AdenineDinucleotide by KineticMethods (original) (raw)

Methods are described for detection of lactate dehydrogenase (LDH) inhibitors in preparations of reduced nicotlnamide adenine dinucleotide. They are (a) comparison of values by kinetic methods with those measured for highly purified NADH and (b) examination of Lineweaver-Burk plots. Chrornatographic inhomogenelties are correlated with deviant values for the kinetic constants of NADH preparations. Lineweaver-Burk plots that curve upward at the high concentrations or have a larger or smaller than normal slope may indicate the presence of inhibitor. As determined in bicarbonate buffer (0.11 mol/liter, pH 7.9) by use of 0.600 mmol/liter pyruvate and NADH freshly separated from impurities by chromatography on diethylamlnoethyl-cellulose, the Km (apparent) of NADH at 25#{176}C has the value 8.11 ± 0.71 mol/liter (SD, n = 28) with LDH-1 (pig heart, 2.48 ± 0.05 U per milliliter of reaction mixture, or 41.3 ± 0.8 nmol/liter per second). Under similar conditions, the Km (apparent) of NADH has the value of 8.57 ± 1.58 mol/liter(SD, n = 21) with LDH-5 (pig muscle, 1.77 ± 0.03 U/mI of reaction mixture), or 29.4 ± 0.6 nmol/liter per second). At infinite substrate concentrations with the same pH, buffer, and temperature, the Km (apparent) for NADH was 26.0 ± 0.63 moI/liter with LDH-1 and 23.2 ± 4.6 tmol/llter with LDH-5.