METHOD FOR HIGH-SENSITIVITY DETECTION OF AMINO ACID DERIVATIVE

Abstract: A method of detecting amino acid derivatives easily with a high sensitivity in order to conduct amino acid sequencing starting with the N-terminus of a protein. The method comprises preparing a phenylthiocarbamylamino acid derivative by the reaction of a 2-anilino-5-oxothiazolinylamino acid derivative with a volatile primary amine X-NH₂ (wherein X represents a halogenated alkyl group) and detecting the derivative thus prepared with the use of a gas chromatograph equipped with an ECD.

Description: 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, conventionally protein is 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 as shown 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 as shown in Fig. 4 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. US-A-4865994 discloses a detection method for amino acid derivatives in which 2-anilino-5-thiazolinone derivatives of amino acids are reacted with radioactive iodine isotope-labelled amino compounds or fluorescent amino compounds to form phenylthiocarbamyl amino acid derivatives, which can be detected by a radioactive detector or fluorescence spectrophotometer. The method achieves high yield (over 80%) and high sensitivity (1-10 fmol and 0.1-1 pmol for radioactive and fluorescent methods respectively). Analytical Biochemistry (volume 107, p140-149, 1980) discloses a method for the chemical ionization mass spectrometric analysis of 2-anilino-5-thiazolinone amino acids obtained from the Edman degradation of proteins and peptides. With regard to the conventional method of detecting the PTH amino acid derivative by the ultraviolet absorption spectroscopy, though the detecting means is rather simple, this is not suitable for 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 labelled with the radioactive isotope, this has rather limited application in view of ill effect to a 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, 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. DISCLOSURE OF THE INVENTION For solving the above noted drawback, according to the invention, in order to analyze 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 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 the high 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 shows a gas chromatograph for detecting a PTC phenylalanine derivative, where (a), (b) and (c) show analysis results of the PTC phenylalanine derivative in the amounts of 5 pmol, 500 fmol and 50 fmol, respectively. BEST FORM OF PRACTICING THE INVENTION The detailed description is given for the invention in conjunction with the attached drawings. The embodiment 1 indicates that the detection sensitivity of an amino acid derivative is significantly high according to the invention. First, Fig. 1 shows steps of the inventive method of this embodiment. An amino compound of hydrocarbon containing a halogen is applied to an ATZ amino acid derivative obtained based on process of Edman degradation to form a PTC amino acid derivative. Next, this PTC amino acid derivative is identified by a gas chromatograph provided with an ECD. The PTC phenylalanine amino acid derivative is prepared by the following steps. First, a dipeptide of Phe-Gly is treated according to Edman degradation to obtain an ATZ phenylalanine derivative. This process of the Edman degradation is known, and is therefore not described in detail here. Then, the ATZ phenylalanine derivative is filled in a test tube of ⊘4 × 40 mm, and is dried. This test tube is placed into another test tube of ⊘13 × 100 mm provisionally filled with 100 µl of 2,2,2-trifluoroethylamine (TFEA). The outer test tube is vacuum-sealed to carry out reaction for 20 minutes at 80°C. After opening the test tube, an excess TFEA is removed by blow of nitrogen gas. Fig. 5 is a gas chromatograph for detecting the PTC phenylalanine amino acid derivative prepared by the above noted condition. The gas chromatography is carried out according to the following condition. Instrument (detector): HP5890 (ECO) Carrier gas (flow rate): helium (1.5 ml/minute) Column: ULTRA #2 (crosslinked 5% phenylmethyl silicone) length 25 m, internal diameter 0.32 mm, film thickness 0.17 µm Oven temperature: Initial temp. 50°C 1 minute : Rinsing rate 10°C/minute : End temp. 300°C 15 minutes Inlet opening temperature: 250°C Detector temperature: 300°C A peak (a) is obtained when 5 pmol of the sample PTC phenylalanine derivative is analyzed, another peak (b) is obtained when 500 fmol is analyzed, and still another peak (c) is obtained when 50 fmol is analyzed. Based on these results, the detection limit of the present invention is compared with that of the conventional method utilizing the PTH derivative (PTH method). As shown in the Table 1, the detection sensitivity of the invention is extremely high (1 pmol = 1×10 -12 mol, 1 fmol = 1×10 -15 mol). Detection method Detection limit PTH method 1 pmol Inventive method 50 fmol or less The embodiment 2 shows that reaction yield rate between ATZ derivative and TFEA is high in the inventive highly sensitive detection method of the amino acid derivative. In this embodiment, the dipeptide of Phe-Gly is used. The PTC phenylalanine derivative is prepared in the same manner as the embodiment 1. In this embodiment, the PTC phenylalanine derivative is analyzed by using a micro-bore fast liquid chromatography (HPLC). The used condition of HPLC is as follows. HPLC instrument: Applied Biosystems 130A Column: Applied Biosystems Spheri-5RP-18 (⊘1.0 mm × 50 mm) Eluent: 32% acetonitrile containing 0.1% trifluoroacetic acid Column temperature: 55°C Detection wave length: 254 nm Table 2 shows a yield of the PTC phenylalanine while the quantity of ATZ derivative is varied. Quantity of ATZ derivative (pmol) Yield (pmol) (%) 100 85 85 10 8.5 85 1 0.81 81 0.15 0.078 78 0.05 0.035 70 As shown, even though the starting material is 50 fmol, the yield of 70% can be obtained such that the high reactivity is maintained for analyzing a trace amount of sample. In the conventional method of using the PTH derivative, the reaction yield rate of the ATZ derivative is 40% at 1 pmol, 10% at 100 fmol and 4% at 50 fmol. INDUSTRIAL APPLICABILITY An aim of the present invention is a highly sensitive and ready detection of an amino acid derivative in the amino acid sequence analysis of protein or peptide from the N terminal. According to a significant aspect of the invention, in order to analyze an amino acid sequence of protein from an amino terminal, an ATZ amino acid derivative is reacted with a volatile primary amine represented by a general formula X-NH 2 (X denotes a hydrocarbon containing halogen) to produce a PTC amino acid derivative. The PTC amino acid derivative is detected by a gas chromatograph provided with an ECD. By this, the high yield reaction is promoted to realize highly sensitive and ready detection of the amino acid derivative. In such a reaction system, the sample is reacted with the reactive reagent composed of the primary amine having the volatile nature, which facilitates removal of an excess the reagent after the reaction. The highly sensitive detection method of the amino acid derivative according to the invention has a great industrial value.

Claims: A highly sensitive detection method of an amino acid derivative, characterized in that 2-anilino-5-thiazolinone amino acid derivative is reacted with a volatile primary amine represented by a general formula X-NH 2 (X denotes a hydrocarbon containing a halogen) to produce a phenylthiocarbamyl amino acid derivative, and the phenylthiocarbamyl amino acid derivative is detected by a gas chromatograph provided with an electron capture detector. A highly sensitive detection method of an amino acid derivative according to claim 1, characterized in that the 2-anilino-5-thiazolinone amino acid derivative is obtained by Edman degradation such that a protein or a peptide is reacted with a phenylisothiocyanate to produce a phenylthiocarbamyl protein or a phenylthiocarbamyl peptide, which is thereafter reacted with an acid.

Inventors: KAMO MASAHARU, SANO MITSURU, TSUGITA AKIRA, KAMO, MASAHARU, SANO, MITSURU, TSUGITA, AKIRA, KAMO, MASAHARU CHIBA HEIGHTS 201, SANO, MITSURU KURABO INDUSTRIES LTD., TSUGITA, AKIRA ISHIZUKA BLDG. 305
Applicants: KURASHIKI BOSEKI KK, SEIKO INSTR INC, KURABO INDUSTRIES LTD., SEIKO INSTRUMENTS INC.
Application year: 1993
Main classification: G01N33
IPC codes: G01N33