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Novel products in hyaluronan digested by bovine testicular hyaluronidase

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Abstract

When the products of hyaluronan (HA) digested by bovine testicular hyaluronidase (BTH) were analyzed by high-performance liquid chromatography (HPLC), minor peaks were detected just before the main even-numbered oligosaccharide peaks. The amount of each minor peak was dependent on the reaction conditions for transglycosylation, rather than hydrolysis, by the BTH. Mainly based on HPLC and MS analysis, each minor peak was found to correspond to its oligosaccharide with one N-acetyl group removed from the reducing terminal N-acetylglucosamine. Enzymatic studies showed that the N-deacetylation activity was closely related to reaction temperature, pH, and the concentration of NaCl contained in the buffer, and glycosaminoglycan types and chain lengths of substrates. These findings strongly suggest that the N-deacetylation reaction in minor peaks was due to a novel enzyme contaminant in the BTH, N-deacetylase, that carries out N-deacetylation at the reducing terminal N-acetylglucosamine of oligosaccharides and is dependent on HA hydrolysis by BTH.

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Abbreviations

BTH:

Bovine testicular hyaluronidase

Ch:

Chondroitin

Ch4S:

Chondroitin 4-sulfate

Ch6S:

Chondroitin 6-sulfate

HA:

Hyaluronan

HPLC:

High-performance liquid chromatography

NDST:

N-Deacetylase/N-sulfotransferase

TLC:

Thin-layer chromatography

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Acknowledgments

This work was supported by Grants-in Aid (Nos. 15370041 and 17770106) for Scientific Research from the Ministry of Education, culture, Sports, Science, and Technology of Japan and Cooperation for Innovative Technology and Advanced Research in Evolution Area (CITY AREA).

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Authors and Affiliations

  1. Department of Glycotechnology, Center for Advanced Medical Research, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, 036-8562, Japan

    Fengchao Chen, Ikuko Kakizaki, Masanori Yamaguchi, Kaoru Kojima, Keiichi Takagaki & Masahiko Endo

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  1. Fengchao Chen
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  2. Ikuko Kakizaki
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  3. Masanori Yamaguchi
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  4. Kaoru Kojima
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  5. Keiichi Takagaki
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  6. Masahiko Endo
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Correspondence to Fengchao Chen.

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Chen, F., Kakizaki, I., Yamaguchi, M. et al. Novel products in hyaluronan digested by bovine testicular hyaluronidase. Glycoconj J 26, 559–566 (2009). https://doi.org/10.1007/s10719-008-9200-2

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  • DOI: https://doi.org/10.1007/s10719-008-9200-2

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