Chemistry: analytical and immunological testing – Peptide – protein or amino acid – Amino acid or sequencing procedure
Patent
1994-05-09
1996-07-23
Redding, David A.
Chemistry: analytical and immunological testing
Peptide, protein or amino acid
Amino acid or sequencing procedure
436161, 436172, 530405, 530409, G01N 3300
Patent
active
055388965
DESCRIPTION:
BRIEF SUMMARY
TECHNOLOGICAL FIELD
The present invention relates to an analysis method of amino acid sequence from an amino (N) terminal of protein.
BACKGROUND TECHNOLOGY
As shown in FIG. 2, for identification of an amino acid derivative in the last stage of Edman degradation, which is a sequence analysis method of a protein from the N terminal, protein is conventionally reacted with phenylisothiocyanate (PITC) to obtain phenylthiocarbamyl (PTC) protein, which is treated with an acid to produce 2-amino-5-thiazolinone (ATZ) amino acid derivative, which is further treated with an acid to form phenylthiohydantoin (PTH) amino acid derivative, which is detected by ultraviolet absorption spectroscopy as a known analysis method (P. Edman, Acta Chem. Scand. 10, 761 (1956)).
Further, another method is disclosed in Japanese Patent Application Laid-Open No. 61-264264, and shown herein in FIG. 3, in which an ATZ amino acid derivative is reacted with an amino compound labeled with a radioactiveisotope to form a PTC amino acid derivative, which is separated by thin-layer chromatography for detection.
Moreover, still another method is disclosed in Japanese Patent Application Laid-Open No. 63-196858 and shown in FIG. 4 herein in which an ATZ amino acid derivative is reacted with a fluorescent amino compound to form a PTC amino acid derivative, which is separated by high speed liquid chromatography for detection.
With regard to the conventional method of detecting the PTH amino acid derivative by ultraviolet absorption spectroscopy, while the detecting means is rather simple, this sequencing method is not suitable for the highly sensitive analysis which is recently demanded for treating a trace amount of protein or very trace amount of peptide.
Further, with regard to the developed highly sensitive analysis method utilizing the amino compound labeled with the radioactiveisotope, this has rather limited application in view of ill effects to the environment and, in particular, to the human body.
Moreover, with regard to the other developed highly sensitive analysis method utilizing the fluorescent amino compound, it is necessary to add a large quantity of a fluorescent reagent for maintaining a reaction rate when treating a trace amount of sample in such a liquid phase reaction. Further, it is known that the reaction yield rate decreases as a density of the sample, i.e., the ATZ derivative of protein or peptide decreases even though a large quantity of the reactive reagent is added. In such a case, the excess fluorescent amino compound which remains after the reaction with the ATZ amino acid derivative disturbs quantitative analysis of the product. Further, since the fluorescent compound is nonvolatile, it is complicated to remove the same after the reaction with the ATZ amino acid derivative.
In view of this, an object of the present invention is to provide a new method of promoting a high yield rate reaction to readily obtain the PTC amino acid derivative for highly sensitive detection.
SUMMARY OF THE INVENTION
For solving the above noted drawback, according to the present invention, in order to analyze the amino acid sequence from an N terminal of a protein, an ATZ amino acid derivative is reacted with a volatile primary amine represented by a general formula X-NH.sub.2 (X denotes a hydrocarbon containing a halogen) to thereby produce a PTC amino acid derivative. Then, the PTC amino acid derivative is detected by a gas chromatograph provided with an electron capture detector (ECD).
By this means, the high yield rate reaction is promoted to readily detect the PTC amino acid derivative with a high degree of sensitivity.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a step diagram showing the inventive analysis method. FIG. 2 shows one conventional analysis method of detection by ultraviolet absorption spectroscopy. FIG. 3 shows another conventional analysis method utilizing an amino compound labeled with a radioactiveisotope. FIG. 4 shows a still another conventional analysis method utilizing a fluorescent amino compound. FIG. 5 s
REFERENCES:
patent: 4865994 (1989-09-01), Tsugita et al.
patent: 5051369 (1991-09-01), Tsugita et al.
Kamo Masaharu
Sano Mitsuru
Tsugita Akira
Kurabo Industries Ltd.
Redding David A.
Seiko Instruments Inc.
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