Immunochemical and molecular investigation of fetal hemoglobin by synthetic overlapping peptides representing the entire gamma chain

Research Project

Project/Area Number	02454216
Research Category	Grant-in-Aid for General Scientific Research (B)
Allocation Type	Single-year Grants
Research Field	Legal medicine
Research Institution	Akita University
Principal Investigator	YOSHIOKA Naofumi Akita University School of Medicine, Professor, 医学部, 教授 (80108935)
Co-Investigator(Kenkyū-buntansha)	SHIDARA Yoshihiro Akita University School of Medicine, Assistant, 医学部, 助手 (40187357) 村田誠秋田大学, 医学部, 助手 (00166290)
Project Period (FY)	1990 – 1991
Project Status	Completed (Fiscal Year 1991)
Budget Amount *help	¥7,000,000 (Direct Cost: ¥7,000,000) Fiscal Year 1991: ¥1,000,000 (Direct Cost: ¥1,000,000) Fiscal Year 1990: ¥6,000,000 (Direct Cost: ¥6,000,000)
Keywords	Adult hemoglobin / Fetal hemoglobin / Synthetic peptide / Antigenic site / Epitope
Research Abstract	Fetal hemoglobin (Hb F) is mainly composed of alpha and gamma subunits and the total amounts of amino acid of gamma chain are 146 residues. The antigenic sites of alpha and beta chains are already reported but not of gamma chain. About 73 % of homologies of amino acid sequence are existing between gamma and beta chain. It is considered to be interesting to define the localization of antigenic sites (antibody binding sites) on gamma chain molecule and then to make a comparison of antigenicity with beta chain. In this study, according to the known primary amino acid structure of gamma chain, 10 peptides with 20 residues each were synthesized by semi-automatic solid-phase synthesiser. The strategy relied on the synthesis of the entire polypeptide chain in 20-residue peptides, each overlapping its two adjacent neighbours by six residues on both sides. Antigenicity of each peptide was assayed by means of regular ELISA and dot-ELISA methods. Several peptides exhibited considerable antibody binding capacity and were unambiguously active. These peptides were : 1-20, 15-34, 29-48, 71-90 and 113-132 with polyclonal rabbit anti Hb F sera, 57-76, 113-132 and 127-146 with monoclonal antibodies to Hb alpha and beta chain, and 57-76 and 127-146 with monoclonal antibody to Hb A. By these results, it is concluded that there are at least three antibody binding sites on gamma chain molecule and residues 71-90 can be a specific antigenic site for gamma chain comparing homology with amino acid structure of beta chain.