JPH04229195A - Determination of amino acid sequence - Google Patents

Abstract

PURPOSE:To provide an easily operable process for the determination of an amino acid sequence. CONSTITUTION:Amino acids are successively eliminated from the carboxyl terminal of a peptide fragment by carboxypeptidase treatment. The eliminated amino acid is made to react with t-RNA specific to said amino acid in the presence of an aminoacyl t-RNA synthetase and the synthesized aminoacyl t-RNA is detected to easily determine the amino acid sequence.

Description

[Detailed description of the invention]

0001

FIELD OF THE INVENTION The present invention relates to a method for analyzing amino acid sequences. More specifically, the present invention relates to a method for analyzing amino acid sequences by sequentially removing amino acids from the carboxyl terminal group of a peptide fragment using carboxypeptidase and detecting the removed amino acids.

0002

BACKGROUND OF THE INVENTION Determination of the primary structure of proteins plays an important role in the fields of molecular biology, biochemistry, medicine, and the like. As a method for determining the primary structure of a protein, 195
Since the Edman decomposition was established by Edman in 2000,
Devices that automate this Edman decomposition have been developed. That is, conventionally, the mainstream method for analyzing amino acid sequences has been the Edman method, which detects amino acids that are sequentially degraded from the N (amino) terminal group of a peptide fragment.
Phenylisothiocyanate (PITC) in the presence of alkali
) and treated with TFA (trifluoroacetic acid) to
The terminal group is a thiazolinone derivative (PTZ-amino acid)
, PTZ-amino acid is heated and rearranged to a stable phenylthiohydantoin derivative (PTH-amino acid), and TL
This method detects PTH-amino acids using C or reverse phase HPLC. On the other hand, although hydrazine decomposition and tritium labeling methods are known as methods for determining only amino acids with carboxyl terminal groups, a method for analyzing amino acid sequences from carboxyl terminal groups is not yet known.

0003

[Problem to be solved by the invention] In the conventional method of decomposition from these N-terminal groups, the reaction conditions are strong, and certain PTH amino acid (serine, threonine, etc.) derivatives are easily decomposed. , a control amino acid is required, but when detecting using a control amino acid, the TL
Since the chemical properties of amino acids in C are similar, there was a problem in that many types of developing solvents had to be used. In addition, reverse phase HPLC is also performed by comparing the elution time with a standard sample, but if the peaks are close, the analysis must be repeated, which is very complicated.

[0004] In recent years, there has been a demand for rapid and simple determination of the primary structure of proteins or peptides, but conventional methods require complicated analysis operations and take time. Problems were pointed out, such as: Therefore, an object of the present invention is to provide a simple method for analyzing amino acid sequences that does not require comparison with a reference amino acid standard sample.

[0005]

[Means for Solving the Problems] As a result of intensive studies to solve the above-mentioned problems, the present inventor sequentially removed amino acids from the carboxyl terminal group of a peptide fragment, and then removed the aminoacyl bond with t-RNA. Aminoacyl t-R, which is the reaction product formed between amino acids,
The inventors have discovered that the above problem can be solved by detecting NA, and have completed the present invention.

That is, the gist of the present invention is to sequentially remove amino acids from the carboxyl terminal group of a peptide fragment by treatment with carboxypeptidase, and then combine the removed amino acids with a t-RNA specific to the amino acid and an aminoacyl t-RNA.
- An amino acid sequence analysis method characterized by reacting with RNA synthetase and detecting the resulting reaction product.

The carboxypeptidase used in the present invention is an enzyme that hydrolyzes a peptide bond from the carboxyl terminal of a peptide, and carboxypeptidase Y or P (for example, manufactured by Boehringer Mannheim, product number 238139) is usually used. Enzyme treatment with carboxypeptidase is usually performed at 0% for 5 nmol of peptide fragment, although it depends on the amount of peptide fragment used as a sample.
．． Add 5-1 U and conduct at 37°C for 30 minutes. Amino acids are sequentially removed and liberated from the carboxyl terminal group by enzymatic treatment, so the enzyme treatment is carried out for an appropriate period of time (usually every 2 minutes, for example).
Sampling is performed every (approximately 0 times).

[0008] Aminoacyl t-RNA synthetase and one type of t-RNA are added to each sampled enzyme treatment solution, and the reaction is usually carried out at 37°C for about 20 minutes to form a bond between the specific amino acid and t-RNA. Forms an aminoacyl bond. The aminoacyl t-RNA synthetase to be added is a known enzyme that has the function of binding an amino acid to its corresponding t-RNA.
Zymology Vol. 29 p547 (197
Alanyl-t-RNA-synthetase described in 4) can be used. The amount added is usually twice the amount of the substrate. The t-RNA to be added is known to be t-RNA corresponding to each of the 20 types of amino acids, "Chemistry of Nucleic Acids III (Biochemistry Experiment Course 2)" edited by the Japanese Biochemical Society, Tokyo Kagaku Doujin p. 485-488 (1977 ) are easily available, such as those listed in . One type of t-RNA is added to each enzyme treatment solution, and the amount added is usually 0.5μ.
It is M. Therefore, normally, the enzyme-treated solution obtained from one sampling is dispensed into 20 tubes, and each t-R
NA and aminoacyl t-RNA synthetase are added and reacted.

[0009] Since each t-RNA is specific for each amino acid, in a reaction solution to which a t-RNA specific for the amino acid detached from the carboxyl terminal group is added, aminoacyl t-RNA synthetase An aminoacyl bond is formed between the amino acid and t-RNA, and a specific aminoacyl t-RNA can be obtained.

[0010] Next, by separating each of these reaction products by HPLC or the like and detecting the absorption peak derived from t-RNA at 260 nm, it is possible to detect the free amino acid desorbed by a conventional method. The amino acid sequence can be analyzed by performing these operations over time and analyzing the amino acids that are sequentially eliminated from the carboxyl terminal group.

[0011] The method of the present invention can be carried out by manual operation.
Alternatively, it can also be carried out using an apparatus that automatically analyzes amino acid sequences using the method of the present invention. One embodiment of the application as an automatic analyzer is the flowchart shown in FIG. First, a peptide fragmentation reaction section 1 in which amino acids are sequentially eliminated from the carboxyl terminal group using carboxypeptidase, and aminoacyl t-RNA synthesis in which aminoacyl t-RNA synthetase and t-RNA are extracted and reacted with the eliminated amino acids. 2, a detection section 3 for detecting synthesized aminoacyl-t-RNA, and a data processing section 4.

0012

[Examples] The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited to these Examples in any way. Example 1 0.5 ml each of 1 nmol/ml peptide fragment in 21 1.5 ml Eppendorf tubes, and 0.05 mol/l sodium acetate (pH 3.7) as a buffer.
．． 4 ml, add 0.1 ml of carboxypeptidase P (manufactured by Boehringer Mannheim) 0.05 U/ml,
Sample 0.1 ml at 30°C every 2 minutes 10 times, and treat at 65°C for about 1 minute to inactivate the enzyme. Next, add aminoacyl t-R to each sampled solution.
NA synthase (Method in Enzymolo)
gy Vol. 29 p547 (1974)) 0.05
Add 0.1 ml of ATP to a final concentration of 10 nm.
ol, and then added 20 types of t-RNA to 1
Add 10 nmol of each species to each tube and allow to react at 37°C for 10 minutes (t-RNA is not added to the remaining tube). The resulting reaction product was separated by HPLC and t at 260 nm.
- From the absorption peak derived from RNA, the elution time is determined for each t-
Calculate the RNA using a PC to determine the order of the amino acid sequence. In this case, an absorption peak (260 nm) derived from the t-RNA that has formed an aminoacyl bond with the amino acid that is eliminated from the carboxyl terminal group is detected, and the remaining 20 nm
Not detected in book tubes.

[0013]

Effects of the Invention Compared with the conventional chemical decomposition method from the N-terminal group, the method of the present invention uses an enzyme for decomposition, so the reaction conditions are milder and no side reactions are involved. Furthermore, t-RNA is specific for each amino acid, and if the t-RNA added in advance is limited by the method of the present invention, only one type of amino acid will be bound to t-RNA by aminoacyl t-RNA synthetase. . Therefore, amino acids bound to t-RNA can be analyzed by detecting absorption at 260 nm. In this way, in the method of the present invention, T
There is no need for separation using various developing solvents such as LC, and there is no need for standard amino acid samples used in HPLC, so operations are simple and analysis can be performed easily.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing a flowchart of an automatic amino acid sequence analyzer using the method of the present invention.

[Explanation of symbols]

1 Peptide fragmentation reaction part 2 Aminoacyl t-RNA synthesis section 3 Detection part 4 Data processing section

Claims (1)

[Claims] Claim 1: Amino acids are sequentially eliminated from the carboxyl terminal group of a peptide fragment by treatment with carboxypeptidase, and then the eliminated amino acids are reacted with t-RNA and aminoacyl t-RNA synthetase specific for the amino acid. , an amino acid sequence analysis method characterized by detecting the obtained reaction product.