Patel R N
Arch Biochem Biophys. 1987 Jan;252(1):229-36. doi: 10.1016/0003-9861(87)90027-0.
An anaerobic procedure was developed for the purification of the flavin:NADH oxidoreductase (flavoprotein) component of methane monooxygenase to homogeneity. The molecular weight of the flavoprotein determined by gel filtration was about 40,000, and by sedimentation equilibrium analysis, about 38,000. The purified flavoprotein is a monomeric protein with a sedimentation constant (S20,W) value of about 2.1 S. The absorption spectrum of the flavoprotein has a peak at 460 nm and shoulder at 395 nm. The fluorescent excitation and emission spectra of the fluorescent component of flavoprotein had peaks at 450, 370, and 530 nm, respectively. A FAD was identified as a prosthetic group of flavoprotein by thin-layer chromatography. The flavoprotein contained about 1 mol of FAD and 2 mol each of iron and acid-labile sulfide per mole of protein. The flavoprotein was directly reduced by NADH under anaerobic conditions. The formation of neutral flavin semiquinone was detected during anaerobic titration of flavoprotein by NADH and also as a free radical signal at a g value of 2.004 by EPR spectroscopy. The iron sulfur cluster has g values of 2.04, 1.96, and 1.87, yielding a g average of 1.96, characteristic of a Fe2S2 center. Antibody prepared against the flavoprotein reacted with flavoprotein and inhibited methane monooxygenase activity.
开发了一种厌氧方法，用于将甲烷单加氧酶的黄素：NADH氧化还原酶（黄素蛋白）组分纯化至同质。通过凝胶过滤测定的黄素蛋白分子量约为40,000，通过沉降平衡分析约为38,000。纯化的黄素蛋白是一种单体蛋白，沉降常数（S20,W）值约为2.1 S。黄素蛋白的吸收光谱在460 nm处有一个峰值，在395 nm处有一个肩峰。黄素蛋白荧光组分的荧光激发和发射光谱分别在450、370和530 nm处有峰值。通过薄层色谱法鉴定FAD为黄素蛋白的辅基。每摩尔蛋白质，黄素蛋白含有约1摩尔FAD、2摩尔铁和2摩尔酸不稳定硫化物。在厌氧条件下，黄素蛋白被NADH直接还原。在NADH对黄素蛋白进行厌氧滴定过程中检测到中性黄素半醌的形成，并且通过电子顺磁共振光谱在g值为2.004处也检测到自由基信号。铁硫簇的g值为2.04、1.96和1.87，g平均值为1.96，这是Fe2S2中心的特征。针对黄素蛋白制备的抗体与黄素蛋白反应并抑制甲烷单加氧酶活性。
