Rivanol, Resin and the Isolation of Thrombins

Abstract

HUMAN, cow, and horse thrombins were quickly fractionated from citrate- or bio-activated prothrombin preparations by precipitation of residual prothrombin and inert protein(s) with the cationic dye 6,9-diamino-2-ethoxyacridine lactate (often referred to as rivanol, available under the trade name ‘Ethodin’ from the Winthrop Laboratories, New York, New York). This is a general procedure previously applied in purifying γ-globulin¹, β1-metal-combining globulin², and cæruloplasmin³. A more efficient yet rapid fractionation, however, was achieved by the adsorption of preparations containing thrombin on filter cakes or short columns of ‘IRC-50’ (‘XE-64-Rivanol’), this resin form being prepared by stirring ‘XE-64-Na⁺’ with an excess of rivanol. After the impurities, including other rivanol-soluble proteins, were washed off, the thrombins were eluted with 0.15 M calcium chloride. The thrombins were recovered from all eluates by acetone precipitation.