THE JOURNAL OF BIOLOGICAL CHEMWFRY

Vol. 236, No. 5, May 1961

Printed in U.S.A.

Glucose 64’hosphate Dehydrogenase (Zwischenferment)

I. ISOLATION OF THE CRYSTALLINE ENZYME FROM YEAST*

ERNST A. NOLTMANN,~ CLARK J. GUBLER,~ AND STEPHEWA. KUBY

From the Institute for Enzyme Research, University of Wisconsin, Madison, Wisconsin

(Received for publication, October 31, 1960)

The discovery of n-glucose g-phosphate dehydrogenase proximately 30%) were determined in triplicate and required
(Ztischenferment) by Warburg and Christian (1, 2) in 1931 amounts were calculated in terms of dry DEAE-SF.
paralleled their pioneer work on triphosphopyridine nucleotide. Versene (disodium ethylenediaminetetraacetate) and bento-
Since the early attempts by the German workers to isolate nite, U.S.P. (Powder), Lot No. 782300, were obtained from the
this enzyme (2-4), great effort has been expended to purify it Fisher Scientific Company; the latter was used without further
from a variety of sources (e.g. 5-14). The most highly purified treatment.
of these preparations was reported by Glaser and Brown in 1955 Diisopropyl fluorophosphate was a gift of Merck and Com-
(9). pany, Inc. A 0.1 M solution of this extremely poisonous reagent
In the present paper, the isolation of crystalline n-glucose was made with dried redistilled propanol and stored in sealed
6-phosphate dehydrogenase from dried brewers’ yeast is de- ampules at 0’. For each preparation, a fresh ampule was opened
scribed. Further studies in this series will deal with the physico- to minimize the possibility of hydrolytic decomposition.
chemical properties of the protein. All other reagents, including the ammonium sulfate, were of
analytical grade and met the American Chemical Society specifi-
MATERIALS AND METHODS cations.
Unless otherwise indicated, twice distilled deionized water
“Dried Brewers’ Yeast for Enzyme Work” was purchased
was used for preparing all reagents including the dialysis fluids.
from Anheuser-Busch, Inc., St. Louis, specified as the Anheuser-
Busch strain of Xaccharomyces carlsbergensis. It was stored at Measurements of enzymatic activity were made by following
-10” and used within a 2-month period after delivery. the change in absorbancy at 340 rnp (l-cm light path) with a
The crystalline barium salt of n-glucose-6-P and “98% pure” modified Beckman model DUR spectrophotometer equipped
TPN were obtained from the Sigma Chemical Company; the with a Leeds and Northrup Speedomax recorder. The tempera-
former was converted to the sodium salt after removal of the ture was maintained at 30” by pumping water from a constant
barium as BaS04, and both reagents were neutralized and as- temperature bath through the thermospacers of the spectro-
sayed enzymatically. photometer. The reaction mixture had the following composi-
Glycylglycine and DEAE-cellulose were products of the Cali- tion: 2.5 ml of 0.1 M glycylglycine buffer, pH 8.0, 0.1 ml of 0.03
fornia Corporation for Biochemical Research. The Cellex-D M glucose-6-P, 0.1 ml of 0.01 M TPN, 0.2 ml of 0.15 M magne-
anion exchange cellulose (DEAE-SF),’ had an exchange capacity sium sulfate. The reaction was initiated by the addition of
of 0.6 meq per, g. (It should be particularly noted that all 0.1 ml of properly diluted enzyme. All dilutions were made
washing and eluting volumes, which in the course of the procedure with ice-cold 0.05 M Versene, pH 8.0; for specific activities
are given in milliliters per gram of dry DEAE-cellulose, are based greater than 2000 units per mg, in addition to the Versene, 1
on that exchange capacity!) Several different lots were investi- mg per ml of crystalline bovine plasma albumin (Armour Labo-
gated with the same results. Before use, the DEAE-SF was ratories) proved necessary to stabilize the highly diluted (see
cycled through H20-NaOH-HQO-HCl-HzO-NaOH-H20, with a Fig. 1) enzyme.
lo-fold excess of base and acid, respectively, and each washing One unit of glucose-6-P dehydrogenase is defined as that
was performed until neutrality. Finally, it was freed from as amount of enzyme, per 1 ml of reaction mixture, which catalyzes
much liquid as possible on a Buchner funnel and stored as a moist the reaction between TPN and glucose-6-P and (under the con-
filter cake in a tightly stoppered bottle at 0”. Dry weights (ap- ditions described above) requires a time of 1 minute to cause an
increase of absorbancy at 340 ml between the limits of 0.05 and
* This work has been supported in part by grants from the 0.15. The conditions are such that the initial velocity is pro-
National Science Foundation and the National Institutes of portional to the enzyme concentration (i.e. first order with re-
Health.
t Aided by a travel grant from the Deutsche Forschungsge- spect to enzyme concentration) and that an initial increment of
meinschaft. the total reaction is held constant. Therefore, it can be shown
$ Special Research Fellow of the National Institutes of Health that e = constant x l/At, i.e. the enzyme concentration (e) in
and Advanced Research Fellow, American Heart Association. arbitrary units is inversely proportional to the time (At) as
Present address, Department of Chemistrv.“. Brigham
- Young Uni-
versity, Provo, ‘Utah. shown in Fig. 1. For routine measurements, it is convenient to
1 The abbreviation used is: DEAE-SF, diethylaminoethyl ex- use enzyme dilutions, which yield At values between 1 and 2
change groups on a Solk-Floe cellulose lattice. minutes (i.e. 1 to 0.5 unit per ml of reaction mixture).

1225

This is an Open Access article under the CC BY license.

1226 Glucose-6-P Dehydrogenase. I Vol. 236, No. 5

Specific activity is expressed in terms of units per milligram of design as shown in Fig. 2, which is a modification of a method
protein. If it is desired to convert to micromoles of TPNH used previously (17) but not described. Visking thin wall
formed per milligram of protein per minute, the above defined seamless cellulose tubing with a 36/32-inch inflated diameter has
enzyme units per milligram of protein should be multiplied by been found convenient for efficient dialysis of volumes from 0.5
the factor 0.0161 (derived from the molar absorbancy index of to 2 liters.
6.22 x lo3 cm-r X M-’ (15) and the above defined unit Tvhich is
ISOLATION PROCEDURE
in terms of 1 ml of reaction mixture). It should be noted that
the reaction velocity is linear up to only about 0.2 absorbancy. Unless otherwise stated, all steps were carried out in a cold
Protein was determined by the calorimetric biuret procedure room at 2-4” or in an ice bath. pH determinations were made
of Gornall et al. (16). A factor of 32.0 mg/lO ml (or 6.4 mg/2.0 with a Beckman model G pH meter calibrated before each meas-
ml) reaction mixture per unit of absorbancy at 540 rnp (l-cm urement at the temperature of the sample.
light path) was used as an average biuret value for the purpose Except for the crystallizing steps, solid ammonium sulfate was
of determining the protein concentration throughout the purifi- slowly added to the vigorously stirred solutions. After dissolu-
cation procedure. The precise value for the isolated crystalline tion of the salt, the stirring was continued for an additional
protein will be reported in the next paper of this series. In lo-minute period, with care to avoid excessive formation of foam,
those cases in which interfering material was present, the protein and the mixture allowed to stand for at least 10 minutes before
was first precipitated with 10% trichloroacetic acid, and then centrifugation. Required amounts of ammonium sulfate were
the biuret procedure carried through. calculated by the formula
Large capacity dialysis was performed with a flow dialysis
0.515 x v, x (SZ - 81)
w=
1 - (0.272 X 8,)

in Ivhich w equals the weight of ammonium sulfate in g, VI is the

volume of the solution in milliliters at fraction saturation &, and
Sz represents the fraction saturation desired at 0” (17).
Ammonium sulfate concentrations (8”) of dissolved ammonium
sulfate precipitated pellets (of final volume V”) were estimated
by the formula:

I I I I assuming that the volume increase due to the dissolved pellet

0.01 0.02 0.03
e (yg ENZYME PER ml REACTION MIXTURE) (Au) is of the same ammonium sulfate saturation as the pre-
vious fraction (8’). Obviously, application of the formula de-
FIG. 1. Enzyme concentration as a reciprocal function of time pends upon quantitative recovery of the liquid volumes.
for a given extent of reaction. The conditions and concentrations
of the reaction mixture components are described in “Materials All solvent additions (alcohol, acetone, saturated ammonium
and Methods.” The crystalline enzyme was diluted in 0.05 M sulfate solutions) were made under the assumption that the
Versene, pH 8.0, containing 1 mg of albumin per ml. volumes are additive, and were calculated by the formula
Vl(C2 - Cl)
tl=
1 - cz

in which v equals the volume of solvent required in milliliters,

RESERVOlR BOTTLES
VI is the volume of the solution at concentration C1 (or fraction
saturation X1), and Cz represents the concentration (or fraction
saturation SJ desired.
Fraction I-Dried brewers’ yeast, 3 kg, is suspended in 30
liters of 0.2 M ammonium sulfate, pH 8.9, at room temperature
(793 g of (NH4)$04 + 200 ml of concentrated ammonium hy-
droxide made up to 30 liters with deionized distilled water). The
suspension is stirred for 40 minutes with a heavy duty me-
,NFL”ENT CONTROL
chanical stirrer, during which time the pH drops to 8.7. The
mixture is then incubated without stirring for 12 hours (over-
night) at 26 f 2”.
Fraction II-After this period of autolysis (the pH is approxi-
mately 8.5), the mixture is transferred to the cold room and
brought to 0.48 saturation with ammonium sulfate, assuming the
saturation to be 0.05 initially. The pH decreases further to
FIG. 2. Diagram of a large capacity flow dialysis. The dialy- about 8.3 and is adjusted to 5.5 with ice-cold 2 N H&04 which
sis vessel and each of the reservoir bottles contain 20 liters. The is also 1.87 M (0.48 saturation) with respect to ammonium
flow rate is initiallv adiusted such that the reservoir bottles will
be emptied within”6 to “8 hours. The stirring should provide effi- sulfate. The sulfuric acid, of which about 1350 ml are required,
cient mixing and proper agitation of the dialysis sacs. is added at a rate of approximately 150 ml per minute, while
May 1961 E. A. Noltmann, C. J. Gubler, and S. A. Kuby 1227

the reaction mixture is stirred vigorously. The precipitated Fraction V-The initial conditions for the ethanol fractionation
protein together with the insoluble cell debris is removed by are: 10 mg of protein per ml, 0.005 M magnesium sulfate, 0.1 M
centrifugation at 1300 X g for 20 minutes in large capacity magnesium acetate. For convenience, a calculated volume of
centrifuges (e.g. International SR-3 or refrigerated International 0.005 M MgS04 is added to Fraction IV, followed by an aliquot
model 13L). (0.1 of required final volume) of a solution, which is 1.0 M
The supernatant liquid, which is still turbid, is clarified by fil- with respect to magnesium acetate and 0.05 M with respect to
tration. Unwashed Celite 503: 5 g per liter of supernatant magnesium sulfate, to yield the desired final concentrations.6
liquid, is added and thoroughly suspended. Filtration is con- The pH of this mixture (about 6.3) is adjusted to 5.5 with ice-
ducted on two stainless steel Buchner funnels3 with a diameter of cold 1 N H2S04 (about 100 ml are required).
33 cm through one layer of coarse4 filter paper (above) and 1 After the solution is chilled to -1” in an -8” bath, 0.25
layer of Whatman No. 1 filter paper (below). The volume re- volume of 95% ethanol (chilled to -loo) is added with me-
covery of the clear, golden yellow filtrate (Fraction II) is chanical stirring, at a rate such that the temperature does not
approximately 30 liters. exceed 0”. Stirring is continued, with care to avoid formation
Fraction IN-The filtrate is made 5 X 1O-3 M with respect of foam, until the temperature has dropped to - 5”. The milky
to silver nitrate by dissolving the calculated amount of crystalline precipitate is then collected by centrifugation at 10,000 x g
AgN03 in 150 ml of cold distilled water and adding this solution and at a flow rate of about 100 ml per minute in the continuous
to the vigorously stirred Fraction II. The pH, about 5.4, is flow rotor (CFR-2) of the Lourdes LRA centrifuge, the tempera-
adjusted to 4.2 f 0.05 with ice-cold 2 N HzS04-1.87 M (NH4)zS04 ture being maintained between -6” and -8” during centrifuga-
(approximately 450 ml are required) in the same manner as de- tion. The small cream-colored precipitate is suspended in a
scribed for Fraction II, and the suspension is allowed to stand volume of ice-cold 0.1 M magnesium acetate, which is equal to
in the cold room for 3 hours with gentle stirring. The precipitate one-tenth the volume of Fraction IV after dilution to 10 mg per
is then collected with three refrigerated Sharples Super centri- ml. Extraction is allowed to take place by stirring the sus-
fuges running at 40,000 r.p.m., while the suspension is siphoned in pension for 30 minutes in an ice bath. The insoluble material
at a rate of approximately 200 ml per minute for each centrifuge. is removed by centrifugation for 15 minutes at 15,000 x g,
The pellet is suspended in 0.05 M Versene, pH 8.0 (one-eighth and the clear supernatant liquid (light greenish yellow) is
the volume of Fraction II), with a Waring Blendor at low speed designated as Fraction V.
regulated with a variable autotransformer (Powerstat). Ini- Fraction VI-After Fraction V is analyzed for protein, it
tially, the pellet dissolves, but with time a considerable amount is diluted to 5 mg per ml, if necessary, and bentonite is
of material, probably denatured, comes out of solution. The added with stirring, at a ratio of 3.0 g of bentonite per g of pro-
mixture is stirred gently overnight and shielded against light. tein. Gentle stirring is continued for 15 minutes and the
The next day, the precipitated material is centrifuged off at adsorbent removed by centrifugation for 40 minutes at 15,000 x
1300 x g (International SR-3) for 45 minutes. The supernatant g. The slightly colored supernatant liquid (Fraction VI) is
liquid (yellow and slightly opalescent) is retained, and the retained.
pellet is extracted a second time with 0.05 M Versene, pH 8.0 Fraction VII--Fraction VI is made 0.66~saturated with
(2% the volume of Fraction II). The very thick suspension this ammonium sulfate, and the resultant semicrystalline-like mate-
time is centrifuged for 20 minutes at 15,000 x g (e.g. Servall or rial is centrifuged off at 15,000 X g for 20 minutes and dis-
Lourdes refrigerated centrifuges with GSA or VRA rotor, re- carded. The ammonium sulfate saturation of the supernatant
spectively) to recover the largest possible volume. Both ex- liquid is increased to 0.86; this second precipitate is collected by
tracts are combined (Fraction III), yielding a volume of about centrifugation at 15,000 X g for 8 hour and dissolved in 0.05 M
one-seventh that of Fraction II. Versene, pH 8.0 (one-tenth the volume of Fraction VI).
Fraction IV-The ammonium sulfate saturation of Fraction The protein solution is made 10m4 M with respect to diiso-
III is estimated as indicated above (averaging approximately propyl fluorophosphate by the addition of an ice-cold 0.1 M
0.06) and increased to 0.48. The small turbidity, which ap- solution, and dialyzed for 8 hours against 30 liters of 0.01 M
pears, is removed by centrifugation for 20 minutes at 15,000 x sodium succinate, pH 5.6. A trace of turbidity, which may
g. The supernatant liquid (yellow and opalescent) is made appear after the dialysis, is removed by centrifugation at
0.67-saturated with ammonium sulfate, and the heavy precipitate 15,000 x g for 15 minutes. The clear yellow supernatant liquid,
is collected by centrifugation under the same conditions as Fraction VII, has about one-sixth the volume of Fraction VI.
before. The pellet is dissolved in freshly prepared 0.05 M Fraction WIT--The next step consists of two parts, firstly,
Versene-0.01 M cysteine, pH 8.0, (one-fifth the volume of Fraction addition of manganese to remove further inactive material and,
III). The resulting solution, slightly turbid and deeply yellow secondly, precipitation of the enzyme with acetone in the pres-
in color, is dialyzed for 10 hours against a total of 60 liters of 0.005 ence of manganese.
M magnesium sulfate by flow dialysis (see “Materials and Meth- Final concentrations required for the first part are: 5 mg of
ods” and Fig. 2). The small precipitate, which forms during protein per ml, 5 X 1O-3 M manganese (ous) acetate, and 2.5 X
dialysis, is removed by centrifugation for 15 minutes at 15,- 1O-4 M manganese (ous) sulfate. These conditions are achieved
000 x g. in a manner analogous to Fraction V by using a 1 M solution of
The protein concentration of the clear, golden yellow superna-
tant fluid (Fraction IV, approximately one-third the volume of 5 An initial turbidity, which appears on dilution with 0.005 M
MgSO,, disappears after addition of the magnesium acetate. It
Fraction III) is determined.
is important to add first the calculated volume of 0.005 M MgS04
2 Johns-Manville Company. before the 1 M magnesium acetate-O.05 M MgSOd, otherwise a
3 Industrial Service Laboratories, Milwaukee. small precipitate may appear, which dissolves rather slowly at the
4 No. 51G, Filpaco Industries, Inc., Chicago. final dilution and will contaminate the alcohol precipitate.
 Glucose-6-P Dehydrogenase. I Vol. 236, No. 5

manganese acetate which is also 0.05 M with respect to manga- mg per ml), at least 1 hour is allowed for precipitation, after
nese sulfate. The manganese solution is added after dilution which time the precipitate is collected by centrifugation at
to the desired protein concentration with 0.01 M sodium succi- 15,000 X g for 1 hour. The pellet is dissolved in 0.001 M
nate, pH 5.6. The pH, which decreases to about 5.3, is ad- Versene, pH 7.0 (1 ml per g of dry DEAE-SF used) to give Frac-
justed to 5.14 f 0.02 with ice-cold 1 N H&Oh (approximately tion X.
0.35 ml is required). The mixture is then centrifuged for 15 Fraction X17-The protein concentration of Fraction X is
minutes at 15,000 x g, and a brownish gelatinous pellet is dis- determined and diluted if necessary to 10 mg per ml with 0.001
carded. M Versene, pH 7.0. The ammonium sulfate saturation is
To the supernatant liquid, an additional aliquot of 1 M manga- estimated as described before and increased to 0.64. After 1
nese acetate-O.05 M manganese sulfate solution is added to hour, a small but significant turbidity is removed by centrifuga-
make a final concentration of 0.02 M manganese acetate. The tion at 25,000 x g. The ammonium sulfate saturation of the
pH of the mixture is then adjusted to 5.5 f 0.02 with ice-cold supernatant liquid is brought to 0.78 and the mixture kept for
1 N NaOH (about 0.5 ml is required). Any turbidity, which at least 4 hour before centrifugation at 25,000 x g for 30 minutes
may appear at this point, is centrifuged off in the same way as (e.g. Servall or Lourdes centrifuges, SS-34 or 9RA rotor, re-
before and discarded. spectively).
The supernatant liquid is chilled to - 1” in a -8” bath and Crystallization-The 0.78 saturation ammonium sulfate pellet
acetone (at -10”) is slowly added to give a final concentration is dissolved in 0.01 M TPN, pH 7.3 (about 2% to & the volume
of 11% (volume per volume). The temperature should not of Fraction X after dilution to 10 mg of protein per ml). The
exceed 0”. The mixture is kept for 10 minutes at 0” and the ammonium sulfate saturation is estimated, and saturated (at
precipitate collected by centrifugation at 15,000 x g for 20 0’) ammonium sulfate solution of pH 7.3 is slowly added with
minutes. The supernatant liquid, which is very faintly gentle swirling to bring the saturation to 0.58. The final pro-
yellowish brown in color, is discarded, and the gelatinous yellow tein concentration should lie between 25 and 30 mg per ml at
pellet is dissolved with some difficulty in 0.05 M ammonium this point.* Immediately after the 0.58 ammonium sulfate
citrate, pH 8.0 (one-tenth the volume of Fraction VII). The saturation is reached, the enzyme solution is centrifuged for 5
protein concentration of the golden yellow solution (Fraction minutes at 25,000 X g to remove any traces of insoluble fibrous
VIII) is determined. material. The clear light yellow liquid is then transferred
i%ction IX-Fraction VIII is diluted to 10 mg of protein into a 3-ml tube with a round bottom, which allows smooth
per ml with 0.05 M ammonium citrate, pH 8.0, and made 0.60 swirling without causing formation of foam, and is kept at 0”.
satu_rated with ammonium sulfate. After removal of a yellow Within several hours a considerable increase in viscosity oc-
precipitate by centrifugation (20 minutes at 25,000 x g), the curs, and, simultaneously, on cautious swirling, a very im-
ammonium sulfate saturation of the supernatant liquid is pressive silkiness can be observed with indirect light.
brought to 0.73, and a very light colored precipitate is collected In the course of the next 3 to 4 days, the ammonium sulfate
by centrifugation as above. It is dissolved in 0.05 M ammonium saturation is gradually increased to 0.62 (about 0.01 per day)
citrate, pH 8.0 (one-third the volume of Fraction VIII) and by the addition of saturated ammonium sulfate solution, pH
dialyzed overnight against 6 liters of 0.002 M dibasic ammonium 7.3. Within 36 to 48 hours after the last increase, 80% or more
phosphate (pH 7.8). The light yellow dialysate is Fraction IX. of the active protein will have crystallized.
Fraction X-After the total protein of Fraction IX is deter-
7 If the procedure is carried through on twice the scale (60
mined, a 35-fold amount of DEAE-SF (prepared as described in liters), centrifugation of the ethanol precipitate requires a longer
“Materials and Methods” and calculated as dry material) is time, which results in further precipitation of inert protein and
suspended in cold distilled water and transferred to a coarse therefore in slightly less purification and in a slightly lower spe-
sintered glass funnel (6.5 cm in diameter), inserted tightly in a cific activity of all the subsequent steps. It is therefore nec-
essary to introduce a second adsorption on DEAE-SF, because
suction flask. The DEAE-SF is stirred to give a thick homoge- crystallization can not be induced readi1.y below a specific ac-
neous slurry, which is finally packed to a wet cake of about 0.3 ti;ity of 20,000 units per mg. In those c&es, the 0.78-saturated
cm height per g of dry DEAE-SF by use of gentle suction. ammonium sulfate oellet of Fraction XI is dissolved in 0.05 M
Fraction IX is allowed to pass into the bed of DEAE-SF ammonium citrate, pH 8.0, and dialyzed again overnight against
without suction. After 10 minutes, the cake is washed with 0.002 M (NH4)2HP04, pH 7.8. DEAE-SF equal to 25 times the
amount of total protein of Fraction XI is equilibrated this time
0.025 M (NH4)zHP04 (100 ml per g of dry DEAE-SF), with use with 500 ml per g of dry DEAE-SF of 0.005 M (NHd)zHPOd, pH
of gentle suction. Elution of the enzyme by 0.15 M (NHd)zHPOh, 7.8, before adsorption of the enzyme. The DEAE-SF is then
pH 7.8, follows with a total of 14 ml of per g of dry DEAE-SF. washed as described for Fraction X and the enzyme eluted with
The first two volumes (2 ml per g of DEAE-SF) do not contain 0.1 M (NH,)J’OI (25 ml per g of dry DEAE-SF), divided into
three portions in a manner similar to that described for Fraction
any enzyme and are discarded. The remaining 12 volumes
X. The combined eluates are concentrated and refractionated
are divided into three portions of 6, 4, and 2 volumes, respec- with ammonium sulfate, with use of the same conditions as de-
tively. Very little suction is applied to the flask to avoid scribed for Fractions X and XI.
formation of foam, and after each of these three eluting portions 8 It has been found most convenient to dissolve the pellet first
the cake is pressed out with a flat glass rod. The eluates are in about 0.5 ml of 0.01 M TPN, pH 7.3, and to determine the re-
covered volume. After the protein concentration is accordingly
combined6 and made 0.95.saturated with ammonium sulfate. estimated, it is then diluted with 0.01 M TPN such that the final
Because the protein concentration is relatively low (0.5 to 1 concentration will be between 25 and 30 mg of protein per ml
after the ammonium sulfate concentration has been brought to
6 It is advisable to measure the absorbancy ratio of 280 m/*./ 0.58 saturation. On the other hand, if the protein is diluted too
260 mp which will rise, after the DEAF,-SF treatment, from about much, it will be very difficult to obtain crystals at an ammonium
0.54 to about 1.6, indicating that this step is highly efficient in sulfate concentration below that which precipitates the amor-
removing almost all contaminating nucleic acid. phous material.
May 1961 E. A. Noltmann, C. J. Gubler, and S. A. Kuby 1229

Recrystallizations-The crystalline suspension is centrifuged solved in 0.01 M TPN, pH 7.3 (one-half the volume used for the
for 1 hour at 25,000 X g, and the mother liquor is removed first crystallization), the ammonium sulfate saturation is esti-
cautiously with a transfer pipet. (The specific activity of the mated and brought to 0.55 as described for the first crystalliza-
yellow mother liquor is less than one-third that of the crystal- tion; the second crystals usually appear within 10 to 60
line material.) The crystals are washed with O.&saturated minutes. Over a period of the next 2 to 3 days the ammonium
ammonium sulfate solution of pH 7.3, (one-fourth the volume of sulfate saturation is increased to 0.60; again, more than 80%
the first mother liquor), and immediately centrifuged for 1 hour of the activity of the first crystals can be obtained in crystalline
at 25,000 x g. After the crystalline pellet is once more dis- form.
The second mother liquor is only very light yellow and addi-
tional liquors are essentially colorless.
Further recrystallizations are carried out in the same manner,
except that the washing step is omitted and crystallization is
induced at 0.54 and gradually increased to 0.58 ammonium sul-
fate saturation.

Usually, the third or fourth crystals yield a constant specific

activity of about 42,000 units per mg of protein, which corre-
sponds, under the described conditions of analysis, to a rate of
676 pmoles of TPNH formed per mg of protein per minute. This
velocity may be compared with the values quoted by Glaser and
Brown (9) for their preparation: 70 to 120 pmoles per mg of
protein per minute under their conditions at 25”.
A photomicrograph of the bipyramidal crystals of glucose-6-P
dehydrogenase is shown in Fig. 3.
A summary of the data for a typical preparation (No. 14) is
given in Table I. The crystalline enzyme is obtained after an
approximately 2500-fold purification over the initial extract with
FIG. 3. Crystals of glucose-G-P dehydrogenase. The photo
micrograph was obtained with a Spencer microscope at about 4”. about 10% over-all yield. The reproducibility of the procedure
The magnification is indicated by the 10-r line drawn on the pho- has been found to be satisfactory, with the yield of each step
tograph. deviating near &5% from the data quoted in the table.

TABLE I

Fractionation of glucose-6-P dehydrogenase (Preparation No. 14)

Initially, 3 kg of dried brewers’ yeast.
- -
Purification Recovery
-
VOllUW Protein Total
Fraction tctivity SP&tic activit: OVW Fr0m
Over-all Ipreceding Over-all preceding
step step
_-
ml mdml units unzts/mg %
x 10-s proteir
I. Autolysate............................ 26,880* 15.3 68.3 16.6 (100) (100)
II. (NH& SO1 supernatant, 0.48 saturation. 30,000 8.5 61.2 23.9 1.4 1.4 90 90
III. Extracted AgN03 precipitate.. 4,360 22.7 48.0 48.5 2.9 2.0 70 78
IV. (NH& Sod, 0.48 to 0.67 saturation. 1,470 39.7 44.5 76.3 4.6 1.6 65 93
V, Alcohol precipitate. 605 4.6 38.4 1,380 83 18.1 56 86
VI. Bentonite supernatant.. 585 2.4 31.2 2,250 136 1.6 46 81
VII. (NH& SO,, 066 to 0.86 saturation. 95 8.8 28.5 3,400 205 1.5 42 91
VIII. Acetone precipitate. 18.8 17.3 23.3 7,160 430 2.1 34 82
IX. (NH& Sod, 0.60 to 0.73 saturation. 18.5 9.5 18.7 10,670 640 1.5 27 81
X. Concentrated DEAE-SF eluate. 7.15 13.1 15.8 16,890 1,020 1.6 23 84
XI. (NHd)&Od, 0.64 to 0.78 saturation. 2.4 27.0 14.2 21,960 1,320 1.3 21 90

Crystallizations
1. Crystals............................... 1.44 24.3 11.4 32,550 1,960 1.5 17 80
2. Crystals..........,.................... 1.09 22.1 9.2 38,000 2,290 1.2 13 80
3. Crystals............................... 0.95 20.5 8.1 41,900 2,520 1.1 12 89
4. Crystals............................... 0.93 17.6 6.9 42,100 2,535 1.006 10 85
4. Mother liquor. . 0.86 3.0 1.1 41,500 13
- -
* After subtraction of the volume included by the cell debris (16%).
1230 Glucose-6-P Dehydrogenase. I Vol. 236, No. 5

DISCUSSION and, by its addition, losses due to proteolysis are essentially

The procedure described above for the isolation of glucose-6-P eliminated.
dehydrogenase involves some points of general interest for puri- 7. Apparently, in yeast, glucose-6-P dehydrogenase is present
fication of enzymes from yeast. in a relatively low concentration. Approximately 160 mg of
1. Considerable effort was spent to find optimal conditions enzyme can be extracted from 3 kg of dried Xaccharomyces carls-
for autolysis and, especially, the effect of mechanical disintegra- bergensis, which is about 0.005% of the total dry weight and
tion of the yeast cells on enzyme yield. Best conditions found about 0.04°]0 of the total extractable protein. Therefore, it was
were with a Potter-Elvehjem homogenizer for a thick cell paste, necessary to emphasize yield more than purification. To achieve
which obviously could not be applied to the required scale. the over-all purification of 2500-fold with 10% over-all yield, 10
Combined use of a large porcelain mortar and a Waring Blendor, fractionation steps and 4 crystallizations with an average yield of
or use of a porcelain ball mill, respectively, resulted in uncon- 85% per step were required. For comparison, calculations show
trollable production of COz in the cell suspension and partial that purification procedures involving 14 steps with average
denaturation of the enzyme. It has been found, however, that yields of 80 and 900/, per step result in over-all yields of 4.4 and
only thorough suspension of the dried yeast by efficient mechan- 23 %, respectively.
ical stirring is necessary, if sufficient ionic strength at an alkaline 8. The enzyme at Fraction XI purity (about 50%) could
pH is present; in particular, ammonium ion seems to facilitate only be induced to crystallize by the addition of its coenzyme,
the autolysis. The optimal conditions worked out are described TPN. The presence of TPN results in surprisingly rapid crys-
under Fraction I. tallization of the enzyme at an ammonium sulfate concentration
2. Precipitation of the proteins as heavy metal complexes at far lower than that which is required to precipitate the amor-
an early stage was found to be convenient, as in previous prep- phous enzyme Whether or not TPN is part of the crystalline
arations (17, 18), to reduce the initially large volumes. structure is a subject for current investigation.
3. For the extraction of the silver proteinates, Versene (with
rather poor complexing ability for silver) was used to effect, as SUMMARY
far as possible, a selective extraction of the enzyme. Concen- A procedure is described for the isolation of crystalline n-glu-
tration and pH of the Versene solution were chosen in such a cose 6-phosphate dehydrogenase from brewers’ yeast.
way that most of the enzyme but only part of the nucleic acids
are extracted. However, the procedure after the silver step is Acknowledgment--We wish to express our gratitude to Profes-
designed to make as much use as possible of the presence of this sor H. A. Lardy for his continued interest and support.
amount of nucleic acids. It allows specific precipitation of the
enzyme nucleates at relatively low solvent concentrations REFERENCES
(ethanol or acetone), leaving in solution the bulk of inert protein.
1. WARBURG, O., AND CHRISTIAN, W., Biochem. Z., 238, 131
Simultaneously, it minimizes the volume increase due to solvent (1931).
additions, which is very important for large scale operations. 2. WARBURD, O., AND CHRISTIAN, W., Riochem. Z., 242, 206
4. Although the presence of nucleic acids facilitates the (1931).
purification of the enzyme, their final removal is necessary be- 3. WARBURU, O., ANI) CHRISTIAN, W., Biochem. Z., 254, 438
(1932).
fore crystallization. After the commonly used methods for re- 4. NEGELEIN, E., AND GERISCHER, W., Biochem. Z., 264, 289
moval of nucleic acids (e.g. Mn++, protamine, digestion with (1936).
ribonuclease) were tried with little success, the use of basic 5. KORNBERG, A., J. Biol. Chem., 182, 805 (1950).
DEAE-cellulose anion exchanger in its unequilibrated hydroxyl G. KORNBERC:, A., AND HORECKER, B. L.,in S P. COLOWICK AND
form proved to be very effective. The DEAE-cellulose has all N. 0. KAPLAN (Editors), Methods in enzymology, Vol. I,
Academic Press, Inc., New York, 1955, p. 323.
the useful properties of protamine (high molecular weight, 7. MCNAIR SCOTT, Il. B., AND COHEN, S. S., Biochem. J., 55, 23
basicity, capacity for combining with nucleic acids with the (1953).
formation of an insoluble compound) but lacks the disadvantage 8. KELLY, T. L., NIELSON, E. D., JOHNSON, R. B., AND VEST-
of possible contamination of the preparation by foreign protein. LING, C. S., J. Biol. Chem., 212, 545 (1955).
9. GLASER, L., AND BROWN, D. H., J. Biol. Chem., 216, 67 (1955).
5. As illustrated by the various ranges of ammonium sulfate
10. DEMoss,R. I>., in S. P. COLOWICK AND N. 0. KAPLAN (Edi-
concentration used during the stages of purification, the solubility tors). Methods in enzumoloav. Vol. Z. Academic Press. , Inc..
-I
of the enzyme in ammonium sulfate solution depends upon (a) Xew'York, 1955, p. i28. “’ ’
the presence or absence of certain impurities (e.g. inert protein 11. JAGANNATHAN, V., RANGACHARI, P. N., AND DAMODARAN,
M., Biochem. J., 64, 477 (1956).
and large molecular nonprotein material), (b) specific ion effects
12. SRERE, P., COOPER, J. R., TABACHNICK, M., AND RACKER,E.,
(e.g. Ag+, Mg ++, Mn++), (c) pH, and (d) effective enzyme con- Arch. Biochem. Biophys., 74, 295 (1958).
centration. 13 S~NNICHSEN, N., FR~NDER, H., B~RNIG, H., AND RICHTER,
6. First attempts at isolation met with extreme difficulty to G.. Z. nhvsiol. Chem.. 316, 209 (1959).
prevent inactivation of the enzyme by apparent proteolytic activ- 14 RAD&AKF&INAN, A. ?;., Biochim. et Biophys. Acta, 40, 546
(1960).
ity. A variety of methods were tried tither to stabilize the en-
15. HORECKER, B. I,., AND KORNBERC~, A., J. Biol. Chem., 1’75,
zyme or inhibit the proteolytic activity. The most successful 385 (1948).
methods found were the use of silver at the very early stages 16. GORNALL, A.G., BARDAWILL, C.J.,and DAVID, M. M., J. Biol.
and, after its removal and further purification of the enzyme, the Chem., 177, 751 (1949).
addition of diisopropyl fluorophosphate. This powerful inhibitor 17 NODA, L., AND KUBY, S. A., J. Biol. Chem., 226, 541 (1957).
18 KUBY, S. A., Nooa, L., AND LARDY, H. A., J. Biol. Chem.,
of proteolysis had been used by Stein and Fischer (19) in the 209, 191 (1954).
isolation of oc-amylases. Fortunately, diisopropyl fluorophos- 19. STEIN, E. A., AND FISCHER, E. H., J. Biol. Chem., 232, 867
phate does not inhibit the glucose-6-P dehydrogenase activity (1958).