Isolation of human blood coagulation alpha-factor Xa by soybean trypsin inhibitor-sepharose chromatography and its active-site titration with fluorescein mono-p-guanidinobenzoate.
A method based on active-site affinity chromatography on soybean trypsin inhibitor (SBTI)-Sepharose was developed for isolation of human factor Xa in primarily the undergraded alpha-form. The chromatography procedure separated factor Xa from factor X, the Russel's viper venom proteinase used to activate factor X, and traces of contaminating thrombin. alpha-Factor Xa was unstable at pH 7.6 and 25 degrees 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 Xa 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 Xa in greater than or equal to 88% alpha-form and minimized the heterogeneity due to proteolytic degradation. Characterization of the reaction of fluorescein mono-p-guanidinobenzoate (FMGB) with human and bovine factor Xa 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 Xa concentrations in G/2 0.3, pH 7.4, buffer at 25 degrees C indicated that the ratio of acylation to deacylation rate constants was greater than 200 and that the Km for FMGB was 0.06-0.11 microM, predicting pre-steady-state burst amplitudes of greater than or equal to 96-98% of the active-site concentration at FMGB concentrations greater than or equal to 5 microM. Human factor Xa 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 alpha-factor Xa as low as 20 nM 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.
American Red Cross Blood Services, Southeastern Michigan Region, Detroit 48232.
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Bock
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Craig
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Olson
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Singh
D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D015951:Factor Xa; D005452:Fluoresceins; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007202:Indicators and Reagents; D007700:Kinetics; D012685:Sepharose; D012697:Serine Endopeptidases / Q000302:isolation & purification*; D013050:Spectrometry, Fluorescence; D014361:Trypsin Inhibitors
D005452:Fluoresceins; D007202:Indicators and Reagents; D014361:Trypsin Inhibitors; C050485:fluorescein mono-p-guanidinobenzoate; D012685:Sepharose; D012697:Serine Endopeptidases; D015951:Factor Xa; D002118:Calcium