STUDY ON METHOD FOR AMINO ACID SEQUENCE OF SUB-PICO-MOLE PROTEIN

亚微量蛋白质的N末端测序是细胞和分子生物学领域中需求最大的项目之一。我们开发了一种方法来敏化EDMAN降解的中间产物ATZ-氨基酸。对~(125)I-I-碘组胺和荧光胺等放射性胺类增敏剂的反应效率、产物的灵敏度和操作简便性进行了测试。选择了最合适的试剂氨基荧光素(AF)进行进一步的研究。用高压液相色谱(HPLC)对市售AF试剂进行纯化。考察了AF与ATZ-Leu的反应动力学，确定了最佳试剂配比、反应温度和反应时间。测试了20种氨基酸的ATZ-衍生物的反应产率和20种不同衍生物的稳定性。除Glu、Arg和His(45%：)外，所有氨基酸均有定量产率，20种产物均稳定。在pH=8时，AF衍生物的灵敏度比在pH=5.0时提高了10倍以上。采用碱性不敏感色谱柱，在pH值为8的条件下，对20种氨基酸进行了分离，产物的灵敏度约为0.u摩尔。该方法被应用于商业蛋白质测序仪，对程序进行了较小的修改。对一个标准蛋白和几个未知蛋白进行了100F-1MPOL的测序，每7-10步。在苯异硫氰酸酯残留物、三氟乙酸根和AF中的一个杂质中观察到了3个意想不到的峰。通过改变程序和提纯试剂，这些都大大减少了。为了进一步增敏测序仪，进行了几个系列的基础实验，以建立一种新的测序仪。

N-terminal sequencing of sub-micro quantities of protein is one of the most demanded projects in the fields of cellular and molecular biology. We have developed a method for sensitizing ATZ-amino acids which are intermediate products of Edman degradation. Several sensitizing reagents involving, radioactive amines such as ^<125>I-iodohistamine and fluorescent amines were tested for their reaction efficiency, sensitivities of the products and their ease of handling. The most suitable reagent, aminofluorescein (AF), was chosen for further investigation. Commercial AF reagents were purified by high pressure liquid chromatography (HPLC). The reaction kinetics between AF and ATZ-Leu was surveyed for the optimum ratio of reagent, reaction temperature, and reaction time. ATZ-derivatives of 20 amino acids were tested for their reaction yields and the stability of the 20 different derivatives. Except for Glu, Arg and His(45%:, all amino acids gave guantitative yields and all 20 products were found to be stable. The AF derivatives showed an increase of more than 10 times sensitivity at pH 8 than at pH 5.0. this lead to the use of an alkalineinsensitive column for HPLC separation at pH 8. all 20 amino acid af derivatives were separated by HPLC and the sensitivity of the product was about 0.u fmole. The method was applied to a commercial protein sequencer with minor modifications to the program. Sequencing of 100f- 1 mpol of protein have been achieved for a standard protein and several unknown proteins, each 7 - 10 steps. 3 unexpected peaks were observed which derived from residual phenylisothiocyanate, trifluoroacetic acid and an impurity in AF. These are substantially reduced by changing the program and purifying the reagent. For further sensitization of the sequencer, several lines of fundamental experiments have been performed to established a new sequencer.