| A procedure for the isolation and purification of bovine alpha-thrombin has been developed. Highly reproducible thrombin crystals were obtained using the procedure. The procoagulant alpha-thrombin (MW 38,000) was crystallized from 37-43% saturated ammonium sulfate, pH 5.8. The crystals are tetragonal (a = b = 87.5 (ANGSTROM), c = 101.5 (ANGSTROM)), space group P42(,1)2, with one molecule per asymmetric unit.;A naturally occurring proteolytic form of alpha-thrombin has been characterized, and found to be similar to elastase digested human thrombin. This new form of thrombin (beta2-thrombin) was found to retain significant fibrinogen clotting activity unlike other well characterized proteolytic forms of thrombin.;Diffractometer data from native and several heavy atom derivatives were measured to varying resolution by sealed capillary and flowcell methods.;The crystal structure of carp muscle parvalbumin has been refined at 1.5 (ANGSTROM) resolution to crystallographic residual of R = 0.215. The final structure revealed anomalies (amino terminus, and metal-oxygen ligands) with the structure previously reported by Moews and Kretsinger (1975).;Crystallization conditions for other forms of thrombin namely (a) neuraminidase digested, (b) benzamidine treated and (c) PMSF-inhibited thrombin have also been determined.;The crystal structure of carp parvalbumin with ytterbium ion in the EF site and calcium ion in the CD site has been determined and refined at 1.5 (ANGSTROM) to R = 0.199. A comparative study between the two crystal structures revealed no changes in the overall structure, and the CD metal binding site, but the substituted EF site shows several changes namely (a) shorter metal-oxygen ligands, (b) both the side chain carboxyl oxygens of aspartic 92 ligand the metal, (c) displacement in the substituted metal ion, and (e) higher thermal factors. |
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