氚离子束标记蛋白质水解产物的氨基酸分析

Determination of amino acids in hydrolysate of protein labelled by the tritium ion beam method

叙述了快速、灵敏、正确地分离分析氚标记蛋白质水解产物的方法。氚化核糖核酸酶A、大豆胰蛋白酶抑制剂和弹性蛋白酶用6mol/l HCl在110℃真空玻璃管中水解24 h后进行氨基酸分析,通过梯度洗脱分离出各氨基酸残基,并使之与OPA试剂反应成衍生物。经荧光检测获得每个氨基酸残基的化学量,并用液闪法测量其放射性。实验证明在氚化蛋白质中,所有的氨基酸残基均被氚化,但各个氨基酸残基上的氚分布情况随氨基酸的结构与蛋白质的结构环境影响而不同。

A fast, sensitive and accurate method for the separation and determination of hydrolysate of proteins labelled with tritium is described. Tritium labelled proteins, 3H-ribonuclease A, 3H-soybean trypsin inhibitors and 3H-elastin which dissolved in 6mol/l HCl and sealed in evacuated glass tube, were hydrolyzed for 24 hours at 110℃. The hydrolysate was then concentrated at 50-55℃, and cleaned with SEP-PAK C18 Cartridge. The eluate was injected to Waters Amino Acids Analysis System, the chemical quantity of amino acids was determined with a post-column fluorometric system. Each amino acid was collected respectively and its radioactivity was measured with a liquid scintillation counter. It was confirmed that tritium atoms were labelled at stable position of the amino acids of proteins.