Gaur R K, Paliwal S, Sharma P, Gupta K C
DNA Synthesis Laboratory, CSIR Centre for Biochemicals, Delhi, India.
J Biochem Biophys Methods. 1989 Jun;18(4):323-9. doi: 10.1016/0165-022x(89)90041-9.
A simple and sensitive method for the quantitative determination of free amino groups on solid support is described. This approach is a modification of Ngo's [(1986) J. Biochem. Biophys. Methods 12, 349-354] method reported earlier. The method is based on the reaction of the solid support with an excess of 5'-O-(4,4'-dimethoxytrityl)-thymidine-3'-O-(2,4-dinitrophenyl) succinate (DTDS) in the presence of a catalytic amount of 4-dimethylaminopyridine. After removing the excess reagent, solid support is treated with perchloric acid to release 4,4'-dimethoxytrityl cation into the solution. The released 4,4'-dimethoxytrityl cation, which has a strong absorption at 498 nm (epsilon 498 = 70,000), is then determined spectrophotometrically. A comparative study of DTDS, N-succinimidyl-3-(2-pyridyldithio)propionate and 4,4-dimethoxytrityl chloride is also included. The method was found to be very useful to determine those amino groups which are available for functionalization of solid supports, especially, monitoring the functionalization of solid supports for affinity chromatography and synthesis of biopolymers.
本文描述了一种用于定量测定固体支持物上游离氨基的简单且灵敏的方法。该方法是对Ngo [(1986) J. Biochem. Biophys. Methods 12, 349 - 354] 早期报道方法的改进。该方法基于固体支持物在催化量的4 - 二甲基氨基吡啶存在下与过量的5'-O-(4,4'-二甲氧基三苯甲基)-胸腺嘧啶核苷-3'-O-(2,4 - 二硝基苯基)琥珀酸酯 (DTDS) 反应。去除过量试剂后，用高氯酸处理固体支持物，使4,4'-二甲氧基三苯甲基阳离子释放到溶液中。然后通过分光光度法测定释放出的在498 nm处有强吸收（ε498 = 70,000）的4,4'-二甲氧基三苯甲基阳离子。文中还包含了对DTDS、N - 琥珀酰亚胺基 - 3-(2 - 吡啶基二硫代)丙酸酯和4,4 - 二甲氧基三苯甲基氯的比较研究。该方法被发现对于测定可用于固体支持物功能化的氨基非常有用，尤其是用于监测用于亲和色谱和生物聚合物合成的固体支持物的功能化。
