were prepared in 0.001 M borate The activity toward BAA was measured by the ninhydrin
buffers at pH 7.5, 8.5, 9.0, and 9.5. Salt elution with a loga- method (17) in the presence of 0.02 M BAA, 1.0 X lo-* M mer-
rithmic gradient was used. Gel filtration on Sephadex G-100 captoethanol, 1.0 X 1O-3 M Vcrsene and at 35.0’ and pH 6.50
was carried out on a l.l- X 192.cm column at 25-27’ in 0.1 M (0.1 M phosphate buffer containing 0.2 M sodium chloride).
acetate-O.4 M sodium chloride buffer, pH 6.0. One-milliliter N-Ethylmaleimide was used to prevent interference of the
fractions were collected at a flow rate of 0.35 ml mini cm-t. mercaptoethanol with the ninhydrin reaction (18). Specific
Variations in pH, type of sodium chloride elution gradient, activity of the zero order reaction with respect to substrate was
and column length were all investigated to determine the best expressed in terms of micromoles of BAA hydrolyzed per minute
conditions to use for separation of ficin on CM-cellulose. The per mg of enzyme protein.
best results were obtained by a stepwise elution scheme which Casein-clotting activity was determined spectrophotometri-
differed for I*‘. carica and F. glabrata. After each chromatog- tally at 540 mp. The reaction mixture was the same as used
raphy, the resin was removed from the column, washed with 0.5 for measuring proteolytic activity. The reaction, carried out in
M sodium hydroxide-O.5 M sodium chloride and filtered on a a thermostated Beckman model DIJ spectrophotometer at 35.0’,
Buchner funnel. It was washed with deionized water until was started by addition of enzyme and the time required to
free of chloride, with 0.1 M sodium phosphate buffer, pH 7.0, and change the absorbance by 1.00 was measured. This was one-
finally with several volumes of 0.01 1~ sodium phosphate buffer, half the maximum absorbance change attainable under the con-
pH 7.0, for the chromatography of F. glabrata latex or of 0.01 M ditions used and was the point at which the rate of absorbance
phosphate buffer, pH 7.0, containing 0.12 M sodium chloride change was the greatest. There is a linear relationship between
for the chromatography of F. carica latex. The column (2.0 x enzyme concentration and casein-clotting activity under the
45.0 cm) was poured at room temperature in one segment by the conditions described. One unit of casein-clotting activity was
use of an extension tube. It was then moved to a cold room defined as that amount of activity which will produce an ab-
(4’) and washed with 500 ml of the buffer before use. sorbance change of 1.00 in 100 seconds. The specific activity
Ficin was adsorbed to CM-cellulose before addition to the was expressed as units of activity per mg of enzyme protein.
column. CM-cellulose was equilibrated with 0.01 M citric acid- All pH measurements were made with a Beckman model G pH
0.02 M sodium phosphate (Na2HPOd) buffer, pH 4.90, for F. meter.
carica latex and 0.005 i+r citric acid-O.01 M sodium phosphate
RESULTS
buffer, pH 4.90, for F. glabrata latex essentially as described for
the preparation of the column. To 3 g of the filter-dry resin were Enzymatic Activities of LaticesFor latices from 16 varieties of
added 2 ml of the latex (or 10% solution of the dried latex) and F. cc&a, the casein-digesting activity varied between 3.12 and
10 ml of the citrate-phosphate buffer. After 5 minutes, this was 3.90 absorbance units per minute per mg of protein, the casein-
filtered on a small Buchncr funnel. The resin was washed six clotting activity between 3.14 and 5.71 units per mg of protein
additional times with 10 ml of the buffer. This treatment and the activity on BAA varied between 2.01 and 3.42 Mmolcs of
eliminated about 60 to 707, of the material in latex which ab- ammonia produced per minute per mg of protein. The ratios of
2172 Ficus Enzymes.
FIG. 1. Free boundary electrophoretic patterns of latices from three varieties of F. carica L. and from Merck F. glabratadried
latex. Experimental conditions are given in the text. The ascendingboundarieswere photographedafter 4000 seconds. Most of
the movementwastoward the cathode (right sideof pictures). Specificconductivity of the solutionswas113ohmsper cm.
the various activities were alsoreasonablyconstant. The ratios
for casein-clottingto casein-digestingactivities varied between
0.80and 1.86, the ratios of BAA to casein-digestingactivities
between0.59 and 1.14,and the ratios of casein-clotting to BAA
activities between1.05and 2.22. While the ratios of the various
activities of solutions prepared from Merck dried F. g2abrata
latex were within the values found for F. carica, the specificac-
tivities were approximately half those of the F. carica latices.
This hasbeen noted previously (19).
ElectrophoresisLatices of 16 varieties of F. catia and dried
Merck F. glabrata latex were examinedby free boundary electro-
phoresis. Representative electrophoretogramsare shown in
Fig. 1. Most of the movementis toward the cathode. Of the
l&ices investigated, only three pairs (Adriatic and Pied de
Boeuf, Beall and Roeding-3, Earlimont and Martinique) ap-
peared to be identical qualitatively by this technique. There
wasalsoa great deal of variation in the quantitative amountsof
each of the components. It is significant that the components
of F. glabratu latex did not move as rapidly as did those of the
other latices. Removal of the liquid in segmentsand examina-
tion indicated that all of the electrophoretically distinct compo-
nentsin Kadota latex had proteolytic activity. In addition, the
component with the lowest mobility (slight movement toward
the anode) had amylase, peroxidase,and acid phosphataseac-
tivities.
Examination by starch gel and celluloseacetate zone electro-
phoresisandby precipitationwith ammoniumsulfate andethanol
supported the view that there are multiple, proteolytically
active componentsin the various latices. The best results were
obtained on celluloseacetate strips equilibrated with 0.05 M
phosphatebuffer, pH 7.0.
Chromatography-Chromatographyof Merck F. glabrata dried
latex on DEAE-cellulose at pH 9.0 gave five componentswith
proteolytic activity. However, the enzymesare not too stable
at pH 9, as only 60% of the starting activity was recovered.
I Vol. 239, No. 7
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At pH 7.5 and 8.5, all the activity cameoff in the breakthrough
fraction.
Stepwiseelution of ficin from CM-celluloseat pH 7.0 hasgiven
excellent results in our hands. Representative separationson
CM-cellulose are shown in Fig. 2 for four F. carica varieties.
Approximate proteolytic enzyme compositionof severalvarieties
of F. curica is given in Table I. It is obvious that there are a
number of proteolytic enzyme componentsin F. carica ficin and
that the number and amount of each varies from variety to
variety. In Kadota latex, there are at least 10 proteolytic en-
zyme componentswhile in Calimyrna there are only 4 compo-
nents. In Kadota latex, ComponentJ accountedfor 27.9% of
the activity, whereasit is not presentin Calimyrna latex. Com-
ponent B accountedfor 31.1and 22.1Toof the activity in Black
Mission and Conadria latices, respectively, but it wasabsentin
King and Calimyrna latices and wasquite smallin most of the
others. Component F accounted for 55.0 and 40.1% of the
activity of Calimyrna and Beall latices, respectively, but it was
absent in Black Mission, Blanquette, and Conadria latices.
The same results are obtained when Black Mission latex is
separatedby gradient rather than by stepwiseelution from CM-
cellulose.
The recovery of activity from the column was usually 85 to
100% (Table I). The elution volumesfor the different compo-
nents werereproducibleto &3 fractions (10 ml) amongthe latices
except for componentsG, H, and I of Kadota latex and H and I
of King latex. Thesediffered by asmuch as22 fractions (10 ml)
from the results with the other latices, and we are inclined to
believe that they must differ somewhatfrom the componentsin
the other laticeswhich are assignedthesenames.
There is somevariation in the specificactivities on caseinof a
given componentfrom different latices (Table I). There are at
least three explanations for this. The peak fraction of a given
componentwasnot necessarilyhomogeneous sothat the specific
activity wasinfluencedby the degreeof contamination with the
 z
BLACK MISSION t

CALIMYRNA

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FRACTION NO. (IO ML)

FIG. 2. Chromatography of F. carica var. Black Mission, King, phosphate-O.12 M sodium chloride buffer at 4’. Following elution
Stanford, and Calimyrna latices on CM-cellulose. &dialyzed with the starting buffer, stepwise elution was used with the fol-
latex, 2 ml, was adsorbed on 3 g of CM-cellulose equilibrated with lowing changes as indicated by the arrows: f,O.l7 M sodium chlo-
0.01 M citric acid-O.02 M sodium phosphate (Na2HPOI) buffer at ride; d, 0.23 M sodium chloride; and 6, 0.50 M sodium chloride, all
pH 4.90, washed free of unadsorbed materials and placed on top at pH 7.0 and in 0.01 M sodium phosphate. -, absorbance at
of 2.0-3X 45.0-cm columns equilibrated with 0.01 M pH 7.0 sodium 280 m; - - - -, activity on casein at pH 7.0 and 35.0’.

50 150 250 50 150 250

FRACTION NO. (IO ML.)
FIG. 3. Chromatography of several preparations of F. glabrata 6, 1.0 M sodium chloride, all at pH 7.0 and in 0.01 M sodium phos-
latex on CM-cellulose. The material was adsorbed on CM-cel- phate. - - - -, activity on casein at pH 7.0 and 35.0”; -, ab-
lulose equilibrated with 0.005 M citric acid-O.01 M sodium phos- sorbance at 28Omp. a, liquid latex obtained from Merck and Com-
phate (Na2HPOd) buffer at pH 4.90, washed free of unadsorbed pany (0.30 g of protein used); b, Merck dried latex (0.40 g of protein
material, and transferred to the top of CM-cellulose columns used); c, salt-purified material obtained from Merck F. glabrata
(2.0 X 45.0 cm) equilibrated with 0.01 M phosphate buffer, pH 7.0, dried latex by the procedure of Hammond and Gutfreund (5) (0.16
at 4”. After starting buffer elution, the following stepwise elu- g of protein used); d, twice crystallized ficin (0.080 g of protein
tions were performed: 1,0.04 M sodium chloride; 8, 0.10 M sodium used) from Nut.ritional Biochemicals Corporation.
chloride; S, 0.12 M sodium chloride; &0.20 M sodium chloride; and

2173
2174 Ficus Enzymes. I Vol. 239, No. 7

TABLE I
Approximate proteolytic enzyme composition of severalvarietiesof Ficus carica
Proteolytic activity was measured by the method of Kunite as the change in absorbance at 230 rnp per minute per mg of pro-
(16) in the presence of 1% casein and 1.25 X 10v2 M each cysteine tein. The approximate percentage composition was calculated
and Versene at pH 7.0 and 35.0”. Protein was determined by the directly from the absorbance and activity data by assigning to one
spectrophotometric method (13). Specific activity was expressed component all values that fell between two minima values.

Horticultural
group Variety
I- Component -i
Activ-
ity re-
A B C D E F G H I J covery

%
Common Adriatic
Protein (%) 17.3 6.66 5.07 8.34 7.38 13.8 21.8 12.9 0 7.00
Activity (%) 0 3.75 3.40 7.04 7.60 13.8 37.4 12.7 0 14.3 62.6
Specific activity 0 4.80 3.00 3.20 2.77 3.67 6.36 3.25 0 8.65
Beall
Protein (%) 1.79 8.72 0 0 0 41.5 26.5 18.6 0 3.70
Activity (%) 1.50 11.5 0 0 0 40.1 31.2 12.4 0 3.20 99.0
Specific activity 3.31 9.22 0 0 0 5.45 6.59 3.88 0 6.52
Black Mission
Protein (%) 2.42 21.8 0 0 14.0 0 25.4 26.6 0 8.35

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Activity (ye) 1.38 31.1 0 0 9.07 0 30.4 13.0 0 14.9 84.8
Specific activity 3.96 10.1 0 0 2.46 0 6.84 2.73 0 9.96
Blanquette
Protein (ye) 0 2.25 13.6 11.1 0 0 64.4 0 0 8.75
Activity (To) 0 1.10 13.5 9.40 0 0 58.4 0 0 18.8 97.4
Specific activity 0 2.40 7.09 7.89 0 0 4.96 0 0 14.5
California Brown Turkey
Protein (%) 4.35 5.25 10.1 7.95 0 12.4 33.3 22.6 0 5.85
Activity (‘%) 2.54 3.86 9.55 8.37 0 11.0 38.2 15.7 0 10.8 82.5
Specific activity 2.81 5.51 5.74 5.98 0 4.71 6.25 3.72 0 9.85
Kadota
Protein (%) 8.40 5.25 10.2 10.6 6.64 10.8 16.8 12.9 6.72 10.8
Activity (%) 4.82 2.61 7.56 11.4 5.94 10.0 20.8 6.24 4.75 27.9 85.4
Specific activity 2.54 4.19 4.04 5.47 3.24 5.40 5.70 2.70 3.30 14.6

Common X Caprifig Conadria

Protein (%) 2.62 15.2 0 0 22.7 0 35.0 18.4 0 6.20
Activity (%) 1.47 22.1 0 0 18.5 0 37.7 11.0 0 9.05 98.4
Specific activity 3.33 13.9 0 0 4.72 0 7.37 4.26 0 11.0

Smyrna Calimyrna
Protein (%) 6.75 0 0 0 56.2 21.2 14.4 0 0
Activity (%) 4.30 0 0 0 55.0 30.7 9.96 0 0 90.8
Specific activity 2.97 0 0 0 4.46 6.89 3.96 0 0

San Pedro King

Protein (ye) 0 0 11.6 11.5 2.77 24.2 22.4 14.5 9.44
Activity (%) 0 0 12.9 10.4 3.51 20.8 24.6 11.1 16.5 94.8
Specific activity 0 0 7.21 6.00 4.53 6.95 6.62 5.01 13.9

Caprifig Stanford
Protein (%) 3.73 2.80 4.80 2.61 18.5 54.5 7.67 0 3.45
Activity (%) 2.52 3.33 4.64 2.13 16.4 59.5 6.77 0 4.77 93.8
Specific activity 4.12 6.20 5.94 4.83 4.32 5.80 3.96 0 8.46

preceding component(s). This depended on the concentration of CM-cellulose is shown in Fig. 3. There are at least nine compo-
the preceding component(s). The stability of the isolated com- nents with proteolytic activity in this latex. Component F
ponent was, in most cases, a function of its concentration and appears to be a real component as shown by gradient elution of
this varied considerably among the latices. And last, the salt-purified Merck dried latex. Qualitatively, the results from
activity determinations on a given component were not always the liquid F. glabrata latex, and Merck, Enzyme Development
made after the same interval of time after elution from the Corporation, and Paul Lewis dried latices are essentially the
column. same. Quantitatively, there are marked differences (Table II).
The separation of several preparations of F. glabrata latex on The F. ghbrata liquid latex (obtained from Merck) and Merck
July 1964 V. C. Sgarbieri, S. M. Gupte, D. E. Kramer, and J. R. Whitaker 2175

TABLE II
Approximate proteolytic enzyme composition of several preparations of Ficus glabrata
The procedure was as described in Table I. Protein concentration was calculated from absorbance at 280 mp and from El& of 16.6
(Lowry protein determination) found for Component G of crystalline ficin.

-
A B C F G* H I
-_ _-
Latex (Merck)
Protein (%). .............. 2.40 6.74 13.4 3.54 19.9 1.36 5.34 6.28 5.08
Activity (%). ............. 2.02 9.28 15.1 6.07 39.0 1.61 17.4 4.12 5.43
Specific activity ........... 0.75 1.43 2.36 1.94 4.15 1.58 3.19 1.66 3.32

Merck powder
Protein (%). .............. 2.70 6.11 6.92 4.60 12.0 2.07 6.62 6.19 5.01
Activity (%). ............. 4.36 9.10 12.38 4.93 20.5 2.94 27.3 8.50 9.90
Specific activity ........... 2.30 1.68 2.61 2.36 3.44 2.40 3.65 2.12 3.30

Enzyme Development Cor-

poration powder

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Protein (%). .............. 1.31 2.28 4.40 0 6.05 2.64 5.04 10.6 16.2
Activity (%). ............. 0.82 2.15 3.66 0 4.91 2.97 30.6 19.8 35.4
Specific activity ........... 0.84 1.22 1.38 0 2.26 2.60 3.67 2.52 4.10

Paul Lewis powder

Protein (‘g). .............. 4.05 2.22 2.69 0.46 7.95 1.57 5.81 10.7 42.4
Activity (%). ............. 0 0 0 0 1.41 0.92 28.0 10.9 58.9
Specific activity ........... 0 0 0 0 1.04 0.75 3.40 1.41 2.11

Ammonium sulfate-sodium L

chloride purified ficin

Protein (%). .............. 3.03 9.46 15.2 6.85 26.9 3.43 23.7 3.56 3.03
Activity (%). ............. 2.37 8.70 17.8 7.05 33.7 3.56 21.8 2.64 2.10
Specific activity ........... 0.94 2.66 4.04 3.29 4.49 3.48 4.30 2.61 3.00

Crystalline ficin
Protein (%). .............. 3.76 3.00 6.75 0 17.5 2.78 53.0 Trace Trace
Activity (%)t ............. 0 0.80 3.02 0 17.8 2.81 69.9 Trace Trace
Specific activity ........... 0 1.26 1.96 0 4.54 3.57 5.99
- -
* All activity betweenComponentsG and H wasassignedto ComponentG. No such assignment could be made for the protein.
t A peak after Component G contained 5yo of the total activity.

dried latex are essentiallyidentical except for the quantitative salt-purified material appearedto be reasonably homogeneous.
amounts of Component E and the large inert componentwhich Liener (6) and Hammond and Gutfreund (5) reported their
cameoff just after Component G. Component E is one of the preparationswerehomogeneous by electrophoresis and ultracen-
leaststablecomponentsin the latex. In terms of active compo- trifugation.
nents Enzyme Development Corporation and Paul Lewis dried The chromatographyof twice crystallized ficin on CM-cellulose
latices are composedmainly of ComponentsG, H, and I while is shownin Fig. 3d. The preparation used here had an initial
the predominatecomponentsin Merck dried latex are B, C, E, specificactivity on caseinof 2.73, a 280 ml:260 rnp ratio of 1.29
and G. The quantitative differencescould be due to differences and contained 26% non-protein material which absorbedat
in handling the latex or to utilization of different varieties of 280 rnl.rand 24.7% inactive protein which were not adsorbedon
F. glabrata. CM-cellulose at pH 4.90. ComponentG accounted for 69.9%
The results of the chromatography of salt-purified ficin of the total activity of this preparation. ComponentsB, C, E,
(Hammondand Gutfreund procedure (5)) are shownin Fig. 3c. F, H, and I accounted for the other 30% of the activity. The
We achieved a 3.3-fold increasein specific activity by this peak fraction of ComponentG had a specificactivity on casein
procedure while Hammond and Gutfreund reported a 2-fold of 5.99,a 280mp:260 rnp ratio of 1.95and E&,,, of 16.6 (protein
increase. The chromatographic data indicate that this material determined by Lowry method (13)). Thesedata representthe
differs qualitatively from the starting material (Fig. 35) only in first proof that the major componentof crystalline ficin is present
the removal of the four inert componentsbetweenactive Compo- in the original latex and doesnot representa degradationproduct
nents G and H. Quantitatively, there is somedecreasein the formed during the crystallization procedure. It should further
amount of Components H and I present (Table II). By use of be noted that ComponentG is not the major componentin the
the chromatographic procedure described by Liener (6), the original latex (Table II).
2176 Ficus Enzymes. I Vol. 239, h-0. 7

The major activity peak in both F. carica var. Kadota and The effect of time of year on the proteolytic enzyme composition
Merck F. glabrata dried latices is eluted from a Sephadex G-100 of latex has not been examined. All the samples used here were
column in about the same volume as cytochrome c and ribonu- collected in late September or early October. We have not
clease and in a larger volume than is papain. By the gel filtration found any differences among latices collected in three different
method, the molecular weights of the proteolytically active com- years.
ponents of the two latices appear to be similar and near 13,000. The F. curicu latices used here came from all four horticultural
However, the low theoretical plat)e numbers of 200 to 300, calcu- groups (Common, Smyrna, San Pedro, and Caprifig) and from a
lated by the method of Glueckauf (20), indicate that the mate- cross between two horticultural groups. (Conadria is a cross
rial is heterogeneous. Proteins which are reasonably homoge- between Common and Caprifig.) Despite the fact there are
genous give theoretical plate numbers of 900 to 1000 (21). large differences among these groups in the shape of the leaves,
size, color, and shape of the fruit and growth habits, we did not
DISCUSSION
find a proteolytic enzyme composition which was distinctive for
Chromatographically, all the components of F. curica lat,ex a given group. The relationship between genotype and pheno-
appear to be different from those of F. glubratu latex. Compo- type in terms of proteolytic enzyme composition must await
nents A through G of F. glabratu latex are eluted from the CM- more detailed studies.
cellulose column by 0.10 M sodium chloride in 0.01 M phosphate Because of the apparent heterogeneity of ficin and the differ-
buffer, pH 7.0, while none of the components of F. curicu latex ences which were found between species and among varieties of a
are eluted by this procedure. Differences in electrophoretic and given species and since there are more than 1880 named species
precipitation behavior between the components of these two of Ficus, a number of subspecies (27), and at least 700 varieties

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species have also been found. All the components of F. curicu of F. curica (28), it becomes very important that workers in this
latex which are absorbed on CM-cellulose at, pH 4.90, except the area define their source of material and the properties of t.he
first component of Adriatic latex, have proteolytic activity. In purified components so that others may adequately use the in-
F. glabrutu latex, there are three (four in Enzyme Development formation reported. The description of the material should
Corporation and Paul Lewis dried latices) components eluted include the genus, species, variety, and organ from which the
between active Components G and H which do not have proteo- material is collected as recommended by Heinicke and Gortner
lytic activity (Fig. 3u and b). We suspect one or more of these (29). In addition, each proteolytic enzyme component from a
components may be lysozyme as Meyer, Hahnel, and Steinberg given latex must be named. For the moment, since chromatog-
(22) reported that crude F. glabrutu latex contained 65% of the raphy on CM-cellulose appears to give the most definitive results,
activity of crystalline egg white lysozyme. However, we have we suggest that the components be named according to their
been unable to find any lysoayme activity in either the latex or behavior on this resin in phosphate buffer at pH 7.0. For
these components with two preparations of Micrococcus Zysodeikti- example, the first proteolytic enzyme component from Kadota
cus. Meyer et al. (22) pointed out some strains of M. ZysocZeikti- latex would be Ficus curicu var. Kadota Ficin A and the first com-
cus are resistant to Ficus lysozyme. ponent from King latex would be Ficus carica var. King Ficin C.
The question of whether some of the proteolytic enzymes Commercially available crystalline ficin would be largely Ficus
isolated from F. carica and F. gZabruta latices may be artifacts is glabrata Ficin G.
a logical one. We have attempted to answer this in the follow-
SUMMARY
ing ways. The F. caricu latices were collected by us and imme-
diately frozen in Dry-Ice. These latices were never above 4’ Ficin from Ficus glalwata and Ficus caricu latices was found to
except for the short time required to adsorb the enzymes on be heterogeneous by electrophoresis, ammonium sulfate and
CM-cellulose. F. curica var. Kadota latex was held at 35” for 6 ethanol precipitation and by chromatography on carboxymethyl
hours and then chromatographed. The chromatogram was cellulose at pH 7.0. By chromatography on carboxymethyl
identical both qualitatively and quantitatively with that of a cellulose, F. glabrutu latex was found to contain 9 components
control sample which had been treated in the usual fashion. The with proteolytic activity. Purification of ficin according to the
large qualitative and quantitative differences found among the procedure of Hammond and Gutfreund (5) failed to separate
latices from different varieties of F. carica are also against the these active components. Crystalline ficin was found to be
proposition that in the original latex there is one proteolytic largely active Component G of the latex. The number and
enzyme and that during collection and separation the other com- relative amounts of the active components of F. curica varied
ponents are formed by autolysis. Although we have not found a among the varieties studied. F. curica var. Kadota and var.
proenzyme, we have not ruled out the possibility that the various Calimyrna had 10 and 4 proteolytically active components,
components are formed by an activation mechanism similar to respectively. All the proteolytic enzyme components of F.
the one for cr-chymotrypsinogen (23) where as many as 12 compo- glubrata appeared to differ chromatographically from those of
nents have been observed during the activation process (24). F. cc&u.
Rechromatography of each of the components from F. curicu var.
Kadota gave the component back in the original elution volume AcknowZecZgm&.s-We would like to thank Dr. T. W. Hum-
and as the major component (25). It is well established that phreys, Merck and Company, Dr. J. D. Haim, Enzyme Derelop-
there are two proteolytic enzymes in papaya latex and no one has ment Corporation, and Dr. Paul Halmbacher, Paul Lewis
looked carefully for others. Multiple molecular forms of several Laboratories for samples of F. glabratu latex, Dr. Miles Doyle and
other enzyme systems are also well known (26). Dr. Richard Warner for help in collecting latices from varieties
F. curicu var. Kadota latex from three areas and Black Mission of F. caricu, and Mr. Ed Scott for permission to collect Kadota
latex from two areas in California have given identical results. latex from his orchard.
July 1964 V. C. Sgarbieri, S. M. Gupte, D. E. Kramer, and J. R. Whitaker

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ARTICLE:
Ficus Enzymes : I. SEPARATION OF THE
PROTEOLYTIC ENZYMES OF FICUS
CARICA AND FICUS GLABRATA
LATICES

Valdemiro C. Sgarbieri, Shashikant M. Gupte,

Donald E. Kramer and John R. Whitaker
J. Biol. Chem. 1964, 239:2170-2177.

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