Development of the methods for the micro-determination of sugar chains by high performance liquid chromatography.

1.氚标记低聚糖和单糖醇的制备：以人凝血酶原、人IgG、人转铁蛋白、hCG、牛C1q、牛凝血因子、凝血酶原、因子X和因子IX为原料，经肼解、NaB <^3H_4>还原制备放射性天冬酰胺连接低聚糖。用NaB <^3H_4>还原糖蛋白糖链中的单糖，制备了放射性单糖醇.用HPLC微量测定糖链方法的发展：对31个制备用于单糖或寡糖分析的HPLC柱进行了试验，以分离上述得到的放射性碳水化合物，分离的重现性，以及从柱上定量洗脱碳水化合物。通过改变柱温、流速、洗脱缓冲液等条件，分析了各色谱柱的性能，成功地实现了单糖醇混合物在80 min内，酸性寡糖在60 min内，在75分钟内中性寡糖的大小达到25个葡萄糖单元，这对于寡糖的单糖测序是有用的，在90 min内测定了与凝集素有亲和性的寡糖，为今后寡糖自动分析仪的研制奠定了基础。

1. Preparation of tritium-labeled oligosaccharides and monosaccharide alcohols.The radioactive asparagine-linked oligosaccharides were prepared from human prothrombin, human IgG, human transferrin, hCG, bovine C1q, and bovine blood coagulation factors, prothrombin, factor X, and factor IX by hydrazinolysis followed by NaB <^3H_4> -reduction. The radioactive monosaccharide alcohols were also prepared by NaB <^3H_4> -reduction of monosaccharides contained in the sugar chains of glycoproteins.2. Development of the methods for micro-determination of sugar chains by HPLC.Thirty one HPLC columns prepared for the analysis of monosaccharides or oligosaccharides were tested for the separation of the radioactive carbohydrates obtained above, for the reproducibility of the separation, and for the quantitative elution of the carbohydrates from the column. The property of each column was analyzed by changing the column temperature, the flow rate, the elution buffer, etc. As the results, we are successful in the developments of the quantitative separations of monosaccharide alcohols mixture within 80 min, of acidic oligosaccharides within 60 min, of the neutral oligosaccharides up to 25 glucose units by their size within 75 min which is useful for the monosaccharide sequencing of the oligosaccharides, and of the oligosaccharides which have affinities to lectins within 90 min. These results will become a basis for the development of automatic analyzers for oligosaccharides in future.