電気泳動法を用いたフィブリノゲン異常症および欠損症の解析

Abstract

Fibrinogen Matsumoto II is a hereditary dysfibrinogenemia with heterozygous missense mutation, γ308 Asn->Lys. The propositus's fibrinogen analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) using the Laemmli's buffer system under reducing conditions and Western blotting revealed two distinct bands corresponding to the γ-chain; one with an apparently normal molecular weight (MW) (47, 500) and the other with a lower MW (45, 500). To better define the characteristics on SDS-PAGE of γ308 Lys mutant γ-chain, we synthesized three fibrinogens with single amino acid substitution at this residue: γ308 Lys, γ308 Ile, γ308 Ala. We found the MW of the γ-chains derived from recombinant fibrinogens, γ308 Asn, γ308 Ala, γ 308Ile, and γ308 Lys, were 48.5, 48.5, 47.0, and 46.0kDa, respectively. These results demonstrated the lower MW of mutant γ-chain derived from plasma fibrinogen of Matsumoto II and Baltimore III (γ308 Asn->Ile) propositus due to the single amino acid substitution. The increased mobility of the mutant γ-chain could be attributed to the binding properties of SDS to the peptide. It is well-known that the increased or decreased SDS binding induces complicated changes in the electrophoretic mobility, however, we speculate the mechanism of the increased mobility of the protein is followings. Since SDS preferentially binds either basic or hydrophobic amino acids, the mutant peptide increased basicity or hydrophobicity bind much more SDS. Then the increased SDS binding may lead to complicated change of conformation and/or net charge of the peptide. Finally these phenomena may result in faster mobility on SDS-PAGE and lead decreased MW of the peptide.