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1991 Fiscal Year Final Research Report Summary

Involvement of molten globule state on the folding process of secretary proteins

Research Project

Project/Area Number 02660094
Research Category

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

Allocation TypeSingle-year Grants
Research Field 応用生物化学・栄養化学
Research InstitutionKyoto University

Principal Investigator

HIROSE Masaaki  Kyoto University, Research Institute for Food Science, Professor, 食糧科学研究所, 教授 (60026523)

Co-Investigator(Kenkyū-buntansha) YAMASHITA Honami  Kyoto University, Research Institute for Food Science, Research associate, 食糧科学研究所, 教務職員
Project Period (FY) 1990 – 1991
Keywordsmolten globule / secretary protein / formation of conformation
Research Abstract

Human serum albumin(HSA), N-terminal half-molecule of ovotransferrin(N-OVT), and ovalbumin(OVA)were reduced and denatured in the presence of dithiothreitol and 8 M urea. The samples were diluted with a neutral buffer, and protein conformation was evaluated by CD-spectrum. With regards to HSA and N-OVT, the refolded, disulfide-reduced form was found to take a partially folded molten globule-like conformation. When oxidized form of gultathione(GSSG)was added to the state, the intrachain protein disulfide bonds were regenerated. These data indicated that HSA as well as N-OVT take a molten globulelike state during oxidative refolding as an intermediate. In contrast, OVA showed the native-like conformation in its disulfide-reduced state as evaluated by CD-spectrum. The reactivity of cysteine sulfhydryls, however, was significantly different between the native form and the refolded, disulfide-reduced form : no reactive sulfhydryl was detected in the former form, but two sulfhydryls were detected in the latter form. By the addition of GSSG to the reduced state, ovalbumin was transformed to the disulfide bonded form with native cyteine pairing(Cys73-CYsl2O). From these data, we concluded that a protein with many disulfide and domain structure, such as HSA and N-OVT, takes a molten globule-like conformation as a intermediate for oxidative refolding, while a protein with single disulfide and single domain, such as OVA, takes a naive-like conformation in the disulfide-reduced state.

  • Research Products

    (8 results)

All Other

All Publications (8 results)

  • [Publications] Mikami,Bunzo: "Crystallization of and preliminary crystallographic data for the N-terminal half-molecule of ovotransferrin." Journal of Biochemistry. 108. 907-908 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Takahashi,Nobuyuki: "Determination of sulfhydryl groups and disulfide bonds in a protein by polyacrylamide gel electrophoresis." Analytics Biochemistry. 188. 359-365 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Hirose,Masaaki: "Partially folded state of disulfide-reduced N-terminal half-molecule of ovotransferrin as a renaturation intermediate." The Journal of Biologycal Chemistry. 266. 1463-1468 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Takahashi,Nobuyuki: "Reversible denaturation of disulfide-reduced ovalbumin and its reoxidation generating the native cystine cross-link." The Journal of Biologycal Chemistry. 267. (1992)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Mikami Bunzo: "Crystallization of and preliminary crystallographic data for the N-terminal half-molecule of ovotransferrin." Journal of Biochemistry. 108. 907-908 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Takahashi Nobuyuki: "Determination of sulfhydryl groups and disulfide bonds in a protein by polyacylamide gel electrophoresis." Analytical Biochemistry. 188. 359-365 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Hirose Masaaki: "Partially folded state of disulfide-reduced N-terminal half-molecule of ovotransferrin as a renaturation intermediate." The Journal of Biological Chemistry. 266. 1463-1468 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Takahashi Nobuyuki: "Reversible denaturation of disulfide reduced ovalbumin and its reoxidation generating the native cystine cross-link." The Journal of Biological Chemistry. 267. (1992)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 1993-03-16  

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