Effect of limited modification of amino groups on the reactivity of human factor X a

Abstract

The basis of the specificity of human coagulation factor X a has been probed with a reagent that reacts with nucleophiles, N-succinimidylpropionate. At pH 8.0 and 0.25 mM N-succinimidylpropionate, 0.4 μM factor X a lost approx. 90% of its activity toward prothrombin in 4 min. The decay was first-order, k = 0.64 min −1, which increased to 0.98 min −1 in 1 mM Ca 2+, and the dependence of k upon pH was consistent with primary amines being the target. The rate of modification was unaffected by the presence of a tetrapeptide substrate during modification; likewise, activity toward a tripeptide p-nitroanilide was unaltered during exposure of factor X a to N-succinimidylpropionate with or without Ca 2+. In addition, inhibition by antithrombin III was retained with a somewhat enhanced rate after modification; however, the acceleration of this by heparin was significally less. Kinetic determination of the number of residues modified gave a reaction order of 2.0, while reaction with N- succinimidyl[ 3H]propionate yielded labeled factor X a containing 1.0 mol N-succinimidylpropionate/mol factor X a and 50% normal clotting activity, or 2.0 mol N-succinimidylpropioate/mol and 1% activity, respectively. Thus, one nucleophilic group is required for the reaction of factor X a with prothrombin but not for the hydrolysis of peptides or recognition of antithrombin III. The decay of clotting activity of the factor X zymogen in N-succinimidylpropionate was much slower though still Ca 2+-dependent. Conversely, the reaction of a related compound — N-succinimidyl-(4-hydroxyphenyl)propionate or Bolton-Hunter reagent — with factor X a broadly resembled that of N-succinimidylpropionate but the decay curves indicated more complex kinetics. Therefore, the target groups vary in their accessibility to modification according to the structural characteristics of both the protein and the reagent.