| Research Abstract |
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.
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