Isolation, characterization, and comparison of the S-carboxymethyl heavy (A) and light (B) chain derivatives of cat, dog, rabbit, and bovine plasmins.

Abstract Highly purified preparations of cat, dog, rabbit, and bovine plasminogens, isolated by affinity chromatography on lysine-substituted Sepharose columns, were found to be similar in molecular weight to human plasminogen in acrylamide gel-dodecyl sulfate electrophoresis. These plasminogens showed multiple electrophoretic forms in acrylamide gel electrophoresis in 0.3 m e-aminocaproic acid at pH 8.4 and in acrylamide gel-isoelectric focusing electrophoresis. The isoelectric points of the multiple forms of rabbit and bovine plasminogens were similar to the isoelectric points of the multiple isoelectric forms of human Lys-plasminogen; the cat and dog plasminogen isoelectric forms were similar to one another and more electronegative than the human Lys-plasminogen isoelectric forms. Cat, dog, rabbit, and bovine plasmins (urokinase-activated plasminogens) reacted stoichiometrically with l-1-chloro-3-tosylamido-7-amino-2-heptanone and were completely inhibited. Cm-heavy (A) and Cm-light (B) chain preparations were isolated from reduced and alkylated plasmin preparations from each species by dialysis against 0.002 m ammonium bicarbonate. The incorporation studies with l-1-chloro-3-tosylamido-7-amino-2-heptanone indicated a single "active site" histidine, for each species of plasmin, located in the Cm-light (B) chain. The Cm-light (B) chain derivatives of the cat, dog, rabbit, and bovine plasmins were essentially homogeneous and similar in molecular weight to the human Cm-light (B) chain derivative in acrylamide gel-urea-dodecyl sulfate electrophoresis. These derivatives showed multiple isoelectric forms in acrylamide gel-urea-isoelectric focusing electrophoresis. The isoelectric points of cat and dog Cm-light (B) chain forms were similar to the isoelectric points of the human Cm-light (B) chain forms; the rabbit Cm-light (B) chain forms were more electronegative and the bovine Cm-light (B) chain forms were more electropositive than the human Cm-light (B) chain forms. The Cm-heavy (A) chain preparations of cat, dog, rabbit, and bovine plasmins all contained both major and minor chains in acrylamide gel-urea-dodecyl sulfate electrophoresis. The cat, dog, and rabbit preparations contain a Cm-heavy (A) chain that had the same molecular weight as the major human Cm-heavy (A) chain. The major cat Cm-heavy (A1) chain was similar in molecular weight to the minor human Cm-heavy (A1) chain. The major dog Cm-heavy (A2) chain had a lower molecular weight than both the major and minor human Cm-heavy (A and A1) chains. The bovine Cm-heavy (A3) chain was of lower molecular weight than the dog, Cm-heavy (A2) chain. The Cm-heavy (A) chain derivatives of all four animal species showed multiple isoelectric forms in acrylamide gel-urea-isoelectric focusing electrophoresis with isoelectric points slightly more electronegative than the isoelectric points of the multiple human Cm-heavy (A) chain forms.

gel-isoelectric focusing electrophoresis. The isoelectric points of the multiple forms of rabbit and bovine plasminogens were similar to the isoelectric points of the multiple isoelectric forms of human Lys-plasminogen; the cat and dog plasminogen isoelectric forms were similar to one another and more electronegative than the human Lysplasminogen isoelectric forms. Cat, dog, rabbit, and bovine plasmins (urokinase-activated plasminogens) reacted stoichiometrically with L-1-chloro-3tosylamido-7-amino-2-heptanone and were completely inhibited.
Cm-heavy (A) and Cm-light (B) chain preparations were isolated from reduced and alkylated plasmin preparations from each species by dialysis against 0.002 M ammonium bicarbonate.
The incorporation studies with L-1-chloro-3tosylamido-7-amino-2-heptanone indicated a single "active site" histidine, for each species of plasmin, located in the Cm-light (B) chain.
The The Cm-heavy (A) chain preparations of cat, dog, rabbit, and bovine plasmins all contained both major and minor chains in acrylamide gel-urea-dodecyl sulfate electrophoresis. The cat, dog, and rabbit preparations contain a Cm-heavy (A) chain that had the same molecular weight as the major human Cm-heavy (A) chain.
The major cat Cm-heavy (Al) chain was similar in molecular weight to the minor human Cm-heavy (Al) chain.
The major dog Cm-heavy (AZ) chain had a lower molecular weight than both the major and minor human Cm-heavy (A and Al) chains. The bovine Cmheavy (A3) chain was of lower molecular weight than the dog, Cm-heavy (A2) chain.
The Cm-heavy (A) chain derivatives of all four animal species showed multiple isoelectric forms in acrylamide gel-urea-isoelectric focusing electrophoresis with isoelectric points slightly more electronegative than the isoelectric points of the multiple human Cm-heavy (A) chain forms. l'lasminogans have been isolated from several animal plasmas in a highly purified state by various types of chromatographic mct,hods (l-6).
The molecular weights of bovine and rabbit plasminogens were determined to be between 80,000 and 90,000 (1, 2, 4, 5). Dog (3) and rabbit (5) plnsminogens showed multiple electrophoretic forms in starch gel and acrylamide gel electrophoresis, respectively. Rovine plasminogen activated by urokinase (7) and rabbit plasminogen activated by streptokinase (5) give plasmins which, after reduction and alkylation, show two chains for each species. The Cm-heavy (A) and Cm-light (U) chain derivatives of bovine plasmin have been isolated (7). The molecular weight of the Cm-heavy (A) chain was reported to be 35,000 and the molecular weight of the Cm-light (II) chain was reported to be 23,500. In this report, cat, dog, rabbit, and bovine plasminogens were isolated by an affinity chromato-graphic method using L-lysine-substituted Srpharosc (8). These plasminogens were characterized and compared in several acrylamide gel electrophoretic systems. These zymogens were activated with urakinase and the plasmin-derived Cm-heavy (A) and Cm-light (13) chains were isolated, characterized, and compared with each other and with similar human preparations.
Pooled titrated cat and rabbit plasmas were obtained from Pel-Frcez, Inc. Pooled titrated bovine plasma was obtained from Armour Pharmaceutical Co., Kankakee, Ill., and Pel-Freez, Inc. All plasma was stored frozen for a period of from 3 to 6 months at -20".
Prior to use, each plasma was thawed at 2" and the cryoprecipitate was removed by centrifugation at 4000 rpm for 1 hour.
Plasminogens and Plasmins-Cat, dog, rabbit, and bovine plnsminogeus wre prepared from plasma by a modification of the method described by Dcutsch and Mcrtz using an afEnit,y chromatography method with L-lysine-substituted Sepharose at 2" (8). Human plasminogen was prepared from plasma Fractions III and 1112.3 (g-11).
Plasma Fraction III crontains Gluand Lys-plasminogens whereas plasma Fraction 1112,s contains Lys-plnsminogen only. The eluted plasminogeu was precipitated by adding 3.1 g of ammonium sulfate per 10 ml of solution. After 1X hours at 2", the suspension was centrifuged at 2" at 4000 rpm (International ('entrifuge PR II, Head iYo. 845). The plasminogcu was dissolved in 0.05 M 'l'ris-0.02 hZ lysine buffer-0.1 M SaCI, pH 9.0, at a concentration of approximately 25 mg per ml, and frozcu.
In the assay, maximunl activatiolr of cash species of pla,sminogen was obtained with 700 CTA units of urokinase (11).
Plnsmin was prepared by activat.ing the plasminogens with low molar ratios of urokinase iu a 25 yfi glycerol solution contairling 0.04 hI Tris-0.016 M lysirie-0.08 hl sodium chloride at pH 9.0, at 25", by methods previously described for preparing human plasmin (12). Complete activation of each preparation was obtaiurd with no loss in activity or change in specific activity.
The [3H]TLC'K was 10';; of the total TLCK used. The procedure was similar to that used to prepare the [3HrrLCK derivative of human plasmiu (13). The TLCK-plasmin derivatives wvcre then adjusted to pH 3, dialyzed against 0.001 M HCl at 4', and lyophilized.
Acrylamide Gel Blectrophoresis-ncr~larnitle gel electrophorrsis Ivvas carried out in gel slabs in a lleckman Microzone acrylamide gel system at pH 8.4 and 7.0 (9, 10). The gel slabs (5 and 7.57;) w?re prepared in 0.037 hi Tris-0.29 M glycine buffer, pH 8.4, containing 0.3 M e-aminocaproic acid. Gel slabs (5~7; for galasminogens and 7.5% for Cm-heavy (A) and Cm-light, (I<) chains) were also prepared in 0.1 M phosphate buffer, pH 7.0, O.l$; tlodecyl sulfate, with 6 M urea. Isoelectric focusing in acrylamide gel (5y0 for plasrninogens and 7.5% for Cm-heavy (A) and Cmlight (13) chains), vAth 8 M urea was carried out in gel slabs using a modification of the method described by Vesterberg (14). The detailed prnccdures for all of the acrylamitle gel electrophoretic analyses have been previously described (9, IO). Qualit,ative determinations for carbohydrate were made in certain grls by st.aining for 3 hours with Schiff's stain (15). The gels were washed a11t1 stored in it& acetic acid.

Prepnrafion
OJ Cat, Dog, Rabbit, und Bovine Plasminogens-The rerovcries aud activities of rat, dog, rabbit, and bovine plasminogens, prepared by the affinity chromatography method using a lysine-substituted Srphnrose column (2.5 X 10 cm) are summarized in Table I. Volumes of approximately 500 ml of the various plasmas were added to the column.
.I single symmetrical peak ~vas obtained containing 0.1 to 0.2 mg of protein per ml of plasma applied.
The specific activities for all four species of plasminogen were between 20 and 23 casein units per mg of protein.
These specific activities are similar to the activities of human plasminogen prepared from Fractions III alit1 1 112,3 lay this method (10 each other and with human Lys-plasminogen in acrylamide gel-the plasminogens appeared to have similar, but not identical urea-dodecyl sulfate electrophoresis (Fig. la). All of the molecular weights. plasminogens appear to be essentially homogeneous except for Cat, dog, rabbit, and bovine plasminogens were compared with the bovine plasminogen preparation which contained several each other and to human Glu-and Lys-plasminogens in acrylamminor components, perhaps other plasminogen forms. All of ide gel-e-aminocaproic acid electrophoresis (Fig. lb)  electrophoretic forms seen in the human, rabbit, and bovine plasminogens. A second group of more electronegative forms were also seen in bovine plasminogen.
Cat, dog, rabbit, and bovine plasminogens were also compared to each other and to human plasminogen in acrylamide gel-urea-isoelectric focusing, pH range of 3 to 10 (Fig. Ic). Multiple isoelectric forms were present in every plasminogen preparation.
The patterns were similar to those seen in the scrylamide gel-e-aminocaproic acid electrophoretic pattern (Fig. 16). More cIearly defined bands could be seen in the bovine plasminogen preparation but the isoelectric forms of the dog and cat plasminogens were not as well resolved in the acrylamide gel-urea-isoelectric focusing system. chains of the animal plasmins contain the "active site" histidine, as previously reported for human Lys-plasmin (13). The reduced incorporation of [3H]TLCK per Imole of Cm-light (1~) chain for the animal species could be due to the sensitivity of the radiolabel to reduction and alkylation, or to handling (17). Each Cm-heavy (A) chain preparation was apparently contaminated with small amounts of Cm-light (B) chain derivative.
The cat, dog, rabbit, and bovine plasmin-derived Cm-heavy (A) and Cm-light (B) chains were compared with each other and with the human Lys-plasmin-derived chains in aerylamide gel-urea-dodecyl sulfate electrophoresis (Fig. 1, d and e). The Cm-light (B) chain derivatives of t.hese species were essentially homogeneous, except for the cat Cm-light (B) chain preparation which contained two additional minor components (Fig. Id,  minor dog Cm-heavy (A) chain derivatives were of the same