Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1981 Aug 1;197(2):275–282. doi: 10.1042/bj1970275

Sequential hydrolysis of hyaluronate by beta-glucuronidase and beta-N-acetylhexosaminidase.

M O Longas, K Meyer
PMCID: PMC1163124  PMID: 6459778

Abstract

1. Hyaluronate extracted from rooster comb was digested by a mixture of beta-N-acetylhexosaminidase and beta-glucuronidase with simultaneous dialysis for 96 h. 2. The produjct, yielding 99.6% of a mixture of mono- and oligo-saccharides, was purified by gel chromatography and analysed for glucuronic acid, N-acetylglucosamine and other sugars. 3. The oligosaccharide portion was chromatographed on DEAE-cellulose, and the effluent fractions were analysed for glucuronic acid and N-acetylglucosamine, reduced with NaBH4, digested by beta-N-acetylhexosaminidase and subjected to acid hydrolysis and glucosamine determination. 4. GlcNAc-GlcA-GlcNAc, GlcA-GlcA-GlcNAc and GlcA-GlcA-GlcA-GlcNAc were the oligosaccharides obtained, which resulted from the transferase activity of the enzymes and represented 57% of the digestion products. The results demonstrate that this hyaluronate is an unbranched polymer of approximatey equal amounts of glucuronic acid and N-acetylglucosamine. The data also indicate that if this glycosaminoglycan contains any of the neutral sugars for which it was analysed, their concentration must be less than 0.020% of the sum of the known components.

Full text

PDF
277

Selected References

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

  1. Appel A., Horwitz A. L., Dorfman A. Cell-free synthesis of hyaluronic acid in Marfan syndrome. J Biol Chem. 1979 Dec 10;254(23):12199–12203. [PubMed] [Google Scholar]
  2. BALAZS E. A. Physical chemistry of hyaluronic acid. Fed Proc. 1958 Dec;17(4):1086–1093. [PubMed] [Google Scholar]
  3. BITTER T., MUIR H. M. A modified uronic acid carbazole reaction. Anal Biochem. 1962 Oct;4:330–334. doi: 10.1016/0003-2697(62)90095-7. [DOI] [PubMed] [Google Scholar]
  4. Darke A., Finer E. G., Moorhouse R., Rees D. A. Studies of hyaluronate solutions by nuclear magnetic relaxation measurements. Detection of covalently-defined, stiff segments within the flexible chains. J Mol Biol. 1975 Dec 15;99(3):477–486. doi: 10.1016/s0022-2836(75)80139-2. [DOI] [PubMed] [Google Scholar]
  5. Elson L. A., Morgan W. T. A colorimetric method for the determination of glucosamine and chondrosamine. Biochem J. 1933;27(6):1824–1828. doi: 10.1042/bj0271824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. HAY G. W., LEWIS B. A., SMITH F. THIN-FILM CHROMATOGRAPHY IN THE STUDY OF CARBOHYDRATES. J Chromatogr. 1963 Aug;11:479–486. doi: 10.1016/s0021-9673(01)80949-3. [DOI] [PubMed] [Google Scholar]
  7. Harris R. G., Rowe J. J., Stewart P. S., Williams D. C. Affinity chromatography of -glucuronidase. FEBS Lett. 1973 Jan 15;29(2):189–192. doi: 10.1016/0014-5793(73)80558-7. [DOI] [PubMed] [Google Scholar]
  8. LINKER A., MAYER K. Production of unsaturated uronides by bacterial hyaluronidases. Nature. 1954 Dec 25;174(4443):1192–1193. doi: 10.1038/1741192a0. [DOI] [PubMed] [Google Scholar]
  9. LINKER A., MEYER K., WEISSMANN B. Enzumatic formation of monosaccharides from hyaluronate. J Biol Chem. 1955 Mar;213(1):237–248. [PubMed] [Google Scholar]
  10. Lee Y. C., Johnson G. S., White B., Scocca J. An accelerated system for analysis of neutral sugars in complex carbohydrates. Anal Biochem. 1971 Oct;43(2):640–643. doi: 10.1016/0003-2697(71)90301-0. [DOI] [PubMed] [Google Scholar]
  11. MEYER K. Chemical structure of hyaluronic acid. Fed Proc. 1958 Dec;17(4):1075–1077. [PubMed] [Google Scholar]
  12. MEYER K. The chemistry of the mesodermal ground substances. Harvey Lect. 1955;51:88–112. [PubMed] [Google Scholar]
  13. Morgan W. T., Elson L. A. A colorimetric method for the determination of N-acetylglucosamine and N-acetylchrondrosamine. Biochem J. 1934;28(3):988–995. doi: 10.1042/bj0280988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Niemann R., Buddecke E. Acceptor-specific glucuronyl transfer catalyzed by beta-glucuronidase. Biochim Biophys Acta. 1979 Mar 16;567(1):196–206. doi: 10.1016/0005-2744(79)90186-4. [DOI] [PubMed] [Google Scholar]
  15. PARK J. T., JOHNSON M. J. A submicrodetermination of glucose. J Biol Chem. 1949 Nov;181(1):149–151. [PubMed] [Google Scholar]
  16. Pokorny M., Glaudemans C. P. Purification of N-acetyl beta-D-hexosaminidase from bull epididymis by affinity chromatography. FEBS Lett. 1975 Jan 15;50(1):66–69. doi: 10.1016/0014-5793(75)81042-8. [DOI] [PubMed] [Google Scholar]
  17. REISSIG J. L., STORMINGER J. L., LELOIR L. F. A modified colorimetric method for the estimation of N-acetylamino sugars. J Biol Chem. 1955 Dec;217(2):959–966. [PubMed] [Google Scholar]
  18. Swann D. A. Studies on hyaluronic acid. II. The protein component(s) of rooster comb hyaluronic acid. Biochim Biophys Acta. 1968 May 6;160(1):96–105. doi: 10.1016/0005-2795(68)90068-8. [DOI] [PubMed] [Google Scholar]
  19. Varma R., Varma R. S., Allen W. S., Wardi A. H. On the carbohydrate-protein linkage group in vitreous humor hyaluronate. Biochim Biophys Acta. 1974 Oct 8;362(3):584–588. doi: 10.1016/0304-4165(74)90154-8. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES