WO1993002356A1

WO1993002356A1 - Analysis of carbohydrates and kits therefore

Info

Publication number: WO1993002356A1
Application number: PCT/US1991/004555
Authority: WO
Inventors: Peter Jackson
Original assignee: Astroscan, Ltd.; Glyko, Inc.
Priority date: 1991-07-22
Filing date: 1991-07-22
Publication date: 1993-02-04
Also published as: EP0595806A1; JPH06504363A

Abstract

Carbohydrate substances are separated or distinguished by a method that involved labelling the carbohydrate substances with a labelling reagent containing a hydrazide group to produce fluorescently labelled substances, applying the labelled substances to an electrophoretic gel, and running the gel to cause differential migration of different substances. The currently preferred labelling reagent is Lucifer Yellow CH.

Description

Analysis of Carbohydrates and Kits Therfore

Field of Invention

This invention concerns analysis of carbohydrates.

Background of the Invention International Publication No. W088/10422 discloses, inter alia techniques for analyzing carbohydrate structures or distinguishing or separating carbohydrate substances, involving applying carbohydrate substances to an electrophoretic gel and running the gel to cause differential migration of different substances. The carbohydrate substances may be pre-labelled with a fluorescent labelling reagent, eg amino fluorescein, to impart a charge to the substance, thereby to enable electrophoretic separation, and to enable visualization of the substances after running of the gel. In this case, visualization may be effected with the naked eye, but enhanced sensitivity is obtained by viewing with a charge coupled device (CCD) .

The present invention concerns a development of such techniques, and is based on the use of alternative materials as the fluorescent labelling reagents.

Summary of the Invention

According to the present invention there is provided a method of separating or distinguishing carbohydrate substances, comprising labelling carbohydrate substances with labelling reagent containing a hydrazide group to produce flourescently labelled substances, applying the labelled substances to an electrophoretic gel, and running the gel to cause differential migration of different substances.

Another aspect of the invention pertains to a kit for separating or distinguishing carbohydrate substances, comprising a labelling reagent containting a hydrozide group capable of producing flourescently labelled substances, an electrophoretic gel, and a carbohydrate standard to compare the carbohydrate(s) substance to be tested. The kit may additionally include a charge couples device.

The hydrazide group of the labelling reagent reacts readily with carbohydrates containing a reducing end group, producing a derivate which is both charged and fluorescent and which may be well suited to electrophoretic treatment.

Good practical results have been obtained using Lucifer Yellow CH 6-Amino-2 [(hydrazinocarbonyl)amino] 2,3-dihydro- 1,3-dioxo- lH-benz[de]isoquinoline- 5,8- disulfonic acid (LYCH) as the labelling reagent. The structural formula for the dipotassium salt is shown is Figure 1. Lucifer Yellow CH and its dipotassium salt are available from Aldrich Chemical Company, Inc., Molecular Probes, Inc., Sigma Chemical Company. Also see Stewart.W;Cell.14. 741 (1978), incorporated herein by reference. Modified versions and derivatives of LYCH may give similar results.

A number of other hydrazide group-containing reagents are also known, and tests have been carried out with some. For example, hydrazinoacridone- labelled carbohydrates produced blue fluorescent bands after electrophoretic treatment. The bands became bright turquoise after drying. Use of lissamine rhodamine B sulfonyl hydrazide as labelling reagent produced photographable sugar^' derivative bands after being run on an electrophoretic gel.

The labelled carbohydrate substance must be fluorescent: the labelling reagent may itself be fluorescent, or may become fluorescent after reaction with the carbohydrate substance.

The gel preferably comprises a relatively dense polyacrylamide gel, having a concentration in the range 15% to 60%, preferably 20% to 40%, although in some cases it may be possible or preferable to use gels of lower concentration.

The gel may be either of uniform concentration, or in the form of a gradient gel.

The gel is preferably cross linked, e.g. with N,N' methylenebisacrylamide (bis).

One presently preferred gel comprises a linear polyacrylamide gradient gel having a polyacrylamide concentration (w/v) varying in a continuous gradient from 20% (top) to 40% (bottom). The gel is crosslinked with bis, at a concentration (w/v) varying from 0.53% at the lowest concentration of polyacrylamide to 1.06% at the highest concentration of polyacrylamide.

For good resolution and sensitivity the gel is preferably run using stacking buffer systems (also known as moving boundary electrophoresis, multiphasic zone electrophoresis and other names), using techniques known for working with protein and DNA fragments, e.g. as described in the book "Gel electrophoresis of proteins: a practical approach" edited by B. D. Hames and D. Rickwood, published by IRL Press.

Electrophoresis is conveniently carried out using the discontinuous electrophoretic buffer system based on that described in Neville, Jr. D.M., J. Biol. Chem. 246. 6328-6334.

After running the gel the labelled carbohydrate substances, when illuminated with light of suitable wavelength, e.g. ultra violet, may be visible with the naked eye in some cases, although better resolution and sensitivity may be obtained by imaging with the

CCD. Upon suitable excitation, LYCH fluoresces strongly with a yellow emission and a high quantum yield. The emission is well suited for detection by a CCD, which has greatest sensitivity and quantum efficiency at the red end of the spectrum and lowest sensitivity and quantum efficiency at the blue end of the spectrum. Use of a CCD also has the advantage of giving readily quantitated results very quickly. Good quantitive results are easily available with a CCD due to its wide linear dynamic range. Further, a CCD can be used to view the gel while it is being run.

It is preferred to use a cooled 2-D CCD, operating in slow scan readout. One example of a suitable CCD system is the CCD 2200 Imaging System produced by Astromed Limited, Cambridge, United Kingdom. The CCD is preferably cooled to as least as low as -25'C, with sensitivity being significantly increased by further cooling down as far as -160'C. Typical operation temperatures are in the range -40^*C to -130'C.

The labelling reagent e.g., LYCH may be attached to sites on the carbohydrate substances, after release if necessary from an attached biomolecule. Alternatively, the biomolecule may be modified in known way to enable incorporation of the labelling reagent.

A carbohydrate substance may be labelled with LYCH by incubating the substance with LYCH, possibly in the presence of a reducing agent, e.g. sodium σyanoborohydride. The sodium cyanoborohydride is preferably in solution in dimethylsulphoxide (DMSO) .

For good labelling it is found useful to add the LYCH in solution in a mixture of acetic acid and water, e.g. containing 15 parts by volume of acetic acid to

85 parts by volume of water. LYCH is commercially available, eg form Molecular Probes Inc., Eugene,

Oregon, which supplies the material in the form of the potassium salt.

The rate of migration of substances undergoing electrophoresis varies with the size (molecular weight) and structure of the substances. The invention may thus be used to obtain information on the size and shape of carbohydrate substances, and by comparing results with those for known standards it may be possible partly or fully to characterize an unknown carbohydrate substance. One use of the invention is in elucidating carbohydrate structures, as described in O88/10422, incorporated herein by reference by cleaving an unknown carbohydrate into smaller fragments by use - of glycosidases and identifying the resulting fragments.

The invention is applicable to use on a wide range of carbohydrate structures, including those derived from glycoproteins, proteoglycans, glycolipidε and glycosphingolipids and other biomolecules.

The invention will be further described, by way of illustration, in the following Example and by reference to the accompanying drawings, in which:

Figure 1 illustrates the structure of LYCH in the form of dipotassium salt; and

Figure 2 is a paragraph of an electrophoretic gel showing various sugars labelled with LYCH.

Example

LYCH was obtained as its dipotassium salt from Molecular Probes Inc. A fine suspension (5.21g/l) was made in glacial acetic acid/water (15:85,v/v). Sodium cyanoborohydride (NaCNBH₃) was obtained from Aldrich Chemical Co. and uses as a 0.1M solution in di ethylsulphoxide (DMSO) .

A solution containing 5nmol of each of the following reducing saccharides (all 'D' configuration) was freeze-dried into a icrocentrifuge tube: 6- deoxyglucose, glucose, galactose, N-acetylgalctose, galactosyl alpha 1-4 galactose, lactose, maltose, galactobiose, cellotriose, maltotriose, altotetraose, maltopentaose, maltohexaose and maltoheptaose. To the dried saccharides was added 5ul of the LYCH suspension and 5ul of the sodium cyanoborohydride solution. The resulting solution was mixed well, incubated for 30 minutes at room temperature and freeze-dried at 40^*C for 4h using a centrifugal vacuum evaporator. A suitable volume of a solution (electrophoresis sample buffer) containing 6M-urea in 0.041M Tris base, O.04M boric acid pH8.64 was added to the reaction mixture. The resulting sample was either used immediately or stored at -70^*C until electrophoresed.

The procedure was repeated using a 52.1g/l suspension of LYCH, and controls containing no sugars were also prepared.

The LYCH labelled sugars and controls were subjected to electrophoresis in a polyacrylamide gel. The separation is dependent on the molecular weight and the structure of each sugar. A moving boundary (stacking) buffer system is used to give sharp bands and high resolution. This system is based on the method of Neville referred to above.

Electrophoresis was carried out using a resolving gel consisting of a linear gradient from 20% w/v acrylamide, 0.53% w/v, N,N' -methylenebisacrylamide (bis) to 40% w/v acrylamide, 1.06% w/v bis. A stacking gel was used containing 3.0% w/v acrylamide and 0.08% w/v bis. A discontinuous electrophoretic buffer system based on that described by Neville was used, but with SDS omitted. The gel was cooled by surrounding buffer at 5 to 7'C. The voltages used were 100V (constant) for 30 minutes, followed by 500V (constant) for 60 minutes, followed by 100V (constant) for 90 minutes.

The gel was run with samples in 4 lanes as follows:

Lane 1 saccharides + LYCH (52.1g/l solution). Lane 2 no saccharides + LYCH (52.1g/l solution). Lane 3 saccharides + LYCH (5.21g/l solution). Lane 4 no saccharides + LYCH (5.21g/l solution).

A photograph of the resulting gel illuminated from below by an ultra violet light box is shown in Figure 2. A Wratten No. 8 filter was uses on the camera to reduce ultra violet and blue light coming from the light box. Bands corresponding to sugars are indicated where these are clear.

The LYCH label fluoresces strongly when stimulated with suitable illuminating light (absorbance maximum wavelength = 428 nm) . Preliminary experiments produced some rather fuzzy bands on the gel, which are thought possibly to be caused by impurities in the LYCH.

The foregoing description of preferred embodiments of the present invention has been provided for the purposes of illustration and description. It is not intended to be exhaustive to limit the invention to the precise forms disclosed. Obviously, many, modifications and variations will be apparent to practitioners skilled in this art. The embodiments were chosen and described in order to best explain the principles of the invention and its practical application, thereby enabling others skilled in the art to understand the invention of various embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the following claims and their equivalents.

Claims

hat is Claimed Is:

1. A method of separating or distinguishing carbohydrate substances, comprising labelling carbohydrate substances with a labelling reagent containing a hydrazide group to produce fluoresσently labelled substances, applying the labelled substances to an electrophoretic gel, and running the gel to cause differential migration of different substances.

2. A method of claim 1, wherein the labelling reagent comprises Lucifer Yellow CH.

3. A method of claim 2, wherein the carbohydrate substance is labelled with Lucifer Yellow CH by incubating the substance with Lucifer Yellow CH in the presence of a reducing agent.

4. A method of claim 3, wherein the reducing agent is sodium cyanoborohydride.

5. A method of claim 1, wherein the gel comprises a relatively dense polyacrylamide gel, having a concentration in the range 15% to 60%.

6. A method according to claim 5, wherein the gel has a concentration in the range 20% to 40%.

7. A method of claims 1, 2, 3, 4 or 5, wherein the gel is in the form of a gradient gel.

8. A method of claims 1, 2, 3, 4, 5, 6 or 7, wherein the gel is crosslinked.

9. A method of claims 1, 2, 3, 4, 5, 6, 7 or 8, wherein the gel comprises a linear polyacrylamide gradient gel having a polyacrylamide concentration (w/v) varying in a continuous gradient from 20% (top) to 40% (bottom), the gel being crosslinked with bis, as a concentration (w/v) varying from 0.53% at the lowest concentration of polyacrylamide to 1.06% at the highest concentration of polyacrylamide.

10. A method of claims 1, 2, 3, 4, 5, 6, 7, 8 or 9, wherein the gel is run using a stacking buffer system.

11. A method of claims 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, wherein the labelled carbohydrate substances are imaged in the gel using a charge couples device.

12. A method of claim 11, wherein the charge coupled device is a cooled 20d CCD, operating in slow scan readout.

13. A kit for separating or distinguishing carbohydrate substances, comprising: a) a labelling reagent containing a hydrazide group capable of producing flourescently labelled substances; b) an electrophoretic gel; and c) a carbohydrate standard to compare the carbohydrate(s) substance to be tested.

14. The kit of claim 13, wherein the labelling reagent comprises Lucifer Yellow CH.

15. The kit of claims 13, wherein the kit additionally includes a reducing agent.

16. The kit of claims 15, wherein the reducing agent is sodium cxanoborohydride.

17. The kit of claim 13, wherein the electrophoretic gel is a relatively dense polyacrylamide gel, having a concentration in the range of 15% to 60%.

18. The kit of claim 16, wherein the gel has a concentration in the range of 20% to 40%.

19. The kit of claim 16 or 17 wherein the gel is in the form of a gradient gel.

20. The kit of claims 16, 17 or 18 wherein the gel is cross-linked.

21. The kit of claims 16, 17, 18 or 19 wherein the kit includes a charge couples device.