Calcium and thiol reactivity of human plasma clotting factor XIII

1. The reaction of iodoacetate, 2-chloromercuri-4-nitrophenol and 5,5'-dithiobis-(2-nitrobenzoate) with thrombin-cleaved Factor XIII (i.e. Factor XIII(a)) was accompanied by enzyme inhibition. 2. The reaction with iodoacetate and 5,5'-dithiobis-(2-nitrobenzoate) was absolutely dependent on Ca(2+), and the rate of reaction increased with the Ca(2+) concentration up to very high, non-physiological concentrations. 3. 2-Chloromercuri-4-nitrophenol reacted with Factor XIII(a) in the absence of Ca(2+), but at a much slower rate. 4. Stopped-flow methods were used to quantify the reaction with 5,5'-dithiobis-(2-nitro-benzoate) because of the Ca(2+)-dependent dissociation of Factor XIII(a) (a'(2)b(2)) and subsequent aggregation of the a' chains into turbid precipitates. 5. The 3-carboxy-4-nitrothio-phenolate released was consistent with the reaction of 2 thiol groups/molecule of Factor XIII(a). The isolated b chains of Factor XIII did not react with either of the chromophoric reagents. This indicated that the a' chains of Factor XIII(a) were responsible for the thiol reactivity of the enzyme. 6. The Ca(2+) dependence of the enzyme inhibition by these thiol reagents was very dependent on protein concentration. This is discussed in relation to the Ca(2+)-induced dissociation of Factor XIII(a). 7. The acceptor substrate, casein, decreased the Ca(2+) concentration required for enzyme inhibition by both the mercurial and the aromatic disulphide compounds. Dansylcadaverine did not affect Ca(2+) dependence of inhibition.