Study on the differentiation of physiological function of hemoglobin using artificial mutants

各种动物血红蛋白的运氧功能因其生存环境的不同而不同。在本研究中，我们打算用基于位点定向诱变的蛋白质工程技术合成人成人血红蛋白（Hb A）的人工突变体来模拟其他动物血红蛋白的生理功能。鳄鱼血红蛋白对碳酸氢盐、HCO^3_有特殊反应，但对CO_2分子和2,3-二磷酸甘油酸（DPG）没有反应。为了模拟鳄鱼血红蛋白的功能，我们通过替换1、2、90、94、135、143和144位点上的全部或部分氨基酸残基合成了三个突变体。在不同的溶液条件下测定了这些突变体Hbs的氧平衡曲线，结果表明DPG的影响与预期的一样几乎被消除，但HCO^-_的影响相对于Hb a有所降低。我们还测量了天然凯门鳄和尼罗鳄血红蛋白在相同条件下的氧平衡曲线。此外，我们合成了两个Hb A突变体来模拟牛血红蛋白的功能，这两个突变体本质上降低了氧亲和力，并且对DPG没有反应。根据Perutz和Imai（1980）的假设，在其中一个突变体中，2beta处的His被Phe取代，而在另一个突变体中，1beta处的Val被删除，2beta处的His被Met取代。DPG对这些突变体的作用如预期的那样减弱，但氧亲和力没有降低，反而略有增加。这些结果表明，目前的氨基酸替代不能单独实现鳄鱼Hb对HCO^-_的特异性反应和牛Hb的内在亲和力降低，需要其他替代。本研究是在英国剑桥MRC分子生物学实验室的Nagai博士及其合作者的合作下进行的

The oxygen-transport function of hemoglobin of various animals is differentiated depending on their living environments. In the present study, we intended to mimic the physiological function of other animals' hemoglobins with artificial mutants of human adult hemoglobin (Hb A) which are synthesized by the protein engineering technique based on site-directed mutagenesis.To mimic the function of crocodilian hemoglobin, which specifically responds to bicarbonate, HCO^3_, but does not to molecular CO_2 and 2,3- diphosphoglycerate (DPG), we synthesized three mutants by replacing all or some of the amino acid residures at the sites, 1,2,90,94,135,143, and 144, of the beta chains of Hb A. Oxygen equilibrium curves of these mutant Hbs measured under a variety of solution conditions indicated that the effect of DPG was almost abolished just as expected but the effect of HCO^-_ was rather decreased compared to that for Hb A. We also measured oxygen equilibrium curves of natural Caiman and Nile crocodile hemoglobins under the same conditions as those for the mutant Hbs.Further, we synthesized two mutants of Hb A to mimic the function of bovine hemoglobin which has intrinsically lowered oxygen affinity and does not respond to DPG. According to the hypothesis by Perutz and Imai (1980), the His at 2beta was replaced by Phe in one of the mutants and the Val at 1beta was deleted and the His at 2beta was replaced by Met in the other mutant. The effect of DPG for these mutants was decrease as expected but the oxygen affinity was not lowered and rather slightly increased.These results indicate that the present amino acid replacements alone do not realize the specific response of crocodilian Hb to HCO^-_ and the intrinsically lowered affinity of bovine Hb, and some other replacements are needed.The present study was conducted under cooperation of Dr. K. Nagai and his collaborators at MRC Laboratory of Molecular Biology, Cambridge, U.K.