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Enzymatic preparation of [3H]NADH
ANALYTICAL
BIOCHEMISTRY
88,338-339
Enzymatic
(1978)
Preparation
of [3H]NADH1
Earlier we reported on the activity of the chlorophyll-forming enzyme Mg-Cethyl-(4-desvinyl)-protoporphyrin IX monomethyl ester:NAD+ oxidoreductase of wheat (I). Used as substrates for the enzyme were chemically synthesized Mg-protoporphyrin IX monomethyl ester and [3H]NADH. The chemically synthesized substrates were not the ones of choice, but were used because of the ease of their preparation. The Mg-protoporphyrin IX monomethyl ester was a mixture (proportion unknown) of the 6 and 7 monoesters and contained a fair amount of degradation products. The [3H]NADH usually contained significant amounts of unreacted NAD+ and a high proportion of unknown contaminants. We have subsequently used the following simple, efficient procedure to prepare (2) enzymatically the desired micromole quantities of [3H]NADH of high purity and specific radioactivity. Ten micromoles of NAD+ (Sigma Chemical Co., St. Louis, MO.) is dissolved in 1 ml of 0.5 M Tris (unadjusted). Fifty microliters of an aqueous [ l-311]ethanol solution (New England Nuclear Corp., Boston, Mass.; specific radioactivity: 5 Ci/mol was diluted with ethanol to specific radioactivities of 0.05 to 1 Ci/mol for these studies) is added to make a concentration of 0.5 M in [l-3H]ethanol of the desired specific radioactivity. Four hundred units of alcohol dehydrogenase (EC 1.1.1.1; obtained from the Sigma Chemical Co.) is added to start the reaction. The reaction mixture is incubated at room temperature for 30 min and then 0.5 ml of 95% ethanol is added. The mixture is centrifuged for 5 min on a clinical centrifuge and the precipitate is discarded. To the supernatant fluid is added 0.2 ml of saturated NaCl and 10 ml of 95% ethanol. The mixture is allowed to stand at 0°C for 2 hr. The precipitate ([3H]NADH, as the sodium salt) is collected by (clinical) centrifugation and then washed with absolute ethanol:diethyl ether (l:l), absolute ethanol, diethyl ether, absolute ethanol, and finally once again with diethyl ether. The E3H]NADH is stored frozen for a day or two before use, if desired, or is stored frozen in a vacuum desiccator for somewhat longer times. The yield of [3H]NADH is typically 40 to 50% of theory. The enzymatically prepared [3H]NADH provided only one observable spot and cochromatographed with authentic NADH (Sigma Chemical ’ This work was supported by a grant from the National Science Foundation (Contract No. PCM 75-16131) to R.K.E. 0003-2697/78/0881-0338$02.00/O Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.
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SHORT COMMUNICATIONS
339
Co.) on paper chromatograms [developed with either ethanol:1 M ammonium acetate (pH 7.5), 7:3 (v/v), or 70% isopropanol containing 3 ml of concentrated NH,OH] when examined under uv light. The uv region absorption spectrum was indistinguishable from that of an authentic sample of NADH; the absorbance 260/340 ratio was the same as that reported for NADH by Rafter and Colowick (2). The [3H]NADH preparations were determined to have specific radioactivities identical to that of the starting material (within experimental error) in each case. The enzymatically prepared [“HINADH was usually slightly more active when used as a substrate ([e.g., Ref. (3)] for alcohol dehydrogenase (acetaldehyde reduction) than was our sample of 98% pure commercially available (Sigma) NADH. The [3H]NADH obtained by chemical synthesis [as before (l)] was found, when examined as above, to be a substantially inferior preparation on the micromole scale (in SO-pmol and larger quantities, chemically synthesized [3H]NADH appears to be a somewhat more satisfactory product). Finally, the enzymatically prepared [3H]NADH increased the activity of the Mg-Cethyl-(4-desvinyl)-protoporphyrin IX monomethyl ester:NAD+ oxidoreductase of wheat (1) in a test assay to over five times that of assays containing chemically prepared [3H]NADH as substrate. REFERENCES 1. Ellsworth, R. K., and Hsing, A. S. (1974) Photosynthetica 8, 228. 2. Rafter, G. W., and Colowick, S. P. (1957) in Methods in Enzymology (Colowick. S. P., and Kaplan, N. O., eds.), Academic Press, London and New York. 3. Chaykin, S. (1966) Biochemical Laboratory Techniques, Wiley, New York and London.
R. K. ELLSWORTH~ M. E. ST. PIERRE Department of Chemistry State University of New York Plattsburgh. New York 12901 Received November I I, 1977; accepted
February
28. 1978
’ To whom requests for reprints and correspondence
should be addressed.
BIOCHEMISTRY
88,338-339
Enzymatic
(1978)
Preparation
of [3H]NADH1
Earlier we reported on the activity of the chlorophyll-forming enzyme Mg-Cethyl-(4-desvinyl)-protoporphyrin IX monomethyl ester:NAD+ oxidoreductase of wheat (I). Used as substrates for the enzyme were chemically synthesized Mg-protoporphyrin IX monomethyl ester and [3H]NADH. The chemically synthesized substrates were not the ones of choice, but were used because of the ease of their preparation. The Mg-protoporphyrin IX monomethyl ester was a mixture (proportion unknown) of the 6 and 7 monoesters and contained a fair amount of degradation products. The [3H]NADH usually contained significant amounts of unreacted NAD+ and a high proportion of unknown contaminants. We have subsequently used the following simple, efficient procedure to prepare (2) enzymatically the desired micromole quantities of [3H]NADH of high purity and specific radioactivity. Ten micromoles of NAD+ (Sigma Chemical Co., St. Louis, MO.) is dissolved in 1 ml of 0.5 M Tris (unadjusted). Fifty microliters of an aqueous [ l-311]ethanol solution (New England Nuclear Corp., Boston, Mass.; specific radioactivity: 5 Ci/mol was diluted with ethanol to specific radioactivities of 0.05 to 1 Ci/mol for these studies) is added to make a concentration of 0.5 M in [l-3H]ethanol of the desired specific radioactivity. Four hundred units of alcohol dehydrogenase (EC 1.1.1.1; obtained from the Sigma Chemical Co.) is added to start the reaction. The reaction mixture is incubated at room temperature for 30 min and then 0.5 ml of 95% ethanol is added. The mixture is centrifuged for 5 min on a clinical centrifuge and the precipitate is discarded. To the supernatant fluid is added 0.2 ml of saturated NaCl and 10 ml of 95% ethanol. The mixture is allowed to stand at 0°C for 2 hr. The precipitate ([3H]NADH, as the sodium salt) is collected by (clinical) centrifugation and then washed with absolute ethanol:diethyl ether (l:l), absolute ethanol, diethyl ether, absolute ethanol, and finally once again with diethyl ether. The E3H]NADH is stored frozen for a day or two before use, if desired, or is stored frozen in a vacuum desiccator for somewhat longer times. The yield of [3H]NADH is typically 40 to 50% of theory. The enzymatically prepared [3H]NADH provided only one observable spot and cochromatographed with authentic NADH (Sigma Chemical ’ This work was supported by a grant from the National Science Foundation (Contract No. PCM 75-16131) to R.K.E. 0003-2697/78/0881-0338$02.00/O Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.
338
SHORT COMMUNICATIONS
339
Co.) on paper chromatograms [developed with either ethanol:1 M ammonium acetate (pH 7.5), 7:3 (v/v), or 70% isopropanol containing 3 ml of concentrated NH,OH] when examined under uv light. The uv region absorption spectrum was indistinguishable from that of an authentic sample of NADH; the absorbance 260/340 ratio was the same as that reported for NADH by Rafter and Colowick (2). The [3H]NADH preparations were determined to have specific radioactivities identical to that of the starting material (within experimental error) in each case. The enzymatically prepared [“HINADH was usually slightly more active when used as a substrate ([e.g., Ref. (3)] for alcohol dehydrogenase (acetaldehyde reduction) than was our sample of 98% pure commercially available (Sigma) NADH. The [3H]NADH obtained by chemical synthesis [as before (l)] was found, when examined as above, to be a substantially inferior preparation on the micromole scale (in SO-pmol and larger quantities, chemically synthesized [3H]NADH appears to be a somewhat more satisfactory product). Finally, the enzymatically prepared [3H]NADH increased the activity of the Mg-Cethyl-(4-desvinyl)-protoporphyrin IX monomethyl ester:NAD+ oxidoreductase of wheat (1) in a test assay to over five times that of assays containing chemically prepared [3H]NADH as substrate. REFERENCES 1. Ellsworth, R. K., and Hsing, A. S. (1974) Photosynthetica 8, 228. 2. Rafter, G. W., and Colowick, S. P. (1957) in Methods in Enzymology (Colowick. S. P., and Kaplan, N. O., eds.), Academic Press, London and New York. 3. Chaykin, S. (1966) Biochemical Laboratory Techniques, Wiley, New York and London.
R. K. ELLSWORTH~ M. E. ST. PIERRE Department of Chemistry State University of New York Plattsburgh. New York 12901 Received November I I, 1977; accepted
February
28. 1978
’ To whom requests for reprints and correspondence
should be addressed.