Isolation and characterization of a new hemoglobin derivative cross-linked between the alpha chains (lysine 99 alpha 1----lysine 99 alpha 2).
Isolation and characterization of a new hemoglobin derivative cross-linked between the alpha chains (lysine 99 alpha 1----lysine 99 alpha 2).
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DOI：
10.1016/s0021-9258(18)67605-7
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发表时间：
1986-07
期刊：
影响因子：
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通讯作者：
R. Chatterjee;E. Welty;R. Walder;S. Pruitt;P. Rogers;A. Arnone;J. A. Walder
中科院分区：
文献类型：
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作者：
R. Chatterjee;E. Welty;R. Walder;S. Pruitt;P. Rogers;A. Arnone;J. A. Walder
双（3，5-二溴水杨基）富马酸酯和许多相关的双功能试剂优先与氧合血红蛋白反应，以交联2，3-二磷酸甘油酸结合位点内的β链。在本报告中，我们描述了一种在α链之间交联的新衍生物，该衍生物是在与脱氧血红蛋白的反应中专门形成的。X射线晶体学研究表明，交联位于Lys-99 α 1和Lys-99 α 2之间，跨越四聚体的中心空腔。Lys-99 α 1和Lys-99 α 2位于非常靠近血红蛋白分子中间的带电残基簇内。在氧合血红蛋白中，交联剂完全无法进入该位点。与肌醇六磷酸的竞争实验表明，该化合物通过α链之间的裂缝进入脱氧血红蛋白的中心空腔。尽管存在α链之间的交联，但修饰的血红蛋白保持高度合作。HbXL 99 α的希尔系数为2.6。交联衍生物的氧亲和力降低约2倍;在pH 7.0下，在0.1 M NaCl存在下，P50为13.9 mm Hg，而HbA为6.6 mm Hg。这种差异似乎是由于KR（氧与R态结合的缔合常数）和变构常数L的相对较小的变化所致。令人惊讶的是，oxyHbXL 99 α的等电点与oxyHbA的等电点几乎相同，而在脱氧形式中，交联衍生物的等电点相对于天然血红蛋白降低，如所预期的，这是由于修饰的氨基的两个正电荷的损失。与这些发现一致，HbXL 99 α的碱性玻尔效应降低了50%以上。早期的研究强烈反对Lys-99 α直接导致HbA中玻尔效应的可能性。结构分析表明，在交联衍生物Glu-101 β中，它与氧合血红蛋白中的Lys-99 α非常接近，成为酸性玻尔基团。
Bis(3,5-dibromosalicyl) fumarate and a number of related bifunctional reagents react preferentially with oxyhemoglobin to cross-link the beta chains within the 2,3-diphosphoglycerate-binding site. In this report we describe a new derivative cross-linked between the alpha chains which is formed specifically in the reaction with deoxyhemoglobin. X-ray crystallographic studies show that the cross-link lies between Lys-99 alpha 1 and Lys-99 alpha 2, spanning the central cavity of the tetramer. Lys-99 alpha 1 and Lys-99 alpha 2 are located within a cluster of charged residues very near the middle of the hemoglobin molecule. In oxyhemoglobin, this site is completely inaccessible to the cross-linking agent. Competition experiments with inositol hexaphosphate indicate that the compound enters the central cavity in deoxyhemoglobin through the cleft between the alpha chains. Despite the presence of the cross-link between the alpha chains, the modified hemoglobin remains highly cooperative. The Hill coefficient for HbXL99 alpha is 2.6. The oxygen affinity of the cross-linked derivative is decreased by approximately 2-fold; at pH 7.0 in the presence of 0.1 M NaCl the P50 is 13.9 mm Hg compared to 6.6 mm Hg for HbA. This difference appears to be due to relatively small changes in both KR, the association constant for binding of oxygen to the R state, and the allosteric constant L. Surprisingly, the isoelectric point of oxyHbXL99 alpha is almost identical to that of oxyHbA, whereas in the deoxy form the isoelectric point of the cross-linked derivative is decreased relative to native hemoglobin as expected due to the loss of the two positive charges of the modified amino groups. In agreement with these findings, the alkaline Bohr effect of HbXL99 alpha is decreased by more than 50%. Earlier studies argue strongly against the possibility that Lys-99 alpha is directly responsible for this large fraction of the Bohr effect in HbA. Analysis of the structure suggests that in the cross-linked derivative Glu-101 beta, which is in close proximity to Lys-99 alpha in oxyhemoglobin, becomes an acid Bohr group.