Isolation of human blood coagulation α-factor X a by soybean trypsin inhibitor-Sepharose chromatography and its active-site titration with fluorescein mono- p-guanidinobenzoate

Abstract

A method based on active-site affinity chromatography on soybean trypsin inhibitor (SBTI)-Sepharose was developed for isolation of human factor X a in primarily the undegraded α-form. The chromatography procedure separated factor X a from factor X, the Russell's viper venom proteinase used to activate factor X, and traces of contaminating thrombin. α-Factor X a was unstable at pH 7.6 and 25 °C, undergoing slow proteolytic degradation to functionally heterogeneous products as evidenced by the greater loss of coagulation assay activity compared to activity measured with a chromogenic substrate. The results of monitoring factor X a degradation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were consistent with proteolysis of the light chain as a major component reaction occurring in parallel with slower proteolysis of the heavy chain. The decreased rates of these reactions at pH 6.0 enabled isolation and storage of factor X a in ⩾88% α-form and minimized the heterogeneity due to proteolytic degradation. Characterization of the reaction of fluorescein mono- p-guanidinohenzoate (FMGB) with human and bovine factor X a isolated by SBTI-Sepharose chromatography demonstrated its utility as a sensitive reagent for continuous fluorometric active-site titration. Analysis of the reaction kinetics as a function of FMGB and human factor X a concentrations in Γ/2 0.3, pH 7.4, buffer at 25 °C indicated that the ratio of acylation to deacylation rate constants was >200 and that the K m for FMGB was 0.06–0.11 μ m, predicting pre-steadystate burst amplitudes of ⩾96–98% of the active-site concentration at FMGB concentrations ⩾5 μ m. Human factor X a active-site concentrations were consistent with 82–99% active preparations when compared with the protein concentrations determined from the 280-nm absorbance. Concentrations of human α-factor X a as low as 20 n m could be measured with FMGB, indicating a sensitivity approximately 50 times greater than that measured by spectrophotometric active-site titration with p-nitophenyl p′-guanidinobenzoate.