JPH06504363A - Carbohydrate analysis and kits - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] The present invention relates to the analysis of carbohydrates.

Background of the invention International Publication No. WO 38/10422 describes an additional method for analyzing carbohydrate structures in particular. discloses a technique for identifying or separating carbohydrate substances, which technique Hydrate substances are applied to an electrophoresis gel and the gel is moved to induce differential transfer of different substances. Including causing. Carbohydrate substances can be labeled with fluorescent reagents, e.g. amine fluorescein pre-labeling with a molecule to impart a charge to the substance, thereby enabling electrophoretic separation. and can allow visualization of the substance after running the gel. in this case , visualization can be done with the inner eye, but it is difficult to see with a charge-coupled device (CCD). Even higher sensitivity is obtained.

The present invention relates to the development of such technology and the use of alternative materials as fluorescent labeling reagents. It is based on that.

Summary of the invention According to the invention, a method for separating or identifying carbohydrate substances, comprising labeling a carbohydrate substance with a fluorescent reagent containing a group to produce a fluorescently labeled substance; The labeled substance is applied to an electrophoresis gel, and the gel is run to separate different substances. A method is provided comprising causing a differential movement of.

Another aspect of the invention is a kit for separating or identifying carbohydrate substances, comprising: , labeling reagents capable of producing fluorescently labeled substances, electrophoresis gels, and A kit containing a carbohydrate standard for comparison with the carbohydrate substance to be tested. Regarding the cut. The keyboard may further include a charge coupled device.

The hydrazine group of the labeling reagent readily reacts with carbohydrates containing reducing end groups, resulting in a charged and produce derivatives that are fluorescent and would be well suited for electrophoretic processing. do.

Good practical results have been obtained using Lucifer Yellow as a fluorescent reagent. low) C84 or 6-amino-2-[(hydrazinocarbonyl)amino)- 2,3-dihydro-1,3-dioxo-IH-benz(de)isoquinoline- Obtained using 5,8-sulfonic acid (LYCH). The structural formula of dipotassium salt is It is shown in FIG. Lunofer Yellow CH and its dipotassium salt are available from Aldr. ich Chemical Company.

Inc, Molecular Probes, Inc, Sigma Ch It is available from Chemical Company Riki 1. Stewart, W. , see also Ce11.14.741 (+978) (this is included in the book for reference). (incorporated herein). Modified variants and derivatives of LYCH give similar results. I can do it.

A number of other hydrazine group-containing reagents are also known, some of which have been tested. It was. For example, carbohydrates labeled with human lannoacrytone can be Afterwards, a blue fluorescent band was generated. After drying, this 7< throat turned bright sky blue. Li Using saminrhodamine B sulfonyl hydrazide as a fluorescent reagent, electrophoresis A photosensitive sugar derivative compound was produced after migration on a moving gel.

The labeled carbohydrate material must be fluorescent. Labeling reagent or itself fluorescent or may become fluorescent after reaction with carbohydrate substances.

Preferably the gel has a concentration ranging from 15% to 60%, preferably from 20% to 40%. or, in some cases, a relatively thick polyacrylamide gel containing It may be possible or preferable to use a gel of lower concentration.

The gel can be a homogeneous gel or in the form of a gradient gel.

Kel is preferably, for example, N,N'-methylenehisacrylamide (bis). more cross-linked.

One currently preferred gel is a continuous gradient from 20% (top) to 40% (bottom). Linear polyacrylamide with different polyacrylamide gel concentration (W/V) Comprising a mid-gradient gel. This Kel is the lowest concentration of polyacrylamide. Concentrations varying from 0.53% to 1.06% at the highest concentration of polyacrylamide (W/V) and crosslinked with bis.

For good separation and sensitivity, gels are suitable for handling proteins and DNA fragments. B, D, Ham emitted by known techniques such as IRL Press es and Tori, Rickwood-edited book “Gel electrophore” sis　ofl)roteins:　a　practical　approac h”, preferably using a concentrating buffer system. (also known as moving interface electrophoresis, multiphase zone electrophoresis and other names).

Electrophoresis is conveniently performed by Neville, Jr., D.M., J., Biol. , Chem.

Discontinuous electrophoresis buffers based on those described in 246.6328-6334 It is carried out using a system.

After electrophoresing the gel, if you irradiate it with light of an appropriate wavelength, such as ultraviolet light, in some cases Although labeled carbohydrate substances cannot be seen with the naked eye, they can be detected using a CCD. Better separation and sensitivity can be obtained by imaging. for appropriate excitation Therefore, LYCH emits yellow light and strong fluorescence with high quantum yield. Light emission is CCD with maximum sensitivity and quantum yield at the red end of the spectrum, The lowest sensitivity and quantum yield exist at the blue end of the spectrum. The use of COD is very It also has the advantage of providing quickly and easily quantized results.

Furthermore, excellent quantitative results are readily available with CCDs due to their wide linear dynamic range. It is possible. Furthermore, CCDs can be used to observe gels while they are running. I can be there.

It is preferable to use a cooled 2-D CCD and operate with slow scan readout. . An example of a suitable COD system is Astromed Limlted.

CCD manufactured by Cambridge, United Kingdom 2200 Imaging system. The CCD preferably has at least -25°C can be cooled to any low temperature, and by further cooling to -160°C Sensitivity is significantly increased. Typical working temperatures range from -40°C to -130°C It is.

The labeling reagent, e.g. It can be attached to sites on hydrated materials. Or, those who know about biomolecules. They may be modified in a method to enable the incorporation of labeled reagents.

The carbohydrate material is optionally treated with a reducing agent, e.g. sodium cyanoborohydride. By incubating the substance with LYCH in the presence of LYCH Can be labeled. Sodium cyanoborohydride is preferably dimethyl In the form of a solution in sulfokid (DMSO). For good labeling, acetic acid and of a solution in a mixture of water, for example a mixture containing 15 parts by volume of acetic acid and 85 parts by volume of water. It turns out that adding LYCH is the best way to grow rice. LYCII is Mo1 for example. Commercially available from Probes Inc., Eugene, Oregon and they supply the material in the form of potassium salt.

The migration speed of substances subjected to electrophoresis depends on the substance's size (molecular weight) and structure. It's different. Therefore, using the present invention, information regarding the size and shape of carbohydrate substances can be obtained. can be obtained, and by comparing with the results of known standards, unknown carbohydrates can be determined. It may be possible to partially or completely characterize chemical substances. One of the inventions The use of As shown, glucosidase was used to cleave the unknown carbohydrate into smaller pieces. , and by identifying the resulting fragments, we uncovered the carbohydrate structure. It is.

The present invention relates to glycoproteins, proteoglycans, glycolipids, and glycosphingolipids. A wide range of carbohydrate structures, including those derived from biomolecules in available.

The invention is illustrated by way of example by way of the following examples and by reference to the accompanying drawings. Explain.

Figure 1 shows the structure of LYCH in the dipotassium salt form.

Figure 2 is a photograph of an electrophoresis gel showing various sugars labeled with LYCH.

Example LYCH is available as dipotassium salt from Molecular Probes Inc. Obtained from.

A fine suspension (5.21 g/l) in glacial acetic acid/water (15:85.v/v) Prepared. Sodium cyanoborohydride (NaCNBH3) is available from Aldrich Chemical Co. in dimethyl sulfoxide (DMSO). Use as a 0.1N1 solution.

Lyophilize a solution containing 5 nanomoles each of the following reducing sugars (all in D configuration) in an ultracentrifuge tube. It was dry. 6-dioxyglucose, glucose, galactose, N-acetylglucose Lactose, galactonol alpha-1,4-galactose, lactose, maltose , galactose, cellotriose, maltotriose, maltotetraose , maltopentaose, maltohexaose and maltoheptaose. Freeze drying Add 5 μl of LYCH suspension and 5 μl of sodium cyanoborohydride solution to the dried sugar. Added. Mix the resulting solution well, incubate for 30 minutes at room temperature, and The reaction mixture was lyophilized for 4 hours at 40"C using a centrifugal vacuum evaporator. 0.04]M Tris base, 6M urea in 0.04M boric acid (pH 8,64). An appropriate amount of a solution (sample buffer for electrophoresis) was added. Use the obtained sample immediately or stored at -70''C until electrophoresis.

Repeat the above procedure using 52.1 g/l LYCH suspension and no sugar. A control without was also prepared.

LYCH-labeled sugars and controls were subjected to electrophoresis in polyacrylamide gels. Ta. Separation depends on the molecular weight and structure of each sugar. Moving interface (for concentration) buffer system Use to give sharp bands and high resolution. This system is based on the Nevi mentioned above. Based on the method of lle.

Electrophoresis was performed using 20% w/v acrylamide, 0.53% w/v N, N'- 40% W/V acrylamide from methylenebisacrylamide (bis), 1,0 It was performed using a separating gel consisting of a linear gradient of 6% W/V Hiss. 3.0 A concentrating kettle containing % W/V acrylamide and 0.08% W/V bis was used. N A discontinuous electrophoresis buffer system based on that described by Or, however, SDS was deleted. The gel was cooled with ambient buffer at 5-7°C.

The voltage used was 100V (constant voltage) for 30 minutes, then 500V (constant voltage). for 60 minutes, and then for 90 minutes at 100V (constant voltage).

The gel was run using the following samples in 4 lanes:

Lane 1 sugar + LYC)! (52.1 g/i solution).

Lane 2 Sugar-free + LYC) I (52.1 g/I! Warm solution.

Lane 3 Sugar + LYCH (5.21 g/l solution).

Lane 4 No sugar + LYCH (5,21 g/l solution).

A photograph of the gel resulting from irradiation from below with an ultraviolet lighting box is shown in FIG.

Wratten N on the camera to reduce ultraviolet and blue light from the lighting box o, 8 filters were used. The hunt equivalent to sugars is where they are distinct. shown.

When stimulated with appropriate irradiation light (maximum absorbance wavelength = 428 nm), the LYCH label emits strong fluorescence. Preliminary experiments showed that << caused by impurities in LYCH It showed some faint bunts on the gel, which is believed to be true.

The above description of preferred embodiments of the invention has been presented for purposes of illustration and description. So It is not intended to limit the invention to the precise form disclosed. clearly , many improvements and modifications will be apparent to those skilled in the art. Principles of the invention and the practical use thereof, and thereby enable those skilled in the art to understand the various embodiments of the present invention. to make it understandable and suitable for the specific intended use. Embodiments have been selected and described, with various modifications. The scope of the present invention is defined by the following claims. and their equivalents.

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Claims (21)

[Claims] 1. A method for separating or identifying carbohydrate substances, the label containing a hydrazine group labeling a carbohydrate substance with a reagent to produce a fluorescently labeled substance; The substances are applied to an electrophoresis gel, and the gel is run to determine the differences between the different substances. A method comprising causing movement. 2. The labeling reagent is Lucifer Yellow CH 2. The method of claim 1, comprising: 3. The carbohydrate material is converted to Lucifer Yellow CH in the presence of a reducing agent. labeled with Lucifer Yellow CH by incubation with Method for request 2. 4. 4. The method of claim 3, wherein the reducing agent is sodium cyanoborohydride. 5. The gel is a relatively thick polyacrylic material having a concentration in the range of 15% to 60%. 2. The method of claim 1, comprising a Ruamide gel. 6. 6. The method of claim 5, wherein said gel has a concentration in the range of 20% to 40%. 7. 6. The method of claim 1, 2, 3, 4 or 5, wherein the gel is in the form of a gradient gel. 8. 8. The method according to claim 1, 2, 3, 4, 5, 6 or 7, wherein the gel is crosslinked. Law. 9. The gels differ in a continuous gradient from 20% (top) to 40% (bottom). Linear polyacrylamide gradient with polyacrylamide gel concentration (w/v) comprising a gel, said gel having a minimum concentration of 0.53% polyacrylamide. Different concentrations (w/v) from to 1.06% at the highest concentration of polyacrylamide 9. The method of claim 1, 2, 3, 4, 5, 6, 7 or 8, wherein the crosslinking is carried out by bis. 10. Claims 1, 2, 3, 4, wherein the gel is run using a concentration buffer system. Method 5, 6, 7, 8 or 9. 11. The labeled carbohydrate substance is imaged in a gel using a charge coupled device. , 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 methods. 12. The charge-coupled device is operated at a cooled 20 dCC with slow scan readout. 12. The method of claim 11, wherein D. 13. A kit for separating or identifying carbohydrate substances, comprising: a) a fluorescent label; a labeling reagent containing a hydrazine group capable of producing a substance; b) Electrophoresis gel: and c) Carbohydrate standards for comparison with the carbohydrate substance to be tested. A kit consisting of. 14. 14. The kit of claim 13, wherein the labeling reagent is Lucifer Yellow CH. 15. 14. The kit of claim 13, wherein said kit further comprises a reducing agent. 16. 16. The kit of claim 15, wherein the reducing agent is sodium cyanoborohydride. . 17. The electrophoresis gel has a concentration in the range of 15% to 60%. 14. The kit of claim 13, which is a polyacrylamide gel. 18. 17. The kit of claim 16, wherein said gel has a concentration ranging from 20% to 40%. 19. 18. The kit of claim 16 or 17, wherein the gel is in the form of a gradient gel. 20. 19. The kit of claim 16, 17 or 18, wherein the gel is crosslinked. 21. Claim 16, 17, 18 or 18, wherein the kit comprises a charge coupled device. 19 kits.