Kitagishi K, D'Ambrosio C, Yonetani T
Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia 19104-6089.
Arch Biochem Biophys. 1988 Jul;264(1):176-83. doi: 10.1016/0003-9861(88)90583-8.
Asymmetric Fe-Co hybrid hemoglobins (Hbs) containing one, two, and three Co(II) protoporphyrins IX per tetramer have been synthesized. These asymmetric Fe-Co hybrids have been prepared by appropriate combinations of subunits of HbA, HbS, Co(II)HbA, Co(II)HbS, and Fe-Co hybrid Hbs and crosslinking with bis(3,5-dibromosalicyl)fumarate, followed by purification with molecular sieve and ion-exchange chromatography. Electron paramagnetic resonance (EPR) spectra have been measured for the crosslinked asymmetric Fe-Co hybrids to investigate the intermediate ligation states of Hb. The electronic state of the Co(II) ion provides a useful probe to estimate how ligation to iron-containing subunits changes the structure of the cobalt-substituted heme vicinity as described previously (T. Inubushi and T. Yonetani (1983) Biochemistry 22, 1894-1900). The crosslink does not affect the EPR spectra of Fe-Co hybrids. EPR spectra of the alpha (Co) subunits in crosslinked hybrids in the fully deoxy state exhibit a mixture of a broad EPR component and a narrow EPR component which correspond to distorted and undistorted Co(II) coordinate states, respectively, and the ratio of the components varies with pH. EPR spectra of the di-liganded asymmetric hybrid, [alpha (Co) beta (Co)]A-[alpha (Fe-CO) beta (Fe-CO)]sXL, and tri-liganded hybrid, [alpha (Co) beta (Fe-CO)]A-[alpha (Fe-CO) beta (Fe-CO)]sXL, show that the coordination state of the alpha (Co) ion in these hybrids is changed to the completely undistorted state in the pH range between 6.5 and 8.8 upon ligation of carbon monoxide (CO). Mono-liganded hybrids, [alpha (Co) beta (Fe-CO)]A-[alpha (Co) beta (Co)]sXL and [alpha (Fe-CO) beta (Co)]A-[alpha (Co) beta (Co)]sXL show intermediate EPR spectra at pH 6.5-7.6, which represent a mixture of the broad and narrow EPR components. The broad component is converted into the narrow one more extensively upon the ligation to the beta (Fe) subunit than to the alpha (Fe) subunit. However, the coordination state of an alpha (Co) subunit in mono-liganded hybrids corresponds to a completely undistorted coordination structure at pH 8.2-8.8. The EPR signal of the alpha (Co) ion is sensitive to the release of alkaline Bohr proton(s) and consequently to the allosteric quaternary structural change of Hb.
已合成了每四聚体分别含有一个、两个和三个钴（II）原卟啉IX的不对称铁 - 钴杂合血红蛋白（Hb）。这些不对称铁 - 钴杂合体是通过将HbA、HbS、钴（II）HbA、钴（II）HbS和铁 - 钴杂合Hb的亚基进行适当组合，并用双（3,5 - 二溴水杨基）富马酸交联，然后通过分子筛和离子交换色谱法纯化而制备的。已对交联的不对称铁 - 钴杂合体进行了电子顺磁共振（EPR）光谱测量，以研究Hb的中间配位状态。如前所述（T. Inubushi和T. Yonetani（1983）Biochemistry 22, 1894 - 1900），钴（II）离子的电子状态为估计与含铁亚基的配位如何改变钴取代血红素附近的结构提供了一个有用的探针。交联不影响铁 - 钴杂合体的EPR光谱。完全脱氧状态下交联杂合体中α（Co）亚基的EPR光谱显示出宽EPR成分和窄EPR成分的混合物，它们分别对应于扭曲和未扭曲的钴（II）配位状态，并且这些成分的比例随pH值变化。双配位不对称杂合体[α（Co）β（Co）]A - [α（Fe - CO）β（Fe - CO）]sXL和三配位杂合体[α（Co）β（Fe - CO）]A - [α（Fe - CO）β（Fe - CO）]sXL的EPR光谱表明，在pH值介于6.5和8.8之间时，这些杂合体中α（Co）离子的配位状态在一氧化碳（CO）配位后变为完全未扭曲状态。单配位杂合体[α（Co）β（Fe - CO）]A - [α（Co）β（Co）]sXL和[α（Fe - CO）β（Co）]A - [α（Co）β（Co）]sXL在pH 6.5 - 7.6时显示出中间EPR光谱，这代表宽EPR成分和窄EPR成分的混合物。与α（Fe）亚基配位相比，与β（Fe）亚基配位时，宽成分更广泛地转变为窄成分。然而，单配位杂合体中α（Co）亚基的配位状态在pH 8.2 - 8.8时对应于完全未扭曲的配位结构。α（Co）离子的EPR信号对碱性玻尔质子的释放敏感，因此对Hb的变构四级结构变化敏感。
