Materials and Methods

The zymogen formof factor XIII was prepared from citrat-ed, outdated blood-bank human plasma by the salt gradient elution DEAE-cellulose chromatographic procedure of Lorand and Gotoh (1970), with 1 mM EDTA included in all solutions used. As a final step, 2-6 ml of the purified protein solution was applied to a 2.5 X 90 cm column of Sepharose 6-B, equilibrated with 50 mM Tris-chloride of pH 7.5 containing 1 mM EDTA. On elution with the same at a rate of 20 ml/hr, monitored at 280 nm, the active peak emerging at V^/V0= 1.4 was concentrated by precipitation with 40% saturation of ammo-nium sulfate; it was then dialyzed against 50 mM Tris-chloride (pH 7.5)-1 mM EDTA and stored at 4 as a 1.7% protein solu-tion. A smaller peak (Fe/Fo= 1.8) containedonly b subunits. Bovine thrombin, used for the activation of factor XIII, was obtained by applying thecontents of three vials of Parke-Davis Thrombin Topical to a cellulose phosphate column (12 X 2.4 cm) which hadbeen equilibrated with 50 mM potassium phos-phate (pH 7.0)(Chou, 1970). On elution with this buffer, a strong yellow band was removed at about 80 ml of effluent and the column was further treated with 250 ml of the buffer. Thrombin was eluted by applying a mixture of 250 mM potas-sium phosphate and 0.5 M sodium chloride at pH 7.0. The en-zyme was dialyzed against 1.0 M potassium chloride (pH 7.0) and was stored at 4.

The limited proteolytic activation (Lorand and Konishi, 1964; Lorand et al., 1968; Schwartz et al., 1971) of the isolat-ed factor XIII zymogen was typically brought about by allowing 1.8-2.0 mg of this protein to react with 6-7 NIH units of thrombin in 0.3-0.5-ml solutions of 50 mM Tris-acetate buffer of pH 7.5. After an activation period of 20 min (at 25), aliquots (ca. 20/d) corresponding to approximately 70/xg of. the original zymogen were taken for alkylation with iodoacetam-ide.