JPS6052758A - Fixing method of low molecular peptide - Google Patents

Abstract

PURPOSE:To enable detection with peptide having about 1,000mol.wt. as well by using the vapor of formaldehyde, polyfunctional aldehyde or polyfunctional isocyanate for fixing reactively the peptide. CONSTITUTION:Isoelectric point electrophoresis using angiotensin II having 1046 dalton mol.wt. as a sample and polyacrylamide gel as a base is performed. The gel of the one-side open type in which polyacrylamide gel is fixed at one side to a glass plate by using methacryloxypropyl methoxy silane is used. The base gel subjected to the isoelectric point electrophoresis is treated with the vapor of gultaraldehyde and the molecules of the separated peptide (angiotensin II) are crosslinked to each other to fix the separated peptide in the base gel. Bi- or more multifunctional aldehydes such as paraformaldehyde and formaldehyde in addition to gluearaldehyde and bi- or more multifunctional isocyanate such as hexamethylene diiosocyanate, tolylene diisocyanate, etc. for isocyanates are usable as the reagent for fixing.

Description

Detailed Description of the Invention [Field of Application of the Invention] The present invention relates to proteins and peptides, particularly those having a molecular weight of 1,000 to 1,000.
Concerning a method for fixing separated peptides in electrophoretic analysis of low molecular weight peptides of several thousand daltons.

[Background of the invention]

Generally, proteins separated in a support gel by electrophoresis are detected by first precipitating and fixing the whole protein in the support gel with a buffer containing 50 timethanol, and then detecting the protein using a dye. However, in this method, low molecular weight peptides are not sufficiently immobilized and are easily washed away from the support gel. Therefore, in electrophoretic analysis of low molecular weight peptides, in order to avoid the problem of peptide fixation, attempts have been made to add fluorescence to the peptide using a fluorescent reagent such as trinulfonylpine isocyanate and then perform electrophoresis. (Protein/Nucleic Acids/Enzymes 27 (12) 1488-1490 (1982)
.

However, in the method of labeling the peptide with a fluorescent reagent before electrophoresis, (1) the isoelectric point of the peptide changes because the peptide is modified with lysine residues; (2) The molecular weight changes because it is modified with a reagent with a large molecular weight. Due to these problems, it has not been widely used.

[Purpose of the invention]

The purpose of the present invention is to immobilize low-molecular-weight peptides with a molecular weight of 1,000 to several thousand daltons in a polyacrylamide gel support separated by electrophoresis, and to submit a complete method for staining and detecting the low-molecular-weight peptides. S2 also known as Vc.

[Summary of the invention]

As mentioned above, if a sample is electrophoresed that has been made to have a fluorescent gold by complete chemical modification of the peptide, the isoelectric point and molecular weight will change. On the other hand, when performing electrophoresis, the isoelectric point and molecular weight of the vegutide can be determined as it is by precipitating the peptide in the support with methanol, fixing it, and staining it, but the fixation is incomplete. The focus of the present invention is to enable detection of separated peptides without changes in isoelectric point or molecular weight. Therefore, after electrophoretic separation of peptides, it is important to fix the peptides within the support gel without diffusing into the solution from the support gel during fixation and staining of the peptides. Therefore, for example, glutara peptide was immobilized. That is,
Since the fixation operation is performed in a gas phase rather than in an aqueous solution, the peptide does not come out of the support gel during the fixation operation.

Furthermore, since the molecular weight of the low-molecular-weight peptide is increased using chemical bonds, the peptide is tightly fixed within the support gel.

In addition to the glutaraldehyde described above, similar results can be obtained using formaldehyde, polyhydric aldehyde or polyhydric incyanate vapors.

[Embodiments of the invention]

An embodiment of the present invention will be described below.

Angiotensin pentamolecular weight 1046 daltons (2,6 m
Isoelectric focusing was carried out using a sample of 4 μt (g/Tnt) as the entire support of the polyacrylamide gel. Polyacrylamide gel (width 4 nl m, thickness 1 mm, length g cm, gel concentration 9% 9 Ampholyte Ampholine L
A single-sided open type gel with one side fixed to glass using 1) H3, 5-92%) methacryloxyglopylmethoxysilane manufactured by K B was used. Gluta, the support used for isoelectric focusing, was treated with saturated glutaraldehyde vapor at 50°C for 10 minutes to immobilize the molecules of the separated peptide (angiotensin ■) in the g7JDmL support gel. Next, the support gel was washed for 1 minute with 50% methanol and 0.07M phosphate buffer (pH 7, 5°C, 20°C). Subsequently, 50 timethanol/o containing 0.1mM hemin, 7M phosphate buffer (1) H7,5) was added for 20 minutes.
The mixture was treated for 1 minute to allow hemin to be adsorbed onto the protein.

50% methanol/0.07M phosphate buffer (pH 7,
5)? After washing the gel for 2 minutes, 50 timethanol-0.07M phosphate buffer (pH 7,
Process in step 5). Add hydrogen peroxide to give a 5 minute diameter of hydrogen peroxide t-0.1M to develop color if the protein sponge in the gel. 1
After developing color for 5 minutes, the gel was transferred to a 0.07 M IJ acid buffer containing 4 μg/mtk of catalase extracted and crystallized from bovine liver to decompose hydrogen peroxide and stop the color reaction.

In this way, according to the present invention, by combining the method of immobilizing angiotensin N2 in an isoelectrically focused angiotensin N2 support gel using glutaraldehyde vapor sound, and the coloring method using the peroxidase activity surrounding hemin,
One clear spot was detected at a position of 7 to 7.5 points.

Fixing reagents that can be used in the present invention include, in addition to glutaraldehyde, difunctional or higher polyvalent aldehydes such as formaldehyde, formaldehyde, and isocyanates such as hexamethyme diisocyanate and trivalent diisocyanate. Polyhydric cyanates having more than two functionalities can be used.

When using these, fixation reagents with high vapor pressure at room temperature can be reacted at room temperature and normal pressure;
If the vapor pressure is low, heat under reduced pressure before use.

The present invention specifically discloses a method for fluorescent detection of proteins σ]-f11 separated by the above method or by conventional methods.

That is, in a method for detecting proteins separated in a support gel by electrophoresis, all 8 tributophane residues contained in the protein in the support gel are oxidized with hydrogen peroxide and dioxane, and then converted into -formylquinine V-nin. , hydrolyze the formyl group with an acidic aqueous solution to generate kynurenine,
A method for detecting isolated protein spots, which is characterized by measuring kynurenine fluorescence, is fully disclosed.

The present invention relates to a method for detecting protein spots separated by electrophoresis in a support, and more particularly, to a method for rapidly and highly sensitively detecting all separated proteins by imparting fluorescence to proteins. The present invention is particularly effective in the field of medical specimen testing.

Conventionally, the detection of separated proteins in electrophoresis using polyacrylamide gel as a support, which has been used as a means of separating and analyzing proteins, is generally carried out by staining the proteins in the gel with Coomassie Brilliant Blue 250. ing. In this method, the entire gel is immersed in the above-mentioned dye solution, and the dye is diffused into the gel and adsorbed onto the protein. Next, it consists of two steps in which excess dye not adsorbed to the protein is removed by diffusion. Both processes depend on free diffusion of the dye, so it takes a long time (about 2 days). Respect.

The above method is effective in detecting proteins electrophoretically separated in a polyacrylamide gel support. It is a revolutionary new technology that significantly shortens the time from the current two days (about 1 hour and 40 minutes), has high sensitivity, and has an extremely low bank ground.

As mentioned above, when detecting a protein electrophoretically separated in a support gel, the dye molecules of Coumasieve + IJ Ant Blue 250 are diffused into the support gel, and then diffused into areas where there is no protein. The dye present is gradually diffused away and detected as a spot of protein-dye aggregate. In the present invention, the following points were taken into consideration when detecting the minute 111 protein at high speed and with low grass ground and high sensitivity.
The goal is to eliminate the dye diffusion step. In other words, if all amino acid residues, which are the constituent components of protein molecules, are chemically modified within the support gel to become a fluorescent substance, there is no need to add dye from outside the support and disperse it within the support gel. do not have. Furthermore, since the fluorescent substance is derived from amino acid residues constituting proteins, it is impossible for fluorescent substances to be emitted in areas of the support gel where proteins are not present, and there are no substances that must be removed by diffusion. .

The present invention takes the above points into consideration. Tryptophan residue, which is one of the amino acid residues that make up all proteins, is purple.
Oxidative chemical modification with hydrogen peroxide-dioxane followed by acid treatment produces kynurenine, a derivative of tryptophan. Quinu V Near+”1340-380 nm
It absorbs light and emits strong fluorescent light on the longer wavelength side of about 120 nm. Therefore, if the remaining tryptophan in the protein separated in the support gel is oxidized and converted to kynurenine, protein sporali within the support gel can be detected by measuring fluorescence.

The present invention will be described in detail below.

First, using 10 μt of human serum using a conventional method, the first dimension was for isoelectric point separation (gel concentration: 4%, ampholyte: 2%), the second dimension was for molecular weight separation, and polyacrylamide gel was used as a support (gel concentration: 4%). ~20%.

All two-dimensional electrophoresis was carried out with a size of 110 x 110 x 0.5 mm). The polyacrylamide gel used was a one-sided open type gel with one side fixed to a glass plate with methalyloxyglobylmethoxysilane. This gel was treated with saturated glutaraldehyde vapor at 50° C. for 10 minutes to fix the separated proteins on the support gel. Next KO, 1% SDS, 104
1.4-Dioxa7,10ti MMnC4tk
The support gel f is immersed in 150 mt of 0.1M carbonate buffer (pH 8,8) containing

After standing for 30 minutes, 31 hydrogen peroxide 1.72 m/, and 1
．． Add a mixture of 5 m/- of 4-dioxane and heat for 15 minutes.
React at ℃. At this point, tryptophan is oxidized to N'-formylkynurenine. Next, add 3 layers of support gel.
An aqueous solution containing 0 thimethanol and 10 thiacetic acid [soaked and left for 30 minutes to convert N'-formylkynurenine to kynurenine. Next, the gel soaking solution was mixed with 30 timethanol and 0.06
Chi S DS, 0.6 including 0.03 Chi Bridge-35
After changing to M IJ acid buffer (pH 7.5) and leaving it for 15 minutes, irradiate it with 340-380 nm light and move it to 4600 m.
Protein sporatra was confirmed by detecting fluorescence before and after.

When we compared the two-dimensional electrophoresis image obtained in this way in a process of about 1 hour and 40 minutes with the image stained with Kumasheep IJ IJ Ant Blue R250 over 2 days, there was a difference in the balance of shading. They were essentially identical separated images. In addition, 460% of the protein-free area (bank ground) in the polyacrylamide gel support
No fluorescence around nm was observed at all, and no fluorescence was observed from sources other than proteins such as ampholytes used for isoelectric focusing. As described above, according to the present invention, the detection of proteins in a polyacrylamide gel support, which conventionally required a long time, can be carried out quickly and with high sensitivity. It is particularly effective in fields such as medical sample testing.

That is, it is possible to quickly and sensitively analyze proteins in a polyacrylamide gel support, which conventionally required a long time. In particular, this detection method utilizes the fluorescence of kynurenine derived from amino acid residues constituting proteins, so fluorescence is emitted only in areas where proteins are present. No influence of ampholytes used in isoelectric focusing was observed, and disk methods such as isoelectric focusing, SDS polyacrylamide gel electrophoresis, concentration gradient gel electrophoresis, two-dimensional electrophoresis, etc.

In electrophoresis using polyacrylamide gels, regardless of the slab method, this is an extremely excellent method that is not affected by bank grounds and can reliably detect only separated proteins.

〔Effect of the invention〕

As described above, according to the present invention, even low-molecular peptides with a molecular weight of around 1000 can be reliably immobilized within the support gel on which electrophoresis has been performed. Furthermore, if used in combination with a coloring method in which hemin is adsorbed onto a peptide and all peroxidase activity is added to the peptide, even peptides with a molecular weight of around 1000 can be detected.

Especially in the field of medical specimen testing such as urine etc.

Claims (1)

[Claims] 1. In the method of immobilizing low-molecular-weight peptides separated into a support gel by electrophoresis, the peptide is reacted and immobilized using formaldehyde, polyvalent aldehyde, or polyvalent isocyanate vapor. A method for immobilizing low-molecular-weight peptides.