Miura R, Sugiyama T, Yamano T
J Biochem. 1979 May;85(5):1107-13.
Adrenodoxin of bovine adrenocortical mitochondria was spin-labeled with two different spin-labeling reagents, N-(2,2,5,5-tetramethyl-3-carbonylpyrroline-1-oxyl)imidazole (I) and N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide (II), without major loss of its activity for electron transport from NADPH to cytochrome c. The EPR spectrum of adrenodoxin spin-labeled with either of the reagents showed a pattern typical of a moderately immobilized spin label. When adrenodoxin was treated with (I), approximately two amino acid residues per molecule were spin-labeled, whereas a single residue was labeled by (II). While assition of NADPH to adrenodoxin spin-labeled with (I) did not diminish the EPR signal intensity, addition of the reductant to the labeled adrenodoxin in the presence of adrenodoxin reductase caused slow reduction of the spin label, the rate of which was dependent on the aerobicity. Addition of adrenodoxin reductase to adrenodoxin spin-labeled with (I) or (II) resulted in the appearance of a more immobilized component in the EPR spectrum. The ratio of the more immobilized component to the less immobilized component was saturated at a molar ratio of one to one. Addition of cytochrome P-450scc to adrenodoxin labeled with (I) had similar effects on the EPR spectrum.
牛肾上腺皮质线粒体的肾上腺皮质铁氧还蛋白用两种不同的自旋标记试剂，即N-（2,2,5,5-四甲基-3-羰基吡咯啉-1-氧基）咪唑（I）和N-（1-氧基-2,2,6,6-四甲基-4-哌啶基）马来酰亚胺（II）进行自旋标记，其从NADPH到细胞色素c的电子传递活性没有明显损失。用这两种试剂中的任何一种自旋标记的肾上腺皮质铁氧还蛋白的电子顺磁共振（EPR）谱显示出中等固定化自旋标记的典型模式。当用（I）处理肾上腺皮质铁氧还蛋白时，每个分子大约有两个氨基酸残基被自旋标记，而用（II）标记时只有一个残基被标记。虽然向用（I）自旋标记的肾上腺皮质铁氧还蛋白中加入NADPH不会降低EPR信号强度，但在肾上腺皮质铁氧还蛋白还原酶存在的情况下向标记的肾上腺皮质铁氧还蛋白中加入还原剂会导致自旋标记缓慢还原，其速率取决于需氧性。向用（I）或（II）自旋标记的肾上腺皮质铁氧还蛋白中加入肾上腺皮质铁氧还蛋白还原酶会导致EPR谱中出现一个更固定化的成分。更固定化成分与较不固定化成分的比例在摩尔比为1:1时达到饱和。向用（I）标记的肾上腺皮质铁氧还蛋白中加入细胞色素P-450scc对EPR谱有类似影响。
