CHEMICAL AND ENZYMIC DETECTION OF PROTEIN CROSS-LINKS . MEASUREMENT OF EPSILON-(GAMMA-GLUTAMYL)LYSINE IN FIBRIN POLYMERIZED BY FACTOR 8

A chemical and an enzymic method were compared with respect to their ability to detect crosslinks in human fibrin. The former method consisted of cyanoethylation followed by acid hydrolysis, a procedure under which ϵ-amino cross-linked lysine yields free lysine, whereas lysine elsewhere in the molecule yields an N-carboxyethyl derivative; 1-2 moles of e-amino cross-linked lysine was found per mole of fibrin polymerized by factor XIII; much less was found when polymerization was competitively inhibited with glycine ethyl ester; very little was found when polymerization was prevented by removing Ca2+ with EDTA. Very low levels of e-amino cross-linked lysine were detected in fibrin prepared from factor XIII poor human fibrinogen, but the level was greatly increasedby the addition of human factor XIII. Amounts of ϵ-(γ-glutamyl)lysine separated from total enzymic hydrolysates of fibrin formed under each of these conditions were in quantitative agreement with those of c-amino cross-linked lysine measured by the chemical method. In addition to providinga direct demonstration of the ϵ-(γ-glutamyl)lysine cross-link in polymerized human fibrin, this agreement indicated (1) that lysine is not cross-linked to any acceptor other than glutamate and (2) that the cyanoethylation technique is a valid procedure for detecting ϵ-lysyl cross-links in proteins. © 1969, American Chemical Society. All rights reserved.