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Journal of Biochemistry Advance Access originally published online on February 23, 2009
Journal of Biochemistry 2009 145(6):739-749; doi:10.1093/jb/mvp031
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© The Authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Evaluation of Conditions for Release of Mucin-Type Oligosaccharides from Glycoproteins by Hydrazine Gas Treatment

Yukinobu Goso*, Daigo Tsubokawa and Kazuhiko Ishihara

Department of Biochemistry, Kitasato University Graduate School of Medical Sciences, Sagamihara 228-8555, Japan

*To whom correspondence should be addressed: Fax: +81-42-778-8441; E-mail: gosoy{at}nifmail.jp

Received February 2, 2009; Accepted February 17, 2009


  Abstract

By using commercially available anhydrous hydrazine in the gas-phase, mucin-type oligosaccharides were released from porcine gastric mucin (PGM) and bovine fetuin. The data indicated that a certain amount of the oligosaccharides from PGM were further degraded. Despite this, the HPLC elution profile of the anthranilic acid (AA)-derivatized oligosaccharides obtained by the treatment with hydrazine at 65°C for 6 h resembled those obtained from the alkaline-borohydride treatment, except for the additional disaccharide fractions derived from the core 1 side of the oligosaccharides by further degradation. The other degraded products derived from the core 2 side could not be derivatized by AA, therefore, not visible by fluorescence detection. Liberation of the oligosaccharides was incomplete by the hydrazine treatment for 6 h. Although almost complete liberation was achieved by extending the treatment to 18 h, the degraded products also increased. In this case, the addition of barium oxide to the reaction vessel decreased the degree of further degradation. Results similar to PGM were obtained from bovine fetuin, but with less degradation. Application of this method for the analysis of rat gastric mucin (RGM) obtained from the corpus and antral region revealed that RGM has a large oligosaccharide portion on the core 1 side.

Key Words: anthranilic acid, fetuin, hydrazine, mucin-type oligosaccharide, porcine gastric mucin, rat gastric mucin

Abbreviations: AA, anthranilic acid; GalNAc-ol, N-acetylgalactosaminitol; MALDI-TOF/MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; PGM, porcine gastric mucin; RGM, rat gastric mucin


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