Cleavage of fibrin-derived D-dimer into monomers by endopeptidase from puff adder venom (Bitis arietans) acting at cross-linked sites of the .gamma.-chain. Sequence of carboxy-terminal cyanogen bromide .gamma.-chain fragments

Abstract

Puff adder venom contains a protease capable of cleaving the .gamma.-chain of cross-liked D-dimer, derived from the plasmin digestion of fibrin, into apparently symmetrical monomers. The cross-linked .gamma.-chains are separated in the process without apparent loss of mass and without loss of the substitutent at the glutamine cross-link site, if fluorescent D-dimer (the lysine analogue dansylcadaverine used as substitutent) is used as substrate (Purves, L. R., Purves, M., Lindsey, G. G. and Linton, N. J. (1986) S. Afr. J. Sci. 82, 30]. The .gamma.-chain from puff adder venom digested D-monomer was isolated and cleaved by cyanogen bromide, and the carboxy-terminal peptide was isolated and sequenced. The carboxy-terminal peptide composition indicated a lower content of histidine, leucine, and glycine than expected. Manual microsequencing by gas-phase Edman degradation demonstrated that two amino-terminal ends were present. By use of the known sequence of the human fibrinogen .gamma.-chain, the sequencing data could be resolved into a dipeptide cross-linked between lysine-406 and either glutamine-398 or -399 (residues 6 and 13 or 14 from the carboxy-terminal end of the .gamma.-chain) with the loss of residues 401-404 that occur between the cross-link sites of both antiparallel cross-linked .gamma.-chains. D-dimer is therefore separated into monomers by cleavage of the .gamma.-chain between the cross-link sites. Two symmetrical fragments are produced consisting of a cross-linking dipeptide with the loss of four amino acids. There is heterogeneous loss of 401HLGG406 and possibility also 399Q and 400H in subfractions of the peptide. The predominant cross-linked glutamine appears to be residue 399, 13 residues from the carboxy-terminal end.