Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1981 Dec;148(3):861–868. doi: 10.1128/jb.148.3.861-868.1981

Partial purification and characterization of the multiple molecular forms of staphylococcal clotting activity (coagulase).

M W Reeves, M C Drummond, M Tager
PMCID: PMC216285  PMID: 7309679

Abstract

The clotting activity of Staphylococcus aureus strain 104 was purified 46,000-fold, but absolute purity was not achieved. Carbohydrate content of the purified material was not more than 5%. Elution of clotting activity from denaturing and nondenaturing polyacrylamide gels revealed the presence of four distinct molecular forms. Molecular weights of the forms were approximately 31,500, 34,800, 44,800, and 56,800 as determined by gel filtration in 8 M urea, by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis, and by calculation with determined values for the Stokes radius and sedimentation coefficient. Molecular weights determined on sodium dodecyl sulfate-urea gels were found to decrease as the gel concentration increased, suggesting that the amount of sodium dodecyl sulfate bound was less than normal. Estimated frictional ratios for the forms showed that they differ in shape from one another and that they are all highly asymmetrical. Each of the forms had an isoelectric point between pH 5.44 and 5.47 when focused in 6% polyacrylamide gels for 9 h; however, prolonged focusing altered the isoelectric point of the forms to within the range of pH 4.35 to 4.65. The multiple clotting forms were not artifacts of the purification procedure and did not appear to be products of the proteolytic degradation of a larger protein.

Full text

PDF
861

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Andrews P. Estimation of the molecular weights of proteins by Sephadex gel-filtration. Biochem J. 1964 May;91(2):222–233. doi: 10.1042/bj0910222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Andrews P. The gel-filtration behaviour of proteins related to their molecular weights over a wide range. Biochem J. 1965 Sep;96(3):595–606. doi: 10.1042/bj0960595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. BARNES E. H., MORRIS J. F. A quantitative study of the phosphatase activity of Micrococcus pyogenes. J Bacteriol. 1957 Jan;73(1):100–104. doi: 10.1128/jb.73.1.100-104.1957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bas B. M., Muller A. D., Hemker H. C. Purification and properties of staphylocoagulase. Biochim Biophys Acta. 1975 Jan 30;379(1):164–171. doi: 10.1016/0005-2795(75)90018-5. [DOI] [PubMed] [Google Scholar]
  5. DAVIS B. J. DISC ELECTROPHORESIS. II. METHOD AND APPLICATION TO HUMAN SERUM PROTEINS. Ann N Y Acad Sci. 1964 Dec 28;121:404–427. doi: 10.1111/j.1749-6632.1964.tb14213.x. [DOI] [PubMed] [Google Scholar]
  6. DUTHIE E. S., HAUGHTON G. Purification of free staphylococcal coagulase. Biochem J. 1958 Sep;70(1):125–134. doi: 10.1042/bj0700125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. DUTHIE E. S. Variation in the antigenic composition of staphylococcal coagulase. J Gen Microbiol. 1952 Nov;7(3-4):320–326. doi: 10.1099/00221287-7-3-4-320. [DOI] [PubMed] [Google Scholar]
  8. Davison P. F. Proteins in denaturing solvents: gel exclusion studies. Science. 1968 Aug 30;161(3844):906–907. doi: 10.1126/science.161.3844.906. [DOI] [PubMed] [Google Scholar]
  9. Fish W. W., Mann K. G., Tanford C. The estimation of polypeptide chain molecular weights by gel filtration in 6 M guanidine hydrochloride. J Biol Chem. 1969 Sep 25;244(18):4989–4994. [PubMed] [Google Scholar]
  10. Fukui S., Tanaka A., Kawamoto S., Yasuhara S., Teranishi Y., Osumi M. Ultrastructure of methanol-utilizing yeast cells: appearance of microbodies in relation to high catalase activity. J Bacteriol. 1975 Jul;123(1):317–328. doi: 10.1128/jb.123.1.317-328.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. HENDERSON A., BRODIE J. INVESTIGATIONS ON STAPHYLOCOCCAL COAGULASE. Br J Exp Pathol. 1963 Oct;44:524–528. [PMC free article] [PubMed] [Google Scholar]
  12. Kanai M., Raz A., Goodman D. S. Retinol-binding protein: the transport protein for vitamin A in human plasma. J Clin Invest. 1968 Sep;47(9):2025–2044. doi: 10.1172/JCI105889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kellen J. Isoenzymes of lactate-dehydrogenase in micro-organisms. Nature. 1965 Aug 14;207(998):783–784. doi: 10.1038/207783b0. [DOI] [PubMed] [Google Scholar]
  14. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. MADOFF M. A., WEINSTEIN L. Purification of staphylococcal alpha-hemolysin. J Bacteriol. 1962 Apr;83:914–918. doi: 10.1128/jb.83.4.914-918.1962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. MARTIN R. G., AMES B. N. A method for determining the sedimentation behavior of enzymes: application to protein mixtures. J Biol Chem. 1961 May;236:1372–1379. [PubMed] [Google Scholar]
  18. MIALE J. B., WINNINGHAM A. R., KENT J. W. Staphylococcal isocoagulases. Nature. 1963 Jan 26;197:392–392. doi: 10.1038/197392a0. [DOI] [PubMed] [Google Scholar]
  19. MURRAY M., GOHDES P. Purification of staphylococcal coagulase. Biochim Biophys Acta. 1960 Jun 3;40:518–522. doi: 10.1016/0006-3002(60)91392-5. [DOI] [PubMed] [Google Scholar]
  20. Martley F. G., Jarvis A. W., Bacon D. F., Lawrence R. C. Typing of coagulase-positive staphylococci by proteolytic activity on buffered caseinate-agar, with special reference to bacteriophage nontypable strains. Infect Immun. 1970 Oct;2(4):439–442. doi: 10.1128/iai.2.4.439-442.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. ORNSTEIN L. DISC ELECTROPHORESIS. I. BACKGROUND AND THEORY. Ann N Y Acad Sci. 1964 Dec 28;121:321–349. doi: 10.1111/j.1749-6632.1964.tb14207.x. [DOI] [PubMed] [Google Scholar]
  22. Owen W. G., Penick G. D., Yoder E., Poole B. L. Evidence for an ester bond between thrombin and heparin cofactor. Thromb Haemost. 1976 Feb 29;35(1):87–95. [PubMed] [Google Scholar]
  23. PARK J. T., JOHNSON M. J. A submicrodetermination of glucose. J Biol Chem. 1949 Nov;181(1):149–151. [PubMed] [Google Scholar]
  24. Reichert L. E., Jr, Rasco M. A., Ward D. N., Niswender G. D., Midgley A. R., Jr Isolation and properties of subunits of bovine pituitary luteinizing hormone. J Biol Chem. 1969 Oct 10;244(19):5110–5117. [PubMed] [Google Scholar]
  25. Righetti P., Drysdale J. W. Isoelectric focusing in polyacrylamide gels. Biochim Biophys Acta. 1971 Apr 27;236(1):17–28. doi: 10.1016/0005-2795(71)90144-9. [DOI] [PubMed] [Google Scholar]
  26. Rosenberg J. S., McKenna P. W., Rosenberg R. D. Inhibition of human factor IXa by human antithrombin. J Biol Chem. 1975 Dec 10;250(23):8883–8888. [PubMed] [Google Scholar]
  27. Rosenberg R. D., Damus P. S. The purification and mechanism of action of human antithrombin-heparin cofactor. J Biol Chem. 1973 Sep 25;248(18):6490–6505. [PubMed] [Google Scholar]
  28. Rydén A. C., Lindberg M., Philipson L. Isolation and characterization of two protease-producing mutants from Staphylococcus aureus. J Bacteriol. 1973 Oct;116(1):25–32. doi: 10.1128/jb.116.1.25-32.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. SIEGEL L. M., MONTY K. J. DETERMINATION OF MOLECULAR WEIGHTS AND FRICTIONAL RATIOS OF MACROMOLECULES IN IMPURE SYSTEMS: AGGREGATION OF UREASE. Biochem Biophys Res Commun. 1965 May 3;19:494–499. doi: 10.1016/0006-291x(65)90152-x. [DOI] [PubMed] [Google Scholar]
  30. Shapiro A. L., Viñuela E., Maizel J. V., Jr Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem Biophys Res Commun. 1967 Sep 7;28(5):815–820. doi: 10.1016/0006-291x(67)90391-9. [DOI] [PubMed] [Google Scholar]
  31. Siegel L. M., Monty K. J. Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductases. Biochim Biophys Acta. 1966 Feb 7;112(2):346–362. doi: 10.1016/0926-6585(66)90333-5. [DOI] [PubMed] [Google Scholar]
  32. Tager M., Drummond M. C. Staphylocoagulase. Ann N Y Acad Sci. 1965 Jul 23;128(1):92–111. doi: 10.1111/j.1749-6632.1965.tb11632.x. [DOI] [PubMed] [Google Scholar]
  33. Tipper D. J., Strominger J. L. Isolation of 4-O-beta-N-acetylmuramyl-N-acetylglucosamine and 4-O-beta-N, 6-O-diacetylmuramyl-N-acetylglucosamine and the structure of the cell wall polysaccharide of Staphylococcus aureus. Biochem Biophys Res Commun. 1966 Jan 4;22(1):48–56. doi: 10.1016/0006-291x(66)90601-2. [DOI] [PubMed] [Google Scholar]
  34. Tirunarayanan M. O. Investigations on the enzymes and toxins of staphylococci. Stimulation of coagulase biosynthesis by glucose and bicarbonate. Acta Pathol Microbiol Scand. 1966;68(2):273–280. doi: 10.1111/apm.1966.68.2.273. [DOI] [PubMed] [Google Scholar]
  35. Vesterberg O., Wadström T., Vesterberg K., Svensson H., Malmgren B. Studies on extracellular PROTEINS FROM Staphylococcus aureus. I. Separation and characterization of enzymes and toxins by isoelectric focusing. Biochim Biophys Acta. 1967 Apr 11;133(3):435–445. doi: 10.1016/0005-2795(67)90547-8. [DOI] [PubMed] [Google Scholar]
  36. Weber K., Kuter D. J. Reversible denaturation of enzymes by sodium dodecyl sulfate. J Biol Chem. 1971 Jul 25;246(14):4504–4509. [PubMed] [Google Scholar]
  37. ZOLLI Z., Jr, SANCLEMENTE C. L. PURIFICATION AND CHARACTERIZATION OF STAPHYLOCOAGULASE. J Bacteriol. 1963 Sep;86:527–535. doi: 10.1128/jb.86.3.527-535.1963. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES