Chromatography, physical characteristics, and disulfide bonds of euglobulin plasminogen

Abstract

Euglobulin plasminogen was chromatographed on diethylaminoethyl cellulose (DEAE) with a 6 M urea-tris solvent system. Specific activity was increased from 86 to 140 casein units and α-, β 1c-, β 1a-, and γ-globulin impurities were removed. Euglobulin plasminogen was then chromatographed on DEAE with tris solvent only. Specific activity did not increase, and a slow diffusing β 2-globulin accompanied the β 2-plasminogen band upon immunoelectrophoresis of the eluate containing plasminogen. The appearance of this slow diffusing β 2-globulin was attributed to aggregation at 4 ° on the evidence of increasing opalescence and viscosity, and the appearance of a quickly sedimenting component on ultracentrifugation coincident with the appearance of the slow diffusing β 2-globulin on immunoelectrophoresis. This aggregate was dispersed by 6 M urea but not by ϵ-amino caproic acid. We also compared some of the physical characteristics of this euglobulin plasminogen and pseudoglobulin plasminogen. Titration of unreduced plasminogen with parahydroxy mercuribenzoate (CMB) revealed no free sulfhydryl groups. Plasminogen reduced by 6 M urea and 0.2 M MEA had an average of 11:42 titratable SH groups or an equivalent of 6 disulfide linkages. Reduction of the first five of these bonds resulted in a simultaneous loss of both the activator and plasmin activities of plasminogen. This reduction also increased the specific viscosity (0.2 – 0.35), decreased the specific optical rotation ([ α] 589 25−90 to −110 °), and decreased the absolute sedimentation coefficient (1.55 × 10 −13 to 1.00 × 10 −13) of plasminogen. Chromatography of reduced plasminogen on Sephadex G75 gave an initial single homogenous peak. These data suggest that both the tertiary structure and the two enzymic activities of plasminogen are changed simultaneously by reduction of its disulfide bonds. Since this comprehensive effort at chromatography and an enzymic and physical study of plasminogen did not result in a physical delineation of activator from plasminogen moieties, and since the activities of both activator and plasmin are similarly affected by diisopropylfluorophosphate and disulfide bond reducing agents, we have concluded that these two activities reside either in the same molecule or in two very similar molecules.