Urmila Duhan1. 1. Center for Molecular Biology of Oral Diseases, College of Dentistry, Chicago, IL, USA. uduhan@uicalumni.org
Abstract
BACKGROUND: The reactive center loop (RCL) of native antithrombin is partially inserted in the main serpin body. It must be fully exposed for optimal inhibitory function. OBJECTIVE: To test the hypothesis that P(14)-s2B interaction affects loop insertion in antithrombin. By mutating Phe(274) to Tyr(274), the objective was to introduce P(14)-s2B interaction in antithrombin. METHODS: Site-directed mutagenesis and affinity chromatography were used to obtain purified recombinant protein. Antithrombin's ability to form sodium dodecyl sulfate (SDS)-stable complex with thrombin, stoichiometry of thrombin inhibition, second-order rate constant for thrombin and factor Xa (fXa) inhibition (M(-1) s(-1)), and heparin dissociation constant (K(D); tryptophan fluorescence emission spectra) were determined. RESULTS AND CONCLUSION: A marginal, but inconclusive, difference between the wild type and the mutant was observed. The result highlights the variable effect of P(14)-s2B interaction in different serpins. Alternate hypothesis for achieving loop expulsion is proposed.
BACKGROUND: The reactive center loop (RCL) of native antithrombin is partially inserted in the main serpin body. It must be fully exposed for optimal inhibitory function. OBJECTIVE: To test the hypothesis that P(14)-s2B interaction affects loop insertion in antithrombin. By mutating Phe(274) to Tyr(274), the objective was to introduce P(14)-s2B interaction in antithrombin. METHODS: Site-directed mutagenesis and affinity chromatography were used to obtain purified recombinant protein. Antithrombin's ability to form sodium dodecyl sulfate (SDS)-stable complex with thrombin, stoichiometry of thrombin inhibition, second-order rate constant for thrombin and factor Xa (fXa) inhibition (M(-1) s(-1)), and heparin dissociation constant (K(D); tryptophan fluorescence emission spectra) were determined. RESULTS AND CONCLUSION: A marginal, but inconclusive, difference between the wild type and the mutant was observed. The result highlights the variable effect of P(14)-s2B interaction in different serpins. Alternate hypothesis for achieving loop expulsion is proposed.