JP2568612Y2 - Edman reactor - Google Patents

Description

[Detailed description of the invention]

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an Edman reactor, and more particularly to an Edman reactor for supplying a sample after the Edman reaction to an amino acid analyzer.

[0002]

2. Description of the Related Art In order to determine the amino acid sequence of a protein, a protein primary structure analyzer is used. The protein primary structure analyzer mainly includes an Edman reaction unit for performing an Edman reaction, and an amino acid analyzer for analyzing amino acids from a sample after the Edman reaction.

The Edman reaction is used when the primary structure of a protein or peptide, that is, the amino acid sequence is determined sequentially from the N-terminus. In this reaction, first, the pretreated sample is placed in the reaction chamber and placed at the N-terminal.
Reacting phenylisothiocyanate (PITC)
After producing PTC-protein (coupling reaction) , the N-terminal peptide of the sample was cleaved with trifluoroacetic acid vapor, and A
Form TZ amino acids (cleavage reaction). Next, released A
The TZ-amino acid is extracted into a conversion flask or the like (extraction reaction). Furthermore, AT with trifluoroacetic acid aqueous solution
The Z-amino acid is converted to a stable PTH-amino acid (conversion reaction). The converted PTH-amino acid is supplied from the conversion flask to the analysis processing section, where the amino acid is separated and detected.

[0004] In the above conversion reaction, the formed ATZ-amino acid is stored in a conversion flask, and for example, a 25% aqueous trifluoroacetic acid solution is supplied thereto under a pressure of an inert gas or the like. This completes the conversion reaction. The aqueous trifluoroacetic acid solution is usually stored in a glass container.

[0005]

In the Edman reactor, a 25% aqueous trifluoroacetic acid solution used for the conversion reaction is contained in a glass container. However, the aqueous solution of trifluoroacetic acid dissolves the glass serving as a container with time, and the eluate accumulates in the flow path of the conversion flask. For this reason, the accumulated matter hinders the flow of the sample, and stable performance cannot be ensured for a long period of time. Therefore, it is necessary to wash with a solution for washing out the accumulated matter.

[0006] An object of the present invention is to eliminate accumulation of eluate from a container in a flow channel and to eliminate the need for washing.

[0007]

The Edman reactor according to the present invention is an Edman reactor for supplying a sample after the Edman reaction to an amino acid analyzer, which cuts an N-terminal peptide bond of a protein sample, It has a reaction part for forming an ATZ-amino acid, and a polyethylene resin container for storing an aqueous solution of trifluoroacetic acid.
It has a conversion unit for converting to amino acids, and a sample supply unit for supplying PTH-amino acids to the amino acid analyzer.

[0008]

In the Edman reaction apparatus according to the present invention, first, the N-terminal peptide bond of the protein sample is cleaved in the reaction section to form an ATZ-amino acid. Next, an aqueous solution of trifluoroacetic acid is supplied to the formed ATZ-amino acid to be converted to a PTH-amino acid.

The aqueous solution of trifluoroacetic acid is polyethylene
Since it is stored in a fat container , the components of the container do not elute. Therefore, the channel for supplying the PTH-amino acid is not clogged. Also, inexpensive polyethylene resin
Uses a vessel and is economically excellent.

[0010]

1 and 2 show an apparatus 1 for analyzing the primary structure of a protein to which an embodiment of the Edman reaction apparatus according to the present invention is applied. The protein primary structure analyzer 1 mainly includes an Edman reaction device 6 including a first reagent supply unit 2, a reaction chamber 3, a conversion flask 4, and a second reagent supply unit 5, and a PTH-amino acid analysis device 7. ing.

The first reagent supply unit 2 includes a reagent bottle 11 of ethyl acetate, a reagent bottle 12 of n-heptane, a reagent bottle 13 of an aqueous solution of trimethylamine, a reagent bottle 14 of an n-heptane solution of phenylisothiocyanate, A reagent bottle 15 of n-butyl and a reagent bottle 16 of trifluoroacetic acid (100% concentration) are provided. Reagent bottle 11-1 6 is made of glass. Each reagent bottle 1
1-16 is connected to one end of the gas supply passage 18 having an electric solenoid valve 17. The other end of each gas supply path 18 is connected to a supply device 19 that supplies an inert gas such as nitrogen or argon. An upper end of each of the reagent bottles 11 to 16 is connected to one end of a gas exhaust path 20 having an electromagnetic valve 20a. Further, each reagent bottle 11, 12, 14, 1
5, one end of a reagent supply path 21 is disposed at the bottom. On the other hand, trimethylamine and trifluoroacetic acid
Normally, the gas is supplied into the reaction chamber 3 in a gaseous state.
6 is located at the top. The other end of each reagent supply path 21 is connected to a solenoid valve 22. Each solenoid valve 2
2 are interconnected in a row. The solenoid valve 22 located at one end (the upper end in FIG. 1) of the solenoid valve group
a includes a gas supply passage 25 from the inert gas supply device 19.
Are connected at one end. On the other hand, the other end of the solenoid valve group (FIG. 1)
(The lower end in FIG. 2) is connected to one end of a reagent introduction passage 33 for introducing a reagent into the reaction chamber 3. This reagent introduction path 33 has a liquid detection sensor 34.

The reaction chamber 3 includes an upper member 3a.
And a lower member 3b, between which a filter 3c for holding a sample is disposed. The lower end of the reaction chamber 3 can be connected to an upstream end of a sample moving tube 35 communicating with the conversion flask 4. At the upstream end of the sample moving tube 35, a liquid sensor 36 and a flow path switching valve 37 are provided.
The flow path switching valve 37 is provided with a drain flow path 37a.

As shown in FIG. 2, the conversion flask 4 has a reagent introduction pipe 38 for introducing a reagent from the second reagent supply section 5 in addition to the sample moving pipe 35, and a gas exhaust pipe having an electromagnetic valve. 39 are connected. The reagent introduction pipe 38 is provided with a pair of liquid detection sensors 30 and 31. Also, at the lower end of the conversion flask 4,
A sample introduction tube 40 for introducing a sample into the amino acid analyzer 7 is connected. The sample introduction tube 40 has a switching valve 41.

The second reagent supply unit 5 includes a glass reagent bottle 50 containing a mixed solution of acetonitrile and water,
And a reagent bottle 51 of a 25% aqueous trifluoroacetic acid solution. As shown in FIG. 3, the reagent bottle 51 is a polyethylene container, and has a screw portion 51b for hermetically sealing the opening portion 51a, and a lid 5 screwed to the screw portion 51b.
1c. One end of a gas supply path 53 having an electromagnetic valve 52 is connected to upper portions of the reagent bottles 50 and 51. The other end of each gas supply path 53 is connected to an inert gas supply device 54. In addition, each reagent bottle 50,
51, a gas exhaust passage 55 having an electromagnetic valve 55a
Is connected. Further, the tip of each reagent supply path 56 extending from each reagent bottle 50, 51 is connected to an electromagnetic valve 57. The solenoid valves 57, 57 are connected in a straight line, and the other end of the sample introduction path 38 is connected to one side (the lower end in FIG. 2) of the solenoid valve 57. The other solenoid valve 57
Is connected via a solenoid valve 58 to a gas supply passage 58a for supplying a pressure-controlled inert gas. Further, a gas supply path 59 a for supplying an inert gas whose flow rate is controlled is connected to the electromagnetic valve 58 via the electromagnetic valve 59.

The PTH-amino acid analyzer 7 has a six-way valve 60. One end of the sample introduction tube 40 is connected to the six-way valve 60. The six-way valve 60 is connected to a pump 61, a column 62, a detector 63, and a fraction collector 64 for high-performance liquid chromatography. Further, the detector 63 includes a data processing device 65.
It is connected to the.

The protein primary structure analyzing apparatus 1 comprises:
The control unit (not shown) is configured to automatically control the electromagnetic valve and the flow path switching valve. Next, the operation of the above embodiment will be described. As a preparation stage, each of the reagent bottles 11 to 11 in the first reagent supply unit 2 and the second reagent supply unit 5
A predetermined amount of each reagent is supplied to 16 and 50. The reagent bottle 51 contains a predetermined amount of a 25% aqueous trifluoroacetic acid solution that has been measured in advance. Since the 25% aqueous trifluoroacetic acid solution has volatility, it is moved and stored in an airtight state by the lid 51c. When installing the reagent bottle 51 in the second reagent supply section 5, the lid 51 c is removed, and one end of each of the reagent supply paths 53, 55, and 56 is arranged in the reagent bottle 51.

Next, the upper members 3a and 3b of the reaction chamber 3 are divided and a pretreated sample is placed on the filter 3c. Next, the operation moves to an analysis operation. First, the reagent introduction path 33 is filled with phenylisothiocyanate (PITC) from the reagent bottle 14 of the first reagent supply unit 2. The internal pressure of the reagent bottle 14 is increased by the inert gas from the gas supply path 18, and this pressure causes the reagent supply path 21
Send PITC in. The PITC sent into the reagent supply path 21 is filled into the reagent introduction path 33 via the electromagnetic valve 22.

When the filling is completed, the electromagnetic valve 22a located at the upper end of FIG. 1 is opened, and the inert gas from the gas supply passage 25 flows into the reagent introduction passage 33. The PITC supplied into the reagent introduction path 33 is jetted toward the filter 3c by the gas from the gas supply path 25, and is supplied to the sample arranged on the filter 3c. In the sample placed on the filter 3c, PITC is reacted with the terminal amino group of the protein in a trimethylamine (TMA) atmosphere, and phenylthiocarbamyl protein (PTC protein) is produced in high yield. The trimethylamine is supplied from the reagent bottle 13 into the reaction chamber 3 in a gaseous state.

Excess reagents and by-products during the PTC-protein production reaction are removed from reagent bottles 11 and 12 by PITC
Washed with ethyl acetate and n-heptane supplied as above. The cleaning liquid here is supplied to the flow path switching valve 37.
Through the drain passage 37a.

[0020] After washing, the reaction chamber 3,
100 from the reagent bottle 16 as a protein cleavage reaction reagent
% Trifluoroacetic acid (TFA) is supplied in gaseous state. The electromagnetic valves 17 and 22b are opened, 20a is closed, and an inert gas is supplied from the gas supply device 19 into the reagent bottle 16 via the gas supply path 18. Thus, the TFA in the reagent bottle 16 is supplied to the reagent supply path 21 and the solenoid valve 22b.
From the reagent introduction path 33 through the reaction chamber 3
Supplied to

When TFA is supplied to the reaction chamber 3, the sample on the filter 3c is cleaved at the N-terminal peptide bond of the PTC-amino acid to give 2-anilino-amino acid.
5-Thiazolinoneamine acid (ATZ-amino acid) is produced. The ATZ-amino acid is extracted by n-butyl chloride from the reagent bottle 15, and the extract is introduced into the conversion flask 4 through the sample transfer tube 35.

The ATZ-amino acid extract in the conversion flask 4 contains the reagent bottle 51 of the second reagent supply unit 5.
To supply a 25% aqueous TFA solution. This 25% TF
Aqueous A solution converts ATZ-amino acids to stable 3-phenyl-2-thiohydantoin amino acids (PTH-amino acids). This completes the Edman reaction.

The PTH-amino acid in the conversion flask 4 is dissolved by a mixture of acetonitrile and water from the reagent bottle 50. The solution with a mixture of this Acetonitrile and water is introduced into the sample outlet passage 66 which is connected through a sample introduction tube 40 into six-way valve 60 of the amino acid analyzer 7. When switching the six-way valve 60, solution of acetonitrile and water is injected into the column 62 of the high-performance liquid chromatography. Then, the PTH-amino acid separated in the column 62 is detected by the detector 63, and the data is processed by the data processing device 65. Thereby, the N-terminal amino acid in the sample is identified. P that did not enter the sample introduction path 66
The TH-amino acid is supplied to the sample tube 64 of the fraction collector 64 through the six-way valve 60 and the collection path 67.
a.

By repeating such a series of analysis operations, the amino acid sequence in the sample is sequentially elucidated, and the amino acid sequence is determined. Since polyethylene is supplied relatively inexpensively, polyethylene containers are suitable for disposable use and are easier to handle as reagent bottles of 25% TFA aqueous solution .

(Experimental Examples) (a) In the above Examples, the number of months passed in a state where a 25% aqueous solution of trifluoroacetic acid was stored in a container made of polyethylene and glass having the same capacity as shown in Tables 1 to 3. After a set number of months, the yield and the amount of PTH-amino acid recovered were repeatedly examined for each container. Tables 1 to 3 show the results.
Shown in P in the section of the container material used is polyethylene,
G represents glass.

The 25% aqueous trifluoroacetic acid solution prepared is stored in a polyethylene container and a glass container at 4 ° C. Next, a constant temperature room (24-25
(° C.) using a reagent after a predetermined period.

[0027]

[Table 1]

[0028]

[Table 2]

[0029]

[Table 3]

From Tables 1 to 3, there is no difference in the yield and the amount of PTH-amino acid recovered from the glass container in general when the polyethylene container is used, and the conventional glass container is changed to a polyethylene container. It is judged that there is no adverse effect. (B) The chromatograms of the sample numbers 9, 11, and 12 of the experimental example are shown in FIGS. FIG. 4 shows the results after performing the conversion reaction during the third repetition of the repetitive analysis. FIG. 5 shows the results of the first analysis in the repeated reaction.

From FIGS. 4 and 5, it can be seen that the same separation was performed based on the retention time of the sample and the shape of the peak, and no elution of the polyethylene container material was observed on the chromatogram.

[0032]

[Effect of the Invention] Since the Edman reactor according to the present invention has a resin container for storing an aqueous solution of trifluoroacetic acid, the elution of glass from a glass container containing an aqueous solution of trifluoroacetic acid as in the prior art can be achieved. Also, the eluate does not block the flow path leading to the amino acid analyzer.

[Brief description of the drawings]

FIG. 1 is a schematic structural diagram of an example of a protein primary structure analyzer.

FIG. 2 is a schematic configuration partial view thereof.

FIG. 3 is a perspective view of a reagent bottle.

FIG. 4 is a chromatogram showing the results of a third analysis of the samples described in Table 3.

FIG. 5 is a chromatogram showing the results of the first analysis corresponding to FIG.

[Explanation of symbols]

 Reference Signs List 3 reaction chamber 4 conversion flask 5 second reagent supply section 7 PTH-amino acid analyzer 51 25% trifluoroacetic acid aqueous solution reagent bottle

Claims (1)

(57) [Scope of request for utility model registration] 1. An Edman reaction device for supplying a sample after Edman reaction to an amino acid analyzer, comprising: a reaction portion for cleaving an N-terminal peptide bond of a protein sample to form an ATZ-amino acid; Made of polyethylene resin containing fluoroacetic acid aqueous solution
An Edman reactor having a container , a conversion unit for converting the ATZ-amino acid to a PTH-amino acid using the trifluoroacetic acid aqueous solution, and a sample supply unit for supplying the PTH-amino acid to the amino acid analyzer .