Studies on the Isolation of the Multiple Molecular Forms of Human Plasminogen and Plasmin by Isoelectric Focusing Methods

Abstract Human plasminogen and diisopropyl phosphorofluoridate or l-1-chloro-3-tosylamido-7-amino-2-heptanone-treated plasmin, prepared from plasma Fraction III2,3 by ion exchange and gel filtration methods, and the equimolar human plasminstreptokinase complex show multiple molecular forms in acrylamide gel electrophoresis at pH 8.4 in either 0.3 m e-aminocaproic acid or 8 m urea. Eight similar bands were found in the gel patterns of the zymogen and enzyme. The S-carboxymethyl heavy (A) chain derivative of plasmin did not show multiple molecular forms in acrylamide gel electrophoresis in either 0.3 m e-aminocaproic acid or 8 m urea. However, the S-carboxymethyl light (B) chain derivative showed multiple forms in acrylamide gel electrophoresis but in 8 m urea only. Plasminogen prepared from either pooled plasma, or plasma Fractions III or III2,3, by affinity chromatography on lysine-substituted-Sepharose columns showed the same multiple molecular forms in acrylamide gel electrophoresis in 0.3 m e-aminocaproic acid as were found in our highly purified plasminogen preparations but with different quantitative distributions. Affinity chromatography of only plasma Fraction III2,3 gave preparations with specific potential proteolytic activities which were equivalent in activity to our standard preparations. The multiple molecular forms of plasminogen and plasmin prepared from plasma Fraction III2,3 by ion exchange and gel filtration methods were isolated by isoelectric focusing methods. Eight plasminogen forms were obtained with isoelectric points ranging from pH 6.4 to pH 8.5. All of the forms had similar specific potential proteolytic activities. Each isoelectric form contained two closely related acrylamide gel electrophoretic components (doublet bands), both common to either adjacent isoelectric forms. Eight diisopropylphosphoryl-plasmin forms were isolated with isoelectric points ranging from pH 7.0 to pH 8.5. Radiolabeling and specific antibody were used to determine specific plasmin forms. Each plasmin isoelectric form also contained two closely related acrylamide gel electrophoretic components (doublet bands), both common to either adjacent isoelectric forms. Plasminogen isoelectric forms pH values 7.2 and 7.5 and diisopropylphosphoryl-plasmin isoelectric forms pH values 7.4 and 7.7 show the same acrylamide gel electrophoretic mobilities, respectively; the zymogen and enzyme forms differ in isoelectric point by approximately 0.2 pH unit. Plasminogen form pH 7.2 and its derived diisopropylphosphoryl-plasmin form show the same acrylamide gel electrophoretic mobilities. The S-carboxymethyl heavy (A) and light (B) chain derivatives of plasmin were studied in isoelectric focusing. Three major light (B) chain fractions were isolated with isoelectric points of pH values 5.8, 5.9, and 6.0. The amount of each of the three major acrylamide gel electrophoretic forms in each isoelectric fraction was different; none of the fractions showed a single pure form. The heavy (A) chain derivative gave a single form with an isoelectric point of pH 4.9. The plasminogen isoelectric forms pH values 7.5 and 7.8, after conversion to plasmin, followed by reduction and alkylation, each gave light (B) chain preparations containing all of the major acrylamide gel electrophoretic forms. No changes in acrylamide gel electrophoretic mobility could be found in any of the multiple molecular forms of zymogen, enzyme, or light (B) chain derivative following isoelectric focusing or other preparative procedures.