ESR- und CD-Spektren von pH-abhängigen Konformationsisomeren des Hämoglobin(III)
ESR- und CD-Spektren von pH-abhängigen Konformationsisomeren des Hämoglobin(III)
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ESR- 和 CD-Spektren von pH-abhängigen Konformationsisomeren des Hämoglobin(III)
DOI：
10.1111/j.1432-1033.1970.tb00999.x
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发表时间：
1970
期刊：
影响因子：
5.4
通讯作者：
A. Wollmer
中科院分区：
文献类型：
--
作者：
K. Gersonde;A. Wollmer
天然马血红蛋白(III)形成两种不同的依赖于pH的洗涤剂缔合物。这两个关联与蛋白质的两个构象异构体的存在有关[11]。通过直接验证天然血红蛋白上的构象异构体(III)，应排除观察到的两种缔合物都只来自一种血红蛋白构象的可能性，该构象在洗涤剂结合过程中经历了随pH变化的蛋白质结构。
ESR和CD技术证实了天然血红蛋白的两个异构体的存在。根据这些发现，血红蛋白(III)是一种异向性变构蛋白，其中一个(室温)或两个(77°K)质子结合部位与第6配体的结合部位通过构象相互作用。这种相互作用可以通过两种异构体的不同信号(g‘1=2.68，g’2=2.16，g‘3=1.82；g“1=2.68，g”2=2.14，g“3=1.77)直接显示在羟基衍生物的ESR谱上。
变构反应和质子化反应被描述为一个循环过程。借助于计算机模拟计算了模型曲线，从而可以确定实际的变构和质子化pk值。假设变构pK值为7.2和7.0，质子化pK值为8.3和8.5，对340和290 nm处的椭圆度随pH变化的模拟结果与实验数据吻合。
在所涉及的pH范围内，α-螺旋含量保持不变，构象的变化只影响三级结构。
由于常见的pH依赖关系，另外三个低自旋信号(g1=2.91，g2=2.23，g3=1.95)除了高自旋信号(g‘=5.9，g’=2.0)外，还必须归因于H2O-Hb(III)，这些信号表征了该络合物物理高自旋-低自旋平衡中的低自旋部分。
此外，还讨论了构象活性Cd带的来源。
The native horse haemoglobin(III) forms two different pH-dependent detergent associates. These two associates were related to the existence of two conformation isomers of the protein [11]. By aid of the direct verification of the conformation isomers on the native haemoglobin(III) the possibility should be excluded that the observed associates both originate from only one haemoglobin conformation, which undergoes a pH-depending transformation of the protein structure in the course of the detergent association.
ESR und CD techniques confirm the existence of two isomers of the native haemoglobin. According to these findings haemoglobin(III) is a heterotropic allosteric protein, in which one (at room temperature) or two (at 77° K) proton binding sites interact with the binding site of the 6th ligand mediated by conformation. This interaction can directly be demonstrated in the ESR spectrum of the OH-derivative by the appearance of distinct signals for both isomers (g′1= 2.68, g′2= 2.16, g′3= 1.82; g″1= 2.68, g″2= 2.14, g″3= 1.77).
The allosteric and protolytic reactions are described as a circuit process. By aid of computer simulation model curves are calculated, which allow determination of the actual allosteric and protolytic pK-values. A simulation of the pH-dependency of the ellipticity at 340 and 290 nm fits the experimental data, if allosteric pK-values of 7.2 and 7.0 and protolytic pK-values of 8.3 and 8.5 are presumed.
The α-helix content remaining constant in the pH-range concerned, the change of conformation affects the tertiary structure only.
Because of the common pH-dependency the further three low spin signals (g1= 2.91, g2= 2.23, g3= 1.95) have to be attributed to the H2O-Hb(III) in addition to the high spin signals (g′= 5.9, g′= 2.0), which characterize the low spin part in the physical high spin-low spin equilibrium of this complex.
Further the origin of the conformational active CD bands is discussed.