Physical characterization of platelet thrombospondin

Abstract

A hydrodynamic model of the high molecular weight platelet glycoprotein thrombospondin is formulated from physical solution measurements of molecular weight, partial specific volume, sedimentation coefficient, and intrinsic viscosity. Simultaneous sedimentation equilibrium analysis of thrombospondin in buffered saline prepared in H2O and D2O yielded values of 420,000 and 0.714 ml/g for the molecular weight and partial specific volume, respectively. A sedimentation coefficient of 8.6 S was found to be independent of the protein concentration over the range 0-6 mg/ml. The intrinsic viscosity was determined to be 40 ml/g at 20 degrees C for native thrombospondin and 52 ml/g for thrombospondin in the presence of 6 M guanidine-HCl. Based on these values thrombospondin is best described as a prolate ellipsoid with an axial ratio of 9.3. This model agrees well with the electron microscopic image of thrombospondin as a nodular rod (7 X 65 nm)-shaped molecule with an axial ratio of 10. Sedimentation equilibrium analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that thrombospondin is comprised of three 140,000-dalton polypeptide chains. The percentage of residues in alpha-helix was calculated to be only 3% from the circular dichroism spectrum.