Matsuno Kanae, Suzuki Shigeo
School of Pharmacy, Kinki University, 3-4-1 Kowakae, Higashi-Osaka 577-8501, Japan.
Anal Biochem. 2008 Apr 1;375(1):53-9. doi: 10.1016/j.ab.2008.01.002. Epub 2008 Jan 9.
A simple and rapid fluorimetric method was developed for detection and quantitative analysis of sialic acids in glycoproteins. Sialic acid residues in glycoproteins were specifically oxidized with periodate at 0 degrees C for 45 min. Formaldehyde generated from carbon 9 (C-9) of sialic acid was converted specifically to fluorescent dihydropyridine derivative with acetoacetanilide and ammonia at room temperature for 10 min. The reaction products indicate intense fluorescence with excitation and emission maxima at 388 and 471 nm, respectively. When the reaction was conducted in approximately a 1-ml volume, the linearity of the calibration exhibited between 2 and 180 microg of bovine fetuin, or between 0.3 and 27 nmol of N-acetylneuraminic acid, as a model glycoprotein. The limit of detection, based on three times the standard deviation of the reagent blank, was 0.5 microg of fetuin. The proposed method was applied to determination of sialic acids in various glycoprotein samples. This proposed method is simple and obviates the heating and extraction steps. It is highly specific to sialic acids in glycoproteins and indicates no fluorescence of neutral glycoproteins.
开发了一种简单快速的荧光法，用于检测和定量分析糖蛋白中的唾液酸。糖蛋白中的唾液酸残基在0℃下用高碘酸盐特异性氧化45分钟。唾液酸C-9位产生的甲醛在室温下与乙酰乙酰苯胺和氨特异性转化为荧光二氢吡啶衍生物，反应10分钟。反应产物在激发波长和发射波长分别为388和471nm时显示出强烈荧光。当反应在约1ml体积中进行时，以牛胎球蛋白为模型糖蛋白，校准曲线的线性范围为2至180μg牛胎球蛋白，或0.3至27nmol N-乙酰神经氨酸。基于试剂空白三倍标准偏差的检测限为0.5μg胎球蛋白。该方法应用于各种糖蛋白样品中唾液酸的测定。该方法简单，无需加热和提取步骤。它对糖蛋白中的唾液酸具有高度特异性，中性糖蛋白无荧光显示。
