1992 Fiscal Year Final Research Report Summary
Study on the differentiation of physiological function of hemoglobin using artificial mutants
| Project/Area Number |
03670037
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
General physiology
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| Research Institution | Osaka University |
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Principal Investigator |
IMAI Kiyohiro Osaka Univ. Med. Sch. Dept. of Physiol. Assoc. Prof., 医学部, 助教授 (50028528)
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| Co-Investigator(Kenkyū-buntansha) |
MIYAZAKI Gentaro Osaka Univ., Fac. of Eng. Sci., Dept. of Biophys Eng., Res. Assoc., 基礎工学部, 教務職員 (50166146)
WATANABE Manabu Osaka Univ. Med. Sch. Dept. of Physiol. Res. Assoc., 医学部, 助手 (30182950)
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| Project Period (FY) |
1991 – 1992
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| Keywords | Artificial mutants / Hemoglobin / Site-directed mutagenesis / Protein engineering / Functional differentiation / Function mimicry / Crocodile hemoglobin / Bovine hemoglobin |
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| Research Abstract |
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.
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Research Products
(6 results)
