1988 Fiscal Year Final Research Report Summary
Practical Application of Fluorescence Labeling to Component Sugar Analysis of Glycoproteins
| Project/Area Number |
62880019
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| Research Category |
Grant-in-Aid for Developmental Scientific Research
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| Allocation Type | Single-year Grants |
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| Research Field |
物質生物化学
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| Research Institution | Osaka University |
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Principal Investigator |
HASE Sumihiro Osaka University College of Science, Associate Prof., 理学部, 助教授 (80028232)
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| Co-Investigator(Kenkyū-buntansha) |
KONDO Akihiro Takara Chuzo Pharmaceutical Research Lab., Research worker, 薬品研究所, 研究員
KATO Ikunoshin Takara Chuzo Pharmaceutical Research Lab., Director, 薬品研究所, 所長
IKENAKA Tokuji Osaka University College of Science, Prof., 理学部, 教授 (20028088)
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| Project Period (FY) |
1987 – 1988
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| Keywords | Fluorescence labeling / Component sugar analysis / ピリジルアミノ化 |
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| Research Abstract |
A method for determination of component sugars of glycoproteins by pyridylamination was reported (H. Takemoto, et al. 1985). The method includes acid hydrolysis of glycoprotein, N-acetylation of amino groups, pyridylamination of the reducing ends of the monosaccharides with NaBH_3CN, 2-aminopyridine, and HCl, purification of pyridylamino (PA-) derivatives of sugars by gel chromatography (HPLC), and the analysis of PA-sugars by reversed-phase HPLC. The pyridylamination conditions were improved for the purpose of the making of an automatic sugar analyzer. 1) Volatile reagents such as dimethylamine. borane complex, and 2-aminopyridine-acetic acid were used intead of NaBH_3CN and 2-aminopyridine-HCl. 2) The reaction conditions were chosen to obtain maximum yield of PA-sugars (neutral sugars 70% and N-acetylaminosugars 87%). 3) Nitrogen flow under reduced pressure was developed to evaporate the reagents effectively. Using the new reaction conditions and the evaporation procedre, purification of PA-sugars by gel chromatography (HPLC) could be omitted. An automatic pyridylamination apparatus using the new conditions was constructed and PA-sugars thus obtained were analyzed by HPLC equipped with an automatic injector and a data processor. Reasonable results were obtained by using 1-10 nmol of sugars of acid hydrolysates of glycoproteins.
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