Equilibria Involved in Prothrombin- and Blood-Clotting Factor X-Membrane Binding

The study of prothrombin- and factor X-membrane interaction by light-scattering intensity measurements at 90° 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 [formula omitted] where P and PL are protein and phospholipid, P_ica and PL_jca are the calcium complexes, P′_ica is the protein after undergoing a calcium dependent transition, and P′•PL_i+y+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 calcium binding measurements indicate m values of 3.2 ± 1.5 and 1.1 ± 1.5 at 0.5 and 1.2 mM calcium, respectively. The total number of functional calcium ions in the complex (i + j + m) is 6 to 9 based on Hill coefficients for the reactions and direct calcium binding measurements. In reaction 3, the maximum stoichiometry of calcium 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 calcium concentration needed to initiate protein-membrane binding.