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[24] Glucose 6-phosphate dehydrogenase from erythrocytes
126
DEHYDROGE~ASES AND OXIDASES
[24]
[24] Glucose 6 - P h o s p h a t e D e h y d r o g e n a s e from E r y t h r o c y t e s
By
ROBERT G. LANGDON
Glucose 6-phosphate + TPN+ ~ 6-phosphogluconate + TPNH -b H+ Assay Method Principle. The reduction of TPN ÷ to T P N H results in the appearance of an absorption band at 340 m~. In the presence of saturating concentrations of G-6-P and TPN, the rate of change of absorbancy at this wavelength is proportional to the enzyme concentration.
Reagents Tris-chloride buffer, 1 M, pH 7.5 Glucose 6-phosphate, 2.5 X 10-2 M. Dissolve 97.5 mg of the dipotassium salt of glucose 6-phosphate trihydrate in 10 ml of water. TPN, 2 X 10-s M. Dissolve a quantity of TPN equivalent to 16.7 mg of the free acid in 9 ml water. Adjust the pH to 7.5. Adjust the volume to 10 ml. MgCl~, 0.2M. Dissolve 4.1 g MgC12"6 H20 in sufficient water to give a final volume of 100 ml All buffers and reagents should be prepared in water distilled from a hard glass or quartz still.
Procedure. The assay is carried out at room temperature (25°). To a 3-ml quartz cell having a 1-cm path length is added sufficient glass-distilled water to yield a final volume of 3 ml after additions of all reagents and enzymes have been made; 0.1 ml buffer, 0.1 ml G-6-P, 0.1 ml TPN, and 0.1 ml MgC12 are then added. These are mixed and the cell is placed in a spectrophotometer adapted for recording of absorbancy as a function of time. A volume of enzyme solution containing approximately 0.1-0.2 enzyme units is then quickly added and mixed, and the rate of change of absorbancy at 340 m# is recorded. The velocity of the reaction during the first 10 seconds following mixing is taken to represent the initial velocity. De'finition of Enzyme Unit and Specific Activity. One unit of enzyme activity is defined as that quantity which catalyzes the reduction of 1 micromole of TPN per minute under the above assay conditions. This corresponds to an absorbancy change of 2.07 per minute. Protein is measured by the optical method of Warburg and Christian 1 or by the 10. Warburg and W. Christian, Bioehem. Z. 310, 384 (1941). See also E. Layne, Vol. III, p. 451.
[24]
GLUCOSEa-PHOSPHATE DEHYDROGENASE ERYTHROCYTES
127
colormetric method of Lowry.2 Specific activity is defined as the units of enzyme per milligram of protein. Purification Procedure The purification procedure given here is essentially that described by Chung and Langdon.3 A similar procedure yielding a slightly less pure product has been described by Kirkman. 4 During the purification it is important to use reagent grade chemicals and distilled water which has been redistilled from a hard-glass still or passed through a mixed-bed ion exchange column.
Preparation o] Adsorbents Commerial ion exchange cellulose is prepared for chromatography as follows: Each 100g of the material is suspended in 2 liters of water and titrated with 1 M KH2P04 or K~HP04 to the desired pH value. After the suspension has settled for a few minutes, any fine particles are removed by decantation and the remainder is filtered through fine-mesh nylon in a Biichner funnel. The damp cake is washed twice in a similar manner with water and 3 times in the desired buffer. It is then stored in buffer at 0 ° until used. Calcium phosphate gel is prepared as described by Keilin and Hartree2
Preparation o] Erythrocytes The erythrocytes from 18 pints of human blood, collected in standard ACD solution by a blood bank and stored at 4 ° for 3-5 weeks, are allowed to settle and the supernatant plasma is removed by suction. The thick suspension is then centrifuged at low speed, and the erythrocytes are washed four times with 0.15 M KC1 in 5 X 10-3 M potassium phosphate buffer, pH 7.0, containing 10.4 M EDTA. The erythrocytes may be stored at 4 ° for a short time prior to use. Step I. Preparation of Hemolyzate. The erythrocytes are mixed with an equal volume of distilled water, frozen solidly by immersing the container in a dry ice bath, and then thawed in a water bath at 10°. Most subsequent operations are carried out in a cold room at 4 °. Step 2. First DEAE-Cellulose Treatment. The hemolyzate from each 6 pints of blood is diluted with water to 2 liters and mixed with 150g of DEAE-cellulose suspended in 2 liters of 5 X 10-3 M potassium phosphate, 20. H. Lowry, :N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). See also E. Layne, Vol. III, p. 448. 3A. E. Chung and R. G. Langdon,J. Biol. Chem. 238~ 2309 (1963). 4H. N. Kirkman, J. Biol. Chem. 237, 2364 (1962). ~D. Keilin and E. F. Hartree, Proc. Roy. Soc. B124, 397 (1938).
128
DEHYDROGE~CASES AND OXIDASES
[24]
pH 7.0, containing 10°4M EDTA. After the suspension has been gently stirred for 30 minutes, it is poured into an 8 X 60 cm column. The packed cellulose is washed with an additional 4 liters of the buffer solution, which elutes most of the hemoglobin. Then 0.3 M KC1 in the same buffer is passed through the column; fractions are collected and assayed for enzyme activity. Those fractions containing enzyme are pooled. Step 3. Ammonium Sulfate Precipitation. The enzyme is precipitated by the addition of 351 g of solid ammonium sulfate per liter of solution obtained in the preceding step. It is collected by centrifugation, dissolved in 5 X 10-2 M potassium phosphate, pH 7.0, containing 10-~ M EDTA, and the ammonium sulfate precipitation is repeated. After solution in 5 X 10-~ M potassium phosphate, pH 6.0, containing 10~ M EDTA, the solution is passed through a Sephadex G-25 column 6 equilibrated with the same buffer in order to free it from ammonium sulfate. Stvp ~. Calcium Phosphate Gel Adsorption. Calcium phosphate gel is added to the enzyme solution in a ratio of 2 mg gel per milligram protein; after the gel has stood for several minutes, it is collected by centrifugation. The supernatant solution is assayed for enzyme activity, and successive additions of gel are made until the enzyme has been almost completely adsorbed. The supernatant fluid is discarded, and the enzyme is eluted from the gel by suspending it in an equal volume of 0.12 M potassium phosphate, pH 7.0; after 30 minutes the suspension is centrifuged and the supernatant fluid is removed and saved. The gel phase is treated twice more in an identical fashion with 0.12 M phosphate buffer, and the supernatant fractions are pooled. Step 5. Ammonium Sulfate Precipitation. The enzyme is precipitated by the addition of 390 g of solid ammonium sulfate per liter of solution. It is collected by centrifugation and redissolved in a few milliliters of 5 X 100SM potassium phosphate, pH 6.0, containing 100~ M EDTA, and the ammonium sulfate is removed by passing the enzyme solution through a Sephadex G-25 column equilibrated with the same buffer. Step 6. CM-Cellulose Chromatography. The protein concentration in the Sephadex eluate is adjusted to 2-5 mg per milliliter by addition of more 0.005 M phosphate buffer; sufficient T P N is then added to give a final concentration of 2 X 10~ M. A 4.5 X 40 cm column containing 60 g (dry weight) of carboxymethyl cellulose equilibrated with 5 X 100SM potassium phosphate, pH 6.0, 100~ M EDTA is prepared. The enzyme is applied to the column and an additional 1000 ml of the buffer solution is allowed to pass through the column; this eluate is discarded. A linear 6j. Porath and P. Flodin, Nature 183, 16,57 (1959).
[24]
GL~COSE6-PHOSPHATE DEHYDROGENASE ERYTHROCYTES
129
ionic strength gradient is begun with 2000 ml of buffer in the mixing chamber and 2000 ml of 1 M KC1 in buffer in the other container. Fractions of 12 ml volume are collected and each is assayed for protein and enzyme. G-6-P dehydrogenase is eluted between 0.05 and 0.15M KC1 concentration. Fractions of higher specific activity are pooled. Step 7. Calcium Phosphate Gel Adsorption. To the pooled fractions from the preceding step calcium phosphate gel is added at a gel to protein ratio of 6:1. The adsorbed enzyme is eluted from the gel by the addition of small increments of 0.12M potassium phosphate pH 7.0 until no additional enzyme appears in the eluate. Step 8. CM-Cellulose Chromatography. The ionic composition of the solution is changed to 5 X 10-3 M potassium phosphate, pH 6.0, 10-4 M EDTA by passing it through a Sephadex G-25 column equilibrated with this buffer; sufficient TPN is added to the eluate to yield a 2 X 10~ M solution of this nueleotide. This solution is then added to a 1.2 X 22 cm column of CM-cellulose previously equilibrated with buffer. The column is washed with 50 ml of buffer and then is developed with a linear ionic strength gradient using 100 ml of buffer and 100 ml of 0.5 M KC1 in buffer. Fractions of 2 ml volume are collected and assayed for both protein and glucose 6-phosphate dehydrogenase. The center fractions of the peak are combined.
Step 9. Calcium Phosphate Gel Adsorption and DEAE-Cel~ulose Chromatography. Enzyme in the pooled fractions is concentrated by adsorption to and elution from calcium phosphate gel as in step 7. The potassium phosphate concentration is then reduced to 0.05 M by addition of 1.4 volumes of water; the solution is made 10-4 M in EDTA and 2 X 10-6M in TPN by addition of concentrated solutions of these reagents. The enzyme solution is then added to a 1.2 X 22 cm column of DEAE-cellulose which has been prewashed by passing through it 100 ml of 0.05M potassium phosphate, pH 7.0, containing 10-9 M EDTA and 2 X 10-6 M TPN. After the enzyme is adsorbed, the column is washed with an additional 50 ml of this solution. The enzyme is eluted by a linear ionic strength gradient using 100 ml of buffer and 100 ml of 0.5 M KC1 in buffer. Fractions of 2 ml volume are collected. Step 10. Final Calcium Phosphate Gel Adsorption. The enzymatieally active fractions from step 9 are pooled and passed through a Sephadex G-25 column previously equilibrated with 5 X 10-8 M potassium phosphato pH 6.0, 10-9 M EDTA. The enzyme is adsorbed to and eluted from calcium phosphate gel as in step 7. The final product may be stored at 0 ° for several weeks or for prolonged periods at --20 ° .
130
DEHYDROGENASES AND OXID).SES
[24]
PURIFICATION OF GLUCOSE 6-PHOSPHATE DEHYDROGENASE
Stepa 1. 2. 3. 4. 5. 6. 7. 8. 9.
Hemolyzate DEAE chromatography (NH4)2SO4precipitation Gel adsorption (NH~)2SO4precipitation CM-cellulose chromatography Gel adsorption CM-cellulose chromatography Gel adsorption and DEAEcellulose chromatography 10. Gel adsorption
Enzyme units
Enzyme specific activity
Cumulative purification
Cumulative yield (%)
5170 1810 1450 1230 970 955 538 571 434
0. 0026 0.123 0.21 0.31 0.60 7.55 11.1 23.1 93
0 47.2 81 119 230 2,900 4,270 8,900 35,800
100 35 28 25 19 18 10 11 8.4
390
113
43,500
7.5
a The number of each step corresponds to that in the text. Properties of E r y t h r o c y t e G - 6 - P Dehydrogenase
Purity. E n z y m e having a specific activity of 113 units per milligram appears homogeneous on free and starch gel electrophoresis. In the analytical ultracentrifuge, one m a j o r component comprising approxim a t e l y 80% of the protein is observed. One minor peak, which is believed to represent the monomeric form, is also present2 Physical Properties. The sedimentation constant determined by the moving boundary method is 7.1 S. The diffusion coefficient as determined by the porous diaphram method 7 is 3.4 X 10 -7 cm 2 sec -1. From these values the calculated molecular weight is 190,000 and the frictional ratio is 1.6. 3 K i r k m a n , 4 using different methods, has concluded t h a t the molecular weight of the enzyme is 105,000. Bound Coenzyme. T h e enzyme as prepared contains 2 moles of T P N which are very tightly bound, s,9 These are reduced to enzyme-bound T P N H when the enzyme is treated with glucose 6-phosphate. The bound nucleotide m a y be removed from the enzyme by t r e a t m e n t with acid a m m o n i u m sulfate. The apoenzyme dissociates into catalytically inactive subunits having approximately one-half the molecular weight of the holoenzyme. The enzyme-bound T P N m a y also be destroyed by treatment of the enzyme with snake (Agkistroden piscivorus) venom which contains an active triphosphopyridine nucleotidase. Under appropriate C. E. Mize, T. E. Thompson, and R. G. Langdon, d. Biol. Chem. 237, 1596 (-1962). aA. E. Chung and R. G. Langdon, J. Biol. Chem. 238, 2317 (1963). 9H. N, Kirkman and E. M. Hendrickson, d. Biol. Chem. 237, 2371 (1962).
[25]
GLUCOSE a-PHOSPHATE DEHYDROGENASE--CLINICAL ASPECTS
131
circumstances, 8,9 the apoenzyme and coenzyme recombine with restoration of the physical and catalytic properties of the native enzyme. Enzyme Structure. Two NH2-terminal amino acids, alanine and tyrosine, have been detected2 Therefore, the enzyme has at least two nonidentical polypeptide subunits. Stability. The purified native enzyme is stable in solution for several weeks at 0 °, and for prolonged periods at --20 °. It may also be kept in solution for several hours at 40 ° without appreciable loss of activity. In t~he absence of added TPN, the apoenzyme is very unstable at 40°; within 1 hour at this temperature most of the catalytic properties of the protein are irreversibly lost. Presumably the instability 1° of glucose 6-phosphate dehydrogenase in crude hemolyzates is due to a nucleotidasecatalyzed destruction of enzyme-bound T P N with production of the thermally unstable apoenzyme. Kinetic Properties. At pH 7.6 the Km for TPN has been reported to be 4.2 X 10-aM and for G-6-P to be 3.5 X 10-~M. 4 Assuming enzyme which has a specific activity of 113 units mg -1 to be 80% pure and that the molecular weight of the functional unit is 190,000, it may be calculated that the turnover number of the enzyme is approximately 21,500 min -1. The enzyme is inhibited by p-ch!oromercuribenzoate; it is protected against this agent by T P N but not by DPN, 2'-AMP, or several closely related compounds. It has also been reported that the enzyme is inhibited by pregneno!one and other steroids 1~ in a noncompetitive manner. 10p. A. Marks, Cold Spring Harbor Symp. Quant. Biol. ~6, 343 (1961). l~p. A. Marks and J. Banks, Proc. Natl. Acad. Sci. U.S. 46, 1483 (1960).
[25]
Glucose 6-Phosphate Dehydrogenase--Clinical
By
Aspects
PAUL A. MARKS
Glucose 6-phosphate -5 TPN + ~ 6 phosphogluconolactone -k TPNH q- H +
Clinical Significance A deficiency in glucose 6-phosphate dehydrogenase occurs as a genetically determined trait associated with an increased susceptibility to hemolytic anemia following ingestion of a large variety of drugs and other chemical agents, including sulfonamides, nitrofurantoins, primaquine, salicylates, naphthaline, and methylene blue; the lava bean; during certain viral and bacterial infections; and in association with
DEHYDROGE~ASES AND OXIDASES
[24]
[24] Glucose 6 - P h o s p h a t e D e h y d r o g e n a s e from E r y t h r o c y t e s
By
ROBERT G. LANGDON
Glucose 6-phosphate + TPN+ ~ 6-phosphogluconate + TPNH -b H+ Assay Method Principle. The reduction of TPN ÷ to T P N H results in the appearance of an absorption band at 340 m~. In the presence of saturating concentrations of G-6-P and TPN, the rate of change of absorbancy at this wavelength is proportional to the enzyme concentration.
Reagents Tris-chloride buffer, 1 M, pH 7.5 Glucose 6-phosphate, 2.5 X 10-2 M. Dissolve 97.5 mg of the dipotassium salt of glucose 6-phosphate trihydrate in 10 ml of water. TPN, 2 X 10-s M. Dissolve a quantity of TPN equivalent to 16.7 mg of the free acid in 9 ml water. Adjust the pH to 7.5. Adjust the volume to 10 ml. MgCl~, 0.2M. Dissolve 4.1 g MgC12"6 H20 in sufficient water to give a final volume of 100 ml All buffers and reagents should be prepared in water distilled from a hard glass or quartz still.
Procedure. The assay is carried out at room temperature (25°). To a 3-ml quartz cell having a 1-cm path length is added sufficient glass-distilled water to yield a final volume of 3 ml after additions of all reagents and enzymes have been made; 0.1 ml buffer, 0.1 ml G-6-P, 0.1 ml TPN, and 0.1 ml MgC12 are then added. These are mixed and the cell is placed in a spectrophotometer adapted for recording of absorbancy as a function of time. A volume of enzyme solution containing approximately 0.1-0.2 enzyme units is then quickly added and mixed, and the rate of change of absorbancy at 340 m# is recorded. The velocity of the reaction during the first 10 seconds following mixing is taken to represent the initial velocity. De'finition of Enzyme Unit and Specific Activity. One unit of enzyme activity is defined as that quantity which catalyzes the reduction of 1 micromole of TPN per minute under the above assay conditions. This corresponds to an absorbancy change of 2.07 per minute. Protein is measured by the optical method of Warburg and Christian 1 or by the 10. Warburg and W. Christian, Bioehem. Z. 310, 384 (1941). See also E. Layne, Vol. III, p. 451.
[24]
GLUCOSEa-PHOSPHATE DEHYDROGENASE ERYTHROCYTES
127
colormetric method of Lowry.2 Specific activity is defined as the units of enzyme per milligram of protein. Purification Procedure The purification procedure given here is essentially that described by Chung and Langdon.3 A similar procedure yielding a slightly less pure product has been described by Kirkman. 4 During the purification it is important to use reagent grade chemicals and distilled water which has been redistilled from a hard-glass still or passed through a mixed-bed ion exchange column.
Preparation o] Adsorbents Commerial ion exchange cellulose is prepared for chromatography as follows: Each 100g of the material is suspended in 2 liters of water and titrated with 1 M KH2P04 or K~HP04 to the desired pH value. After the suspension has settled for a few minutes, any fine particles are removed by decantation and the remainder is filtered through fine-mesh nylon in a Biichner funnel. The damp cake is washed twice in a similar manner with water and 3 times in the desired buffer. It is then stored in buffer at 0 ° until used. Calcium phosphate gel is prepared as described by Keilin and Hartree2
Preparation o] Erythrocytes The erythrocytes from 18 pints of human blood, collected in standard ACD solution by a blood bank and stored at 4 ° for 3-5 weeks, are allowed to settle and the supernatant plasma is removed by suction. The thick suspension is then centrifuged at low speed, and the erythrocytes are washed four times with 0.15 M KC1 in 5 X 10-3 M potassium phosphate buffer, pH 7.0, containing 10.4 M EDTA. The erythrocytes may be stored at 4 ° for a short time prior to use. Step I. Preparation of Hemolyzate. The erythrocytes are mixed with an equal volume of distilled water, frozen solidly by immersing the container in a dry ice bath, and then thawed in a water bath at 10°. Most subsequent operations are carried out in a cold room at 4 °. Step 2. First DEAE-Cellulose Treatment. The hemolyzate from each 6 pints of blood is diluted with water to 2 liters and mixed with 150g of DEAE-cellulose suspended in 2 liters of 5 X 10-3 M potassium phosphate, 20. H. Lowry, :N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). See also E. Layne, Vol. III, p. 448. 3A. E. Chung and R. G. Langdon,J. Biol. Chem. 238~ 2309 (1963). 4H. N. Kirkman, J. Biol. Chem. 237, 2364 (1962). ~D. Keilin and E. F. Hartree, Proc. Roy. Soc. B124, 397 (1938).
128
DEHYDROGE~CASES AND OXIDASES
[24]
pH 7.0, containing 10°4M EDTA. After the suspension has been gently stirred for 30 minutes, it is poured into an 8 X 60 cm column. The packed cellulose is washed with an additional 4 liters of the buffer solution, which elutes most of the hemoglobin. Then 0.3 M KC1 in the same buffer is passed through the column; fractions are collected and assayed for enzyme activity. Those fractions containing enzyme are pooled. Step 3. Ammonium Sulfate Precipitation. The enzyme is precipitated by the addition of 351 g of solid ammonium sulfate per liter of solution obtained in the preceding step. It is collected by centrifugation, dissolved in 5 X 10-2 M potassium phosphate, pH 7.0, containing 10-~ M EDTA, and the ammonium sulfate precipitation is repeated. After solution in 5 X 10-~ M potassium phosphate, pH 6.0, containing 10~ M EDTA, the solution is passed through a Sephadex G-25 column 6 equilibrated with the same buffer in order to free it from ammonium sulfate. Stvp ~. Calcium Phosphate Gel Adsorption. Calcium phosphate gel is added to the enzyme solution in a ratio of 2 mg gel per milligram protein; after the gel has stood for several minutes, it is collected by centrifugation. The supernatant solution is assayed for enzyme activity, and successive additions of gel are made until the enzyme has been almost completely adsorbed. The supernatant fluid is discarded, and the enzyme is eluted from the gel by suspending it in an equal volume of 0.12 M potassium phosphate, pH 7.0; after 30 minutes the suspension is centrifuged and the supernatant fluid is removed and saved. The gel phase is treated twice more in an identical fashion with 0.12 M phosphate buffer, and the supernatant fractions are pooled. Step 5. Ammonium Sulfate Precipitation. The enzyme is precipitated by the addition of 390 g of solid ammonium sulfate per liter of solution. It is collected by centrifugation and redissolved in a few milliliters of 5 X 100SM potassium phosphate, pH 6.0, containing 100~ M EDTA, and the ammonium sulfate is removed by passing the enzyme solution through a Sephadex G-25 column equilibrated with the same buffer. Step 6. CM-Cellulose Chromatography. The protein concentration in the Sephadex eluate is adjusted to 2-5 mg per milliliter by addition of more 0.005 M phosphate buffer; sufficient T P N is then added to give a final concentration of 2 X 10~ M. A 4.5 X 40 cm column containing 60 g (dry weight) of carboxymethyl cellulose equilibrated with 5 X 100SM potassium phosphate, pH 6.0, 100~ M EDTA is prepared. The enzyme is applied to the column and an additional 1000 ml of the buffer solution is allowed to pass through the column; this eluate is discarded. A linear 6j. Porath and P. Flodin, Nature 183, 16,57 (1959).
[24]
GL~COSE6-PHOSPHATE DEHYDROGENASE ERYTHROCYTES
129
ionic strength gradient is begun with 2000 ml of buffer in the mixing chamber and 2000 ml of 1 M KC1 in buffer in the other container. Fractions of 12 ml volume are collected and each is assayed for protein and enzyme. G-6-P dehydrogenase is eluted between 0.05 and 0.15M KC1 concentration. Fractions of higher specific activity are pooled. Step 7. Calcium Phosphate Gel Adsorption. To the pooled fractions from the preceding step calcium phosphate gel is added at a gel to protein ratio of 6:1. The adsorbed enzyme is eluted from the gel by the addition of small increments of 0.12M potassium phosphate pH 7.0 until no additional enzyme appears in the eluate. Step 8. CM-Cellulose Chromatography. The ionic composition of the solution is changed to 5 X 10-3 M potassium phosphate, pH 6.0, 10-4 M EDTA by passing it through a Sephadex G-25 column equilibrated with this buffer; sufficient TPN is added to the eluate to yield a 2 X 10~ M solution of this nueleotide. This solution is then added to a 1.2 X 22 cm column of CM-cellulose previously equilibrated with buffer. The column is washed with 50 ml of buffer and then is developed with a linear ionic strength gradient using 100 ml of buffer and 100 ml of 0.5 M KC1 in buffer. Fractions of 2 ml volume are collected and assayed for both protein and glucose 6-phosphate dehydrogenase. The center fractions of the peak are combined.
Step 9. Calcium Phosphate Gel Adsorption and DEAE-Cel~ulose Chromatography. Enzyme in the pooled fractions is concentrated by adsorption to and elution from calcium phosphate gel as in step 7. The potassium phosphate concentration is then reduced to 0.05 M by addition of 1.4 volumes of water; the solution is made 10-4 M in EDTA and 2 X 10-6M in TPN by addition of concentrated solutions of these reagents. The enzyme solution is then added to a 1.2 X 22 cm column of DEAE-cellulose which has been prewashed by passing through it 100 ml of 0.05M potassium phosphate, pH 7.0, containing 10-9 M EDTA and 2 X 10-6 M TPN. After the enzyme is adsorbed, the column is washed with an additional 50 ml of this solution. The enzyme is eluted by a linear ionic strength gradient using 100 ml of buffer and 100 ml of 0.5 M KC1 in buffer. Fractions of 2 ml volume are collected. Step 10. Final Calcium Phosphate Gel Adsorption. The enzymatieally active fractions from step 9 are pooled and passed through a Sephadex G-25 column previously equilibrated with 5 X 10-8 M potassium phosphato pH 6.0, 10-9 M EDTA. The enzyme is adsorbed to and eluted from calcium phosphate gel as in step 7. The final product may be stored at 0 ° for several weeks or for prolonged periods at --20 ° .
130
DEHYDROGENASES AND OXID).SES
[24]
PURIFICATION OF GLUCOSE 6-PHOSPHATE DEHYDROGENASE
Stepa 1. 2. 3. 4. 5. 6. 7. 8. 9.
Hemolyzate DEAE chromatography (NH4)2SO4precipitation Gel adsorption (NH~)2SO4precipitation CM-cellulose chromatography Gel adsorption CM-cellulose chromatography Gel adsorption and DEAEcellulose chromatography 10. Gel adsorption
Enzyme units
Enzyme specific activity
Cumulative purification
Cumulative yield (%)
5170 1810 1450 1230 970 955 538 571 434
0. 0026 0.123 0.21 0.31 0.60 7.55 11.1 23.1 93
0 47.2 81 119 230 2,900 4,270 8,900 35,800
100 35 28 25 19 18 10 11 8.4
390
113
43,500
7.5
a The number of each step corresponds to that in the text. Properties of E r y t h r o c y t e G - 6 - P Dehydrogenase
Purity. E n z y m e having a specific activity of 113 units per milligram appears homogeneous on free and starch gel electrophoresis. In the analytical ultracentrifuge, one m a j o r component comprising approxim a t e l y 80% of the protein is observed. One minor peak, which is believed to represent the monomeric form, is also present2 Physical Properties. The sedimentation constant determined by the moving boundary method is 7.1 S. The diffusion coefficient as determined by the porous diaphram method 7 is 3.4 X 10 -7 cm 2 sec -1. From these values the calculated molecular weight is 190,000 and the frictional ratio is 1.6. 3 K i r k m a n , 4 using different methods, has concluded t h a t the molecular weight of the enzyme is 105,000. Bound Coenzyme. T h e enzyme as prepared contains 2 moles of T P N which are very tightly bound, s,9 These are reduced to enzyme-bound T P N H when the enzyme is treated with glucose 6-phosphate. The bound nucleotide m a y be removed from the enzyme by t r e a t m e n t with acid a m m o n i u m sulfate. The apoenzyme dissociates into catalytically inactive subunits having approximately one-half the molecular weight of the holoenzyme. The enzyme-bound T P N m a y also be destroyed by treatment of the enzyme with snake (Agkistroden piscivorus) venom which contains an active triphosphopyridine nucleotidase. Under appropriate C. E. Mize, T. E. Thompson, and R. G. Langdon, d. Biol. Chem. 237, 1596 (-1962). aA. E. Chung and R. G. Langdon, J. Biol. Chem. 238, 2317 (1963). 9H. N, Kirkman and E. M. Hendrickson, d. Biol. Chem. 237, 2371 (1962).
[25]
GLUCOSE a-PHOSPHATE DEHYDROGENASE--CLINICAL ASPECTS
131
circumstances, 8,9 the apoenzyme and coenzyme recombine with restoration of the physical and catalytic properties of the native enzyme. Enzyme Structure. Two NH2-terminal amino acids, alanine and tyrosine, have been detected2 Therefore, the enzyme has at least two nonidentical polypeptide subunits. Stability. The purified native enzyme is stable in solution for several weeks at 0 °, and for prolonged periods at --20 °. It may also be kept in solution for several hours at 40 ° without appreciable loss of activity. In t~he absence of added TPN, the apoenzyme is very unstable at 40°; within 1 hour at this temperature most of the catalytic properties of the protein are irreversibly lost. Presumably the instability 1° of glucose 6-phosphate dehydrogenase in crude hemolyzates is due to a nucleotidasecatalyzed destruction of enzyme-bound T P N with production of the thermally unstable apoenzyme. Kinetic Properties. At pH 7.6 the Km for TPN has been reported to be 4.2 X 10-aM and for G-6-P to be 3.5 X 10-~M. 4 Assuming enzyme which has a specific activity of 113 units mg -1 to be 80% pure and that the molecular weight of the functional unit is 190,000, it may be calculated that the turnover number of the enzyme is approximately 21,500 min -1. The enzyme is inhibited by p-ch!oromercuribenzoate; it is protected against this agent by T P N but not by DPN, 2'-AMP, or several closely related compounds. It has also been reported that the enzyme is inhibited by pregneno!one and other steroids 1~ in a noncompetitive manner. 10p. A. Marks, Cold Spring Harbor Symp. Quant. Biol. ~6, 343 (1961). l~p. A. Marks and J. Banks, Proc. Natl. Acad. Sci. U.S. 46, 1483 (1960).
[25]
Glucose 6-Phosphate Dehydrogenase--Clinical
By
Aspects
PAUL A. MARKS
Glucose 6-phosphate -5 TPN + ~ 6 phosphogluconolactone -k TPNH q- H +
Clinical Significance A deficiency in glucose 6-phosphate dehydrogenase occurs as a genetically determined trait associated with an increased susceptibility to hemolytic anemia following ingestion of a large variety of drugs and other chemical agents, including sulfonamides, nitrofurantoins, primaquine, salicylates, naphthaline, and methylene blue; the lava bean; during certain viral and bacterial infections; and in association with