Rabbit Plasma Inhibitor of the Activated Species of Blood Coagulation Factor X PURIFICATION AND SOME PROPERTIES

Abstract A naturally occurring inhibitor of activated blood coagulation Factor X has been isolated from pooled rabbit plasma and purified by the combination of Sephadex G-200 gel filtration, DEAE-Sephadex A-50, and DEAE-cellulose chromatography. On microzone electrophoresis the purified product traveled as an α2-globulin, and on 7.5% polyacrylamide gel disc electrophoresis at pH 9.5 it traveled as a single component close to transferrin. With exclusion chromatography on Sephadex G-75, G-100, and G-200 it emerged in the same elution volume as crystalline bovine serum albumin. On analytical disc electrophoresis, the inhibitor stained positive for glycoprotein with the periodic acid-Schiff technique. It contained 4.1% hexose, 4.6% sialic acid, and was soluble in 2.5% but not in 5% trichloroacetic acid. Greater than 80% of its original activity persisted at 56° in the 1st hour and gradually diminished to 20% by the 6th hour. The pH optimum of the inhibitor activity was between 7 and 9, and the activity was most stable at pH 6 to 8. Optimum inhibitor activity was at 37° and nondetectable at 1°. Preparative disc electrophoresis of the purified inhibitor on 15% polyacrylamide gel at pH 8.1 caused extensive aggregation of the eluted inhibitor protein with a concomitant total loss of biological activity within 48 hours of storage, even at -60°.