Heme Orientation of Cavity Mutant Hemoglobins (His F8 --> Gly) in Either alpha or beta Subunits: Circular Dichroism, (1) H NMR, and Resonance Raman Studies.
Heme Orientation of Cavity Mutant Hemoglobins (His F8 --> Gly) in Either alpha or beta Subunits: Circular Dichroism, (1) H NMR, and Resonance Raman Studies.
复制标题
α 或 β 亚基中空腔突变血红蛋白（His F8 --> Gly）的血红素取向：圆二色性、(1) H NMR 和共振拉曼研究。
作者：
Nagai;M.;Nagai;Y.;Aki;Y.;Sakurai;H.;Mizusawa;N.;Ogura;T.;Kitagawa;T.;Yamamoto;Y. and Nagatomo;S.
天然人成人血红蛋白（Hb A）大部分血红素取向正常，而重组Hb A （rHb A）表达inE。共包含血红素的正反方向。具有正常血红素的Hb A在Soret和260 nm区域显示出正的圆二色性（CD）带，而具有反血红素的rHb A在Soret区域显示出负的圆二色性（CD）带，并在260 nm区域显示出减少的CD带。为了研究α或β亚基的近端组氨酸（His F8）在决定血红素取向中的作用，我们制备了两个空腔突变体rHb（α h87g）和rHb(β h92g)，在咪唑存在下用甘氨酸取代His F8。两种空腔突变体Hbs的CD光谱均未显示出Soret负带，而是在Soret和260 nm区域显示出强强度的正带，表明空腔突变体Hbs中几乎不存在反向血红素。我们通过1h NMR和共振拉曼（RR）光谱证实，空腔突变Hbs在突变亚基和天然亚基中主要具有正常血红素取向。这些结果表明，α和β亚基中的血红素Fe‐His F8连锁都影响血红素取向，并且一种亚基的血红素取向与互补亚基的血红素取向相同有关。本研究表明，除了常用的1h NMR技术外，CD和RR光谱也为检测Hb A的血红素旋转紊乱提供了强有力的工具。手性学报（自然科学版），2016。©2016 Wiley期刊公司
Native human adult hemoglobin (Hb A) has mostly normal orientation of heme, whereas recombinant Hb A (rHb A) expressed inE. colicontains both normal and reversed orientations of heme. Hb A with the normal heme exhibits positive circular dichroism (CD) bands at both the Soret and 260‐nm regions, while rHb A with the reversed heme shows a negative Soret and decreased 260‐nm CD bands. In order to examine involvement of the proximal histidine (His F8) of either α or β subunits in determining the heme orientation, we prepared two cavity mutant Hbs, rHb(αH87G) and rHb(βH92G), with substitution of glycine for His F8 in the presence of imidazole. CD spectra of both cavity mutant Hbs did not show a negative Soret band, but instead exhibited positive bands with strong intensity at the both Soret and 260‐nm regions, suggesting that the reversed heme scarcely exists in the cavity mutant Hbs. We confirmed by1H NMR and resonance Raman (RR) spectroscopies that the cavity mutant Hbs have mainly the normal heme orientation in both the mutated and native subunits. These results indicate that the heme Fe‐His F8 linkage in both α and β subunits influences the heme orientation, and that the heme orientation of one type of subunit is related to the heme orientation of the complementary subunits to be the same. The present study showed that CD and RR spectroscopies also provided powerful tools for the examination of the heme rotational disorder of Hb A, in addition to the usual1H NMR technique.Chirality 28:585–592, 2016. © 2016 Wiley Periodicals, Inc.