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

Research Project

Project/Area Number 03670037
Research Category

Grant-in-Aid for General Scientific Research (C)

Allocation TypeSingle-year Grants
Research Field General physiology
Research InstitutionOsaka University

Principal Investigator

IMAI Kiyohiro  Osaka Univ. Med. Sch. Dept. of Physiol. Assoc. Prof., 医学部, 助教授 (50028528)

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)
Project Period (FY) 1991 – 1992
Project Status Completed (Fiscal Year 1992)
Budget Amount *help
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1992: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1991: ¥1,400,000 (Direct Cost: ¥1,400,000)
KeywordsArtificial mutants / Hemoglobin / Site-directed mutagenesis / Protein engineering / Functional differentiation / Function mimicry / Crocodile hemoglobin / Bovine hemoglobin / 生理機能の分化 / 人工変異蛋白質 / アミノ酸置換 / ワニ / 炭酸水素イオン / 酸素親和性
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.

Report

(3 results)
  • 1992 Annual Research Report   Final Research Report Summary
  • 1991 Annual Research Report

Research Products

(8 results)

All Other

All Publications (8 results)

  • [Publications] K.IMAI: "Site-directed mutagenesis in haemoglobin.Functional role of tyrosine-42(C7)α at the α1-β2 interface" Journal of Molecular Biology. 218. 769-778 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] K.ISHIMORI: "Site-directed mutagenesis in hemoglobin:Functional and structural role of inter-and intrasubunit hydrogen bonds as studied with 37β and 145β mutations" Biochenistry. 31. 3256-3264 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] 今井 清博: "ヘモグロビンの改変" 細胞工学. 12. 64-66 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] I. Imai, K. Fushitani, G. Miyazaki, K. Ishimori, T. Kitagawa, Y. Wada, H. Morimoto, I. Morishima, D. T.-B. Shih & J. Tame: "Site-directed mutagenesis in hemoglobin. Functional role of tyrosine-42(C7)alpha at the alpha1-beta2 interface" J. Mol. Biol. 218. 769-778 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] K. Ishimori, K. Imai, G. Miyazaki, T. Kitagawa, Y. Wada, H. Morimoto & I. Morishima: "Site-directed mutagenesis in hemoglobin: Functional and structural role of inter- and intrasubunit hydrogen bonds as studied with 37beta and 145beta mutations" Biochemistry. 31(12). 3256-3264 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] K. Imai: "Renovation of hemoglobin(in Japanese)" Cell Technology. 12(1). 64-66 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] 今井 清博: "ヘモグロビンの改変" 細胞工学. 12. 64-66 (1993)

    • Related Report
      1992 Annual Research Report
  • [Publications] 宮崎 源太郎: "遺伝子工学によるヘモグロビンの酸素親和性の分子進化の研究" 生物物理. 31. S60 (1990)

    • Related Report
      1991 Annual Research Report

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Published: 1991-03-31   Modified: 2016-04-21  

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