ARCHIVES OF BIOCHEMISTRY
AND BIOPHYSICS 169,362-363
An Improved
Procedure Dehydrogenase
for Rapid
(1975)
Isolation
from
Human
OT Glucose
6-Phosphate
Erythrocytes’
Coupling of NE-(aminohexyl)-adenosine 2’,5’-bisphosphate to BrCN-activated agarose was exploited to develop a simple procedure by which homogeneous glucose B-phosphate dehydrogenase can be isolated in good yield and in a short time (2 days) from human erythrocytes. The method involves three steps, i.e., chromatography on DEAE-Sephadex, chromatography on phosphocellulose and affinity chromatography on the above ligand-matrix complex. This procedure is applicable for the purification of glucose 6-phosphate dehydrogenase from single donors. A new procedure suited for the rapid purification ot glucose 6-phosphate dehydrogenase (GGPD. EC 1.1.1.49) from human erythrocytes is reported. A substantial improvement over the lengthy techniques of purification of this protein had been already obtained in our laboratory by means of chromatography on a NADP-agarose complex (1). We had, however, experienced some difficulties. especially poor reproducibility of the affinity chromatography step: these difficulties were later rationalized in terms of marked modifications in NADP structure occurring during the carbodiimide-directed coupling of the dinucleotide to the matrix (2). Therefore, a more convenient affinity adsorbent, Nb-(B-aminohexyl)adenosine 2’.5’-bisphosphate was synthesized in our laboratory (2) and coupled to BrCN-activated agarose. Synthesis of the same compound was obtained independently by Brodelius et al. (X). by a slightly different method. Use of this matrix-bound effector allowed us to overcome the variability in the recoveries of GGPD activity and to develop a refined and simple procedure by which homogeneous GGPD can be isolated in good yield and in a short time (2 days) from human erythrocytes. The present method is applicable for the purification of GGPD from single donors and can, therefore, be conveniently employed for the study of structural and functional modifications in genetic variants of this enzyme. Forty milliliters of agarose (Sepharose 4B. Pharmacia) were activated for 40 min at 10°C with 4 g of BrCN, keeping the pH constant at 11 with 5 N NaOH. The gel was washed with :X)0 ml of cold 0.1 M NaHC03, drained on a Biichner funnel and added to an aqueous solution 01F OO i ml of 15 mM NG-(6.aminohexyl)-adenosine 2’.5’.bisphosphate (2), pH 8.8. After gentle stirring at 4°C for 60 h, the mixture was filtered, ‘This work was supported by grants from the Italian C.N.R. and by Research Grant GM 19526 from the N.I.H. to the University of Genoa. We are grateful to Dr. L. Radin for making available the analytical centrifuge to us.
and the gel was washed with 50 ml of water. The filtrate and this wash were combined (liquid phase): the amount of matrix-bound ligand was calculated by difference after evaluating the concentration of free Ne-(6-aminohexyl)-adenosine 2’5.bisphosphate in the liquid phase and found to be 3.5-4.0 Fmol per g of gel. The ligand-agarose complex was then sequentially washed with 200 ml of water, 500 ml of 5 mM HCl, 500 ml of 0.5 M NaCl, and finally with water to neutral pH. All operations of purification of GGPD were performed at 4°C. starting from 2 liters of pooled erythrocytes (kindly provided by Dr. R. Castagna. Istituto Sieroterapico Milanese, Milan. Italy). All buffers contained 1 mM EDTA and 0.2’? &mercaptoethanol (volivol): additional components are indicated below. The enzyme was purified within 2 days and the following details are reported according to this time schedule. Day I. Washing of the erythrocytes (two times). hemolysis, batchwise chromatography on DEAESephadex and phosphocellulose were carried out as reported previously (1). The eluate from phosphocellulose was precipitated by addition of solid ammonium sulfate (360 g/liter) and the pellet, obtained by centrifugation for I5 min at 12,OOOg,was dissolved in 50 ml of 0.1 M Na acetate, pH 6.0, containing 1 mM e-aminohexanoic acid and 2 PM NADP. The enzyme was then dialyzed overnight against 100 vol of the same buffer, yet containing 0.5 mM rather than 1 mM c-aminohexanoic acid and lacking NADP. Da.v 2. The dialyzed enzyme, cleared by centrif’upation. was diluted with 3 vol of 0.1 M Na phosphate in 0.1 M Na acetate, pH 6.0 (Buffer A). The solution was applied to a column (10 K 1.2 cm, i.d.) of the ligand-agarose complex, prepared as described above and equilibrated with Buffer A prior to use. The flow rate was 70 ml/h and no GGPD activity was detected in the effluent. The gel was sequentially washed, at a flow rate of 150 ml/h, with 50 ml of Buffer A. then with 50 ml of 0.1 M phosphate, pH 8.0 (Buffer B), and finally with approximately 500 ml of’ Buffer B. con-
362 Copyright 0 1975by Academic Press, Inc. All rights of reproduction in any form reserved.
COMMUNICATIONS TABLE PURIFICATION
OF GLCCOSE
&PHOSPHATE
I
DEHYDKOGENASEFROM HVMAN ERYTHHOCYTES
Activitv’
Step
363
Protein”
(mg/ml)
Hemolysate DEAE-Sephadex Phosphocellulose Affinity
eluate eluate
chromatography
step
Units/ml
Total units
0.5
1950
3.4 1.9
1360 1900
5I.3
71.2
1000
0.41
166
Specific activity (unitsimg)
Yield
0.00:1
100
0.27 2.6 174
Days
97 70
1I
51
2t
‘I Assayed as reported previously (41. /’ Determined by the method of Lowry et al. (5) or from absorbance at 280 nm. assuming that I mg/ml would give a value of 1.0. taining 0.15 M KCl, until the absorbance at 280 nm was zero. Elution was obtained by applying to the column 25 ml of Buffer B. containing 0.1 M KC1 and 0.2 mM NADP, at a flow rate of 50 ml/h. A summary of the purification procedure is shown in Table I. Final GGPD preparations appeared to be homogeneous according to the criteria of polyacrylamide gel electrophoresis. sedimentation patterns in the analytical centrifuge, and values of specific activity t 11. The above procedure has been successfully scaled to purify GGPD from smaller amounts of erythrocytes. In this case, however, the phosphocellulose step should be replaced by gel chromatography on Sephadex G-100, after precipitating with ammonium sulfate the eluate from DEAE-Sephadex. Thus. starting from 200 ml of packed erythrocytes collected from single donors. approximately 0.5 mg of pure GGPD were obtained. Attempts to achieve a further simplication of the present procedure by omitting the phosphocellulose step were unsuccessful. Preparations of the enzyme obtained without this step were found to be contaminated with substantial amounts of another protein. showing a higher electrophoretic mobility than that of dimeric and monomeric GGPD. This protein appears. therefore. to be selectively recognized by the matrixbound adenosine 2’.5’-hisphosphate and by the eluting ligand. i.e.. NADP: it is desorbed only to a very limited extent by NAD and thus shows the same NADP specificity as human GGPD (61. Its sedimentation coefficient was found to be 4.05 * 0.1 S, both at pH 6.0 and 8.4; furthermore it appears to be composed of a single type of polypeptide chain whose molecular weight, as determined by Na dodecyl sulfate disc gel electrophoresis in the presence of d-mercaptoethanol. is 32.000 + ‘1 -.OOO. On the basis of catalytic activity we were unable to identify this protein with any of the following enzymes: malate dehydrogenase, isocitrate
dehydrogenase. phosphogluconate dehydrogenase, lactate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase. glutamate dehydrogenase. glutathione reductase. NAD(P)H-methemoglobin reductase. NADPH-triose phosphate oxidoreductase (7). glucose phosphate isomerase, NAD kinase, and NAD(P)glycohydrolase. Experiments are in progress in our laboratory in order to further characterize this un known protein. REFERENCES 1. DE FLORA. A., GICLIANO. F.. AND MORELI‘I. A. ( 1973 Ital. J. Biochem. 22, 258-270. 2. MORELLI. A., AND BENATTI, U. (19741 Ital. J. Biothem. 23, 279-291. 3. BRODELIVS, P., LARSSOK;.P. 0.. AND MOSISACH, K. (1974). Eur. J. Biochem. 47, 81-89. 4. WRIGLEY, N. G.. HEATHER, .J. V.. BONSIWOHE, A., AND DE FLORA, A. (192). J. Mol. Biol. 68, 4tw499. 5. LOWRY, 0. H., ROSEBHOLY~H.N. J.. FARR, A. L.. AND RANDALI., R. J. (1951) J. Rio/. (‘hem. 193, 265.-“T5. I I 6. BONSIGNORE.A.. AND DE FLORA, A. (1972) in Current Topics in Cellular Regulation (Horecker, B. L., and Stadtman, E. R., eds.), Vol VI, pp. 21-62. Academic Press. New York. 7. WOOD. T. (1974) Biochem. J. 138, 71.-76. A. DE FLORA A. MORELLI U. BENATTI F. GIVI.IANO Institute of Biochemistry Uniuersit,\fl of Genoa 26232 Genoa. Ital? Received Februarv 18. 1975
AND BIOPHYSICS 169,362-363
An Improved
Procedure Dehydrogenase
for Rapid
(1975)
Isolation
from
Human
OT Glucose
6-Phosphate
Erythrocytes’
Coupling of NE-(aminohexyl)-adenosine 2’,5’-bisphosphate to BrCN-activated agarose was exploited to develop a simple procedure by which homogeneous glucose B-phosphate dehydrogenase can be isolated in good yield and in a short time (2 days) from human erythrocytes. The method involves three steps, i.e., chromatography on DEAE-Sephadex, chromatography on phosphocellulose and affinity chromatography on the above ligand-matrix complex. This procedure is applicable for the purification of glucose 6-phosphate dehydrogenase from single donors. A new procedure suited for the rapid purification ot glucose 6-phosphate dehydrogenase (GGPD. EC 1.1.1.49) from human erythrocytes is reported. A substantial improvement over the lengthy techniques of purification of this protein had been already obtained in our laboratory by means of chromatography on a NADP-agarose complex (1). We had, however, experienced some difficulties. especially poor reproducibility of the affinity chromatography step: these difficulties were later rationalized in terms of marked modifications in NADP structure occurring during the carbodiimide-directed coupling of the dinucleotide to the matrix (2). Therefore, a more convenient affinity adsorbent, Nb-(B-aminohexyl)adenosine 2’.5’-bisphosphate was synthesized in our laboratory (2) and coupled to BrCN-activated agarose. Synthesis of the same compound was obtained independently by Brodelius et al. (X). by a slightly different method. Use of this matrix-bound effector allowed us to overcome the variability in the recoveries of GGPD activity and to develop a refined and simple procedure by which homogeneous GGPD can be isolated in good yield and in a short time (2 days) from human erythrocytes. The present method is applicable for the purification of GGPD from single donors and can, therefore, be conveniently employed for the study of structural and functional modifications in genetic variants of this enzyme. Forty milliliters of agarose (Sepharose 4B. Pharmacia) were activated for 40 min at 10°C with 4 g of BrCN, keeping the pH constant at 11 with 5 N NaOH. The gel was washed with :X)0 ml of cold 0.1 M NaHC03, drained on a Biichner funnel and added to an aqueous solution 01F OO i ml of 15 mM NG-(6.aminohexyl)-adenosine 2’.5’.bisphosphate (2), pH 8.8. After gentle stirring at 4°C for 60 h, the mixture was filtered, ‘This work was supported by grants from the Italian C.N.R. and by Research Grant GM 19526 from the N.I.H. to the University of Genoa. We are grateful to Dr. L. Radin for making available the analytical centrifuge to us.
and the gel was washed with 50 ml of water. The filtrate and this wash were combined (liquid phase): the amount of matrix-bound ligand was calculated by difference after evaluating the concentration of free Ne-(6-aminohexyl)-adenosine 2’5.bisphosphate in the liquid phase and found to be 3.5-4.0 Fmol per g of gel. The ligand-agarose complex was then sequentially washed with 200 ml of water, 500 ml of 5 mM HCl, 500 ml of 0.5 M NaCl, and finally with water to neutral pH. All operations of purification of GGPD were performed at 4°C. starting from 2 liters of pooled erythrocytes (kindly provided by Dr. R. Castagna. Istituto Sieroterapico Milanese, Milan. Italy). All buffers contained 1 mM EDTA and 0.2’? &mercaptoethanol (volivol): additional components are indicated below. The enzyme was purified within 2 days and the following details are reported according to this time schedule. Day I. Washing of the erythrocytes (two times). hemolysis, batchwise chromatography on DEAESephadex and phosphocellulose were carried out as reported previously (1). The eluate from phosphocellulose was precipitated by addition of solid ammonium sulfate (360 g/liter) and the pellet, obtained by centrifugation for I5 min at 12,OOOg,was dissolved in 50 ml of 0.1 M Na acetate, pH 6.0, containing 1 mM e-aminohexanoic acid and 2 PM NADP. The enzyme was then dialyzed overnight against 100 vol of the same buffer, yet containing 0.5 mM rather than 1 mM c-aminohexanoic acid and lacking NADP. Da.v 2. The dialyzed enzyme, cleared by centrif’upation. was diluted with 3 vol of 0.1 M Na phosphate in 0.1 M Na acetate, pH 6.0 (Buffer A). The solution was applied to a column (10 K 1.2 cm, i.d.) of the ligand-agarose complex, prepared as described above and equilibrated with Buffer A prior to use. The flow rate was 70 ml/h and no GGPD activity was detected in the effluent. The gel was sequentially washed, at a flow rate of 150 ml/h, with 50 ml of Buffer A. then with 50 ml of 0.1 M phosphate, pH 8.0 (Buffer B), and finally with approximately 500 ml of’ Buffer B. con-
362 Copyright 0 1975by Academic Press, Inc. All rights of reproduction in any form reserved.
COMMUNICATIONS TABLE PURIFICATION
OF GLCCOSE
&PHOSPHATE
I
DEHYDKOGENASEFROM HVMAN ERYTHHOCYTES
Activitv’
Step
363
Protein”
(mg/ml)
Hemolysate DEAE-Sephadex Phosphocellulose Affinity
eluate eluate
chromatography
step
Units/ml
Total units
0.5
1950
3.4 1.9
1360 1900
5I.3
71.2
1000
0.41
166
Specific activity (unitsimg)
Yield
0.00:1
100
0.27 2.6 174
Days
97 70
1I
51
2t
‘I Assayed as reported previously (41. /’ Determined by the method of Lowry et al. (5) or from absorbance at 280 nm. assuming that I mg/ml would give a value of 1.0. taining 0.15 M KCl, until the absorbance at 280 nm was zero. Elution was obtained by applying to the column 25 ml of Buffer B. containing 0.1 M KC1 and 0.2 mM NADP, at a flow rate of 50 ml/h. A summary of the purification procedure is shown in Table I. Final GGPD preparations appeared to be homogeneous according to the criteria of polyacrylamide gel electrophoresis. sedimentation patterns in the analytical centrifuge, and values of specific activity t 11. The above procedure has been successfully scaled to purify GGPD from smaller amounts of erythrocytes. In this case, however, the phosphocellulose step should be replaced by gel chromatography on Sephadex G-100, after precipitating with ammonium sulfate the eluate from DEAE-Sephadex. Thus. starting from 200 ml of packed erythrocytes collected from single donors. approximately 0.5 mg of pure GGPD were obtained. Attempts to achieve a further simplication of the present procedure by omitting the phosphocellulose step were unsuccessful. Preparations of the enzyme obtained without this step were found to be contaminated with substantial amounts of another protein. showing a higher electrophoretic mobility than that of dimeric and monomeric GGPD. This protein appears. therefore. to be selectively recognized by the matrixbound adenosine 2’.5’-hisphosphate and by the eluting ligand. i.e.. NADP: it is desorbed only to a very limited extent by NAD and thus shows the same NADP specificity as human GGPD (61. Its sedimentation coefficient was found to be 4.05 * 0.1 S, both at pH 6.0 and 8.4; furthermore it appears to be composed of a single type of polypeptide chain whose molecular weight, as determined by Na dodecyl sulfate disc gel electrophoresis in the presence of d-mercaptoethanol. is 32.000 + ‘1 -.OOO. On the basis of catalytic activity we were unable to identify this protein with any of the following enzymes: malate dehydrogenase, isocitrate
dehydrogenase. phosphogluconate dehydrogenase, lactate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase. glutamate dehydrogenase. glutathione reductase. NAD(P)H-methemoglobin reductase. NADPH-triose phosphate oxidoreductase (7). glucose phosphate isomerase, NAD kinase, and NAD(P)glycohydrolase. Experiments are in progress in our laboratory in order to further characterize this un known protein. REFERENCES 1. DE FLORA. A., GICLIANO. F.. AND MORELI‘I. A. ( 1973 Ital. J. Biochem. 22, 258-270. 2. MORELLI. A., AND BENATTI, U. (19741 Ital. J. Biothem. 23, 279-291. 3. BRODELIVS, P., LARSSOK;.P. 0.. AND MOSISACH, K. (1974). Eur. J. Biochem. 47, 81-89. 4. WRIGLEY, N. G.. HEATHER, .J. V.. BONSIWOHE, A., AND DE FLORA, A. (192). J. Mol. Biol. 68, 4tw499. 5. LOWRY, 0. H., ROSEBHOLY~H.N. J.. FARR, A. L.. AND RANDALI., R. J. (1951) J. Rio/. (‘hem. 193, 265.-“T5. I I 6. BONSIGNORE.A.. AND DE FLORA, A. (1972) in Current Topics in Cellular Regulation (Horecker, B. L., and Stadtman, E. R., eds.), Vol VI, pp. 21-62. Academic Press. New York. 7. WOOD. T. (1974) Biochem. J. 138, 71.-76. A. DE FLORA A. MORELLI U. BENATTI F. GIVI.IANO Institute of Biochemistry Uniuersit,\fl of Genoa 26232 Genoa. Ital? Received Februarv 18. 1975
