Equilibriums involved in prothrombin- and blood-clotting factor X-membrane binding

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Abstract
The study of prothrombin- and factor X [bovine]-membrane interaction by light-scattering intensity measurements at 90.degree. is reported. This technique, which uses a fluorometer as a light-scattering photometer, can be applied to measurement of free and membrane-bound protein concentrations, from which equilibrium constants can be obtained. The following equilibria adequately describe the observed properties of prothrombin-membrane interaction .**GRAPHIC**. where P and PL are protein and phospholipid, PiCa and PLjCa are the Ca complexes, P''iCa is the protein after undergoing a Ca dependent transition, and P''.cntdot.PLi+j+mCa is the protein-membrane complex. Several lines of evidence indicate that i, j and m are interrelated and m decreases to 0 when i and j are saturated. In agreement with this, direct Ca binding measurements indicate m values of 3.2 .+-. 1.5 and 1.1 .+-. 1.5 at 0.5 and 1.2 mM Ca, respectively. The total number of functional Ca ions in the complex (i + j + m) is 6-9 based on Hill coefficients for the reactions and direct Ca binding measurements. In reaction 3, the maximum stoichiometry of Ca per acidic phospholipid is 1:2. While the details of factor X-membrane binding were not determined in quite as great detail, the equilibria (identified) appear the same but a major difference is the Ca concentration needed to initiate protein-membrane binding.