- Email: [email protected]
Multimolecular forms of profibrinolysin revealed by a zymogram technique
PRELIMINARY NOTES
609
This work was supported by grants from National Institutes of Health, Bethesda, U.S.A. (HE o7379-o4), the Swedish Medical Research Council (K65-I3X635-Ol, K67-I3X-2227-oI, K67-I9X-52o-o3) and Gunnel och Svante H6gboms stiftelse. Excellent assistance by Mrs. Helga Messel, Sonj a S6derman and Anita Lindholm is acknowledged.
Department of Blood Coagulation Research, Karolinska Institutet, Stockholm (Sweden)
SADAAKI IWANAGA, P E R WALLi~N, NILS J . GR6NDAHL, AGNES HENSCHEN BIRGER BLOMBACK
I 2 3 4 5 6 7 8 9 io 11 12 13 14
t3. BLOMBXCK AND I. YAMASHINA, Arkiv Kemi, 12 (1958) 299. A. HENSCHEN, Arkiv Kemi, 22 (1963) I. A. HENSCHEN, Arhiv Kemi, 22 (1964) 375. F. R. BETTELHEIM AND Z. BAILI~¥, Biochim. Biophys. Acta, 9 (I952) 578. L. LORAND, Biochem. J., 52 (1952) 200. F. R. BETTELHEIM, Biochim. Biophys. Acta, 19 (1956) 21. B. BLOMBXCK, Arkiv Kemi, 12 (1958) 321. B. BLOMBACK, M. BLOMBACK, P. EDMAN AND B. HESSE;L, Biochim. Biophys. Acta, 115 (1966) 371 • P. WALLI~N AND K. BERGSTROM, Acta Chem. Scan&, 12 (1958) 579. 13. BLOMB'&CK AND M. BLOMBXCK, Arkiv Kemi, io (1956) 415 . S. IWANAGA, A. HENSCHEN AND ]~. BLOMBACK, Acta Chem. Scand., 20 (1966) 1183. P. WALLgN AND B. WIMAN, to be published. B. BLOMBACK, S. IWANAGA AND P. WALLI~N, Abstr. 7th International Biochemical Congress, Tokyo, A u g u s t 1967 . B. BLOMBACK, M. BLOMBXCK, B. HESSEL AND S. I\VANAGA, Nature, 215 (1967) I445.
Received August 2ist, 1967 Biochim. Biophys. Acta, 147 (I967) 6o6-6o9
BBA 31 023
Multimolecular forms of profibrinolysin revealed by a zymogram technique The phenomenon of isoenzymes is well recognized as occurring among many enzyme systems such as the dehydrogenases, acid phosphatases and carbonic anhydrase. The identification of these isoenzymes resulted from electrophoresis, particularly on starch gels, with subsequent localization by in situ techniques. Such assay techniques enabled the investigator to identify, most directly, the actual position of the active enzyme in the electrophoresis medium without elution and its possible artifactual effects. Multimolecular forms of profibrinolysin have been hypothesized by various investigators 1-4 from electrophoretic evidence. However, an assay in situ was not available to relieve these investigators of the uncertainty of artifacts produced when the several protein bands were eluted from starch or acrylamide gel. Our development of a novel assay for profibrinolysin or fibrinolysin in situ on any stable electrophoresis medium, as well as the isolation of the most highly active canine profibrinolysin yet reported 5, enables us to suggest the existence of four discrete isoproenzymes of canine profibrinolysin. The assay system consisted of a fibrin-impregnated cellulose acetate strip. This Biochim. Biophys. Acta, 147 (1967) 609 612
610
PRELIMINARY NOTES
strip, containing a suitable activator, such as urokinase, was applied to the electrophoresis medium (i.e. starch gel, cellulose acetate, or polyacrylamide) over the areas of protein migration. The proenzyme, after diffusion into the fibrin-acetate strip, was activated and caused lysis of the fibrin in the matrix of the acetate strip. After washing the fibrin acetate strip to remove soluble lysis products, the unlysed fibrin was stained with Ponceau S. This resulted in the appearance of clear zones of fibrinolysis which were directly comparable to the stained protein zones produced in tile second half of the electrophoresis lnediunl. Details of this system and the results follow. Strips of cellulose acetate (12.5 cm / 2.5 cm, Serometrics, Inc., Chicago Heights, Ill.) were hydrated in Tris-citrate, pH 8.65, containing o.2 o~ ,'o bovine fibrinogen. The strips were blotted and placed in a solution of thrombin (IO N.I.H. units/ml) for 5 min and blotted. The strips were then heated for I5 min at 85 ° in Tris--citrate to inactivate any proteolytic components present in the fibrin-acetate strips. When assaying for fibrinolysin, no activator need be added, otherwise, just prior to application, the fibrin-acetate strips were placed in a solution of urokinase (o.43 Michigan arbitrary unit/ml). When using starch gels, at the end of the electrophoresis, the gel was cut in half. One half was stained for protein with imido-black, to the other half was applied the fibrin-acetate strip. Diffusion and lysis were allowed to proceed at room temperature for 2o min. The fibrin strips were then washed thoroughly in saline and stained in Ponceau S (o.2 % in 3 % trichtoroacetic acid) for 7 rain and cleared of excess stain in 7 ?'o acetic acid. The white zones of lysis were clearly visible against the pink background of stained, unlysed fibrin. Utilizing this i~z s i t u assay technique, four distinct bands of profibrinolysin were apparent when our highly purified canine profibrinolysin (135oo centiunits/mg protein) was submitted to electrophoresis on starch gel at pH 8.65 (Fig. I). The purified profibrinolysin was homogeneous according to ultracentrifugal and immunologic criteria. No protein bands other than the active ones appeared in the zymogram to illustrate the high purity of the proenzyme product. The separate proenzyme bands were excised from the gel and subjected directly to electrophoresis on a second starch gel (Fig. 2), showing clearly their individuality
Fig. i. A. Starch-gel electrophoresis of pooled canine profibrinolysin c h r o m a t o g r a p h e d on h y d r o x y apatite. E q u i v a l e n t results were obtained from a single donor source. 25o V d.c. applied for 4.5 h at room temperature. Tris-citrate buffer at p H 8.65. B. F i b r i n - a c e t a t e e n z y m o g r a m of pooled canine isoproenzymes subjected to electrophoresis on starch gel.
t3iochim. Biophys. Acla, 147 (1967) 6o9-612
PRELIMINARY NOTES
61I
and capacity for activation to fibrinolysin. When activator was absent from the fibrinacetate strip, lysis did not occur. This indicates that none of the protein zones contained spontaneous fibrinolytic activity. Furthermore, eleetrophoresis did not cause activation of the proenzyme.
I:ig. 2. A. Starch-gel electrophoresis of excised canine profibrinolysin c h r o m a t o g r a p h e d on h y d r o x y a p a t i t e i s o p r o e n z y m e s indicating r e t e n t i o n of i n d i v i d u a l mobility. 25o V d.c. applied for 4.5 h at r o o m t e m p e r a t u r e . B. Fibrin a c e t a t e e n z y m o g r a m of (A) d e m o n s t r a t i n g r e t e n t i o n of i n d i v i d u a l isoproenzymes.
To ascertain that these multimolecular forms were not present as a consequence of plasma pooling, profibrinolysin was isolated from a single donor and subjected to starch-gel analysis. Again 4 bands showed up on starch-gel electrophoresis. Apparently multimolecular forms of canine profibrinolysin do exist and represent distinct molecules possessing individuality of certain physicochemical characteristics while exhibiting common enzymatic reactivity. Explanations as to the origin of these multiple proenzyme components remain obscure. Whether they are the products of chemical fractionation has yet to be demonstrated; clearly they are not produced by the electrophoresis on starch gel. It is also possible that these isoproenzymes represent profibrinolysin molecules at various stages of m a t u r i t y or that separate cellular origins m a y produce this variance. BARNHART AND RIDDLE 6 have shown b y immunofluorescence that one cellular site for profibrinolysin synthesis is eosinophils in the bone marrow. The specific granules (lysosomes) of eosinophils also yield a substance that requires activation by urokinase before digesting fibrinL Further work on these possible isoproenzymes is necessary for a reasonable selection of an explanation. The isolation and characterization of each of these four isoproenzymes is being undertaken in our laboratory, as well as investigations of equine, bovine, and human isoproenzymes, which will be reported elsewhere. This investigation was supported by the National Institutes of Health, grant Nos. I-FL-GM-33, 527-Ol and HE-o4712.
Department of Physiology, Wayne State University, School of Medicine, Detroit, Mich. (U.S.A.)
P A U L J. H E B E R L E I N ~[ARION I. BARNHART
Biochim. Biophys. dcla, 147 (1967) 6o9-612
612 i
2 3 4 5 6 7
PRELIMINARY NOTES
ALKJAERSIG, ]3iochem. J., 93 (I964) 171. N. SLOTTA AND J. D. (;ONZ~XLEZ, Thromb. Diath. Haemorrhag., 12 (1964) 126. T. MERTZ AYD J. Y. S. CHA~,', Can. J. Biochem. Physiol., 41 (1963) 1811. B. ~VALLI~;N,Arhiv Kemi., 19 (I962) 469. J. HEBERLEIN ANO M. I. BARNHART, Thrombos. Diath. Haemorrhag., i8 (1967) 286. I. BARNHART AND J. M. RIDDLE, Blood, 21 (1963) 3o3 . I. BARNHART, C. QUINTANA, H. L. LENON, G. B. BLUM AND J. ~I. RIDDLE, Ann. N . Y . Acad. Sci., in th e press. N. K. E. P. PM. M.
Received September 4th, 1967 Biochim. Biophys. Acta, 147 (1967) 6o9-612
BBA 31025
A m i n o acid sequence in rat pancreatic ribonuclease The amino acid sequence in rat pancreatic ribonuclease (EC 2.7.7.16) was determined using 12o mg of tile purified enzyme 1. The following procedures were used successively: performic acid oxidation2; either tryptic or chymotryptic hydrolysis; peptide fractionation on Sephadex G-25 (ref. 3), Dowex i (ref. 4), and Dowex 50 ; amino acid analysis of the peptides; hydrolysis of the larger peptides by chymotrypsin, trypsin, papain or dilute HC1; E d m a n degradation with identification of the new N-terminal amino acids as their dimethylaminonapbthalenesulfonyl (DNS) derivatives 5 and in some cases identification of the amino acids removed as their phenylthiohydantoin derivatives; and carboxypeptidase digestion. All amino acids could be located unambiguously: Fig. I shows the sequence obtained. It also shows some partial sequences of the horse enzyme, which in contrast to the rat enzyme--contains carbohydrate (A. J. SCHEFFER AND J. J. BEIXTEXIA, unpublished results). It is obvious that the best alignment of the rat enzyme with the beef enzyme sequence is that shown in Fig. I. For the sake of clarity the numbering of the beef enzyme has been retained; consequently the rat enzyme is counted from --3 at the N-terminal. The disulfide bridges were not determined; they are very probably identical with those in the beef enzyme. The positions of the amide groups at residue 71 rather than 7 ° and at IOI rather than lO3 are probable, but not definitely established. It is striking that the rat and beef enzymes, which are physically and enzymically very similar, have different amino acids at 33 % of the positions. This is much greater than the differences between the mammalian eytochromes c (ref. 6); it is even larger than the differences between the fl- and y-chains of human hemoglobin 7. In spite of the differences at one-third of the positions all amino acids shown to be part of the active site, or implicated in activity or conformation, are in exactly the same positions in both enzymes. Moreover, replacement of the amino acids in the three-dimensional structure of the beef enzyme (as determined b y X - r a y diffraction s,9) b y those of the rat enzyme does not lead to any structural changes, as far as can be predicted at the moment. Glycine-38 in the rat enzyme, which replaces 13iochim. Biophys. Acta, 147 (1967) 612-614
609
This work was supported by grants from National Institutes of Health, Bethesda, U.S.A. (HE o7379-o4), the Swedish Medical Research Council (K65-I3X635-Ol, K67-I3X-2227-oI, K67-I9X-52o-o3) and Gunnel och Svante H6gboms stiftelse. Excellent assistance by Mrs. Helga Messel, Sonj a S6derman and Anita Lindholm is acknowledged.
Department of Blood Coagulation Research, Karolinska Institutet, Stockholm (Sweden)
SADAAKI IWANAGA, P E R WALLi~N, NILS J . GR6NDAHL, AGNES HENSCHEN BIRGER BLOMBACK
I 2 3 4 5 6 7 8 9 io 11 12 13 14
t3. BLOMBXCK AND I. YAMASHINA, Arkiv Kemi, 12 (1958) 299. A. HENSCHEN, Arkiv Kemi, 22 (1963) I. A. HENSCHEN, Arhiv Kemi, 22 (1964) 375. F. R. BETTELHEIM AND Z. BAILI~¥, Biochim. Biophys. Acta, 9 (I952) 578. L. LORAND, Biochem. J., 52 (1952) 200. F. R. BETTELHEIM, Biochim. Biophys. Acta, 19 (1956) 21. B. BLOMBXCK, Arkiv Kemi, 12 (1958) 321. B. BLOMBACK, M. BLOMBACK, P. EDMAN AND B. HESSE;L, Biochim. Biophys. Acta, 115 (1966) 371 • P. WALLI~N AND K. BERGSTROM, Acta Chem. Scan&, 12 (1958) 579. 13. BLOMB'&CK AND M. BLOMBXCK, Arkiv Kemi, io (1956) 415 . S. IWANAGA, A. HENSCHEN AND ]~. BLOMBACK, Acta Chem. Scand., 20 (1966) 1183. P. WALLgN AND B. WIMAN, to be published. B. BLOMBACK, S. IWANAGA AND P. WALLI~N, Abstr. 7th International Biochemical Congress, Tokyo, A u g u s t 1967 . B. BLOMBACK, M. BLOMBXCK, B. HESSEL AND S. I\VANAGA, Nature, 215 (1967) I445.
Received August 2ist, 1967 Biochim. Biophys. Acta, 147 (I967) 6o6-6o9
BBA 31 023
Multimolecular forms of profibrinolysin revealed by a zymogram technique The phenomenon of isoenzymes is well recognized as occurring among many enzyme systems such as the dehydrogenases, acid phosphatases and carbonic anhydrase. The identification of these isoenzymes resulted from electrophoresis, particularly on starch gels, with subsequent localization by in situ techniques. Such assay techniques enabled the investigator to identify, most directly, the actual position of the active enzyme in the electrophoresis medium without elution and its possible artifactual effects. Multimolecular forms of profibrinolysin have been hypothesized by various investigators 1-4 from electrophoretic evidence. However, an assay in situ was not available to relieve these investigators of the uncertainty of artifacts produced when the several protein bands were eluted from starch or acrylamide gel. Our development of a novel assay for profibrinolysin or fibrinolysin in situ on any stable electrophoresis medium, as well as the isolation of the most highly active canine profibrinolysin yet reported 5, enables us to suggest the existence of four discrete isoproenzymes of canine profibrinolysin. The assay system consisted of a fibrin-impregnated cellulose acetate strip. This Biochim. Biophys. Acta, 147 (1967) 609 612
610
PRELIMINARY NOTES
strip, containing a suitable activator, such as urokinase, was applied to the electrophoresis medium (i.e. starch gel, cellulose acetate, or polyacrylamide) over the areas of protein migration. The proenzyme, after diffusion into the fibrin-acetate strip, was activated and caused lysis of the fibrin in the matrix of the acetate strip. After washing the fibrin acetate strip to remove soluble lysis products, the unlysed fibrin was stained with Ponceau S. This resulted in the appearance of clear zones of fibrinolysis which were directly comparable to the stained protein zones produced in tile second half of the electrophoresis lnediunl. Details of this system and the results follow. Strips of cellulose acetate (12.5 cm / 2.5 cm, Serometrics, Inc., Chicago Heights, Ill.) were hydrated in Tris-citrate, pH 8.65, containing o.2 o~ ,'o bovine fibrinogen. The strips were blotted and placed in a solution of thrombin (IO N.I.H. units/ml) for 5 min and blotted. The strips were then heated for I5 min at 85 ° in Tris--citrate to inactivate any proteolytic components present in the fibrin-acetate strips. When assaying for fibrinolysin, no activator need be added, otherwise, just prior to application, the fibrin-acetate strips were placed in a solution of urokinase (o.43 Michigan arbitrary unit/ml). When using starch gels, at the end of the electrophoresis, the gel was cut in half. One half was stained for protein with imido-black, to the other half was applied the fibrin-acetate strip. Diffusion and lysis were allowed to proceed at room temperature for 2o min. The fibrin strips were then washed thoroughly in saline and stained in Ponceau S (o.2 % in 3 % trichtoroacetic acid) for 7 rain and cleared of excess stain in 7 ?'o acetic acid. The white zones of lysis were clearly visible against the pink background of stained, unlysed fibrin. Utilizing this i~z s i t u assay technique, four distinct bands of profibrinolysin were apparent when our highly purified canine profibrinolysin (135oo centiunits/mg protein) was submitted to electrophoresis on starch gel at pH 8.65 (Fig. I). The purified profibrinolysin was homogeneous according to ultracentrifugal and immunologic criteria. No protein bands other than the active ones appeared in the zymogram to illustrate the high purity of the proenzyme product. The separate proenzyme bands were excised from the gel and subjected directly to electrophoresis on a second starch gel (Fig. 2), showing clearly their individuality
Fig. i. A. Starch-gel electrophoresis of pooled canine profibrinolysin c h r o m a t o g r a p h e d on h y d r o x y apatite. E q u i v a l e n t results were obtained from a single donor source. 25o V d.c. applied for 4.5 h at room temperature. Tris-citrate buffer at p H 8.65. B. F i b r i n - a c e t a t e e n z y m o g r a m of pooled canine isoproenzymes subjected to electrophoresis on starch gel.
t3iochim. Biophys. Acla, 147 (1967) 6o9-612
PRELIMINARY NOTES
61I
and capacity for activation to fibrinolysin. When activator was absent from the fibrinacetate strip, lysis did not occur. This indicates that none of the protein zones contained spontaneous fibrinolytic activity. Furthermore, eleetrophoresis did not cause activation of the proenzyme.
I:ig. 2. A. Starch-gel electrophoresis of excised canine profibrinolysin c h r o m a t o g r a p h e d on h y d r o x y a p a t i t e i s o p r o e n z y m e s indicating r e t e n t i o n of i n d i v i d u a l mobility. 25o V d.c. applied for 4.5 h at r o o m t e m p e r a t u r e . B. Fibrin a c e t a t e e n z y m o g r a m of (A) d e m o n s t r a t i n g r e t e n t i o n of i n d i v i d u a l isoproenzymes.
To ascertain that these multimolecular forms were not present as a consequence of plasma pooling, profibrinolysin was isolated from a single donor and subjected to starch-gel analysis. Again 4 bands showed up on starch-gel electrophoresis. Apparently multimolecular forms of canine profibrinolysin do exist and represent distinct molecules possessing individuality of certain physicochemical characteristics while exhibiting common enzymatic reactivity. Explanations as to the origin of these multiple proenzyme components remain obscure. Whether they are the products of chemical fractionation has yet to be demonstrated; clearly they are not produced by the electrophoresis on starch gel. It is also possible that these isoproenzymes represent profibrinolysin molecules at various stages of m a t u r i t y or that separate cellular origins m a y produce this variance. BARNHART AND RIDDLE 6 have shown b y immunofluorescence that one cellular site for profibrinolysin synthesis is eosinophils in the bone marrow. The specific granules (lysosomes) of eosinophils also yield a substance that requires activation by urokinase before digesting fibrinL Further work on these possible isoproenzymes is necessary for a reasonable selection of an explanation. The isolation and characterization of each of these four isoproenzymes is being undertaken in our laboratory, as well as investigations of equine, bovine, and human isoproenzymes, which will be reported elsewhere. This investigation was supported by the National Institutes of Health, grant Nos. I-FL-GM-33, 527-Ol and HE-o4712.
Department of Physiology, Wayne State University, School of Medicine, Detroit, Mich. (U.S.A.)
P A U L J. H E B E R L E I N ~[ARION I. BARNHART
Biochim. Biophys. dcla, 147 (1967) 6o9-612
612 i
2 3 4 5 6 7
PRELIMINARY NOTES
ALKJAERSIG, ]3iochem. J., 93 (I964) 171. N. SLOTTA AND J. D. (;ONZ~XLEZ, Thromb. Diath. Haemorrhag., 12 (1964) 126. T. MERTZ AYD J. Y. S. CHA~,', Can. J. Biochem. Physiol., 41 (1963) 1811. B. ~VALLI~;N,Arhiv Kemi., 19 (I962) 469. J. HEBERLEIN ANO M. I. BARNHART, Thrombos. Diath. Haemorrhag., i8 (1967) 286. I. BARNHART AND J. M. RIDDLE, Blood, 21 (1963) 3o3 . I. BARNHART, C. QUINTANA, H. L. LENON, G. B. BLUM AND J. ~I. RIDDLE, Ann. N . Y . Acad. Sci., in th e press. N. K. E. P. PM. M.
Received September 4th, 1967 Biochim. Biophys. Acta, 147 (1967) 6o9-612
BBA 31025
A m i n o acid sequence in rat pancreatic ribonuclease The amino acid sequence in rat pancreatic ribonuclease (EC 2.7.7.16) was determined using 12o mg of tile purified enzyme 1. The following procedures were used successively: performic acid oxidation2; either tryptic or chymotryptic hydrolysis; peptide fractionation on Sephadex G-25 (ref. 3), Dowex i (ref. 4), and Dowex 50 ; amino acid analysis of the peptides; hydrolysis of the larger peptides by chymotrypsin, trypsin, papain or dilute HC1; E d m a n degradation with identification of the new N-terminal amino acids as their dimethylaminonapbthalenesulfonyl (DNS) derivatives 5 and in some cases identification of the amino acids removed as their phenylthiohydantoin derivatives; and carboxypeptidase digestion. All amino acids could be located unambiguously: Fig. I shows the sequence obtained. It also shows some partial sequences of the horse enzyme, which in contrast to the rat enzyme--contains carbohydrate (A. J. SCHEFFER AND J. J. BEIXTEXIA, unpublished results). It is obvious that the best alignment of the rat enzyme with the beef enzyme sequence is that shown in Fig. I. For the sake of clarity the numbering of the beef enzyme has been retained; consequently the rat enzyme is counted from --3 at the N-terminal. The disulfide bridges were not determined; they are very probably identical with those in the beef enzyme. The positions of the amide groups at residue 71 rather than 7 ° and at IOI rather than lO3 are probable, but not definitely established. It is striking that the rat and beef enzymes, which are physically and enzymically very similar, have different amino acids at 33 % of the positions. This is much greater than the differences between the mammalian eytochromes c (ref. 6); it is even larger than the differences between the fl- and y-chains of human hemoglobin 7. In spite of the differences at one-third of the positions all amino acids shown to be part of the active site, or implicated in activity or conformation, are in exactly the same positions in both enzymes. Moreover, replacement of the amino acids in the three-dimensional structure of the beef enzyme (as determined b y X - r a y diffraction s,9) b y those of the rat enzyme does not lead to any structural changes, as far as can be predicted at the moment. Glycine-38 in the rat enzyme, which replaces 13iochim. Biophys. Acta, 147 (1967) 612-614