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The preparation of stereospecific tritium-labeled reduced nicotinamide adenine dinucleotide phosphate
ANALYTICAL
BIOCHEMISTRY
The Preparation
51, 632636
of Stereospecific
Nicotinamide
Adenine
R. I. FREUDENTHAL, Chemistry
(1973)
Tritium-Labeled Dinucleotide
J. A. KEPLER
Reduced
Phosphate AND
C. E. COOK
and Life science Division, Research Triangle Institute, Research Triangle Park, North Carolina R709 Received July 10, 1972; accepted October
18, 1972
A method has been developed for the preparation of a high specific activity stereospecifically labeled tritiated NADPH. In this procedure, tritium is enzymatically transferred from d-isocitric acid-aH (8 Ci/mmole) to the A face of a pyridine nucleotide during its stereospecific reduction, resulting in the formation of NADPH-4A-“H (2 Ci/mmole).
The stereospecificity of the enzyme-catalyzed transfer of a hydrogen from a reduced pyridine nucleotide to a number of different substrates has been determined (l-3). The preparation and use of pyridine nucleotides labeled with hydrogen isotopes has made possible both the identification of the 4-position of the nicotinamide ring as the site of the transferra,ble hydrogen (4,5), and the determination of the stereochemistry of the products resulting from the hydrogen transfer (6-8). Although initial preparations of tritium-labeled reduced nicotinamide adenine dinucleotide phosphate (NADPH-4-3H) were of relatively low specific activity (9), the tritium content, has been increased in more recently reported preparations. Krakow et al. report the preparation of NADPH-4A-3H having a specific activity of approximately 1.3 mCi/ mmole (6) while Bjorkhem and Danielsson describe the preparation of NADPH-4A-“H of approximately 4.2 mCi/mmole (8). The present communication describes an enzymatic method for preparing NADPH4A-3H having a specific activity of 2 Ci/mmole, almost 500 times higher than that obtained by any of the previously described procedures. MATERIALS
AND
METHODS
NADP, oxalosuccinate and isocitrate dehydrogenase were purchased from Sigma. Sodium borotritide (specific activity, 42 Ci/mmole) was obtained from New England Nuclear, and Bio-Gel P-2 from BioRad 632 Copyright @ 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.
STEREOSPECIFIC
RADIOLABELED
NADPH
633
Laboratories. ChromAR 1000 chromatography medium was purchased from Mallinckrodt and precoated silica gel thin-layer chromatography plates from Brinkmann. Chemical Synthesis of Isocitric Acid-~2-~H. Sodium borotritide (4.5 mg, 0.119 mmole, specific activity 42 Ci/mmole) was added to a solution of triethyloxalosuccinate (121 mg, 0.428 mmole) in 1 ml of ethanol. The solution was stirred at, ambient temperature for 1 hr after which 0.5 ml of concentrated hydrochloric acid was added. The solut.ion was diluted with 10 ml of methylene chloride and washed with 4 ml of water. The organic layer was separated and then dried by adding sodium sulfate to the solution. Removal of the solvent gave 119 mg of crude triethyl isocitrate-2-3H. The crude product was purified by chromatography on two 20 X 20cm ChromAR 1000 sheets with 5% acetone in carbon tetrachloride. The product was eluted from the ChromAR strip with chloroform to give 61 mg (0.22 mmole; specific activity, 9.2 Ci/mmole) of triethylisocitrate2-3H. A very small aliquot of this material was chromatographed on a silica gel H plate in benzene:ethyl acetate, 2:l. The developed t’hinlayer plate was scanned on a Packard Model 7201 radiochromatogram scanner which showed the ester to be better than 98% radiochemically pure. The triethylisocitrate-2-3H (61 mg) was hydrolyzed to isocitric acidzZ-~H by dissolving it in 1 ml of 1 N sodium hydroxide and stirring at ambient temperat,ure for 15 hr. At the end of this time 0.05 ml of acetic acid was added and the solut,ion diluted to 25 ml. A very small aliquot of the resulting isocitric acid-2-3H solution was chromatographed on silica gel H plates in a solvent system containing ether: formic acid: water, 5: 2: 1. A radiochromatogram of the developed plate indicated that the isocitric acid-2-“H was greater than 98% radiochemically pure. However, the tritiated product is composed of 4 stereoisomers, d- and lisocitric acid and d- and I-alloisocitric acid, and only the d-isocitrate is oxidized by isocitrate dehydrogenase. The isocitric acid-2-3H solution was used in the next step. Enzymatic Synthesis of NADPH-4A-JH. One hundred ,umoles of the tritiated isocitrate preparation (820 mci), of which 25 pmoles is true substrate, was incubated wit’h 25 pmoles of NADP, 50 units of isocitrate dehydrogenase, and 250 pmoles of MnCl, for 3 min in a 0.1 M Tris buffer, pH 7.2 (37°C). The reaction was stopped by heating the incubate at 72” for 3 min. The heat-denatured protein was precipitated by centrifugation and the resulting clear supernatant was applied to a BioGel P-2 column (2 X lOO-cm) and eluted with a 0.05 M potassium phosphate buffer, pH 9.1.
634
FREUDENTHAL,
KEPLER,
AND
COOK
RESULTS
A typical elution pattern of NADPH-4A-3H and isocitric acid from a Bio-Gel P-2 column is shown in Fig. 1. The elution of NADPH-4A-3H was determined spectrophotometrically. The reduced pyridine nucleotide has a strong absorption peak at 340 nm, with a molar extinction coefficient of 6.2 X lo3 (12). The elution pattern of the isocitrate-3H and alloisocitrateJH was determined by column chromatography in the absence of radiolabeled NADPH, followed by liquid scintillation counting. Of the 20 mg NADP incubations, 14.4 mg was recovered as NADPH-4A-3H. A number of smaller batches of NADPH-4A-3H have also been prepared. When fractions 24 through 26 (Fig. 1) were pooled, a specific activity of 2 Ci/mmole was obtained as determined by liquid scintillation counting and uv spectroscopy at 340 nm. The product was determined to be at least 90% pure by thin-layer chromatography. A very small aliquot was spotted on a precoated cellulose plate which was then developed in isobutyric acid: NH,OH (cont.) : water (96: 1: 22), The developed plate was scanned on a radiochromatogram scanner which showed that the NADPH-4A-3H peak contained greater than 90% of the radioactivity present on the plate. The remaining impurities had no effect on the enzymatic reactions in which this radiolabeled cofactor
RADIOACTIVITY.-@ NADPH-4A-3H &-A
5 x I k
4.0
3.0
- 0.15
2.0
- 0.10
I .o
-0.05
20
22
24
26 FRACTION
RIG. 1. The elution pattern NADPH4A-3H from tritiated through 26 were pooled to Ci/mmole.
26
30
32
34
36
36
NUMBER
of a Bio-Gel P-2 column showing the l-isocitrate and CL and I-alloisocitrate. obtain NADPH4A-3H with a specific
separation Fractions activity
of 24 of 2
STEREOSPECIFIC
RADIOLABELED
was used. At this stage the pooled material can be diluted with nonlabeled NADPH or concentration. The pooled material was l-dram vials. The vials were lyophilized to Stored dry, the NADPH-4A-3H was stable
635
NADPH
from the Bio-Gel P-2 column to a desired specific activity divided into l-ml aliquots in dryness and stored at - 15°C. for at least 4 months.
DISCUSSION
The procedure just described involving the combination of chemical synthesis and enzymatic catalysis has resulted in the preparation of a stereochemically tritiated reduced pyridine nucleotide of high specific activity. It is now well established that the enzyme isocitrate dehydrogenase transfers a hydrogen from the C-2 of isocitric acid to face A of NADPH (10,ll) _ The preparations of NADPH-4A-3H produced in this laboratory resulted in a specific activity of approximately 2 Ci/mmole, indicating an isotope effect factor of 4 for the transfer of tritium from d-isocitrate to position 4A of NADPH by isocitrate dehydrogenase. This high specific activity has many uses, including: (i) the enzymatic biosynthesis of high specific activity tritiated metabolites of substrates whose metabolism includes pyridine nucleotide mediated stereospecific reduction; and (ii) the study of the stereochemistry of specific enzymatic assays used in the determinat,ion of nonlabeled enzyme-reducible compounds in biological fluids, based on the principle of selectively transferring the tritium atom to t.he compound during its enzymatic reduction. In the last example, the high specific activity would allow for the quantitative measurement of compounds in concentrations as low as 1 rig/ml plasma. Furthermore, substitution of sodium borodeuteride for the tritide would permit synthesis of stereochemically pure NADPH-4A-‘H for specific deuterium labelling of compounds. This radiolabeled coenzyme has been successfully used in this laboratory in studies involving the enzymatic reduction of certain synthetic steroids. ACKNOWLEDGMENTS Supported by G. D. Searle and Company Tox Program, NIGMS, NIH.
and by Contract
PH-43-65-1057,
Pharm-
REFERENCES 1. PULLMAN, M. E., SAN PIETRO, A., AND COLOWICK, S. P. (1954) J. Viol. 206, 129. 2. LOEWUS, F. A., VENNESLAND, B., AND HARRIS, D. L. (1955) J. Amer. sot. 77, 3391. 3. VENNESLAND, B. (1958) Fed. Proc. 17, 1150. 4. LEVY, H. R., AND VENNESLAND, B. (1957) J. Biol. Chem. 228, 85. 5. SAN PIETRO, A. (1955) J. Biol. Chem. 217, 579.
Chem.
Chem.
636
FREUDENTHAL,
KEPLER,
AND
COOK
6. WKOW, G., LUWWIEO, J., MATEIES, J. H., TOSI, L., UDAKA, S., AND LAND, B. (1963) Biochemistry 2, 1009. 7. BEAUS, O., AND BJORKHEM, I. (1967) Eur. J. Biochem. 2, 503. 8. BJORKHEM, I., AND DANIELSSON, H. (1970). Eur. J. Biochem. 12, 80. 9. PASTORE, E. J., AND FRIEDKIN, J. (1961) J. Biol. Chem. 236, 2314. 10. NAKAMOTO, T., AND VENNESLAND, B. (1960) J. Biol. Chem. 235, 202. 11. ENGLARD, S., AND COLOWICK, S. P. (1957) J. Biol. Chem. 226, 1047. 12. HORECKER, B. L., AND KORNBERG, A. (1948) J. Biol. Chem. 175, 385.
VENNES.
BIOCHEMISTRY
The Preparation
51, 632636
of Stereospecific
Nicotinamide
Adenine
R. I. FREUDENTHAL, Chemistry
(1973)
Tritium-Labeled Dinucleotide
J. A. KEPLER
Reduced
Phosphate AND
C. E. COOK
and Life science Division, Research Triangle Institute, Research Triangle Park, North Carolina R709 Received July 10, 1972; accepted October
18, 1972
A method has been developed for the preparation of a high specific activity stereospecifically labeled tritiated NADPH. In this procedure, tritium is enzymatically transferred from d-isocitric acid-aH (8 Ci/mmole) to the A face of a pyridine nucleotide during its stereospecific reduction, resulting in the formation of NADPH-4A-“H (2 Ci/mmole).
The stereospecificity of the enzyme-catalyzed transfer of a hydrogen from a reduced pyridine nucleotide to a number of different substrates has been determined (l-3). The preparation and use of pyridine nucleotides labeled with hydrogen isotopes has made possible both the identification of the 4-position of the nicotinamide ring as the site of the transferra,ble hydrogen (4,5), and the determination of the stereochemistry of the products resulting from the hydrogen transfer (6-8). Although initial preparations of tritium-labeled reduced nicotinamide adenine dinucleotide phosphate (NADPH-4-3H) were of relatively low specific activity (9), the tritium content, has been increased in more recently reported preparations. Krakow et al. report the preparation of NADPH-4A-3H having a specific activity of approximately 1.3 mCi/ mmole (6) while Bjorkhem and Danielsson describe the preparation of NADPH-4A-“H of approximately 4.2 mCi/mmole (8). The present communication describes an enzymatic method for preparing NADPH4A-3H having a specific activity of 2 Ci/mmole, almost 500 times higher than that obtained by any of the previously described procedures. MATERIALS
AND
METHODS
NADP, oxalosuccinate and isocitrate dehydrogenase were purchased from Sigma. Sodium borotritide (specific activity, 42 Ci/mmole) was obtained from New England Nuclear, and Bio-Gel P-2 from BioRad 632 Copyright @ 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.
STEREOSPECIFIC
RADIOLABELED
NADPH
633
Laboratories. ChromAR 1000 chromatography medium was purchased from Mallinckrodt and precoated silica gel thin-layer chromatography plates from Brinkmann. Chemical Synthesis of Isocitric Acid-~2-~H. Sodium borotritide (4.5 mg, 0.119 mmole, specific activity 42 Ci/mmole) was added to a solution of triethyloxalosuccinate (121 mg, 0.428 mmole) in 1 ml of ethanol. The solution was stirred at, ambient temperature for 1 hr after which 0.5 ml of concentrated hydrochloric acid was added. The solut.ion was diluted with 10 ml of methylene chloride and washed with 4 ml of water. The organic layer was separated and then dried by adding sodium sulfate to the solution. Removal of the solvent gave 119 mg of crude triethyl isocitrate-2-3H. The crude product was purified by chromatography on two 20 X 20cm ChromAR 1000 sheets with 5% acetone in carbon tetrachloride. The product was eluted from the ChromAR strip with chloroform to give 61 mg (0.22 mmole; specific activity, 9.2 Ci/mmole) of triethylisocitrate2-3H. A very small aliquot of this material was chromatographed on a silica gel H plate in benzene:ethyl acetate, 2:l. The developed t’hinlayer plate was scanned on a Packard Model 7201 radiochromatogram scanner which showed the ester to be better than 98% radiochemically pure. The triethylisocitrate-2-3H (61 mg) was hydrolyzed to isocitric acidzZ-~H by dissolving it in 1 ml of 1 N sodium hydroxide and stirring at ambient temperat,ure for 15 hr. At the end of this time 0.05 ml of acetic acid was added and the solut,ion diluted to 25 ml. A very small aliquot of the resulting isocitric acid-2-3H solution was chromatographed on silica gel H plates in a solvent system containing ether: formic acid: water, 5: 2: 1. A radiochromatogram of the developed plate indicated that the isocitric acid-2-“H was greater than 98% radiochemically pure. However, the tritiated product is composed of 4 stereoisomers, d- and lisocitric acid and d- and I-alloisocitric acid, and only the d-isocitrate is oxidized by isocitrate dehydrogenase. The isocitric acid-2-3H solution was used in the next step. Enzymatic Synthesis of NADPH-4A-JH. One hundred ,umoles of the tritiated isocitrate preparation (820 mci), of which 25 pmoles is true substrate, was incubated wit’h 25 pmoles of NADP, 50 units of isocitrate dehydrogenase, and 250 pmoles of MnCl, for 3 min in a 0.1 M Tris buffer, pH 7.2 (37°C). The reaction was stopped by heating the incubate at 72” for 3 min. The heat-denatured protein was precipitated by centrifugation and the resulting clear supernatant was applied to a BioGel P-2 column (2 X lOO-cm) and eluted with a 0.05 M potassium phosphate buffer, pH 9.1.
634
FREUDENTHAL,
KEPLER,
AND
COOK
RESULTS
A typical elution pattern of NADPH-4A-3H and isocitric acid from a Bio-Gel P-2 column is shown in Fig. 1. The elution of NADPH-4A-3H was determined spectrophotometrically. The reduced pyridine nucleotide has a strong absorption peak at 340 nm, with a molar extinction coefficient of 6.2 X lo3 (12). The elution pattern of the isocitrate-3H and alloisocitrateJH was determined by column chromatography in the absence of radiolabeled NADPH, followed by liquid scintillation counting. Of the 20 mg NADP incubations, 14.4 mg was recovered as NADPH-4A-3H. A number of smaller batches of NADPH-4A-3H have also been prepared. When fractions 24 through 26 (Fig. 1) were pooled, a specific activity of 2 Ci/mmole was obtained as determined by liquid scintillation counting and uv spectroscopy at 340 nm. The product was determined to be at least 90% pure by thin-layer chromatography. A very small aliquot was spotted on a precoated cellulose plate which was then developed in isobutyric acid: NH,OH (cont.) : water (96: 1: 22), The developed plate was scanned on a radiochromatogram scanner which showed that the NADPH-4A-3H peak contained greater than 90% of the radioactivity present on the plate. The remaining impurities had no effect on the enzymatic reactions in which this radiolabeled cofactor
RADIOACTIVITY.-@ NADPH-4A-3H &-A
5 x I k
4.0
3.0
- 0.15
2.0
- 0.10
I .o
-0.05
20
22
24
26 FRACTION
RIG. 1. The elution pattern NADPH4A-3H from tritiated through 26 were pooled to Ci/mmole.
26
30
32
34
36
36
NUMBER
of a Bio-Gel P-2 column showing the l-isocitrate and CL and I-alloisocitrate. obtain NADPH4A-3H with a specific
separation Fractions activity
of 24 of 2
STEREOSPECIFIC
RADIOLABELED
was used. At this stage the pooled material can be diluted with nonlabeled NADPH or concentration. The pooled material was l-dram vials. The vials were lyophilized to Stored dry, the NADPH-4A-3H was stable
635
NADPH
from the Bio-Gel P-2 column to a desired specific activity divided into l-ml aliquots in dryness and stored at - 15°C. for at least 4 months.
DISCUSSION
The procedure just described involving the combination of chemical synthesis and enzymatic catalysis has resulted in the preparation of a stereochemically tritiated reduced pyridine nucleotide of high specific activity. It is now well established that the enzyme isocitrate dehydrogenase transfers a hydrogen from the C-2 of isocitric acid to face A of NADPH (10,ll) _ The preparations of NADPH-4A-3H produced in this laboratory resulted in a specific activity of approximately 2 Ci/mmole, indicating an isotope effect factor of 4 for the transfer of tritium from d-isocitrate to position 4A of NADPH by isocitrate dehydrogenase. This high specific activity has many uses, including: (i) the enzymatic biosynthesis of high specific activity tritiated metabolites of substrates whose metabolism includes pyridine nucleotide mediated stereospecific reduction; and (ii) the study of the stereochemistry of specific enzymatic assays used in the determinat,ion of nonlabeled enzyme-reducible compounds in biological fluids, based on the principle of selectively transferring the tritium atom to t.he compound during its enzymatic reduction. In the last example, the high specific activity would allow for the quantitative measurement of compounds in concentrations as low as 1 rig/ml plasma. Furthermore, substitution of sodium borodeuteride for the tritide would permit synthesis of stereochemically pure NADPH-4A-‘H for specific deuterium labelling of compounds. This radiolabeled coenzyme has been successfully used in this laboratory in studies involving the enzymatic reduction of certain synthetic steroids. ACKNOWLEDGMENTS Supported by G. D. Searle and Company Tox Program, NIGMS, NIH.
and by Contract
PH-43-65-1057,
Pharm-
REFERENCES 1. PULLMAN, M. E., SAN PIETRO, A., AND COLOWICK, S. P. (1954) J. Viol. 206, 129. 2. LOEWUS, F. A., VENNESLAND, B., AND HARRIS, D. L. (1955) J. Amer. sot. 77, 3391. 3. VENNESLAND, B. (1958) Fed. Proc. 17, 1150. 4. LEVY, H. R., AND VENNESLAND, B. (1957) J. Biol. Chem. 228, 85. 5. SAN PIETRO, A. (1955) J. Biol. Chem. 217, 579.
Chem.
Chem.
636
FREUDENTHAL,
KEPLER,
AND
COOK
6. WKOW, G., LUWWIEO, J., MATEIES, J. H., TOSI, L., UDAKA, S., AND LAND, B. (1963) Biochemistry 2, 1009. 7. BEAUS, O., AND BJORKHEM, I. (1967) Eur. J. Biochem. 2, 503. 8. BJORKHEM, I., AND DANIELSSON, H. (1970). Eur. J. Biochem. 12, 80. 9. PASTORE, E. J., AND FRIEDKIN, J. (1961) J. Biol. Chem. 236, 2314. 10. NAKAMOTO, T., AND VENNESLAND, B. (1960) J. Biol. Chem. 235, 202. 11. ENGLARD, S., AND COLOWICK, S. P. (1957) J. Biol. Chem. 226, 1047. 12. HORECKER, B. L., AND KORNBERG, A. (1948) J. Biol. Chem. 175, 385.
VENNES.