Molecular Weight and Amino Acid Composition of Equine Thrombin

抄録

Equine thrombin was obtained in a homogeneous state from prothrombin by activation with tri-sodium citrate, followed by gel filtration through a Sephadex G-100 column and by chromatography with DEAE cellulose. This preparation was shown to be chromatographically homogeneous and the homogeneity was further confirmed by electrophoresis and by ultracentrifugal analysis. The sedimentation and diffusion constants, s⁰_{20, W} and D°_{20, W}, and the partial specific volume were determined to be 3.87S, 8.66×10^-7cm²/sec and 0.686 ml/g, respectively, from which the molecular weight and frictional ratio were calculated to be 34, 600 and 1.16, respectively. The esterase activity of equine thrombin as measured with TAMe as the substrate was 1.7×10^-5 mole/ min/mg of protein, and the Michaelis constant (K_m) was 2.36×10^-4M. The amino acid contents were determined by a common procedure of amino acid analysis. The content of glutamic acid including its amide was lower than that in bovine thrombin, whereas the content of isoleucine was higher. The contents of other amino acids were similar to those reported for bovine thrombin. The sum of the amino acid contents was 263±12 residues per mole, from which the molecular weight was calculated to be 30, 700±1, 200. The about 10% difference between the molecular weight calculated from the amino acid contents and that determined by sedimentation-diffu-sion analysis is due to the content of sugars assumed in the calculation of the amino acid contents. The content of hexose in equine thrombin was 3.7%.