The assay of human plasminogen with casein as substrate.

Casein has been widely used for the assay of proteolytic enzymes since Kunitz (1947) described his procedure for the measurement of the activity of trypsin and chymotrypsin. Several procedures have since been designed for the assay of plasmin, the most widely used being that of Remmert & Cohen (1949). These workers activated plasminogen with excess of streptokinase in the presence of casein and then casein was added to the reaction mixture. After incubation the protein was precipitated and the tyrosine content of the filtrates estimated. A proteolytic unit was defined as the amount of enzyme producing an increase of 450,tg. of acid-soluble tyrosine in an hour at 350 in a medium containing 4 g. of casein/100 ml. For work on the purification of human plasminogen it was intended to use an assay based on that described by Mullertz (1955), who purified casein and measured hydrolysis by the increase in extinction at 275 m,u after precipitating protein with perchloric acid. With our procedure, when a balance sheet was made of the activities of fractions obtained during the course of purification experiments, the total activity exceeded that of the starting material by 12-20 %. Moreover, for any particular preparation, the estimated activity varied with the time allowed for the interaction of the enzyme with the substrate: the longer the incubation the higher the resulting activity. By examining samples taken at short intervals from the incubation mixture, the course of the reaction was found to be triphasic: an initial non-linear region of increasing slope, then a linear region followed by a region of decreasing slope. From this it was clear that an assay based on a single sample taken at a fixed time from the initiation of the reaction would be unreliable because of the initial non-linear phase. Many experiments were made under different conditions, with several plasmin preparations. It was found that a closer agreement between the total activity of the fractions and that of the starting material was obtained when the slope of the linear part of the curve was used for calculating the activity. * Present address: Department of Colloid Science, Free School Lane, Cambridge. The present work was undertaken to establish the validity of an assay based on this calculation.