Investigation of the binding of calcium to prothrombin by two dimensional immuno electrophoresis

The reported binding constant of calcium to purified prothrombin based on equilibrium dialysis is K<104 . This study investigated the ability of EDTA, EGTA, to remove calcium from the normal prothrombin, in the plasma of patients treated with warfarin.Coumarin plasma was obtained from freshly drawn venous blood of patients on warfarin. This plasma contains prothrombin with no GLA’s, which does not bind calcium, and prothrombin with GLA’s, which does bind calcium. The plasma anti coagulated with EDTA or EGTA was subjected to two dimensional immuno electrophoresis (2DIE). In parallel experiments the plasma was dialysed against normal saline containing EDTA 2mM for 24 hours. The dialysis was performed at pH 8,9 and 10, and the dialysed plasmas, subiected to 2DIERESULTS All plasmas, irrespective of the anti coagulant or pH of dialysis exhibited the same two prothrombin peaks on 2DIE, which indicates that the normal prothrombin had retained its bound calcium. This suggests that native prothrombin binds calcium with a K assos>l010 the binding constant for EDTA and calcium at pHl0.0.