JP3420784B2 - Equipment for hydrolyzing proteins or peptides - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an apparatus for automating a chemical process, and more particularly to an apparatus for hydrolyzing a protein or peptide into its constituent amino acids.

[0002]

2. Description of the Related Art Conventionally, for example, protein hydrolysis is
It was done as follows. One is a liquid phase method using azeotropic hydrochloric acid. (S. Moore, H. Stein Methods in Enzymology (SP Colobic,
N. O. Coupler edition) 1963 6th volume 819-831
(Page, Academic Press, NY) In this method, first, azeotropic boiling hydrochloric acid is added to the dry sample at the bottom of the test tube, and the test tube is sealed under reduced pressure while cooling with ice. Then, this ampoule is heated at 105-110 ° C for 24-144.
Heat for hours. Next, the ampoule is opened to remove hydrochloric acid by evaporation.

Further, a high-speed vapor phase method using an acid mixed vapor containing hydrochloric acid and trifluoroacetic acid has been reported. This method is disclosed in Japanese Patent Laid-Open No. 61-151157. In this method, a small test tube whose sample has been dried to the bottom is placed in a test tube containing an aqueous solution containing hydrochloric acid and trifluoroacetic acid, and the tube is sealed in the same manner as the above method. After heating at 158 ° C for 22.5 or 45 minutes,
Open the tube and remove the acid remaining in the small test tube under reduced pressure.

An attempt to automate the hydrolysis process has also been made in US Pat. It is disclosed in Japanese Patent No. 5,106,583. This is to move a slide glass equipped with a sample-supporting porous filter to a hydrolysis station for hydrolysis reaction, and then move this slide glass to a derivatization station for derivatization for amino acid analysis. It is intended to let you do.

[0005]

The conventional hydrolysis method using azeotropic boiling hydrochloric acid requires a long time of 24-144 hours, and it is necessary to remove the acid by a complicated evaporation operation after hydrolysis. . Further, since it is a liquid phase method, it is contaminated with acid, and it is difficult to accurately analyze the composition. This effect is remarkable especially when a small amount of sample is required.

Further, in the high-speed vapor phase method, glasswork requiring skilled techniques is required, individual differences are unavoidable, and there is contamination for humans to engage in each sample handling operation. It remains a drawback. Further, the above-mentioned automation attempt is not suitable for high-speed decomposition utilizing high temperature because both the slide glass and the hydrolysis station are moving parts.

[0007]

The apparatus for hydrolyzing a protein or peptide of the present invention has an opening with a tube at the bottom in order to overcome the above-mentioned drawbacks and perform hydrolysis of the protein or peptide. The container has a container, a plurality of conduits, and a tank having no opening. The tube of the container and the plurality of conduits include a reaction chamber penetrating the tank.

[0008]

By the above means, it becomes possible to automatically perform high-speed hydrolysis at high temperature by acid vapor.

[0009]

EXAMPLES The present invention will be described below based on examples. (Example 1) Here, an example of the reaction chamber of the present invention will be described.

This reaction chamber has a container having an opening with a tube at the bottom, a plurality of conduits, and a tank having no opening, and the tube of the container and the plurality of conduits are The structure penetrates the tank. FIG. 1 shows a sectional view of a reaction chamber 1 which is a place where a hydrolysis reaction is carried out.

An inert gas such as argon is fed into the reaction chamber 1 through the upper conduit 2. The acid solution used for hydrolysis is supplied and discharged through a conduit 3 leading to the lower part of the tank. The supplied acid solution is sent to the bottom 4 of the reaction chamber. This conduit 3 is also the outlet for the inert gas. A sample solution containing a protein or peptide is sent to the container 6 through a pipe 5 connected to the container 6. Further, the pipe 5 is an inflow path of a solution for dissolving an amino acid which is a hydrolyzate, and is also an outflow path of a solution containing an amino acid.

The container 6 is a place where water vapor containing an acid acts on the sample to cause the hydrolysis reaction to proceed. This container 6
For example, glass can be used. The pipe 5, which is connected to the conduit 2, the conduit 3 and the container 6, is fixed to the tank 7 as follows.

A joint 10 having an o-ring 8 and a guide ring 9 is screwed into the tank 7. The conduit 2 has a push screw 12 with a ferrule 11 on the joint 10.
Is fixed by. The method of fixing the pipe 5 connected to the conduit 3 and the container 6 is similar to this.

For the tank 7 and the joint 10, for example, an acid-resistant ceramic can be used. For the ferrule 11, the push screw 12, the conduit 2 and the conduit 3, for example, an acid resistant resin can be used. FIG. 2 shows an example of the configuration of a device for hydrolyzing a protein or peptide of the present invention.

The device 30 has a function of hydrolyzing a sample into amino acids which are its constituents. A sample solution 31 containing a protein or peptide is contained in a sample vial 32. The sampling needle 33 is the tube 3
It is connected to the 6-way switching valve 35 via 4. For continuously collecting sample solutions from multiple sample vials,
Means for operating the sample collection needle or sample vial are well known and will not be described here.

Solvent bottles 36 and 37 are two-way valves 38 and 38, respectively.
It is connected to the four-way joint 40 via 39. An injection cylinder 41 as a transfer means is connected to the four-way joint 40, and a four-way valve 43 is further connected via a two-way valve 42. The 4-way valve 43 also includes a 6-way switching valve 35,
It is connected to the reaction chamber 1 via a two-way valve 44.

The reaction chamber 1 further includes a two-way valve 45 and a two-way valve 4.
6 is connected. On the other hand, the reaction chamber 1 is stored in the block 47. Further, a heater 48 and a fan 49, which are means for adjusting the temperature of the reaction chamber 1, are installed. This device includes a pressure regulator 50 as a pressure regulating means and a branch pipe 51.
An inert gas having a constant pressure, for example, argon is supplied through and.

The solvent bottle 52 is connected to the branch pipe 51 via the two-way valve 53, the two-way valve 55 and the three-way valve 56 via the three-way valve 54, and the waste liquid tank 58 via the two-way valve 57. Each is connected. The three-way valve 59 and the three-way valve 56 are both waste liquid tanks 58.
Are connected to each other and to each other. The waste liquid tank 58 is also connected to the four-way valve 43.

The 6-way switching valve 35 has a loop 60 and is connected to a pipe 61 connected to a liquid feed pump which is a component of the amino acid analysis means and a pipe 62 connected to a column which is also a component of the amino acid analysis means. There is.
The solvent bottles 36, 37, and 52 in the above description constitute storage means.

Two-way valves 38, 39, 42, 44, 4
5, 46, 53, 55, 57, 3-way valve 54, 56, 5
9, 4-way valve 43, 6-way switching valve 35, 4-way joint 4
0 constitutes the flow path control means. Next, the device 3 of the present invention
An example of the operation of 0 will be described based on FIGS. 1 and 2.

First, the sample solution 31 contained in the sample vial 32 is drawn into the pipe 63 between the four-way valve 43 and the two-way valve 42 by the action of the injection cylinder 41. Then, the sample solution is sent to the container 6 of the reaction chamber 1 through the two-way valve 44 and the pipe 5 by switching the four-way valve 43 and the action of the injection cylinder 41. Next, a drying operation is performed. Argon is supplied through the two-way valve 45 and the conduit 2 and sprayed on the sample solution sent into the container 6.

After the sample is dried, the hydrolysis reaction is carried out as follows. As for the acid solution 64 stored in the sample bottle 52, argon is sent in advance by the action of the two-way valve 53 and the two-way valve 57 to perform oxygen removal by bubbling. Then, the two-way valve 53, the three-way valve 54,
The acid solution is sent into the pipe 65 by the action of the three-way valve 56. Further, by the action of the 2-way valve 55, 3-way valve 54, 3-way 56, 3-way valve 59, 2-way valve 46, the acid solution is sent to the reaction chamber bottom 4 in the reaction chamber 1 through the conduit 3. Here, by closing the two-way valve 44, the two-way valve 45, and the two-way valve 46, the reaction chamber 1 is sealed. Next, the heater 48
The reaction chamber 1 is heated by heating the block 47 by the action of. At this time, a part of the acid solution sent to the bottom 4 of the reaction chamber is evaporated, and the water vapor containing the acid acts on the sample in the container 6 to cause the hydrolysis reaction to proceed. Here, the protein or peptide becomes a mixture of amino acids which are its hydrolysates.

As the conditions for carrying out this hydrolysis reaction, for example, hydrochloric acid and trifluoroacetic acid are used, and at 2 ° C at 158 ° C.
The conditions of the high-speed vapor phase method of heating for 2.5 or 45 minutes can be used. After proceeding the reaction for a certain time,
The temperature of the block 47 and the reaction chamber 1 is lowered by the action of the fan 49. Then, the acid solution remaining in the reaction chamber bottom 4 is discharged through the conduit 3 by the action of the two-way valve 46 and the three-way valve 59. Further, the hydrolyzate in the container 6 is dried by supplying argon through the two-way valve 45 and the conduit 2. At this time, the acid solution is collected in the waste liquid tank 58.

After the hydrolyzate has been dried, the solution 66 stored in the solvent bottle 36 is sucked and discharged by the injection cylinder 41, so that the two-way valve 38, the four-way joint 40 and the two-way valve 4 are used.
It is sent to the container 6 through the 2-, 4-way valve 43, the 2-way valve 44, and the pipe 5. Here, the hydrolyzate is dissolved in the solution. Then, by the suction action of the injection cylinder 41, the dissolution liquid in which the hydrolyzate is dissolved is supplied to the pipe 63 between the two-way valve 42 and the four-way valve 43.
Pull in. Further, the solution in which the hydrolyzate is dissolved by the discharge action of the injection cylinder 41 is injected into the loop 60 of the 6-way switching valve 35. As the solution, for example, a dilute hydrochloric acid solution or a citrate buffer solution can be used.

Next, by switching the 6-way switching valve 35,
Amino acid analysis is performed by the amino acid analysis means connected by the pipe 61 and the pipe 62. Amino acid analysis is to separate and quantify a mixture of amino acids or a mixture of amino acid derivatives on a column. This step of amino acid analysis is known and will not be described here.

Next, cleaning is performed. The cleaning liquid 6 stored in the solvent bottle 37 by the action of suction and discharge of the injection cylinder 41.
7 is sent to the container 6 through the 2-way valve 39, 4-way joint 40, 2-way valve 42, 4-way valve 432-way valve 44, and pipe 5. Then, this cleaning liquid is used as a two-way valve 45, a two-way valve 44, a four-way valve 43.
Is discharged to the waste liquid tank 58 by utilizing the pressure of argon. Further, the cleaning liquid 67 stored in the solvent bottle 37 is sucked and discharged by the injection cylinder 41, so that the cleaning liquid 67 is stored in the two-way valve 39, the four-way joint 40, the two-way valve 42, the four-way valve 43, 6
Loop 60 of one-way switching valve, piping 34, sampling needle 33
Pass through. Through these steps, the sample and the portion through which the hydrolyzate of the sample has passed are washed.

By repeating these washing steps, the washing efficiency can be improved. By repeating the above operation, high-speed hydrolysis at high temperature by acid vapor can be continuously performed.

Finally, a block diagram of the apparatus for hydrolyzing a protein or peptide of the present invention, FIG. 3, will be described. Temperature control means 1 for controlling the temperature of the reaction chamber 1
00, a flow path control means 101, a transfer means 102, a storage means 103, and a pressure adjusting means 104.
05 and the amino acid analysis means 106 are connected in this order. Furthermore, the temperature control means 100, the supply means 105, and the amino acid analysis means 106 are respectively the device control means 1
It is controlled according to the operation sequence preset to 07. With this configuration, it is possible to automatically perform high-speed hydrolysis at a high temperature by acid vapor, and simultaneously perform amino acid analysis of the hydrolyzate automatically.

Example 2 Here, another example of the reaction chamber of the present invention will be described. This reaction chamber has a container having an opening with a tube at the bottom, a plurality of conduits, and a tank having no opening, and the tube of the container and the plurality of conduits are the tanks. It is configured to penetrate.

FIG. 4 is a sectional view of the reaction chamber 13 where the hydrolysis reaction is carried out. An inert gas, for example argon, is fed into the reaction chamber 1 through the upper conduit 2. The acid solution used for hydrolysis is supplied through a conduit 14 that leads to the bottom of the tank. The supplied acid solution is sent to the bottom 4 of the reaction chamber. The acid solution used for hydrolysis is discharged through a conduit 15 leading to the bottom of the tank. This conduit 15 is also the outlet for the inert gas. A sample solution containing a protein or peptide is sent to the container 6 through a pipe 5 connected to the container 6. Further, the pipe 5 is an inflow path of a solution for dissolving an amino acid which is a hydrolyzate, and is also an outflow path of a solution containing an amino acid.

The container 6 is a place where water vapor containing an acid acts on the sample to cause the hydrolysis reaction to proceed. This container 6
For example, glass can be used. The method for fixing the conduit 2, the conduit 14, the conduit 15 and the pipe 5 connected to the container 6 is the same as that described in the first embodiment.

For the tank 16 and the joint 10, for example, an acid resistant ceramic can be used. Ferrule 11,
For the push screw 12, the conduit 2, the conduit 14, and the conduit 15, for example, an acid resistant resin can be used.

FIG. 5 shows an example of the construction of an apparatus for hydrolyzing a protein or peptide of the present invention. The device 68 has a function of hydrolyzing a sample into amino acids which are its constituent elements.

A sample solution 31 containing a protein or peptide is contained in a sample vial 32. The sampling needle 33 is connected to a 6-way switching valve 35 via a tube 34. Means for performing the operation of the sample collection needle or the sample vial for continuously collecting the sample solution from a plurality of sample vials are known and will not be described here.

The solvent bottles 36 and 37 are provided with two-way valves 38 and 38, respectively.
It is connected to the four-way joint 40 via 39. An injection cylinder 41 as a transfer means is connected to the four-way joint 40, and a four-way valve 43 is further connected via a two-way valve 42. The 4-way valve 43 also includes a 6-way switching valve 35,
It is connected to the reaction chamber 1 via a two-way valve 44.

The reaction chamber 1 further includes a two-way valve 45 and a two-way valve 6.
9 and the two-way valve 70 are connected. On the other hand, the reaction chamber 1 is stored in the block 47. Further, a heater 48 and a fan 49, which are means for adjusting the temperature of the reaction chamber 1, are installed. This device includes a pressure regulator 50 as a pressure regulating means.
An inert gas having a constant pressure, for example, argon, is supplied through the and the branch pipe 51.

The solvent bottle 52 is connected to the branch pipe 51 via the two-way valve 53, the two-way valve 55 and the three-way valve 56 via the three-way valve 54, and the waste liquid tank 58 via the two-way valve 57. Each is connected. The three-way valve 56 is connected to the waste liquid tank 58. The waste liquid tank 58 is also connected to the four-way valve 43.

The 6-way switching valve 35 is provided with a loop 60 and is connected to a pipe 61 with a liquid feed pump which is a component of the amino acid analysis means and a pipe 62 with a column which is also a component of the amino acid analysis means. There is. The solvent bottles 36, 37, and 52 in the above description constitute storage means.

Two-way valves 38, 39, 42, 44, 4
5, 53, 55, 57, 69, 703 one-way valves 54, 56,
The 4-way valve 43, the 6-way switching valve 35, and the 4-way joint 40 form a flow path control means. Next, an example of the operation of the device 68 according to the embodiment of the present invention will be described with reference to FIGS. 4 and 5.

The operation until the sample is dried is the same as that described in Example 1. After the sample is dried, the hydrolysis reaction is performed as follows. The acid solution 64 stored in the sample bottle 52 is previously stored in the two-way valve 53 and the two-way valve 5.
Argon is sent by the action of 7 and oxygen is removed by bubbling. Next, 2-way valve 53, 3-way valve 5
The acid solution is fed into the pipe 65 by the action of the 4- and 3-way valve 56. Furthermore, the two-way valve 55, the three-way valve 54, the three-way valve 56,
By the action of the two-way valve 69, the acid solution is sent to the reaction chamber bottom 4 in the reaction chamber 1 through the conduit 14. Here, by closing the two-way valve 44, the two-way valve 45, the two-way valve 69, and the two-way valve 70, the inside of the reaction chamber 1 is sealed. Next, by heating the block 47 by the action of the heater 48,
The reaction chamber 1 is heated. At this time, a part of the acid solution sent to the bottom 4 of the reaction chamber is evaporated, and the water vapor containing the acid acts on the sample in the container 6 to cause the hydrolysis reaction to proceed. Here, the protein or peptide becomes a mixture of amino acids which are its hydrolysates.

As the conditions for carrying out this hydrolysis reaction, for example, hydrochloric acid and trifluoroacetic acid are used, and at 2 ° C and 158 ° C.
The conditions of the high-speed vapor phase method of heating for 2.5 or 45 minutes can be used. After proceeding the reaction for a certain time,
The temperature of the block 47 and the reaction chamber 1 is lowered by the action of the fan 49. Then, the acid solution remaining in the reaction chamber bottom 4 is discharged through the conduit 15 by the action of the two-way valve 70. Further, the hydrolyzate in the container 6 is dried by supplying argon through the two-way valve 45 and the conduit 2. At this time, the acid solution is collected in the waste liquid tank 58. The operations for dissolution of the sample, amino acid analysis and washing after the completion of drying of the hydrolyzate are the same as those described in Example 1.

By repeating the above operation, high-speed hydrolysis at high temperature by acid vapor can be continuously carried out. The configuration for controlling the device for hydrolyzing a protein or peptide of the present invention is the same as in Example 1.

[0043]

As described above, the main object of the present invention is, in the apparatus for hydrolyzing a protein or peptide, a container having an opening with a tube at the bottom, a plurality of conduits, and an opening. A high-speed hydrolysis at a high temperature by an acid vapor by automatically providing a reaction chamber having a vessel not having a container, the tube of the container, and the plurality of conduits penetrating the vessel. Is to be done in a specific way.

[Brief description of drawings]

FIG. 1 is a sectional view showing an example of a reaction chamber of the present invention.

FIG. 2 shows an example of the constitution of an apparatus for hydrolyzing a protein or peptide of the invention.

FIG. 3 is a block diagram of an apparatus for hydrolyzing a protein or peptide of the present invention.

FIG. 4 is a sectional view showing an example of a reaction chamber of the present invention.

FIG. 5 shows an example of the constitution of an apparatus for hydrolyzing a protein or peptide of the present invention.

[Explanation of symbols]

1,13 Reaction chamber 2, 3, 14, 15 conduits 4 Reaction chamber bottom 5 tubes 6 containers 7, 16 tanks

─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yasuro Terui 6-31-1, Kameido, Koto-ku, Tokyo Seiko Electronics Co., Ltd. (72) Shigeki Yagi 6-31-1, Kameido, Koto-ku, Tokyo Seiko Electronics Co., Ltd. (72) Inventor Junichi Takahashi 6-31-1 Kameido, Koto-ku, Tokyo Seiko Electronics Co., Ltd. (72) Inventor Shotaro Takahashi 6-31-1 Kameido, Koto-ku, Tokyo Seiko Electronics Co., Ltd. (72) Inventor Kiyoshi Kobayashi 63-11-1 Kameido, Koto-ku, Tokyo Seiko Electronics Co., Ltd. (72) Inventor Yasuhiro Obata 6-31-1 Kameido, Koto-ku, Tokyo Seiko Electronics Co., Ltd. (72) Inventor Toyoaki Uchida 6-31-1, Kameido, Koto-ku, Tokyo Seiko Electronics Co., Ltd. (56) References JP 62-220867 (JP, A) (58) Fields investigated (Int.Cl. ⁷ , DB name) C07C 227/18 C07C 229/06 G01N 33/68

Claims (5)

(57) [Claims] 1. A a container for holding a sample, having a said container
And bath that, a tube passing through the vessel to the lowermost portion of the container, before
A conduit that passes through the tank at the top of the tank, and a conduit at the bottom of the tank.
A tube guided comprising a reaction chamber having a one or more conduits extending through the tank, through the tank to the bottom of the container, the upper portion of the tank
A conduit that penetrates the tank, and is guided to the bottom of the tank.
Connected to each of one or more conduits that penetrate the tank,
An apparatus for hydrolyzing a protein or peptide, characterized by having a flow path control means for sealing or opening the reaction chamber . 2. The apparatus according to claim 1, wherein one or more conduits are guided to a lower portion of the tank and penetrate the tank.
Is a conduit to which the acid solution used for hydrolysis is supplied,
The acid solution used for the hydrolysis is discharged or before
Supplied to the upper part of the storage tank through a conduit penetrating the storage tank
A device for hydrolyzing a protein or peptide, which is a conduit through which an inert gas is discharged . 3. The apparatus for hydrolyzing a protein or peptide according to claim 1, further comprising a temperature adjusting means for adjusting the temperature of the reaction chamber. 4. A container for holding a sample, a tank having the container, and a container for protein or
Is a sample solution containing peptides and is a hydrolyzate.
Inflow of a solution for dissolving an amino acid, or
A pipe for flowing out a solution containing mino acid, and a guide for supplying an inert gas through the tank above the tank.
A pipe and a lower part of the tank that penetrates the tank and is used for hydrolysis.
Acid solution is supplied or discharged, or said inert
A reaction chamber with one or more conduits through which gas is discharged
The apparatus comprising: a pressure adjusting means for adjusting the pressure of the inert gas ;
Storage means for storing the liquid or the solution , and the tamper
And a flow path control means for controlling the transfer of the gas, the acid solution or the solution, and the sample solution. An apparatus for hydrolyzing a protein or peptide, characterized in that the reaction chamber is connected to an amino acid analysis means for separating a mixture of amino acids and quantifying each amino acid via the supply means. 5. The apparatus according to claim 1, further comprising apparatus control means for controlling the temperature control means, the supply means, and the amino acid analysis means in accordance with a preset operation sequence. A device that hydrolyzes proteins or peptides.