High and low oxygen affinity conformations of T state hemoglobin
High and low oxygen affinity conformations of T state hemoglobin
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DOI：
10.1110/ps.20501
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发表时间：
2001-11-01
期刊：
影响因子：
8
通讯作者：
Mozzarelli, A
中科院分区：
文献类型：
--
作者：
Bruno, S;Bonaccio, M;Mozzarelli, A
为了了解与血红蛋白功能和调节相关的三级和四级转换之间的相互作用，通过在湿多孔二氧化硅中封装固定在T四级状态的血红蛋白A获得氧结合曲线。在pH7.0和15 ℃下，在不存在和存在强变构效应物肌醇六磷酸和苯扎贝特的情况下制备的血红蛋白凝胶的半饱和氧压（p50）分别为12.4 +/- 0.2和139 +/- 4 mmHg。这两个值都是在很好的协议，发现在类似的实验条件下，在溶液中的血红蛋白的第一个氧的结合。血红蛋白凝胶的相应希尔系数为0.94 +/- 0.02和0.93 +/- 0.03，表明在莫诺、怀曼和尚格模型的框架中，高和低氧亲和力的三级T态构象已经以纯的形式分离出来。数值略低于1，反映了α-和β-血红素不同的氧亲和力.值得注意的是，在弱效应磷酸盐存在下包封的血红蛋白导致显示中等氧亲和力和0.7至0.8的希尔系数的凝胶。异质氧结合的结果从存在的高和低的氧亲和力T状态的混合物。玻尔效应进行了测量血红蛋白凝胶含有纯的构象，并发现是更明显的高亲和力的T状态和低亲和力的T状态几乎不存在。这些发现表明，T四级状态的功能特性是由两种不同的相互转换构象的贡献造成的，其特征是氧亲和力差异10倍和不同程度的三级波尔效应。在溶液中和结晶状态下观察到的T-状态协同性的非常小的程度可能来自于配体诱导的高亲和力和低亲和力T-状态构象之间的分布扰动。
To understand the interplay between tertiary and quaternary transitions associated with hemoglobin function and regulation, oxygen binding curves were obtained for hemoglobin A fixed in the T quaternary state by encapsulation in wet porous silica gets. At pH 7.0 and 15 degreesC, the oxygen pressure at half saturation (p50) was measured to be 12.4 +/- 0.2 and 139 +/- 4 torr for hemoglobin gels prepared in the absence and presence of the strong allosteric effectors inositol hexaphosphate and bezafibrate, respectively. Both values are in excellent agreement with those found for the binding of the first oxygen to hemoglobin in solution under similar experimental conditions. The corresponding Hill coefficients of hemoglobin gels were 0.94 +/- 0.02 and 0.93 +/- 0.03, indicating, in the frame of the Monod, Wyman, and Changeux model, that high and low oxygen-affinity tertiary T-state conformations have been isolated in a pure form. The values, slightly lower than unity, reflect the different oxygen affinity of alpha- and beta -hemes. Significantly, hemoglobin encapsulated in the presence of the weak effector phosphate led to gels that show intermediate oxygen affinity and Hill coefficients of 0.7 to 0.8. The heterogeneous oxygen binding results from the presence of a mixture of the high and low oxygen-affinity T states. The Bohr effect was measured for hemoglobin gels containing the pure conformations and found to be more pronounced for the high-affinity T state and almost absent for the low-affinity T state. These findings indicate that the functional properties of the T quaternary state result from the contribution of two distinct, interconverting conformations, characterized by a 10-fold difference in oxygen affinity and a different extent of tertiary Bohr effect. The very small degree of T-state cooperativity observed in solution and in the crystalline state might arise from a ligand-induced perturbation of the distribution between the high- and low-affinity T-state conformations.