Association of the A subunits of recombinant placental factor XIII with the native carrier B subunits from human plasma
Radek, J.T.; Jeong, J.M.; Wilson, J.; Lorand, L.
Biochemistry 32(14): 3527-3534
1993
ISSN/ISBN: 0006-2960
PMID: 8466897
DOI: 10.1021/bi00065a002
Accession: 008197786
Interactions of a recombinant human placental protein (rA-2) expressed in yeast and considered to be identical to the catalytic A-2 subunits of factor XIII, the fibrin stabilizing factor zymogen, were examined with the native carrier subunits (B-2) of the factor isolated from human plasma. Nondenaturing electrophoresis and HPLC gel-filtration experiments showed a tight binding of rA-2 to B-2 for forming an ensemble similar to that of plasma factor XIII (A-2B-2). In the presence of excess B-2, however, some higher ordered oligomers (rA-2B-n, where n gt 2) were also seen in electrophoresis. The same technique revealed a microheterogeneity in the rA-2 preparation; nevertheless, all isoforms could bind to B-2. By employing an ELISA procedure for measuring free B-2 in mixtures with rA-2, an apparent binding constant of 4 times 10-7 M-1 was derived for the association of rA-2 With B-2. Fluorescence depolarization was used to monitor the heterologous association of rA-2 with fluorescein-labeled B-2-F as well as the dissociation of the rA-2B-2-F structure. The former was characterized by an increase, and the latter by a decrease, in the fluorescence anisotropy of the system. Binding of rA-2 to B-2-F (pH 7.5, mu = 0.315, 37 degree C) was not influenced by low concentrations of Ca-2+ ( ltoreq 30 mM), and rA-2B-2-F proved to be quite stable under these conditions. Much higher concentrations of Ca-2+ as well as higher ionic strengths, were required to dissociate this assembly. By contrast, release of B-2-F from the thrombin-modified rA-2'B-2-F occurred rapidly in the presence of low concentrations of Ca-2+ at low ionic strength. An apparent binding constant of 5.3 times 10-7 M-1 was obtained for the association of rA-2 with B-2-F. A considerably lower value of 3.4 times 10-6 M-1 was measured for the association of thrombin-activated rA-2' with B-2-F, indicating that the thrombin-catalyzed removal of the activation peptide from rA-2 weakens the strength of heterologous association to the carrier subunits by about 2 kcal/mol. The release of B-2-F subunits from the thrombin-modified rA-2'B-2-F ensemble could also be readily elicited with inclusion of monomeric fibrin. Activation by fibrin is attributed to the fact that thrombin-modified rA-2'F binds to monomeric fibrin (K-assoc = 0.4 times 10-6 M-1).