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

人工突变体对血红蛋白生理功能分化的研究

基本信息

批准号：
03670037
负责人：
IMAI Kiyohiro
金额：
$ 1.22万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

项目摘要

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.

不同动物的血红蛋白的氧运输功能因其生活环境的不同而不同。为了模拟鳄鱼血红蛋白1、2、90、94、135、143和144位氨基酸残基的全部或部分残基，模拟鳄鱼血红蛋白的功能，我们合成了3个突变体，分别与碳酸氢钠、HCO3_3、分子CO2和2，3-二磷酸甘油酸(DPG)反应。在不同溶液条件下测得的HbAβ链的氧平衡曲线表明，DPG的作用几乎如预期的那样消失，但HCO^-_的作用比HbA的作用要小得多。我们还测定了天然的凯曼和尼罗河鳄鱼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HbA的突变体来模拟本质上降低了氧亲和力的牛血红蛋白的功能。根据Perutz和Imai(1980)的假设，在一个突变体中，2beta位的His被Phe取代，而在另一个突变体中，1beta位的Val被缺失，2beta位的His被Met取代。DPG对这些突变体的作用如预期的那样降低，但氧亲和力并没有降低，而是略有增加。这些结果表明，目前的氨基酸替代不能实现鳄鱼Hb对HCO^-_的特异性反应，而牛Hb的亲和力固有地降低，需要其他一些替代。本研究是在英国剑桥MRC分子生物学实验室K.Nagai博士及其合作者的合作下进行的。