A novel assay for factor XIII based on cross-linking of synthetic peptides: analysis of different substrates.
A novel assay for factor XIII is described that utilizes exclusively small synthetic peptides as substrates for the cross-linking reaction catalyzed by activated factor XIII (FXIIIa). The acyl donor substrate (selection peptide) is immobilized on a microplate via biotin while the acyl acceptor substrate (detection peptide) is labeled with the fluorochrome Oregon green to allow sensitive detection without the need for secondary enzyme systems for signal amplification. Starting with an amino acid sequence from the fibrin gamma-chain (GQQHHLGGAKQAGDV) as a prototype peptide, the influence of amino acid exchanges were investigated with respect to their impact on the FXIIIa-catalyzed reaction. It was found that FXIIIa readily accepts a broad range of substrate peptides, with a proline neighboring the essential lysine having the most detrimental effect. The assay appears to be valuable for the molecular characterization of factor XIII and may be used for a deeper investigation into the substrate requirements of this final enzyme of wound repair, and eventually also for the characterization of other transglutaminases.
Kusch
Manuela
M
Paul Ehrlich Institut, Langen, Germany.
Grundmann
Claudia
C
Keitel
Stefanie
S
Seitz
Rainer
R
König
Herbert
H
D003432:Cross-Linking Reagents / Q000138:chemical synthesis / Q000737:chemistry*; D005033:Ethylmaleimide / Q000494:pharmacology; D005176:Factor XIII / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000737:chemistry*; D006801:Humans; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D012680:Sensitivity and Specificity; D013379:Substrate Specificity
D003432:Cross-Linking Reagents; D010455:Peptides; D005176:Factor XIII; D005033:Ethylmaleimide