Howell B F, McCune S, Schaffer R
Clin Chem. 1977 Dec;23(12):2231-7.
We previously observed [Clin. Chem. 22, 1648 (1976)] that values of the Michaelis constant for NADH for the conversion of pyruvate to lactate with lactate dehydrogenase (EC 1.1.1.27) in the presence of 0.1 mol/liter buffers at 25 degrees C showed first-order dependence on enzyme concentration. This is now recognized to be the result of an inhibitory influence exerted by buffers [NH4HCO2, tris(hydroxymethyl)aminomethane, and phosphate] and salts [(NH4)2SO4 and NaCl] present in the reaction mixtures. Inhibition constants for the enzyme/inhibitor complexes formed with these substances are about 0.3 mol/liter for competition of NH4HCO3 with NaOH and 0.4 mol/liter for competition of NH4HCO3 with pyruvate; they are 0.6 mol/liter for NaCl, 1.0 mol/liter for sodium phosphate, 0.3 mol/liter for (NH4)2SO4, and 0.8 mol/liter for tris(hydroxymethyl)aminomethane when these substances compete with NADH. Because of the large molar ratio of buffer to substrate (about 10(9):1) in enzymatic assays, the buffer concentration significantly influences the Michaelis constant, despite the large value for the inhibition constant. Attention to the concentrations of these substances may be required for decreasing variability in clinical assays in which lactate dehydrogenase and possible other enzymes are used.
我们之前观察到[《临床化学》22, 1648 (1976)]，在25℃下，于0.1摩尔/升缓冲液存在的情况下，乳酸脱氢酶（EC 1.1.1.27）催化丙酮酸转化为乳酸时，NADH的米氏常数的值对酶浓度呈一级依赖性。现在认为这是反应混合物中存在的缓冲液[NH₄HCO₂、三（羟甲基）氨基甲烷和磷酸盐]和盐[（NH₄）₂SO₄和NaCl]产生抑制作用的结果。与这些物质形成的酶/抑制剂复合物的抑制常数，对于NH₄HCO₃与NaOH竞争约为0.3摩尔/升，对于NH₄HCO₃与丙酮酸竞争约为0.4摩尔/升；当这些物质与NADH竞争时，NaCl为0.6摩尔/升，磷酸钠为1.0摩尔/升，（NH₄）₂SO₄为0.3摩尔/升，三（羟甲基）氨基甲烷为0.8摩尔/升。由于酶促测定中缓冲液与底物的摩尔比很大（约为10⁹:1），尽管抑制常数的值很大，但缓冲液浓度仍会显著影响米氏常数。在使用乳酸脱氢酶以及可能的其他酶的临床测定中，为降低变异性，可能需要关注这些物质的浓度。
