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

• 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

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.

亚微米量蛋白的N末端测序是细胞和分子生物学领域需求最高的项目之一。我们已经开发了一种敏化ATZ-氨基酸的方法，ATZ-氨基酸是Edman降解的中间产物。测试了几种涉及放射性胺的敏化试剂，例如 ^<125> I-碘组胺和荧光胺的反应效率，产品的敏感性及其易于处理性。选择了最合适的试剂氨基氟乙烯（AF）进行进一步研究。通过高压液相色谱（HPLC）纯化商业AF试剂。调查了AF和ATZ-LEU之间的反应动力学，以达到试剂，反应温度和反应时间的最佳比率。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。 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该方法适用于对程序的较小修饰的商业蛋白质测序蛋白。试剂。

项目成果

A. Tsugita et al.: Proteins: structure and function Approaches for microsequencing. Plenum Pub. Co. New York, 315-322 (1987)

A. Tsugita 等人：蛋白质：结构和功能微测序方法。

A.Tsugita 他: J.Biochem.103. 399-401 (1988)

A. Tsugita 等人：J.Biochem.103（1988）。

次田晧: 化学と生物. 26. 330-337 (1988)

次田晃：化学与生物学。26. 330-337 (1988)

M.Kamo: J.Biochem. 102. 243-246 (1987)

M.Kamo：J.Biochem。

F.Gabrielli: Mol.Cell.Biol.71. 129-134 (1986)

F.Gabrielli：Mol.Cell.Biol.71。