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The clotting activity of Staphylococcus aureus strain 104 was purified 46,000-fold, but absolute purity was not achieved. Carbohydrate content of the purified material was not more than 5%. Elution of clotting activity from denaturing and nondenaturing polyacrylamide gels revealed the presence of four distinct molecular forms. Molecular weights of the forms were approximately 31,500, 34,800, 44,800, and 56,800 as determined by gel filtration in 8 M urea, by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis, and by calculation with determined values for the Stokes radius and sedimentation coefficient. Molecular weights determined on sodium dodecyl sulfate-urea gels were found to decrease as the gel concentration increased, suggesting that the amount of sodium dodecyl sulfate bound was less than normal. Estimated frictional ratios for the forms showed that they differ in shape from one another and that they are all highly asymmetrical. Each of the forms had an isoelectric point between pH 5.44 and 5.47 when focused in 6% polyacrylamide gels for 9 h; however, prolonged focusing altered the isoelectric point of the forms to within the range of pH 4.35 to 4.65. The multiple clotting forms were not artifacts of the purification procedure and did not appear to be products of the proteolytic degradation of a larger protein.

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The coagulase-reacting factor (CRF) of human plasma has been purified, concentrated, and largely freed of prothrombin activity by Seitz filtration, absorbtion on hyflo-amphogel columns, controlled phosphate buffer elution, and fractional ammonium sulfate precipitation. The purified CRF preparations localized between the beta and the gamma globulin position on paper electrophoresis, and the principal of two components observed on analytical ultracentrifugation gave sedimentation rates between 2.75 and 3.07. Highly purified prothrombin preparations of Seegers effectively react with staphylocoagulase to lead to the coagulation of plasma. The electrophoretic and ultracentrifugal properties of the human prothrombin preparations examined differ significantly from those of the purified CRF. Derivatives of prothrombin, involving a loss of prothrombin activity, such as thrombin and "autoprothrombin," do not correspondingly lose in CRF function. The hypothesis is proposed that CRF activity is resident in some component or components of the prothrombin molecule not involved in prothrombin function per se, and that therefore prothrombin and CRF activity may either parallel each other when the molecule is intact or diverge when the smaller units only are involved.

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