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

亚皮摩尔蛋白氨基酸序列测定方法的研究

基本信息

批准号：
61470160
负责人：
TSUGITA Akira
金额：
$ 4.48万
依托单位：
SCIENCE UNIVERSITY OF TOKYO
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1986
资助国家：
日本
起止时间：
1986 至 1988
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-61470160/
关键词：
N-terminal analysis Sensitization with aminofluoresein アミノフロレセイン蛍光末端分析超微量アミノ酸配列蛋白質N末端配列決定法高感度化 0.1fモル蛍光アミノ誘導体 ATZアミノ酸とアミノフロレセンの反応 Bセル転写賦活因子 DNAー蛋白複合体の配列決定法レーザ螢光検出球型シーケンサ SDSゲネ泳動の二重ゲル揮揆性緩衝液のゲル泳動 C未酵素分解(微量)法の開発 500fモル

项目摘要

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.

亚微量蛋白质的N-末端测序是细胞和分子生物学领域最迫切需要解决的问题之一。我们已经开发了一种方法敏化ATZ-氨基酸，这是埃德曼降解的中间产物。几种敏化剂，包括放射性胺，如<125>碘组胺和荧光胺进行了测试，其反应效率，产品的灵敏度和易于处理。选择最适合的试剂氨基荧光素（AF）进行进一步研究。通过高压液相色谱（HPLC）纯化商业AF试剂。研究了AF与ATZ-Leu的反应动力学，确定了最佳反应配比、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，其余氨基酸均能定量产率，20种产物均稳定。AF衍生物在pH 8时的灵敏度比在pH 5.0时增加了10倍以上。这导致在pH 8下使用碱不敏感柱进行HPLC分离。通过HPLC分离所有20种氨基酸AF衍生物，产物的灵敏度为约0微摩尔。将该方法应用于商业蛋白质测序仪，对程序进行微小修改。对一个标准蛋白和几个未知蛋白进行了100 f- 1 mpol的测序，每个步骤7 - 10步。观察到3个非预期峰，它们来自残留的异硫氰酸苯酯、三氟乙酸和AF中的杂质。通过改变程序和纯化试剂，这些峰大大减少。为了进一步提高测序仪的灵敏度，我们进行了几系列的基础实验，以建立一个新的测序仪。