Stoichiometry of binding of high molecular weight kininogen to factor XI/XIa

Factor XI is a dimeric protein and circulates in plasma complexed with high molecular weight kininogen (HMWK). We investigated the binding of HMWK to factor XIa utilizing two active site directed fluorescent probes: nitrobenzoxadiazole aminopentyl methylphosphonofluoridate for serine and dansyl-glu-gly-arg-chloromethyl ketone for histidine. In the presence of saturating amounts of HMWK, the fluorescence of factor XIa-fluorophore was quenched by approximately 28% for each probe. Titrations of the fluorescent factor XIa with HMWK revealed that each subunit of factor XIa binds one molecule of HMWK with a Kd approximately 3.4 X 10(-8)M.