Protein-protein interactions in blood clotting. The use of polarization of fluorescence to measure the dissociation of plasma factor XIIIa.
1. Human plasma Factor XIII (the precursor of fibrin-glutamine-fibrin-lysine endo-gamma-glutamyltransferase) was randomly labelled by incubation with fluorescein isothiocyanate. The biochemical properties of the system were unaltered by the label. The polarization of the fluorescein fluorescence attached to the plasma protein was measured and the following conclusions were reached. 2. Factor XIII (a'2b2) does not dissociate in the protein-concentration range 10-500 microgram/ml either with or without added Ca2+. 3. Factor XIIIa (a'2b2) does not dissociate in the absence of Ca2+ in the protein-concentration range 10-500 microgram/ml. 4. Additions of Ca2+ to Factor XIIIa result in a decreased polarization of fluorescence as the tetramer dissociates. The decrease in polarization was the same amplitude at protein concentrations 10-500 microgram/ml and Ca2+ concentrations 2-66 mM and indicates that the overall process is essentially irreversible. The decrease in polarization consisted of fast and slow exponential phases. Both the rate of the fast phase and the proportion of the reaction it represented increased with Ca2+ concentration. 5. A comparison of the rate of dissociation measured by fluorescence polarization and the rate of appearance of enzyme activity in the presence of a protein substrate suggests that the Factor XIII is autoactivated by a soluble a-subunit-containing molecular forming a tight complex with the substrate.
D001798:Blood Proteins / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D055598:Chemical Phenomena; D002621:Chemistry; D005176:Factor XIII / Q000378:metabolism*; D005452:Fluoresceins; D005453:Fluorescence; D007700:Kinetics; D046911:Macromolecular Substances; D008722:Methods; D013917:Thrombin / Q000378:metabolism
D001798:Blood Proteins; D005452:Fluoresceins; D046911:Macromolecular Substances; D005176:Factor XIII; D013917:Thrombin; D002118:Calcium