Blumenstein M
Biochemistry. 1975 Nov 4;14(22):5004-8. doi: 10.1021/bi00693a034.
31P nuclear magnetic resonance spectra of the pyrophosphate group in NAD+ and NADH were recorded in the presence of beef heart lactate dehydrogenase and rabbit muscle glyceraldehyde-3-phosphate dehydrogenase. At high lactate dehydrogenase concentrations (60 mg/ml), two NADH resonances are observed: a slowly exchanging peak which is shifted to 1.9 ppm downfield (relative to free NADH) and a rapidly exchanging peak with a downfield shift of 0.5-0.6 ppm. At lover concentrations (15 mg/ml) only the rapidly exchanging peak is observed thus indicating that the peak observed at-1.9 ppm is due to coenzyme bound to an aggregated enzyme species. With NAD+, rapid exchange and downfield shifts are observed at both enzyme and concentrations, with shifts of about 1.5 ppm and 0.6 ppm at 60 and 15 mg/ml, respectively. In the presence of glyceraldehydephosphate dehydrogenase, the results are independent of enzyme concentration, and slow exchange and upfield shifts of 0.4-0.6 ppm occur with each coenzyme. These data indicate that the environment of the pyrophosphate group of oxidized and reduced coenzyme is the same for a given dehydrogenase, but is different in one enzyme from the other. The resonances observed with glyceraldehydephosphate dehydrogenase are broader than those observed with lactate dehydrogenase. This is indicative of either shorter relaxation times with the former enzyme, or the presence of multiple, unresolved resonances.
在牛心乳酸脱氢酶和兔肌甘油醛 -3-磷酸脱氢酶存在的情况下，记录了NAD⁺和NADH中焦磷酸基团的³¹P核磁共振谱。在高浓度乳酸脱氢酶（60mg/ml）时，观察到两个NADH共振峰：一个缓慢交换的峰，其向低场位移1.9ppm（相对于游离NADH），以及一个快速交换的峰，其低场位移为0.5 - 0.6ppm。在较低浓度（15mg/ml）时，仅观察到快速交换的峰，因此表明在 -1.9ppm处观察到的峰是由于辅酶与聚集的酶物种结合所致。对于NAD⁺，在两种酶浓度下均观察到快速交换和低场位移，在60mg/ml和15mg/ml时的位移分别约为1.5ppm和0.6ppm。在甘油醛 -3-磷酸脱氢酶存在的情况下，结果与酶浓度无关，并且每种辅酶都会发生缓慢交换和0.4 - 0.6ppm的高场位移。这些数据表明，对于给定的脱氢酶，氧化型和还原型辅酶的焦磷酸基团环境是相同的，但在一种酶与另一种酶中是不同的。用甘油醛 -3-磷酸脱氢酶观察到的共振峰比用乳酸脱氢酶观察到的共振峰更宽。这表明要么前者酶的弛豫时间更短，要么存在多个未解析的共振峰。
