AFFINITY CHROMATOGRAPHY RESOLUTION OF PLASMINOGEN ISOZYMES

Abstract

ABSTRACT. Two major forms of rabbit plasminogen have been isolated by affinity chromatography from rabbit plasma. One form consists of 5 subforms with an isoelectric pI range of 6.20–7.78. The other plasminogen form can also be resolved into 5 subforms, but these possess an isoelectric pI range of 6.95–8.74. All plasminogen forms and subforms can be converted to plasmin by common plasminogen activators such as urokinase, streptokinase, and the streptokinase-human plasmin (ogen) complex. Each major plasminogen form, as well as all isolated subforms, possesses nearly identical molecular weights of approximately 90,000 and is a single chain molecule. In addition, each major form contains identical amino- and carboxyl-terminal amino acids and identical amino terminal amino acid sequences through at least 12 residues. Carbohydrate analyses indicate differences in sialic acid, neutral sugars and hexosamines between the two major forms. Removal of the sialic acid from each form abolishes the electrophoretic differences between the forms and also leads to loss of resolution of some of the component subforms. However, the affinity chromatography resolution of the two major forms is not affected by removal of the sialic acid. Metabolic studies in intact animals demonstrate that both major plasminogen forms possess dissimilar rates of turnover and synthesis and are not interconverted. Pre-steady state kinetic analyses, using specific p-nitrophenolate esters, of the plasmins resulting from the urokinase induced activation of each major rabbit plasminogen form demonstrate that substrates bind more tightly to plasmin form 1 than plasmin form 2. Acylation rate constants for these substrates are very similar for each plasmin form.