JPH01250863A - Method of determining c-terminal amino acid of protein or the like - Google Patents

Abstract

PURPOSE:To establish the method of determining C-terminal amino acid which can deal with increasingly slighter quantities of protein, etc., by treating denatured protein or denatured peptide to convert a terminal carboxyl group to an acetyl group, hydrolyzing the resultant product, converting the liberated amino ketone body to a deriv. and separating and identifying the same. CONSTITUTION:The protein, etc., are subjected to the denaturation treatment by performic acid oxidation, etc., to form the denatured protein, etc., in which the three-dimensional structure is broken down and the C-terminal is exposed. After the protein, etc., are dried, the protein, etc., are treated with acetic anhydride in the presence of an org. ternary base in an anhydrous aprotic solvent to convert the carboxyl group at the terminal to the acetyl group. The amino ketone body corresponding to the terminal amino acid part having the C- terminal is liberated as the mixture with the other amino acid if the product is hydrolyzed by using hydrochloric acid, etc. The desired amino ketone body is separated and identified by a hydrazone deriv., etc.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Field of Industrial Application This invention relates to a method for determining the C-terminal amino acid of proteins, etc. More specifically, the present invention relates to a method for determining the C-terminal amino acid of a protein or peptide as part of determining the primary structure of a protein or the like.

(1 sip) Conventional techniques Methods for determining the amino acid sequence (-substructure) of proteins or peptides produced by protein fragmentation include the amino (N) terminal and carboxyl (C
) A method is used in which amino acids are released one by one from the ends of the peptide chain and sequentially analyzed using the free amino groups and carboxyl groups present at each end. Among these, in the C-terminal analysis method, a method for sequentially removing amino acid residues from the C-terminal group has not been developed. Calpokin vepticase (CPage) -'1' and -P release amino acids from the C-terminus of the peptide chain regardless of the type of amino acid, but the number of lost residues is the limit for determining the sequence order. on the other hand,
A method for determining only the C-terminal group is the hydrazine decomposition method +1. ) Lithium label method 2' and thiohydantoin 3' method.

In the above hydrazine decomposition method, for example, 1
In this method, amino acids other than the C-terminus are converted into hydrazide by heating at 00° C. for 6 to 10 hours (in a vacuum-sealed tube), and free amino acids at the C-terminus are separated and identified.

The above tritium labeling method uses pyridine-tritium water-
In acetic anhydride (3:I.3.v/v) mixture, e.g.
In this method, only the hydrogen atom at the C-α position of the C-terminal amino acid is converted to tritium by leaving the sample at 0°C for 1 to 2 hours, and after hydrolyzing the sample, the tritium-labeled amino acid is separated and identified.

The above thiohydantoin method involves reacting a C-terminal amino acid such as a protein with thiocyanate and/or thiocyanate ion in the presence of acetic anhydride, and then generating a thiohydantoin derivative of the C-terminal amino acid by dilute alkaline decomposition. This is a method of sequential decomposition.

References) 1) Akabori, S., 0hno, ni., a.
nd Narita, K.

(1954), Bull, Chem, Soc.
Japan, 25.2142) Matsuo,
H,, and Narita, -Protei
nSequenceDtermination
'(S, B, Needleman.

ed, ), Springer Verlag Berl
in, 1975. p, 1943) 5tark, G.R.
, (196g), Biochemistry, 7,17
96Meuth, J.L., Harris, D.E.
, Dwelet, F.

E,,Crowl-Powers,l'v1. L...
and Gurd, F.

R, N., (1982), Biochemistr.
y, 21.3750 (c) Problems to be Solved by the Invention However, none of the above methods a) may be ineffective against a specific protein or peptide, and b) may contain amino acids that are impossible or difficult to detect. can be,
C) It has the problem that many by-products are produced and it may be difficult to solve the problem. Furthermore, it is difficult to determine the C-terminal amino acid of trace amounts of proteins or peptides.

The present invention has been made in view of this situation, and aims to provide a method for determining the C-terminal amino acid that can be applied to any protein or peptide and can be applied to miniaturization of proteins or peptides.

(d) Means for Solving the Problems Thus, according to the present invention, a denatured protein or a denatured peptide having a carboxyl group at the terminal which has been denatured in advance is dehydrated in the presence of an organic tertiary base in an anhydrous aprotic solvent. The terminal carboxyl group is converted to an acetyl group by treatment with acetic acid, the resulting product is subjected to hydrolysis, and the free aminoketone corresponding to the terminal amino acid moiety is converted into a hydrazone derivative or an oxime derivative. ,
C of proteins, etc. characterized by separating and identifying this
A method for determining terminal amino acids is provided.

The method of this invention involves the use of proteins or peptides (hereinafter referred to as denatured proteins, etc.) having a previously denatured C-terminus.
(1) A reaction step for converting the terminal carboxyl group of the above-mentioned denatured protein into an acetyl group, (2) Hydrolytic heating to release the terminal aminoketone moiety converted to an acetyl group, and (3) Free aminoketone. It consists of three steps: derivatizing the body, separating it, and identifying it.

The denatured protein etc. to be subjected to the method of the present invention refers to one whose three-dimensional structure has been destroyed by a denaturation treatment in advance and the C-terminus has been exposed as a result. Examples of the modification treatment include performic acid oxidation, S-carboxymethylation, S-aminoethylation, and the like.

The above-mentioned denatured proteins and the like are usually dried before being subjected to the reaction step (1) above. This is because the next reaction step (1) proceeds under anhydrous conditions, and by performing this drying treatment, the reaction proceeds efficiently. This drying treatment can be carried out, for example, by drying the sample with dimethyl sulfoxide (hereinafter referred to as D
For example, it may be dissolved in a solvent such as M SO or hexafluoroisopropanol (hereinafter referred to as HFIP) and dried under reduced pressure. The denatured protein etc. obtained by such anhydrous treatment is then subjected to the reaction step (1). That is, this denatured protein etc. is treated with acetic anhydride in an anhydrous aprotic solvent in the presence of an organic tertiary base, and the terminal carpoxyl group is converted to an acetyl group. This reaction process utilizes the so-called Dakin-West reaction. The anhydrous aprotic solvent used here is selected from those capable of dissolving the target protein, such as DMSO. The above-mentioned organic tertiary base is also usually used after being dried to an anhydrous state. This tertiary base is used as a catalyst and/or a basicity regulator, and includes, for example, dialkylaminopyridine, triethylamine (hereinafter referred to as TEA), and the like. The dialkylaminopyridine is preferably dimethylaminopyridine (hereinafter referred to as DMAP), but is not limited thereto.

Acetic anhydride used in the above reaction is also usually dried to an anhydrous state before use. This acetic anhydride is used as a reaction component.

Conditions for this reaction step include, for example, holding at about 50° C. for 1 to 3 hours in an anhydrous and basic atmosphere. For details, refer to the description of Examples described later. By strictly drying the reaction reagents, reaction components, etc. used in the above reaction step, the reaction yield can be improved and the amount of protein, etc. to be tested can be reduced.

In the method of this invention, the product obtained in the above reaction step is subjected to hydrolysis. The hydrolysis is carried out under acidic conditions due to the nature of the desired hydrolyzate, and is usually carried out using hydrochloric acid, but acids other than hydrochloric acid may be used. Furthermore, this hydrolysis is often carried out under reduced pressure and in a sealed tube from the viewpoint of stability of the hydrolyzate. Examples of the above-mentioned hydrolysis treatment include adjusting the pH to a predetermined value with hydrochloric acid, and then holding it at 110° C. for 12 hours in a sealed tube under reduced pressure. Through this treatment, the product obtained in the reaction step (1) is hydrolyzed, and the aminoketone body corresponding to the terminal amino acid moiety that grows the C-terminus is released together with other amino acids simultaneously released by this treatment. Obtain it as a mixture.

In the method of the present invention, the aminoketone of interest liberated as described above reacts selectively with only this aminoketone, is easily detected from impurities such as amino acids in the pond, and is separated and identified through scale derivatization. This derivatization includes derivatization into hydrazone derivatives or oxime derivatives. Among these, hydrazone derivatives are more effective in the points described below. The separation and identification described above is preferably performed using a chromatography technique. In this case, it is preferable to use liquid chromatography (hereinafter referred to as HPLC) from the viewpoint of automatic analysis of the method of the present invention. As one preferable example of the above separation and identification, the target aminoketone obtained together with impurities as a result of the hydrolysis is induced into 2,4-dinitrophenylhydrazone having an absorption maximum at a specific wavelength, and then separated by HPLC. Examples include a method of purifying the derivative, detecting it based on absorption at a specific wavelength, and identifying it from the retention time. This identification is performed by comparison with the retention time of each amino acid in the standard substance.

In addition, when carrying out automatic analysis using the method of the present invention as described above, it is preferable that the sample of protein, etc. to be provided has a molecular weight of 10,000 to 20,000.

Furthermore, for analysis of aminoketone bodies obtained by hydrolysis, it is also possible to use mass spectrometry (F A B-tv[ass) instead of chromatography or a combination thereof. Furthermore, it is also possible to perform more sensitive detection and analysis by utilizing the unique fluorescence of hydrazone. Treatment with acetic anhydride in the presence of an organic tertiary base in an anhydrous aprotic solvent converts the terminal carboxyl group into an acetyl group, which is then subjected to hydrolysis to liberate an aminoketone corresponding to the terminal amino acid moiety. will be separated and identified after being guided to a hydrazone derivative or oxime derivative.

The present invention will be described in detail below with reference to Examples, but the present invention is not limited thereby.

(f) Preparation of Example Materials Ribonuclease A [BovineP a
ncreas), type I-A] was purchased from Sigma after recrystallizing 5 times (lot 77F-03).
40).

Hexafluoroisopropanol (hereinafter referred to as HFTP) was a product from Central Glass Co., Ltd. and was used as it was. Dimethyl sulfoxide (hereinafter referred to as DMSO) is manufactured by Pierce (
Sillation grade (sil) manufactured by P.
ylation grade) was used as it was. Acetic anhydride was obtained by azeotropically removing acetic acid with toluene and then rectifying it (b, p, 138-13
9℃). Triethylamine (hereinafter referred to as TEA) was dried with anhydrous potassium carbonate, benzoic anhydride was added, distilled, and then rectified (b, p, 89-
90°C). Dimethylaminopyridine (hereinafter referred to as DMA
P) was recrystallized once from dichloromethane (m, p, 112.5-114.5°C). Hydrochloric acid used was 6N, twice distilled. 2.4-
Dinitrophenylhydrane (hereinafter referred to as DNPH) was recrystallized once from ethanol (
m, p, 200-202.5°C). Water for liquid chromatography (hereinafter referred to as HP, LC) was purified using a Milli-Q system manufactured by Waters. HPLC grade acetonitrile was purchased from Katayama Chemical Co., Ltd.
Used as is.

The apparatus HPLC was manufactured by Gilson. Column is Wakosil 5
Cl8S (4.6 mm x 250 mm) was used.

Method Performic acid oxidation or honuclease A 260 μg (approximately 20
nmol) in HFIP50μ(, 50-70
It was dried under reduced pressure at °C. This operation was repeated twice. Dissolve this in 50 μl of DMSO and 50 μg of DMAP (400 μl).
nmol), acetic anhydride 5 μC (50, czmol),
After adding 5tt of TEA (l<40ttmoI) and stirring well, the mixture was reacted at 50'C for 3 hours +i'jl. Thereafter, the reaction was stopped by adding 10μρ of methanol, and the mixture was dried in a concentrating centrifuge.

Add 100μ of 6N hydrochloric acid, seal the tube under reduced pressure, and heat at 110°C.
Hydrolysis was carried out for 12 hours. Thereafter, hydrochloric acid was distilled off under reduced pressure. DNP 820μg (100n100n
was dissolved in 10 μρ of a mixed solvent of ethanolonilic acid=1=1, and the mixture was stirred and reacted at 50° C. for 30 minutes. Then add IO μg of acetone and add 50 μg of acetone.
The reaction was allowed to proceed at ℃ for 30 minutes. Ethanol and acetone were distilled off under reduced pressure, 90 µg of methanol was added, and 10 µQ of this was used as an HPLC sample.

Results When the product obtained by the above method was subjected to HPLC under the following conditions, the chromatogram shown in FIG. 1 was obtained. This contains each standard aminoketone (500 pmol each)
) under the same conditions (but the mobile phase was acetonitrile).
From the chromatogram (FIG. 2) of the PLC analysis results, it was identified as an aminoketone derivative of valine (V). The yield is calculated from the peak height and is 190 pmol.
(10%).

〔Analysis conditions〕

HPLC: GI LSON Column: Wakosil 5 C1g-5 Mobile phase:
lomM formic acid buffer CpH 3,5) Flow rate 1 m
12/min Detection wavelength: 350 nm Aufs=O, (18 In Figure 2, (A) indicates the concentration of acetonitrile (number on the left), and the abbreviations used in this figure are as follows. It shows.

K: Lysine D: Aspartic acid
E: Glutamic acid E
o: Second product of glutamic acid A: Alanine Y: Tyrosine v ・Valine M: Methionine] ・Isoleucine L: Leucine
eucine) Nle: Norleucine F
: Phenylalanine From the above results, according to this method, it is possible to determine the amino acid corresponding to the C-terminus of proteins, etc.

(g) Effects of the invention The method of the invention can be used for proteins or peptides that cannot be determined by conventional methods to determine the amino acid corresponding to the C-terminus, and is a suitable method for determining the primary structure of proteins or peptides. can be provided.

It is also effective for proteins or peptides with a molecular weight of about 10,000 to 20,000. Furthermore, analysis by liquid chromatography becomes possible, and therefore an analysis method that can handle miniaturization of proteins or peptides can be provided.

[Brief explanation of the drawing]

FIG. 1 is a liquid chromatogram diagram of the hydrazone derivative of aminoketone obtained by the method of the present invention, and FIG. 2 is a liquid chromatogram diagram of each hydrazone derivative of El aminoketone. Figure 1

Claims (1)

[Scope of Claims] 1. A denatured protein or a denatured peptide having a carboxyl group at the terminal which has been denatured in advance is treated with acetic anhydride in the presence of an organic tertiary base in an anhydrous aprotic solvent to remove the terminal carboxyl group. It is characterized by converting it into an acetyl group, subjecting the obtained product to hydrolysis, and then converting the released aminoketone corresponding to the terminal amino acid moiety into a hydrazone derivative or oxime derivative, which is then isolated and identified. A method for determining the C-terminal amino acid of a protein, etc. 2. The method for determining a C-terminal amino acid according to claim 1, wherein the hydrazone derivative is separated by chromatography and identified based on the obtained chromatogram. 3. The method for determining a C-terminal amino acid according to claim 2, wherein the hydrazone derivative is 2,4-dinitrophenylhydrazone.