Nonaka Y, Fujii S, Yamano T
J Biochem. 1986 Mar;99(3):803-14. doi: 10.1093/oxfordjournals.jbchem.a135540.
NADPH-adrenodoxin oxidoreductase was titrated with NADPH under anaerobic conditions. As the amount of added NADPH was increased to a ratio to the reductase of 1 : 1, a broad absorbance band from approximately 500 to 900 nm, which is attributed to a charge transfer complex, increased and then sharply decreased after the 1 : 1 ratio was attained. Concomitant with the decrease in the charge transfer band, a peak at 575 nm with a shoulder at 635 nm increased, indicating the formation of a semiquinone. This showed clearly that a semiquinone was formed only when more than the stoichiometric amount of NADPH (It is meant by "the stoichiometric amount of NADPH" that the molar ratio of NADPH to adrenodoxin reductase is equal to one, that is, NADPH/FAD bound to the reductase = 1.) was added. The semiquinone band reached its maximum with an approximately 3-fold excess of NADPH over the reductase, and then gradually decreased. Concurrent with the decrease in absorbance of both the charge transfer complex and the semiquinone, the reaction mixture was bleached, indicating that a pale colored species was produced. 1H NMR studies suggested that the pale colored species was a complex of fully reduced adrenodoxin reductase and NADPH, and that the semiquinone also bound 1 mol of the pyridine nucleotide per mol of the reductase. These data suggest that the semiquinone state of the reductase is observable only when a complex between NADPH and the enzyme in the flavin semiquinone is formed.
在厌氧条件下，用NADPH对NADPH - 肾上腺皮质铁氧化还原蛋白氧化还原酶进行滴定。随着添加的NADPH量增加至与还原酶的比例为1:1时，约500至900nm的宽吸收带（归因于电荷转移复合物）增加，然后在达到1:1比例后急剧下降。伴随着电荷转移带的下降，575nm处的峰和635nm处的肩峰增加，表明形成了半醌。这清楚地表明，仅当添加的NADPH超过化学计量的量时（“NADPH的化学计量的量”是指NADPH与肾上腺皮质铁氧化还原蛋白还原酶的摩尔比等于1，即与还原酶结合的NADPH/FAD = 1）才形成半醌。半醌带在NADPH相对于还原酶过量约3倍时达到最大值，然后逐渐下降。随着电荷转移复合物和半醌的吸光度都下降，反应混合物被漂白，表明产生了浅色物质。1H NMR研究表明，浅色物质是完全还原的肾上腺皮质铁氧化还原蛋白还原酶与NADPH的复合物，并且半醌每摩尔还原酶也结合1摩尔吡啶核苷酸。这些数据表明，仅当在黄素半醌中形成NADPH与酶之间的复合物时，才能观察到还原酶的半醌状态。
