JPH0763766A - Method for deciding amino acid sequence - Google Patents

Abstract

PURPOSE:To easily decide the amino acid sequence of a protein by avoiding an S/N drop due to the rise of background signals caused by the yield of the Edman degradation before one cycle which does not reach 100 % in an arbitrary cycle. CONSTITUTION:In a method for deciding the amino acid sequence of a protein in which a cycle composed of a series of processes for generating a PTC (phenylthiocarbamyl) amino acid derivative by causing a reaction between an ATZ (2-anilino-5-thiazolinon) amino acid derivative and the amino compound expressed by an general formula, X-NH2, and detecting the generated amino acid derivative is repeatedly executed by the same number as the number of residual groups of the amino acid to be analyzed, a plurality of amino compounds is used.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an amino acid sequence analysis method for determining the binding order of amino acids which are units constituting proteins.

[0002]

2. Description of the Related Art Conventionally, as a method for analyzing an amino acid sequence of a protein, as shown in FIG. 2, phenylthiocarbamate (PITC), which is one of the isothiocyanate derivatives, is allowed to act on the protein. By treating the mill (PTC) protein with an acid, 2
-A series of aniline-5-thiazolinone (ATZ) amino acid derivative is produced, and the phenylthiohydantoin (PTH) amino acid derivative obtained by further treating with an acid is identified by a combination of chromatograph and ultraviolet absorptiometry. The method of repeatedly executing the cycle composed of the steps of Steps 1 and 2 as many times as the number of residues of the amino acid to be analyzed, Edman decomposition method, has been used (P. Edma).
n, Acta Chem. Scand. 10, 7
61 (1956)).

On the other hand, as shown in FIG. 3, a method of reacting an ATZ amino acid derivative with an amino compound labeled with a radioisotope and separating the produced PTC amino acid derivative by thin layer chromatography to detect it is disclosed in Japanese Patent Laid-Open No. 61-62. -26426
4 is disclosed. Furthermore, as shown in FIG.
A method of reacting an amino acid derivative with a fluorescent amino compound, separating the produced PTC amino acid derivative by high performance liquid chromatography, and detecting it by a fluorometric method is disclosed in JP-A-63-63.
-1968858.

[0004]

In principle, an infinite amino acid sequence can be determined by the Edman decomposition method. However, in reality, the reaction yield of each step constituting the Edman decomposition method is not 100%, and thus the product of these products, that is, the yield per cycle (repeated yield) is necessarily 100%.
It will be below%. In the amino acid sequence analysis, the main reason why the analysis becomes difficult gradually as the cycle progresses is that the intensity of the signal to be identified decreases with the repetition yield in each cycle, and Increase in background signal (which is called overlap signal) caused by repeated reaction of unreacted amino group terminal (N terminal) in subsequent cycles, and decrease S / N ratio Can be given. Therefore, the analysis becomes difficult as the cycle is repeated. This is unavoidable as long as a single isothiocyanate derivative such as PITC is used in the Edman degradation method.

Here, P is used as an isothiocyanate derivative.
When a compound other than ITC, for example, an isothiocyanate compound having an absorption wavelength in the visible region or an isocyanate compound having fluorescence is used, the repeat yield is further lowered. Further, a method of detecting an produced PTC amino acid derivative by reacting an amino compound with an ATZ amino acid derivative is disclosed. In this method as well, the first half of the step is the Edman degradation method, and unreacted in a certain cycle. Similarly, the background signal generated by the repeated reaction of the amino group terminal (N terminal) in the subsequent cycles increases and the S / N ratio decreases.

Therefore, the present invention is intended to provide a method for easily determining the amino acid sequence of a protein while avoiding an increase in background signal and a decrease in S / N ratio as much as possible.

[0007]

In order to overcome the above-mentioned drawbacks and to easily determine the amino acid sequence of a protein while avoiding a decrease in S / N ratio due to an increase in background signal, the present invention provides: A residue of an amino acid to be analyzed in a cycle composed of a series of steps of reacting an ATZ amino acid derivative with an amino compound represented by the general formula X-NH2 to form a PTC amino acid derivative and detecting the PTC amino acid derivative. In the method of performing the same number of times repeatedly, a plurality of amino compounds, for example,
Two kinds of amino compounds are used alternately, for example, aminofluorescein is used as one of the amino compounds,
For example, as another one of the amino compounds, aminotetramethylrhodamine was used for sequence analysis.

The ATZ amino acid derivative used here causes PITC to act on a protein or peptide to give PT
It is obtained by the Edman degradation method in which an acid is allowed to act after the C protein or PTC peptide is produced.

[0009]

By the above means, it becomes possible to easily determine the amino acid sequence of a protein while avoiding a decrease in S / N ratio due to an increase in background signal.

[0010]

EXAMPLES The present invention will be described below based on examples. (Example 1) FIG. 1 is a process diagram showing an analysis method of the present invention. In the figure, Xn-NH2 is a plurality of amino compounds, Ph-,
Indicates a phenyl group and R- indicates an amino acid side chain. This is the same in each of the following figures.

First, the PITC is allowed to act on the protein,
Generate a PTC protein derivative. Next, this PT
An acid is allowed to act on the C protein derivative to produce an ATZ amino acid derivative and a protein in which one amino-terminal amino acid has been lost. From a mixture of an ATZ amino acid derivative and a protein in which one amino-terminal amino acid has been lost, AT
The Z amino acid derivative is taken out, and the amino compound is caused to act on the PTC amino acid derivative to identify the PTC amino acid derivative.

In this method, a plurality of amino compounds are used in a cycle consisting of this series of steps repeated the same number of times as the number of amino acid residues to be determined. (Example 2) Here, an example in which two kinds of amino compounds are alternately used is shown.

FIG. 5 is a process chart showing the analysis method of the present invention when two kinds of amino compounds are alternately used. Figure 6
FIG. 6 is a process diagram following FIG. 5 showing the analysis method of the present invention when two kinds of amino compounds are used alternately. One of the amino compounds used here is aminofluorescein. The structural formula of this compound is shown in FIG. As another one of the amino compounds, aminotetramethylrhodamine is used. The structural formula of this compound is shown in FIG.

Table 1 shows the maximum excitation wavelength and the maximum fluorescence wavelength of these amino compounds.

[0015]

[Table 1]

The experimental procedure will be described. X1-NH2 in FIG.
Shows aminofluorescein, X2-NH2 in FIG. 6 shows aminotetramethylrhodamine, respectively. First, PITC is allowed to act on a protein to generate a PTC protein derivative. Next, an acid is allowed to act on the PTC protein derivative to produce an ATZ amino acid derivative and a protein in which one amino-terminal amino acid has been lost. ATZ
The ATZ amino acid derivative is taken out from the mixture of the amino acid derivative and the protein in which one amino-terminal amino acid is lost, and aminofluorescein is allowed to act to produce the PTC amino acid derivative, which is then separated by high performance liquid chromatography (HPLC) of one system. Then, the PTC amino acid derivative is identified. The conditions for fluorescence detection used in this identification are an excitation wavelength of 486 nm and a fluorescence wavelength of 513 nm.

Next, PITC is allowed to act on the protein which has lost one amino-terminal amino acid to produce a PTC protein derivative. Next, an acid is allowed to act on the PTC protein derivative to produce an ATZ amino acid derivative and a protein in which one amino-terminal amino acid has been lost. A
An ATZ amino acid derivative is taken out from a mixture of a TZ amino acid derivative and a protein in which one amino-terminal amino acid has been lost, and aminotetramethylrhodamine is allowed to act to produce a PTC amino acid derivative. Separated and identified by HPLC of the system. The conditions for fluorescence detection used in this identification are an excitation wavelength of 544 nm and a fluorescence wavelength of 567 nm.

After that, a series of these operations is repeated. That is, in the odd-numbered cycle, fluorescence detection is performed using aminofluorescein, and the condition is set to the excitation wavelength of 48
6 nm and fluorescence wavelength 513 nm. In the even-numbered cycle, fluorescence detection is performed using aminotetramethylrhodamine, and the conditions are set to an excitation wavelength of 544 nm and a fluorescence wavelength of 567 nm. As a result, PTC amino acid derivatives resulting from the repeated yield of the Edman reaction of less than 100% in the odd cycle are not detected during identification in the even cycle. Similarly, the PTC amino acid derivative resulting from the repetition yield of the Edman reaction in the even-numbered cycle being less than 100% is not detected during the identification in the odd-numbered cycle.

In this way, in any given cycle, the influence of background signals due to the inability to obtain a 100% yield in Edman degradation one cycle before can be avoided. This is because fluorescence detection was performed using aminotetramethylrhodamine in the odd-numbered cycle, and the condition was set to the excitation wavelength of 544n.
m, fluorescence wavelength is 567 nm, fluorescence detection is performed using aminofluorescein in the even-numbered cycle, and the conditions are as follows: excitation wavelength 486 nm, fluorescence wavelength 513
The same is true for the case of nm.

The contents described above can be summarized as follows. A residue of an amino acid to be analyzed in a cycle composed of a series of steps of reacting an ATZ amino acid derivative with an amino compound represented by the general formula X-NH2 to form a PTC amino acid derivative and detecting the PTC amino acid derivative. Multiple amino compounds were used in a method that was run as many times as the number. This allows different detectors such as a detector or a detection wavelength when identifying the PTC amino acid derivative before and after successive cycles. That is, it is possible to avoid the influence of the background signal due to the fact that a 100% yield cannot be obtained in Edman degradation one cycle before in any given cycle.

In this way, it became possible to easily determine the amino acid sequence of the protein while avoiding a decrease in the S / N ratio due to an increase in background signal.

[0022]

As described above, the important point of the present invention is that S / N is increased by the increase of background signal.
It is possible to easily determine the amino acid sequence of a protein while avoiding a decrease in the ratio.

In the examples, an example in which aminofluorescein and aminotetramethylrhodamine are alternately used has been shown, but it is clear that a plurality of other amino compounds, that is, two or more kinds can be used. ,
It is not limited to the embodiment. Therefore, the method for determining the amino acid sequence of a protein or peptide according to the present invention has great industrial value.

[Brief description of drawings]

FIG. 1 is a process chart showing an analysis method of the present invention.

FIG. 2 shows a conventional analysis method using PITC.

FIG. 3 shows a conventional analytical method of reacting an amino compound labeled with a radioisotope.

FIG. 4 shows a conventional analysis method in which a fluorescent amino compound is reacted.

FIG. 5 is a process chart showing the analysis method of the present invention when two types of amino compounds are used alternately.

FIG. 6 is a process chart showing the analysis method of the present invention when two kinds of amino compounds are alternately used.

FIG. 7 shows the structural formula of aminofluorescein.

FIG. 8 shows the structural formula of aminotetramethylrhodamine.

Claims (6)

[Claims] 1. A 2-anilino-5-thiazolinone amino acid derivative is reacted with an amino compound represented by the general formula X-NH2 to obtain a phenylthiocarbamyl amino acid derivative, and the phenylthiocarbamyl amino acid derivative is detected. A method for determining an amino acid sequence, characterized in that at least two or more kinds of amino compounds are used in a method in which a cycle composed of a series of steps is repeatedly performed as many times as the number of residues of the amino acid to be analyzed. 2. A 2-anilino-5-thiazolinone amino acid derivative is reacted with an amino compound represented by the general formula X-NH2 to obtain a phenylthiocarbamyl amino acid derivative, and the phenylthiocarbamyl amino acid derivative is detected. 2. A method of repeatedly performing a cycle constituted by a series of steps, the number of which is the same as the number of amino acid residues to be analyzed, wherein at least two or more kinds of amino compounds are alternately used. A method for determining an amino acid sequence. 3. The 2-anilino-5-thiazolinone amino acid derivative causes a phenylisothiocyanate to act on a protein or peptide to generate a phenylthiocarbamyl protein or a phenylthiocarbamyl peptide, and then to act an acid. , Edman decomposition method,
The method for detecting an amino acid derivative with high sensitivity according to claim 1, which is obtained by 4. The method for determining an amino acid sequence according to claim 1, wherein the amino compound is a fluorescent amine. 5. The method for determining an amino acid sequence according to claim 4, wherein the fluorescent amine is aminofluorescein. 6. The method for determining an amino acid sequence according to claim 4, wherein the fluorescent amine is aminotetramethylrhodamine.