NOTES 3393Incorporation of Nicotinamide4-d into DPN.-One hundred and ten mg. of DPN (Pabst) was incubated at 37' for 3 hours in the presence of 100 mg. of nicotinamide-4-d with 5 ml. of beef spleen DPNase (ca. 750 units). After inactivation of the enzyme by heating the reaction mixture for 10 minutes in a water-bath a t 70", the mixture was cooled and centrifuged. The precipitate was washed with 5 ml. of HzO and the combined supernatants were added to a Dowex-1 formate exchange column (20 X 1 cm.). The column was washed with 200 ml. of H t 0 followed by 0.1 M formic acid which eluted the DPK. Seventy-seven mg. of material rontaining 68% DPN by enzymatic assay was recovered by Lyophilization from the DPN-containing fractions. A portion of this DPN was analyzed for excess deuterium with glycine as a d i l~e n t .~ Reduction of DPN Containing Nicotinamide-4-d.-Forty rng. of the above material containing 27 mg. of pure DPN was reduced in 5 ml. of 1.3'% NaHC03 solution with 30 mg.of Na~S204.~~ After the reaction was complete, the mixture was pipetted into 15 ml. of absolute ethanol together with 0.5 ml. of wash water. After 20 minutes at -20", the mixture was centrifuged free of precipitated salts. The latter were dissolved in 1 ml. of HzO and the salts were reprecipitated with 3 ml. of absolute ethanol. The supernatants were combined, poured into 90 ml. of absolute ethanol and stored a t -15' overnight. The resulting precipitate was centrifuged, washed with ether and dried in vacuo. The dried powder weighed 42 mg. and contained 18 mg. of reduced DPhT by enzymatic assay.Enzymatic Oxidation of Reduced DPN Containing Nicotinamide-4-d.-Thirtv-one ma. of the reduced D P N (containing 13.2 mg. of reduced B P N by enzymatic assayj was oxidized with 0.18 ml. of 0.1 M sodium pyruvate in the presence of crystalline muscle lactic dehydrogenase in 5.0 ml. of 0.1 ;M phosphate buffer, pH 7.4. When the reaction was complete as indicated by the disappearance of the absorption band at 340 mp, the enzyme was destroyed by heating for 1 minute in a boiling water-bath. The reaction mixture was cooled, adjusted to p H 1-2 with 6 N HzSO4 and 50.0 mg. of unlabeled lithium-L-lactate was added (26.9-fold dilution). The solution was centrifuged to remove denatured protein and the lactic acid was recovered by ether extraction and converted to its phenacyl ester which was analyzed for D, all as previously described.* (11) P. Ohlmeyer, Biochem. Z., 297, 66 (1938)