[42]
GLUCOSE-6-PHOSPHATE DEHYDROGENASE
[42] G l u c o s e - 6 - p h o s p h a t e D e h y d r o g e n a s e Bovine Mammary Gland
183
from
By G. R. JULIAN and F. J. REITHEL D-Glucose 6-phosphate + NADP + ~---6-phosphoglucono-~-lactone + NADPH + H +
Assay Method
Principle. The reaction catalyzed results in the formation of N A D P H , and the rate of formation is accurately estimated by measuring the increase in absorbancy at 340 nm. Concentrations of substrates must be nonlimiting in order to ensure that the rate corresponds to the limiting concentration of enzyme being assayed. Reagents Buffer I: a solution 0.1 M in HC1 and 0.1 M in MgCl~ was adjusted to pH 7.2 with Tris. The resultant concentration of Tris was 0.092 M. Water used for this buffer and all other procedures was distilled and deionized. Glucose 6-phosphate, disodium salt, 0.26 mM. Stock solution in buffer I. NADP, sodium salt, 0.56 raM. Stock solution in buffer I. :Enzyme: the volume (in t~l) of enzyme solution used for assay varied according to its concentrations during purification. In every case, however, sufficient buffer I was added to maintain the total assay volume of 1.0 ml.
Procedure. A recording spectrophotometer was used to measure and record the rate of change of absorbancy at 340 nm. The cuvette compartlnent was maintained at 25 ° and 1-ml quartz cuvettes were employed. Appropriate concentrations of substrates were provided by adding a 10-~l aliquot of the N A D P and a 20-~1 aliquot of the glucose 6-phosphate stock solution to a volume of approximately 1 ml of buffer. The glucose-6-phosphate debydrogenase reaction was initiated on addition of enzyme. Volumes of enzyme solution over 20 td were introduced as a droplet on a plastic stick designed as an agitator. Definition o] Unit and Specific Activity. The unit of activity was defined as the amount of enzyme necessary to effect a change of 1.0 unit of absorbance per minute under the conditions specified. Specific activity considered as activity units per milligram of protein
184
OXIDATION REDUCTION ENZYMES
[42l
requires estimation of protein. The method used was based on measurements of absorbance at 280 and at 260 nm but corrected for the presence of thioglyeolate, when present, as follows. The absorbanee ratios A2so/A26o = RB for Tris-thioglycolate (described below) b u f f e r - - 0 . 4 . The same ratio, Rp for protein = 1.58. Assuming an extinction coefficient --2S0~lmg/m~= 1.0, then the following relationship allows one to calculate a conversion factor K, from experimental values of A26o/A28o.
A26o/A2so = 1/RB + K [(RB -- Re)/(E~o glint. R e . RB)] thus, A~80" K = mg p r o t e i n / m l Purification
Procedure 1
Cow udders were obtained from the local abattoir soon after slaughter of the animal and cooled to 0-4 ° with dispatch. The chilled gland was trimmed to remove fat, drained of milk, wrapped in paper or plastic, and frozen at --10% The frozen gland could then be sliced with a mechanical slicer (a commercial butcher's meat slicer was used) and added to buffer II (10 m M thioglycolate, adjusted to pH 7.2 with solid Tris) sufficient to keep the tissue covered as slices were added. In a typical preparation 12 liters of tissue slices required 8 liters of buffer II for extraction. Step 1. Preparation o] Extract. The sliced gland was allowed to stand in the covering buffer for 2 hr at room temperature with occasional stirring. The first buffer extract was removed by draining through a plastic screen that formed the top of a filter table. A second extract was made with 0.75 times the first volume of buffer and finally a third extract with 0.5 times the first volume of buffer, allowing the slices to soak 30 min each time before draining. The combined extracts were centrifuged to separate the large amounts of fat and cell debris entrained. Careful filtration of the clarified extract through plastic screen and cotton toweling yielded a deep red clear extract. Step 2. Ethanol Fractionation. The extract was cooled to 1-2% From a reservoir 95% ethanol, cooled to --20 °, was added to the extract with efficient stirring at a rate of about 50 drops per minute through a capillary tipped manifold of nine outlets. Ethanol was added until the final concentration was 24% by volume. The precipitated protein was collected by continuous centrifugation at 4 ° and the supernatant liquid (containing most of the 6-phosphogluconate dehydrogenase) was discarded. The precipitate was converted to a free-flowing suspension by careful blending or homogenizing in buffer II containing 15% ethanol. About 250 ml of i G. R. Julian, R. G. Wolfe, and F. J. Reithel, J. Biol. Chem. 236, 754 (1961).
[42]
GLUCOSE-6-PHOSPHATE DEHYDROGENASE
185
buffer was required per 100 g of precipitate. The suspension was finally centrifuged for 30 min at 1.3 X 10~ g and 4 ° to obtain a firmly packed, washed, homogeneous sediment. Step 3. Extraction with (NH~).~S04 Solution. A stock solution of saturated (NH~)~SO~ was prepared by dissolving the salt in 10 mM thioglycolic acid, 2 mM in EDTA, to saturation at room temperature. After adjustment to pH 7.2 with Tris, the solution was filtered, freed of 05 by bubbling purified N~ through it, and stored under N._, in carboys at 4 °. This stock was diluted with buffer II to prepare less saturated solutions when needed. In this paper, "saturation" refers to saturation at 4% When percentage saturation was adjusted by adding saturated (NH4) S04 solution. The volume change was ignored. For example, equal volumes of protein solution and saturated (NH4)2S04 solution, when mixed, resulted in a solution considered to be 50% saturated. The protein precipitate of step 2 was suspended in 40% saturated (NH4)2S04 by low speed homogenization to form a smooth homogeneous slurry. Protein from 1 kg of tissue resulted in a volume of about 500 ml at this point. The slurry was centrifuged at 1.3 X 10~ g at 4 ° for 3.0 min, the extract was collected, and the residue was extracted twice more by repeating the described procedure. Step 4. Fractionation by Extraction with (NH4).,S04 Gradient. Celite (Johns-Manville No. 535) was rid of fines by water washing and added to a 9 X 72 cm Lucite column to within 10 cm of the top. This served to measure accurately the amount of Celite used during this step. It was washed with about 9 liters of l0 mM Tris-thioglycolate, pH 7.2, 10% saturated with (NH~)~S04. Fe(III) contamination when present was evident by the formation of a purple complex resulting from its complex with thioglycolate in basic solution. The column contents were washed with a like volume of distilled water, allowed to drain, removed from the column and stored at 4% The extract of step 3 was added to 3 liters of washed Celite and sufficient 40% saturated (NH4)._,S0~ solution was added to produce a slurry fluid enough for efficient stirring. The protein was precipitated on the Celite particles by raising the (NH4),.,S04 concentration to 65% saturation. To achieve this, saturated (NH4)._,S04 solution was added through the capillary-tipped manifold, employed in step 2, with efficient stirring. The supernatant liquid was decanted and the protein-Celite slurry was allowed to settle and compact in the 9 X 72 cm Lucite column. The bed was washed initially with 1 liter of 65% saturated (NH~)2S04 solution. Protein was then gradually extracted with a descending exponential (NH,)2S04 gradient. A 500-ml filter flask served as a mixing chamber. It was filled with 65% saturated (NH~)2S04 and into it was fed 40%
186
OXIDATION--REDUCTION ENZYMES
[42]
saturated (NH~)=,S04 from a 6-liter reservoir. Fractions of 250 ml were collected and enzymic activity appeared in the eluate after about 3.5 liters of eluate had been collected. Those fractions containing substantial enzymic activity were pooled and made 70% saturated with respect to (NH~) 2S04 to precipitate the desired protein. Step 5. Precipitation o] the Enzyme with Zinc Ion. The precipitate from step 4 was collected by centrifugation and dissolved in about 2 liters of buffer II. To this solution was added 80 ml of 0.5 M zinc acetate buffered at p H 7.2 in buffer II. The zinc-containing solution was added very slowly with mild stirring over a period of 6-7 hr. The final concentration of Zn ~+ was 20 mM. The precipitate was collected by centrifugation without delay, F r o m this precipitate the enzyme protein was extracted with 25 m M E D T A in buffer I I at p H 7.2. Again, the protein desired was precipitated from this extract at 70% (NH4)._,S04. Step 6. First Chromatography on Hydroxyapatite. The precipitate of Step 5 was dissolved in 250 ml of buffer I I and added to a column of hydroxyapatite 9.5 cm in diameter and 9.0 cm in height. As soon as the solution had entered the bed, 200 ml of buffer was added to remove (NH4)~S04 and unadsorbed protein. Next a quantity of 200 ml 50 m M K phosphate in buffer I I (pH 7.2) was applied. The collection of fractions was initiated during the next elution with 200 ml of 0.1 M K phosphate in buffer II. In general, enzyme activity was not eluted from the column until 15 m M K phosphate was used for elution. The enzyme in the eluate was precipitated again at 70% (NH4)._,SO~ and collected by centrifugation. At this point, the protein was dissolved in a minimum volume of buffer I I and desalted by passing the solution through a column of Sephadex G-25. Step 7. Purification by Use of DEAE-Cellulose. A 2.2 X 19 cm PURIFICATION OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE (PREPARATION 48)
Fraction 1. 2. 3. 4. 5. 6. 7. 8. 9.
Initial extract Extract of ethanol ppt. (NH4)2SO4 extract Extract before Zn 2÷ ppt. Solution after Zn 2+ removal Recovery from hydroxyapatite Recovery from DEAE-cellulose Sephadex eluate Solution of crystallized enzyme
Units of activity
Specific activity
Yield (%)
41,300 34,200 31,800 19,800 16,000 9,5OO 6,700 2,780 1,180
0.041 0.37 3.32 3.36 5.3 8.4 70 111 420
83 77 48 39 23 16 6.7 2.9
[42]
GLUCOSE-6-PHOSPHATE DEHYDROGENASE
187
DEAE-cellulose column was packed and equilibrated with buffer II. To this column was now adsorbed the desalted enzyme solution of step 6 without delay. To the eolumn was attached a 250-ml mixing chamber filled with buffer II. A 500-ml separatory funnel was fitted to the top of the mixing chamber and filled with 0.2 M NaC1 in buffer II. With this arrangement the enzyme was eluted with a salt gradient. Five-milliliter fractions were collected at a flow rate of 15--20 ml per hour at 4 °. Enzyme usually was present in the eluate after 250 ml had been collected. Step 8. Second Chromatography o'n Hydroxyapatite. Since the total amount of protein at~ this stage was 100-200 mg it was possible to employ a smaller (1.8 X 10 cm) column. The active fractions of step 7 were added to this column and the enzyme was found to be adsorbed. One colunm volume of buffer II was percolated through to remove residual NaC1 and then gradient elution was started at a concentration of 0.1 M K phosphate, increasing to 0.15 M. Step 9. Crystallization. The enzyme eluted in step 8 was precipitated with (NH~)._,SO~, collected by centrifugation and dissolved in a minimal volume of 50 mM Tris-thioglycolate buffer, pH 7.0. This was desalted by passing through a small (1 X 10 cm)column of Sephadex and the enzyme solution was allowed to stand for a few hours at 4 °. Crystals formed, accounting for most of the activity in a solution of 1% protein concentration. Examination by ultracentrifugation showed that at low ionic strength higher molecular weight forms were evident when the enzyrne was highly purified. Comments. The procedure outlined is the result of nearly fifty trials. As described here, our procedure allowed the processing of relatively large amounts of tissue and of protein with reasonable facility. To expedite handling, no attempt was made to obtain a maximum yield at any step, but rather to obtain a moderate yield with dispatch. In some steps, such as that employing Zn -"+ precipitation, speed was necessary to avoid aggregation and hence grave losses due to decrease in solubility of the protein. In others, storage or slow collection of fractions in which protein concentration was low resulted in losses at any temperature. Again, during chromatography on DEAE-eellulose, two distinct peaks of protein and activity were discovered but only the most active was collected. When highly purified enzyme was stored at low ionic strength at any temperature between 4=° and 25 ° the formation of crystals was observed. Repe'~ted crystallization yielded the same activity, but there was increased evidence of aggregation. Storage of crystalline enzyme was accompanied by slow loss of enzyme activity. The use of ethanol precipitation at an early stage allowed nearly eom-
188
OXIDATION--REDUCTION ENZYMES
[431
plete removal of 6-phosphogluconate dehydrogenase that would interfere with accurate assay of the glucose-6-phosphate dehydrogenase. Also it should be noted that the use of DEAE-cellulose dictated the use of buffers of low ionic strength, despite an early recognition that the enzyme protein desired had a rather low solubility in such buffers. Losses during dialysis at this step, where the ionic strength was lowered, were substantial and likely involved some aggregation. Stabilization of the enzyme was achieved partly by use of thioglycolate but principally by using EDTA. Use of Tris-EDTA buffers maintained 90% of the activity for 15 days in one test, 52% of the activity for 76 days in another. However, it was found that EDTA even at concentrations as low as 2 mM interfered with adsorption of the enzyme on hydroxyapatite.
[43] G l u c o s e - 6 - p h o s p h a t e D e h y d r o g e n a s e f r o m Cow Adrenal Cortex I
By
KENNETH W. MCKEaNS
Glucose 6-phosphate -4- NADP+ ~ 6-phospho-$-gluconolactone -4- NADPH + H+ Enzyme Assay
During the purification procedure dehydrogenase activity was measured by monitoring the initial rate of reduction of NADP ÷ or NAD ÷. Increase in absorbance at 340 nm was followed using a Beckman spectrophotometer with recorder. It is convenient to have scale expansion to increase the sensitivity of the method. A stop-flow apparatus with oscilloscope recording is desirable for kinetic studies of the enzyme. The routine assay mixture contains the following components at 37°: 0.1 M Tris-HC1 buffer (pH 8.0), 10 ~M NADP ÷ or 10 mM NAD ÷, 0.1 mM glucose 6-phosphate, 6 mM MgC12, and enzyme. During stages I-IV of enzyme purification, corrections for 6-phosphogluconate dehydrogenase were made by noting the difference in the reaction rate obtained with NADP ÷, 0.1 mM 6-phosphate gluconate, and 0.1 mM glucose 6-phosphate, and the reaction obtained with NADP ÷ and 0.1 mM 6-phosphate gluconate. The reactions were initiated with glucose 6-phosphate or 6-phosphate gluconate. All assays were made with the enzyme diluted 1 (D-Glucose 6-phosphate:NADP÷ oxidoreductase, EC 1.1.1.49).
GLUCOSE-6-PHOSPHATE DEHYDROGENASE
[42] G l u c o s e - 6 - p h o s p h a t e D e h y d r o g e n a s e Bovine Mammary Gland
183
from
By G. R. JULIAN and F. J. REITHEL D-Glucose 6-phosphate + NADP + ~---6-phosphoglucono-~-lactone + NADPH + H +
Assay Method
Principle. The reaction catalyzed results in the formation of N A D P H , and the rate of formation is accurately estimated by measuring the increase in absorbancy at 340 nm. Concentrations of substrates must be nonlimiting in order to ensure that the rate corresponds to the limiting concentration of enzyme being assayed. Reagents Buffer I: a solution 0.1 M in HC1 and 0.1 M in MgCl~ was adjusted to pH 7.2 with Tris. The resultant concentration of Tris was 0.092 M. Water used for this buffer and all other procedures was distilled and deionized. Glucose 6-phosphate, disodium salt, 0.26 mM. Stock solution in buffer I. NADP, sodium salt, 0.56 raM. Stock solution in buffer I. :Enzyme: the volume (in t~l) of enzyme solution used for assay varied according to its concentrations during purification. In every case, however, sufficient buffer I was added to maintain the total assay volume of 1.0 ml.
Procedure. A recording spectrophotometer was used to measure and record the rate of change of absorbancy at 340 nm. The cuvette compartlnent was maintained at 25 ° and 1-ml quartz cuvettes were employed. Appropriate concentrations of substrates were provided by adding a 10-~l aliquot of the N A D P and a 20-~1 aliquot of the glucose 6-phosphate stock solution to a volume of approximately 1 ml of buffer. The glucose-6-phosphate debydrogenase reaction was initiated on addition of enzyme. Volumes of enzyme solution over 20 td were introduced as a droplet on a plastic stick designed as an agitator. Definition o] Unit and Specific Activity. The unit of activity was defined as the amount of enzyme necessary to effect a change of 1.0 unit of absorbance per minute under the conditions specified. Specific activity considered as activity units per milligram of protein
184
OXIDATION REDUCTION ENZYMES
[42l
requires estimation of protein. The method used was based on measurements of absorbance at 280 and at 260 nm but corrected for the presence of thioglyeolate, when present, as follows. The absorbanee ratios A2so/A26o = RB for Tris-thioglycolate (described below) b u f f e r - - 0 . 4 . The same ratio, Rp for protein = 1.58. Assuming an extinction coefficient --2S0~lmg/m~= 1.0, then the following relationship allows one to calculate a conversion factor K, from experimental values of A26o/A28o.
A26o/A2so = 1/RB + K [(RB -- Re)/(E~o glint. R e . RB)] thus, A~80" K = mg p r o t e i n / m l Purification
Procedure 1
Cow udders were obtained from the local abattoir soon after slaughter of the animal and cooled to 0-4 ° with dispatch. The chilled gland was trimmed to remove fat, drained of milk, wrapped in paper or plastic, and frozen at --10% The frozen gland could then be sliced with a mechanical slicer (a commercial butcher's meat slicer was used) and added to buffer II (10 m M thioglycolate, adjusted to pH 7.2 with solid Tris) sufficient to keep the tissue covered as slices were added. In a typical preparation 12 liters of tissue slices required 8 liters of buffer II for extraction. Step 1. Preparation o] Extract. The sliced gland was allowed to stand in the covering buffer for 2 hr at room temperature with occasional stirring. The first buffer extract was removed by draining through a plastic screen that formed the top of a filter table. A second extract was made with 0.75 times the first volume of buffer and finally a third extract with 0.5 times the first volume of buffer, allowing the slices to soak 30 min each time before draining. The combined extracts were centrifuged to separate the large amounts of fat and cell debris entrained. Careful filtration of the clarified extract through plastic screen and cotton toweling yielded a deep red clear extract. Step 2. Ethanol Fractionation. The extract was cooled to 1-2% From a reservoir 95% ethanol, cooled to --20 °, was added to the extract with efficient stirring at a rate of about 50 drops per minute through a capillary tipped manifold of nine outlets. Ethanol was added until the final concentration was 24% by volume. The precipitated protein was collected by continuous centrifugation at 4 ° and the supernatant liquid (containing most of the 6-phosphogluconate dehydrogenase) was discarded. The precipitate was converted to a free-flowing suspension by careful blending or homogenizing in buffer II containing 15% ethanol. About 250 ml of i G. R. Julian, R. G. Wolfe, and F. J. Reithel, J. Biol. Chem. 236, 754 (1961).
[42]
GLUCOSE-6-PHOSPHATE DEHYDROGENASE
185
buffer was required per 100 g of precipitate. The suspension was finally centrifuged for 30 min at 1.3 X 10~ g and 4 ° to obtain a firmly packed, washed, homogeneous sediment. Step 3. Extraction with (NH~).~S04 Solution. A stock solution of saturated (NH~)~SO~ was prepared by dissolving the salt in 10 mM thioglycolic acid, 2 mM in EDTA, to saturation at room temperature. After adjustment to pH 7.2 with Tris, the solution was filtered, freed of 05 by bubbling purified N~ through it, and stored under N._, in carboys at 4 °. This stock was diluted with buffer II to prepare less saturated solutions when needed. In this paper, "saturation" refers to saturation at 4% When percentage saturation was adjusted by adding saturated (NH4) S04 solution. The volume change was ignored. For example, equal volumes of protein solution and saturated (NH4)2S04 solution, when mixed, resulted in a solution considered to be 50% saturated. The protein precipitate of step 2 was suspended in 40% saturated (NH4)2S04 by low speed homogenization to form a smooth homogeneous slurry. Protein from 1 kg of tissue resulted in a volume of about 500 ml at this point. The slurry was centrifuged at 1.3 X 10~ g at 4 ° for 3.0 min, the extract was collected, and the residue was extracted twice more by repeating the described procedure. Step 4. Fractionation by Extraction with (NH4).,S04 Gradient. Celite (Johns-Manville No. 535) was rid of fines by water washing and added to a 9 X 72 cm Lucite column to within 10 cm of the top. This served to measure accurately the amount of Celite used during this step. It was washed with about 9 liters of l0 mM Tris-thioglycolate, pH 7.2, 10% saturated with (NH~)~S04. Fe(III) contamination when present was evident by the formation of a purple complex resulting from its complex with thioglycolate in basic solution. The column contents were washed with a like volume of distilled water, allowed to drain, removed from the column and stored at 4% The extract of step 3 was added to 3 liters of washed Celite and sufficient 40% saturated (NH4)._,S0~ solution was added to produce a slurry fluid enough for efficient stirring. The protein was precipitated on the Celite particles by raising the (NH4),.,S04 concentration to 65% saturation. To achieve this, saturated (NH4)._,S04 solution was added through the capillary-tipped manifold, employed in step 2, with efficient stirring. The supernatant liquid was decanted and the protein-Celite slurry was allowed to settle and compact in the 9 X 72 cm Lucite column. The bed was washed initially with 1 liter of 65% saturated (NH~)2S04 solution. Protein was then gradually extracted with a descending exponential (NH,)2S04 gradient. A 500-ml filter flask served as a mixing chamber. It was filled with 65% saturated (NH~)2S04 and into it was fed 40%
186
OXIDATION--REDUCTION ENZYMES
[42]
saturated (NH~)=,S04 from a 6-liter reservoir. Fractions of 250 ml were collected and enzymic activity appeared in the eluate after about 3.5 liters of eluate had been collected. Those fractions containing substantial enzymic activity were pooled and made 70% saturated with respect to (NH~) 2S04 to precipitate the desired protein. Step 5. Precipitation o] the Enzyme with Zinc Ion. The precipitate from step 4 was collected by centrifugation and dissolved in about 2 liters of buffer II. To this solution was added 80 ml of 0.5 M zinc acetate buffered at p H 7.2 in buffer II. The zinc-containing solution was added very slowly with mild stirring over a period of 6-7 hr. The final concentration of Zn ~+ was 20 mM. The precipitate was collected by centrifugation without delay, F r o m this precipitate the enzyme protein was extracted with 25 m M E D T A in buffer I I at p H 7.2. Again, the protein desired was precipitated from this extract at 70% (NH4)._,S04. Step 6. First Chromatography on Hydroxyapatite. The precipitate of Step 5 was dissolved in 250 ml of buffer I I and added to a column of hydroxyapatite 9.5 cm in diameter and 9.0 cm in height. As soon as the solution had entered the bed, 200 ml of buffer was added to remove (NH4)~S04 and unadsorbed protein. Next a quantity of 200 ml 50 m M K phosphate in buffer I I (pH 7.2) was applied. The collection of fractions was initiated during the next elution with 200 ml of 0.1 M K phosphate in buffer II. In general, enzyme activity was not eluted from the column until 15 m M K phosphate was used for elution. The enzyme in the eluate was precipitated again at 70% (NH4)._,SO~ and collected by centrifugation. At this point, the protein was dissolved in a minimum volume of buffer I I and desalted by passing the solution through a column of Sephadex G-25. Step 7. Purification by Use of DEAE-Cellulose. A 2.2 X 19 cm PURIFICATION OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE (PREPARATION 48)
Fraction 1. 2. 3. 4. 5. 6. 7. 8. 9.
Initial extract Extract of ethanol ppt. (NH4)2SO4 extract Extract before Zn 2÷ ppt. Solution after Zn 2+ removal Recovery from hydroxyapatite Recovery from DEAE-cellulose Sephadex eluate Solution of crystallized enzyme
Units of activity
Specific activity
Yield (%)
41,300 34,200 31,800 19,800 16,000 9,5OO 6,700 2,780 1,180
0.041 0.37 3.32 3.36 5.3 8.4 70 111 420
83 77 48 39 23 16 6.7 2.9
[42]
GLUCOSE-6-PHOSPHATE DEHYDROGENASE
187
DEAE-cellulose column was packed and equilibrated with buffer II. To this column was now adsorbed the desalted enzyme solution of step 6 without delay. To the eolumn was attached a 250-ml mixing chamber filled with buffer II. A 500-ml separatory funnel was fitted to the top of the mixing chamber and filled with 0.2 M NaC1 in buffer II. With this arrangement the enzyme was eluted with a salt gradient. Five-milliliter fractions were collected at a flow rate of 15--20 ml per hour at 4 °. Enzyme usually was present in the eluate after 250 ml had been collected. Step 8. Second Chromatography o'n Hydroxyapatite. Since the total amount of protein at~ this stage was 100-200 mg it was possible to employ a smaller (1.8 X 10 cm) column. The active fractions of step 7 were added to this column and the enzyme was found to be adsorbed. One colunm volume of buffer II was percolated through to remove residual NaC1 and then gradient elution was started at a concentration of 0.1 M K phosphate, increasing to 0.15 M. Step 9. Crystallization. The enzyme eluted in step 8 was precipitated with (NH~)._,SO~, collected by centrifugation and dissolved in a minimal volume of 50 mM Tris-thioglycolate buffer, pH 7.0. This was desalted by passing through a small (1 X 10 cm)column of Sephadex and the enzyme solution was allowed to stand for a few hours at 4 °. Crystals formed, accounting for most of the activity in a solution of 1% protein concentration. Examination by ultracentrifugation showed that at low ionic strength higher molecular weight forms were evident when the enzyrne was highly purified. Comments. The procedure outlined is the result of nearly fifty trials. As described here, our procedure allowed the processing of relatively large amounts of tissue and of protein with reasonable facility. To expedite handling, no attempt was made to obtain a maximum yield at any step, but rather to obtain a moderate yield with dispatch. In some steps, such as that employing Zn -"+ precipitation, speed was necessary to avoid aggregation and hence grave losses due to decrease in solubility of the protein. In others, storage or slow collection of fractions in which protein concentration was low resulted in losses at any temperature. Again, during chromatography on DEAE-eellulose, two distinct peaks of protein and activity were discovered but only the most active was collected. When highly purified enzyme was stored at low ionic strength at any temperature between 4=° and 25 ° the formation of crystals was observed. Repe'~ted crystallization yielded the same activity, but there was increased evidence of aggregation. Storage of crystalline enzyme was accompanied by slow loss of enzyme activity. The use of ethanol precipitation at an early stage allowed nearly eom-
188
OXIDATION--REDUCTION ENZYMES
[431
plete removal of 6-phosphogluconate dehydrogenase that would interfere with accurate assay of the glucose-6-phosphate dehydrogenase. Also it should be noted that the use of DEAE-cellulose dictated the use of buffers of low ionic strength, despite an early recognition that the enzyme protein desired had a rather low solubility in such buffers. Losses during dialysis at this step, where the ionic strength was lowered, were substantial and likely involved some aggregation. Stabilization of the enzyme was achieved partly by use of thioglycolate but principally by using EDTA. Use of Tris-EDTA buffers maintained 90% of the activity for 15 days in one test, 52% of the activity for 76 days in another. However, it was found that EDTA even at concentrations as low as 2 mM interfered with adsorption of the enzyme on hydroxyapatite.
[43] G l u c o s e - 6 - p h o s p h a t e D e h y d r o g e n a s e f r o m Cow Adrenal Cortex I
By
KENNETH W. MCKEaNS
Glucose 6-phosphate -4- NADP+ ~ 6-phospho-$-gluconolactone -4- NADPH + H+ Enzyme Assay
During the purification procedure dehydrogenase activity was measured by monitoring the initial rate of reduction of NADP ÷ or NAD ÷. Increase in absorbance at 340 nm was followed using a Beckman spectrophotometer with recorder. It is convenient to have scale expansion to increase the sensitivity of the method. A stop-flow apparatus with oscilloscope recording is desirable for kinetic studies of the enzyme. The routine assay mixture contains the following components at 37°: 0.1 M Tris-HC1 buffer (pH 8.0), 10 ~M NADP ÷ or 10 mM NAD ÷, 0.1 mM glucose 6-phosphate, 6 mM MgC12, and enzyme. During stages I-IV of enzyme purification, corrections for 6-phosphogluconate dehydrogenase were made by noting the difference in the reaction rate obtained with NADP ÷, 0.1 mM 6-phosphate gluconate, and 0.1 mM glucose 6-phosphate, and the reaction obtained with NADP ÷ and 0.1 mM 6-phosphate gluconate. The reactions were initiated with glucose 6-phosphate or 6-phosphate gluconate. All assays were made with the enzyme diluted 1 (D-Glucose 6-phosphate:NADP÷ oxidoreductase, EC 1.1.1.49).
