Urinary protein C inhibitor binding region in the A alpha-chain of human fibrinogen.

Abstract

This paper describes the binding region of urinary protein C inhibitor (PCI) in the A alpha-chain of human fibrinogen. The A alpha-chain was digested by staphylococcal V8 protease, yielding five main peptides consisting of 10, 11, 11.5, 12 and 14 kDA bands on SDS-PAGE. These peptides were electroblotted to nitrocellulose and PVDF membranes, where ligand blotting against urinary PCI and the N-terminal amino acid sequencing were performed, respectively. The 14 (main band), 13 and 16 kDA bands (faint bands) bound to urinary PCI. These bands were directly sequenced, indicating that all of these N-terminal amino acid sequence was ITRGGST, corresponding to I250 to T256 in the A alpha-chain. The N-terminal amino acids of the 14 kDa band contained I (main peak) and P (small peak). To precisely determine the N-terminal sequence of the 14 kDa peptide, the band on PVDF membrane was eluted with 70% acetic acid and the eluate was further purified by reversed-HPLC. The peak showing urinary PCI binding was sequenced, indicating that its N-terminal amino acid sequence was also ITRGGST. Consequently, we showed that the urinary PCI binding region in the A alpha-chain was from I250 to E357 (or E370), judging by the N-terminal amino acid sequence and the molecular weight of the peptide.