LIMITED PROTEOLYSIS OF FIBRINOGEN αC-REGION REVEALS ITS STRUCTURE

Abstract

Aim. The purpose of our study was to compare hydrolytic action of proteases from Gloydius halys halys, Agkistrodon contortrix contortrix and Calloselasma rhodostoma rhodostoma snake venoms and from Bacillus thuringiensis вар. israelensis IMV B-7465 culture medium on αC-regions of fibrinogen molecule. Methods. Products of hydrolysis were characterized by SDS-PAGE under reducing conditions with following Western-Blot using the mouse monoclonal 1-5А (anti-Aα509-610) and ІІ-5С (anti-Aα20-78) antibody. MALDI-TOF analysis of fibrinogen hydrolysis products was performed using a Voyager-DE. Results. Combination of SDS-PAGE, FPLC and MALDI-TOF analysis enabled to detect the peptide bonds cleaved by studied proteases. In particular proteases from Gloydius halys halys and Agkistrodon contortrix contortrix snake venoms cleaved peptide bond Aα413-414. Action of protease from Calloselasma rhodostoma rhodostoma on fibrinogen led to the formation of hydrolytic product generated from C-terminal portion of Aα-chain that corresponded to fragments generated by enzymes from two other snakes. On the other hand protease from Bacillus thuringiensis вар. israelensis IMV B-7465 culture medium cleaved peptide bond Aα504-505. Conclusions. Use of limited proteolysis technique as the source of additional information for computer modeling allowed us to propose an improved model of 3D-structure of fibrinogen αC-regions. This model takes into account the behavior of αC-regions in the physiological condition and contributes to the general knowledge about fibrinogen structure.