Photo-oxidation of histidine as a probe for aminoterminal conformational changes during fibrinogen-fibrin conversion

Abstract.

Fibrinogen is known to become unclottable when irradiated with light in the presence of methylene blue, the loss of clottability being due to photo-oxidation of the histidine at position 16 of the B<beta> chain. In the present investigation it could be demonstrated that not only this histidine but also the one at position 24 of the A<alpha> chain was modified and that the rates of modification could be modulated by fibrinopeptide release, polymerization inhibition and denaturation. Accordingly, the histidine modifications can be used as probes for surface accessibility of and conformational differences among the various forms of the protein. Both histidines are shielded by the fibrin polymer structure. Fibrinopeptide A release alone leads to maximal protection of the one in the A<alpha> chain, but only partial protection of the one in the B<beta> chain. Subsequent fibrinopeptide B release leads to maximal protection of the one in the B<beta> chain. The differential effects indicate that two conformational changes have occurred. Polymerization inhibition reverses the protective effect. Denaturation leads to maximal and equal modification in all samples as a consequence of the loss of native conformation.