Enzyme Studies on Human Blood. II. The Substrate in the Fibrinogen-Thrombin Reaction.

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Abstract
34 prepns. of fibrinogen fractions were obtained from dried human plasma by the low temp.-ethanol procedure of the Dept. of Physical Chemistry, Harvard Medical School. The purity of these prepns. in respect to clottable protein was determined chemically by 2 methods: thrombin ("He-mostatic Globulin" - Lederle) and heat coagulation at 54-56 C. Fibrinogen fractions, ranging in purity from 60-84%, were made up in 1 or 2% solns. in either Michaelis veronal buffer as modified by Owren, pH 7.3, gamma/2 (ionic strength) 0.154, or a citrate-phosphate buffer, pH 7.2, gamma/2 0.129. Each expt. consisted of clotting time detns. on 1.0, 0.5, 0.25, 0.125, 0.062, 0.031, and 0.016% fibrinogen fraction in the appropriate buffer. The clotting times were determined at 37.5 C upon addition of 0.2 ml. of thrombin to 0.8 ml. of the substrate. In each of the 40 expts. with veronal buffer and 11 expts. with citrate-phosphate buffer, the concn.-clotting time curve was U-shaped. The over all optimum range of the substrate was approx. 0.1-0.3%, calculated as fibrinogen. In the 40 expts. employing veronal buffer it was also observed that the level of the clotting time at the concn. of fibrinogen fraction over 0.25% becomes elevated as the purity of the prepn. increases. This elevation in the reaction time, which could not be accounted for by the absolute increase in the concn. of fibrinogen, is discussed.