JPH0469573A - Apparatus for hydrolyzing protein or peptide - Google Patents

Abstract

PURPOSE:To facilitate an exact analysis and saving of manpower by online executing the processes from transportation up to a hydrolysis by a solid phase- gaseous phase reaction, extraction and injection to an amino acid analyzer. CONSTITUTION:First of all, a sample soln. is dried. The sample soln. is impregnated into a glass filter 6 of a sample holder 9. This sample holder 9 is then stowed into a reduced pressure box 10. The sample holder 9s is thereafter transported to a stage where a hydrolysis reaction is effected. The sample holder 9 in this stage is fixed to an evaporating chamber 15 and glass tube 16 in a heater 14. The acid evaporated to vapor in the evaporating chamber 14 is filled in the space including the inside of the sample holder 9. Steam is supplied into the sample holder 9 to remove the remaining vapor mixed with the acid. The vapor mixed with the acid past a solenoid valve 20 is captured by a trap 22.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an apparatus for hydrolyzing proteins or peptides, which is the first step in analyzing the amino acid composition of proteins or peptides.

[Conventional technology]

Conventionally, amino acid composition analysis has been carried out, for example, as follows.

(S, Moore, H, Stein Mesonos in Enzymology (S, P, Kolovik, N, O.

Capra (ed.), 1963, vol. 6, pages 819-831,
(Academic Press, New York) First, distilled azeotropic 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 on ice. Next, add this ampoule to 1
Heat at 05°C to 110°C for 24 hours to 144 hours. Next, the ampoule is opened and the hydrochloric acid is removed by evaporation. Finally, the hydrolyzed sample is dissolved and injected into the amino acid analyzer. However, the high-speed gas phase hydrolysis method recently developed by Narita et al. (Narita Akira et al., Journal of Biochemistry, 1987, 102S, pages 1593-1597) performs the hydrolysis, which is the first step in compositional analysis, as follows. It is being improved. (2) In order to increase the hydrolysis rate, the ashing temperature was set at 158°C, so that hydrolysis could be completed quickly in 5 to 45 minutes at 22°C. ■Volatile and strongly acidic! A acid, trifluoroacetic acid, was added to improve the hydrolysis rate of hydrophobic proteins. ■By using a gas phase method that uses mixed acid vapor to act on a protein sample, it is possible to prevent the contamination of amino acids contained in the acid itself, enabling accurate amino acid composition analysis, and also to remove residual acids after hydrolysis. Made it simple.

[Problem to be solved by the invention]

The conventional hydrolysis method using distilled azeotropic hydrochloric acid requires a long time of 24 to 144 hours, and requires a complicated evaporation operation to remove the acid after hydrolysis. Furthermore, since it is a liquid phase method, there is contamination from acids, making accurate composition analysis difficult. This effect is particularly noticeable when miniaturization of the sample is required. Furthermore, the gas-phase method developed by Narita et al. also requires skilled glass fabrication techniques, individual differences are unavoidable, and there is contamination due to human involvement in each operation of sample handling. This remains a drawback. Therefore, in order to solve these conventional drawbacks, this invention allows each operation from hydrolysis of a dried protein sample to injection of the hydrolyzate into an amino acid analyzer to be performed continuously without any manual operations. The purpose is to perform continuous processing and to process a large number of samples continuously.

(Means for Solving the Problems) The present invention has been made to eliminate the above-mentioned drawbacks, and provides a transport mechanism for a sample carrier carrying a dried sample of kumbaku substance or peptide, and a transport mechanism for transferring the dried sample to the sample carrier. Pressurization·
A mechanism for supplying acid mixed vapor under heating to perform a hydrolysis reaction by a solid phase-gas phase reaction, a transport mechanism for a sample carrier carrying a hydrolyzed sample, and a mechanism for carrying out a hydrolysis reaction from the sample carrier. a mechanism for extracting the extracted sample; a mechanism for feeding the extracted sample solution to the sample solution injection device of the amino acid analyzer;
The structure includes a fluid supply mechanism used for the hydrolysis, extraction, and liquid feeding.

[For production]

The hydrolysis device configured as described above enables online processing of transportation, hydrolysis by solid phase-gas phase reaction, extraction, and injection into the amino acid analyzer for amino acid composition analysis of proteins or peptides supported on sample carriers. This makes it possible to perform accurate amino acid composition analysis by reducing essential contamination and eliminating individual differences without using any manual operations for each operation, and also saves labor.

Furthermore, it is possible to perform not only continuous processing of a series of operations but also continuous processing of a large number of samples.

〔Example〕

Embodiments of the present invention will be described below based on the drawings.

Figure 1 is a block diagram showing the configuration of the apparatus of the present invention. The apparatus for hydrolyzing proteins or peptides of the present invention comprises a transport mechanism 1 for a sample carrier carrying a dried protein or peptide sample, and A reaction mechanism 2 for hydrolyzing a sample and acid mixed vapor, a transport mechanism 3 for a sample carrier carrying a hydrolyzed sample, and a mechanism for extracting the hydrolyzed sample from the sample carrier and transporting the extracted sample. It consists of an extraction/liquid feeding mechanism 4 that feeds liquid to the amino acid analyzer, a reaction mechanism 2, and a solution supply mechanism 5 to the extraction/liquid feeding mechanism 4. A protein or peptide sample is hydrolyzed into amino acids through mechanisms 1 to 4, and directly injected into an amino acid analysis needle.

Next, the operation of the apparatus for hydrolyzing proteins or peptides of the present invention will be described.

FIG. 2 is a sectional view showing one embodiment of a sample holder used in the present invention. FIG. 3 is a process diagram for explaining the present invention in detail. The sample holder 9 as a sample carrier consists of a rod-shaped glass filter 6 wrapped in a glass tube 7 whose both end surfaces are polished into a tapered shape and further grooved inward from a portion of the taper. The outer periphery between the grooves was covered with a tube 8 made of heat-resistant resin such as fluorocarbon resin or polyether ether kedone.

First, the glass filter 6 of the sample holder 9, which is used to dry the sample solution, is impregnated with the sample solution. Next, this sample holder 9 is stored in a vacuum box 10. This decompression box 10 is connected to a vacuum pump 13 via a solenoid valve 12 equipped with a vacuum pump 11, and the solenoid valve 12 is opened and closed automatically to perform intermittent suction according to a set time program. The solvent in the I4 solution is removed and the sample is dried, and the dried sample is placed in the glass filter 6 of the sample holder 9! It is held for a long time.

Next, the sample holder 9 undergoes a hydrolysis reaction and is transported to a second stage. Here, the sample holder 9 is fixed to the vaporization chamber 15 and the glass tube 16 inside the heater 14. The acid mixed solution 17 purged with oxygen with an inert gas is sucked into the syringe pump 19 through the three-way cock 18, and then the three-way cock 18 is switched and the vaporization chamber 1
Supply the acid mixed solution to 5.

At this time, the vaporization chamber 15 and the sample holder 9 are heated by the heater 4, and the acid vaporized in the vaporization chamber 15 fills the space including the sample holder 9.

At this time, the solenoid valve 20 is closed, but the solenoid valve 20 can also be opened and closed in conjunction with the pressure gauge 21 to keep the pressure inside the sample holder 9 constant. Here, the sample supported on the sample carrier is hydrolyzed by a solid phase-gas phase reaction with the acid mixed vapor. Next, water vapor is supplied into the sample holder 9 to remove the remaining acid mixed vapor. . TL magnetic valve 2
The acid mixed vapor that has passed through the trap 22 is collected by the trap 22.

At this time, the solenoid valve 20 remains open.

Next, the sample holder 9 is transported to a stage where extraction and liquid feeding are performed. Here, the sample holder 9 is fixed by the nozzle 23. Dilute hydrochloric acid 24 is sucked into a syringe 26 via a three-way cock 25. Then, the three-way cock 25 is switched, and the sample is extracted with dilute salt M24. The liquid containing the sample is passed through the six-way valve 27, which is the injection device of the amino acid analyzer.
The sample coil 28 is sent to the sample coil 28 inside. Amino acid analysis following the above operations is well known and will not be described here. The various conditions in this example are summarized in Table 1.

Table 1 Here, the hydrolysis stage will be specifically explained. FIG. 4 (al is an external view of an embodiment of the hydrolysis stage of the present invention when the sample holder is fixed, and FIG. 4fbl is an embodiment of the hydrolysis stage of the present invention when the sample holder is fixed. Cross-sectional view, FIG. 4 (C1 is a cross-sectional view showing one embodiment of the vaporization chamber of the hydrolysis stage of the present invention.

The heater 14 shown in FIG. Heater 3
0, a heat insulating material 31, a cover 32, an air cylinder 35, a movable heating unit 37 consisting of a roller 36, a rubber heater 30, a heat insulating material 31, a cover 32, and a mouth.
The support unit 38 includes a roller 36 and a support unit 38. These units are arranged on the base 39 as shown in FIG. 4 (δ). The support unit 38 is the roller 3
The movable heating unit 37, which is mounted on a carriage 40 equipped with G via a hinge 41, and the carriage 40 are placed on rails 42 using rollers 36, and by operating the air cylinder 35, the sample holder 9 can be fixed (FIG. 4(b)) and released (FIG. 4(a)). A support par 43 and a spring 44 are installed that pass through the truck 40 to position the support unit 41 back when released. The vaporization chamber 15 is constructed by connecting a Pyrex glass tube 45 with branches to a Teflon tube 46 provided with a flange using a napht 47 and a cap screw 48.
It is connected and configured. This Teflon tube 4
6, an acid mixed solution 19 is supplied to the vaporization chamber 15.

FIG. 5 is an external view showing one embodiment of the configuration of the extraction liquid feeding stage of the present invention. The figure shows sample holder 9 on support stand 50.
is shown fixed by the pair of nozzles 23 inserted into the movable nozzle support plate 52 and the fixed nozzle support plate 53, respectively, due to the action of the air cylinder 51. The support stand 50 is connected to a spacer 55 via a hinge 54.

A fixed nozzle support plate 53 and a spacer 55 are fixed on a base 56 as shown in the figure. Two guide bars 57 pass through the fixed nozzle support plate 53.
A release plate 58 is supported at both ends of the guide bar 57. Release plate 5 on the sample holder 9 side
8 and the fixed nozzle support plate 53
A spring 59 is installed in the sample holder 9.
It is the driving source when released.

FIG. 6 fat to +di are cross-sectional views for explaining the operation of the transport mechanism of the sample holder of the present invention.

An example of the device of the present invention includes an autoholder loader 63 consisting of a tray 60 and an air cylinder 62 fixed to the tray 60 via a retainer 61, a hydrolysis stage 29, an extraction liquid feeding stage 49, and a storage case. 6
4 are arranged in this order, and the Bi t'+ holder 9 holds the sample from which the solvent has been removed and has been dried.
is mounted on the tray 60 of the auto holder loader 63 (FIG. 6(a)).

Next, sample holder 9! J<By the action of the air cylinder 62, it is transported from the tray 60 to the hydrolysis stage 29. (Figure 6(b)).

The sample holder 9, which has undergone the hydrolysis process in the hydrolysis stage 29, is raised by the action of an air cylinder 66 fixed to the trolley 40 via a retainer 65, and one end of the support unit 3 connected by the hinge 41 is lifted, and the sample holder 9 is extracted. The liquid is transported to the support stand 50 of the liquid feeding stage 49 (Fig. 6(C)
1).

The sample holder 9 that has undergone the extraction liquid feeding process is attached to the spacer 55 by the hinge 54 by the action of the air cylinder 68 fixed to the spacer 55 and the base 56 via the J retainer 67.
One end of the support stand 50, which is connected via the support stand 50, rises and is stored in the storage case 64 (FIG. 6fdl).

In this way, automatic transportation of the sample holder 9, which is a sample carrier, has been achieved, and all processes from hydrolysis and extraction through solid phase-gas phase reactions to injection into the amino acid analyzer are completely handled in the amino acid composition analysis of proteins or peptides. It can be carried out continuously without any manipulation.

The sample holding part must have a structure that does not react with the sample solution and solutions such as hydrochloric acid used for hydrolysis and extraction, and that can hold these solutions, such as woven glass fibers or porous glass. These are processed into solid wood or filled with glass or ceramic beads.

[Effects of the Invention] As explained above, this invention employs a gas phase hydrolysis method, and allows all operations from sample hydrolysis to injection into an amino acid analyzer to be performed manually in analyzing the amino acid composition of proteins or peptides. Continuous processing is possible without any manipulation, and continuous processing of a large number of samples is possible.In addition, accurate amino acid composition analysis is possible by eliminating manual operations that require skill and individual differences, which saves labor. It is.

It goes without saying that the terms and explanations used herein do not exclude similar terms as long as the features of the present invention are maintained.

[Brief explanation of the drawing]

FIG. 1 is a block diagram showing the configuration of the apparatus of the present invention, FIG. 2 is a sectional view showing an embodiment of a sample holder used in the present invention, and FIG. 3 is a process diagram for explaining the process in detail of the present invention. figure,
FIG. 4 (al is an external view showing an embodiment of the hydrolysis stage of the present invention when the sample holder is released, FIG. 4 (bl is
FIG. 4 is a cross-sectional view showing an embodiment of the hydrolysis stage of the present invention when the sample holder is fixed (C1 is a cross-sectional view showing an embodiment of the vaporization chamber of the hydrolysis stage of the present invention, and FIG. 5 is The external view showing one embodiment of the configuration of the extraction/liquid feeding stage of the present invention, and FIGS. SEIKO ELECTRONICS INDUSTRIES CO., LTD. Agent Patent Attorney Takayuki Hayashi 9

Claims (4)

[Claims] (1) A transport mechanism for a sample carrier carrying a dried protein or peptide sample, and a hydrolysis reaction by solid phase-gas phase reaction by supplying acid mixed vapor to the dried sample under pressure and heat. a mechanism for transporting a sample carrier carrying a sample subjected to hydrolysis; a mechanism for extracting the hydrolyzed sample from the sample carrier; and a sample solution injection device of an amino acid analyzer for the extracted sample solution. 1. An apparatus for hydrolyzing proteins or peptides, comprising: a liquid feeding mechanism; and a fluid feeding mechanism used for the hydrolysis, extraction, and liquid feeding. (2) The sample carrier consists of a rod-shaped glass filter wrapped in a glass tube with both end surfaces polished into a tapered shape and a groove formed on the inside of the tapered part, and between the two grooves of the glass tube. 2. The apparatus for hydrolyzing proteins or peptides according to claim 1, wherein the outer periphery of the apparatus is covered with a cylinder made of a heat-resistant resin such as fluorocarbon resin or polyetheretherketone. (3) The protein or peptide hydration according to claim 1, wherein an inert gas such as argon or nitrogen, water vapor, or a mixture thereof is used to remove the acid mixture from the sample that has undergone hydrolysis. Decomposition equipment. (4) The apparatus for hydrolyzing proteins or peptides according to claim 1, wherein the sample carrier transport mechanism continuously transports one or more sample carriers.