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WO1992011531A1 - Analyse de glucides au moyen de la 2-aminoacridone - Google Patents

Analyse de glucides au moyen de la 2-aminoacridone Download PDF

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Publication number
WO1992011531A1
WO1992011531A1 PCT/US1991/009727 US9109727W WO9211531A1 WO 1992011531 A1 WO1992011531 A1 WO 1992011531A1 US 9109727 W US9109727 W US 9109727W WO 9211531 A1 WO9211531 A1 WO 9211531A1
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Prior art keywords
carbohydrate
aminoacridone
electrophoresis
carbohydrates
gel
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PCT/US1991/009727
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English (en)
Inventor
Peter Jackson
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Astroscan Ltd
Glyko Inc
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Astroscan Ltd
Glyko Inc
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Priority claimed from GB909027990A external-priority patent/GB9027990D0/en
Priority claimed from GB919104412A external-priority patent/GB9104412D0/en
Application filed by Astroscan Ltd, Glyko Inc filed Critical Astroscan Ltd
Publication of WO1992011531A1 publication Critical patent/WO1992011531A1/fr
Anticipated expiration legal-status Critical
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
    • G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
    • G01N27/44726—Arrangements for investigating the separated zones, e.g. localising zones by optical means using specific dyes, markers or binding molecules
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44747—Composition of gel or of carrier mixture

Definitions

  • 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, e.g. 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.
  • a fluorescent labelling reagent e.g. amino fluorescein
  • visualization may be effected with the naked eye, but enhanced sensitivity is obtained by viewing with a charge coupled device (CCD).
  • CCD charge coupled device
  • the present invention concerns a novel development of such techniques, and is based on use of 2-aminoacridone as the fluorescent labelling reagent.
  • Two-dimensional (2-D) gel electrophoresis is a well known procedure generally used to obtain very good resolution of proteins.
  • a first electrophoretic separation in a first dimension is followed by a second electrophoretic separation in a second, transverse direction.
  • 2-dimensional electrophoresis separation of carbohydrates labeled with a fluorophore different than 2-aminoacridone, i.e., 8-aminonapthalene-1,3,6-trisulphonic acid has been described in U.S. patent 4,975,165.
  • the present invention is in part based on the discovery that use of 2-aminoacridone for labelling carbohydrate substances can enable 2-D gel electrophoresis of carbohydrate substances.
  • methods of separating or distinguishing carbohydrate substances comprising labelling carbohydrate substances with 2-aminoacridone, applying the labelled substances to an electrophoretic gel, and running the gel in either one or two dimensions to cause differential migration of different labelled substances.
  • the subject invention also provides for carbohydrate substances labeled by 2- aminoacridone.
  • kits for labeling carbohydrate substance with 2-aminoacridone or separating carbohydrates labeled by 2-aminoacridone are also provided.
  • Figure 1 illustrates the structure of 2- aminoacridone and the reaction thereof with a reducing sugar.
  • Figure 2 is a graph of absorbance versus concentration of 2-aminoacridone, illustrating the degree of labelling of Gal-6-SO 3 with varying quantities of 2-aminoacridone. Each point represents the mean absorbance of four determinations. The standard errors for any value were all less than 6.1% of any mean.
  • Figure 3 is a CCD image illustrating the sensitivity of detection of Gal-6-SO 3 derivatized with 2-aminoacridone.
  • Figure 4 is a graph of fluorescence versus quantity of saccharide, illustrating the variation of the CCD response with the quantity of 2-aminoacridone labelled Gal-6-SO 3 per gel band.
  • Figures 5a and 5b are CCD images of two fluorescent electrophoretograms showing a range of saccharides labelled with 2-aminoacridone and separated by PAGE using the Tris-borate buffer system.
  • Figure 6 is a CCD image of a fluorescent electrophoretogram showing only acidic saccharides labelled with 2-aminoacridone and separated by PAGE using the Tris-HCl/Tris- glycine buffer system.
  • Figure 7 is a CCD image of a fluorescent electrophoretogram showing the positions of artifactual bands and various neutral saccharide derivatives.
  • Figures 8 and 9 are CCD images of 2-D gels obtained using IEF for the first dimension.
  • Figures 10 and 11 are CCD images of 2-D gels obtained using electrophoresis for the first dimension.
  • 2-aminoacridone reacts with the reducing end groups of carbohydrates, producing highly fluorescent derivatives capable of electrophoretic separation.
  • An example of the labeling reaction is provided in figure 1.
  • the 2-aminoacridone itself confers no charge on labelled carbohydrates under the conditions (e.g. of alkaline pH) frequently used for electrophoretic separation.
  • For derivatives of neutral carbohydrates it is thus necessary to include a component, e.g. borate ions, which will confer charge onto the saccharide derivative and so enable separation. This is not necessary for negatively charged acidic saccharide derivatives (but these will nevertheless electrophorese in certain borate- containing systems), so the technique enables neutral and acidic saccharides to be easily distinguished.
  • Carbohydrates labeled by 2-aminoacridone may be represented according to the formula:
  • R is a carbohydrate having a reducing end group.
  • the subject invention also provided for 2-aminoacridone labelled carbohydrates that have been reduced so as bear a charge, this permitting migration in an electric field.
  • a reduced 2-aminoacridone labeled carbohydrate may be represented by the formula.
  • R is a carbohydrate having a reducing end group.
  • carbohydrate includes molecules that are completely carbohydrate and glycoconjugates such as glycoproteins, glycolipids, proteoglycans, glycosphingolipids, and the like.
  • 2-aminoacridone labeled carbohydrates are used as marker standards for the identification of unknown carbohydrates labeled by 2-aminoacridone.
  • 2-aminoacridone labeled carbohydrates of known concentration may be used to quantitate the amount of a carbohydrate of interest in a sample for analysis.
  • 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 20% w/v polyacrylamide gel containing 0.67% w/v bis.
  • the gel may be run using a stacking buffer system (also known as moving boundary electrophoresis, multiphasic zone electrophoresis and other names), using techniques known for working with protein and DNA fragments, e.g.
  • Electrophoresis may also be conveniently carried out using the discontinuous electrophoretic buffer system described in reference 1,which contains borate ions, but with sodium dodecyl sulphate (SDS) omitted throughout.
  • SDS sodium dodecyl sulphate
  • Two particularly suitable electrophoretic buffer systems are: a continuous Tris-borate buffer system as described in reference 2; and a discontinuous Tris-HCL/Tris-glycine buffer system as described by Laemmli (reference 3), with sodium dodecyl sulphate (SDS) omitted throughout.
  • 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 a CCD.
  • CCD's are semiconductor imaging devices that permit the sensitive detection of emitted light.
  • 2-aminoacridone fluoresces strongly with a yellow emission and a high quantum yield both before and after reaction with reducing saccharides.
  • 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 quantitative 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.
  • CCD system is the CCD 3200 Imaging System produced by Astromed Limited, Cambridge, United
  • the CCD is preferably cooled to at least as low as -25oC, with sensitivity being significantly increased by further cooling down as far as -160oC.
  • Typical operation temperatures are in the range -40oC to -120oC.
  • the 2-aminoacridone labelling reagent may be attached to sites on the carbohydrate substances, after release if necessary, from an attached biomolecule.
  • the biomolecule may be modified in known way to enable incorporation of the 2-aminoacridone labelling reagent.
  • a carbohydrate substance may be labelled with 2-aminoacridone by incubating the substance with 2-aminoacridone, possibly in the presence of a reducing agent, e.g. sodium cyanoborohydride.
  • the sodium cyanoborohydride is preferably in solution in dimethylsulfoxide (DMSO).
  • DMSO dimethylsulfoxide
  • 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 PCT Application WO88/10422, by cleaving an unknown carbohydrate into smaller fragments by use of glycosidases and identifying the resulting fragments. Similarly, it may be useful to employ glycosyl transferases, carbohydrate esterases, or epimerases, to elucidate carbohydrate structure.
  • the fluorescent labelling procedure was virtually quantitative and as little as 0.63 pmol could be detected photographically when gels were illuminated by uv light. When gels were viewed using an imaging system based on a charge-coupled-device, as little as 0.2 pmol was detected.
  • a method of separating or distinguishing reducing carbohydrate substances by 2-D gel electrophoresis comprising fluorescently labelling reducing carbohydrate substances by reaction with 2-aminoacridone, and subjecting the labelled carbohydrate substances to electrophoretic separations in 2 dimensions.
  • acidic reducing carbohydrates labelled with 2-aminoacridone (2-AA) are subjected to isoelectric focusing (IEF) in polyacrylamide gel in the first dimension, followed by polyacrylamide gel electrophoresis (PAGE) in the second dimension.
  • IEF isoelectric focusing
  • PAGE polyacrylamide gel electrophoresis
  • the acidic reducing saccharides are derivatized by a reaction at their reducing end groups charged with the fluorophore 2-AA.
  • this fluorophore donates positive charge(s) to the derivative.
  • the amount of positive charge depends on the pH of the solution and arises from the protonation of either the 2-AA ring nitrogen or the secondary amino group generated in the derivatization reaction, or both of these. (Knowledge of the exact pKs of these two groups is not important in understanding the principle or practice of the method.)
  • a consequence is that the derivatives of acidic, but not neutral, saccharides will be zwitterions and suitable for analysis by IEF in a system containing ampholytes with a suitable pH range.
  • IEF is conveniently carried out in low concentration polyacrylamide gel rods using ampholytes with a nominal range of pH 2.5-4 in high proportion. After focusing, the gel rods are extruded from their tubes and placed directly on top of the second dimension polyacrylamide slab gel (preferably 20% w/v).
  • the second dimension separation is conveniently similar to the one-dimensional method for 2-AA derivatized reducing saccharides which is described previously.
  • the first-dimension IEF-gels after positioning end-to-end on the second-dimension gels, are sealed in place with agarose and electrophoresed by vertical slab PAGE in a discontinuous buffer system described by Neville (reference 1) but with SDS omitted throughout.
  • the system operates at alkaline pH where the 2- AA derivatives are both negatively charged and fluoresce.
  • the Neville buffer system is different from either of the buffer systems previously described for the one dimensional electrophoresis separation of 2- aminoacridone labeled carbohydrates. The separation is dependent on the effective size, net charge, structure and the interaction with borate ions of the saccharide derivatives.
  • the method may find application in the rapid analysis of samples of mixtures of reducing saccharides which cannot be resolved by simple one-dimensional methods.
  • the first dimension electrophoresis is carried out in an acidic gel buffer system, followed by second dimension electrophoresis in a continuous buffer system containing borate ions at alkaline pH.
  • the first dimension electrophoresis is carried out using a discontinuous system operating at alkaline pH, without borate ions, followed by second dimension electrophoresis generally as for the preferred method described above (i.e. with borate ions).
  • borate ions may be used in the first dimension separation but not in the second dimension separation.
  • kits for performing the 2-aminoacridone labeling of carbohydrates are also included. These kits for the 2- aminoacridone labeling of carbohydrates may be further used to provide for the electrophoretic separation of 2-aminoacridone labeled carbohydrates and/or the identification of carbohydrates substances labeled by 2- aminoacridone.
  • the kits provide collections of reagents required for performing the 2- aminoacridone labeling of carbohydrates. Suitable kits enable laboratories to conveniently perform 2-aminoacridone labeling. Kits may include reagents for performing tests to identify one or more specific carbohydrate substances. Kits may include carbohydrate identification standards, 2-aminoacridone, instructions, sample containers, polyacrylamide gel reagents, and IEF reagents.
  • kits may include equipment for performing one or two dimensional electrophoresis, such as the gel apparatus CCDs, computers, software for analysis of electrophoresis band patterns, and the like. Reagents in the kits are preferably provided in premeasured amounts. The kits may also include the instructions for carrying out 2- aminoacridone labeling of carbohydrate substances and/or the electrophoretic separation of 2-aminoacridone labeled carbohydrate substances.
  • Saccharides were obtained from either Sigma Chemical Co. (Poole, Dorset, U.K.) or Aldrich Chemical Co. (Gillingham, Dorset, U.K.).
  • the complex oligosaccharides 42 and 43 (See Table 1) were obtained from Biocarb Chemicals (Russel Fine Chemicals, Chester, U.K.) and 2-aminoacridone was from Lambda Probes (Graz, Austria).
  • Sodium cyanborohydride (NaCNBH 3 ) was obtained from Aldrich Chemical Co.
  • Samples usually 1.0 ⁇ l or 2.0 ⁇ l, were electrophoresed in 20% w/v polyacrylamide gels containing 0.67% w/v, N,N'-methylene bisacrylamide. The final concentrations of N,N,N',N'-tetramethylenediamine and ammonium persulphate were 0.1%v/v and 0.1% w/v, respectively.
  • a Hoefer Scientific Instruments (Newcastle Under Lyme, Staffordshire, U.K.) SE600 vertical slab gel electrophoresis apparatus was used with 8 cm (nominal) long glass plates. Pyrex glass was used when gels were viewed by the CCD system. The gel dimensions were 140 mm wide by 0.5 mm thick, the sample well were 2 mm wide.
  • Tris-borate buffer pH 8.3 (reference 2) (the final concentration of Tris (Trizma base, Sigma) was 0.1M, the pH was adjusted with boric acid at approximately 22oC); the other was discontinuous Tris-HCl/Tris-glycine buffer system described by Laemmli (reference 3), with SDS omitted throughout and a resolving gel 65 mm long. In both cases, the gels were cooled by surrounding analyte at +7oC. In the Tris-borate system, the samples were electrophoresed initially at 100V for 30 min, then at 200V for 30 min and finally at 500V for approximately 90 min.
  • Tris-HCl/Tris-glycine system voltages used were 50V for 30 min, 100V for 30 min, 200V for approximately 60 min and 500V for approximately 60 min. The voltages were all held constant. Bromophenol blue was used as a marker and electrophoreses were stopped when it reached 10 mm from the anodic end of the gel. After the electrophoresis, the sample wells were rinsed with water, using a syringe and fine needle, to remove excess 2-aminoacridone which remained therein.
  • 2-aminoacridone was reacted, using the standard conditions, with various quantities of glucose ranging from 1.9 nmol to 52 nmol per reaction tube, each of which contained 0.5 uCi of 14 C glucose.
  • 90 ul of water was added to each tube and 1.0 ul of each mixture applied to a silica-gel TLC plate (Polydram SILG: 20 cm ⁇ 20 cm; Macherey-Nagel) and chromatographed in a solution of butan-1-ol/ethanol/water, (5:3:2, by vol).
  • the chromatogram was autoradiographed using a Cronex 4 X-ray film (DuPont).
  • Known quantities of unchanged 14 C-glucose were chromatographed as standards.
  • the gels were viewed for either 2 seconds, or 10 seconds, or 60 seconds, and the images processed electronically to compensate for any point to point unevenness in the intensity of the illuminating light.
  • the fluorescence of the saccharide bands in the gels was measured by determining the mean number of photons registered/min per pixel in a defined rectangular area covering each band and subtracting the gel background measured on similar adjacent blank areas in the same gel lane.
  • the proportion of 14 C-glucose which was derivatized with 2-aminoacridone in the standard conditions was determined by visual inspection of the autoradiographies of the TLC analyses of the reaction products. For all samples in the range tested, from 1.9 to 52 nmol of glucose per reaction tube, virtually all of the 14 C-glu ⁇ ose was derivatized and appeared as a spot having a mobility significantly greater than that of the unreacted glucose.
  • Figure 3 illustrates serially diluted samples of Gal-6-SO 3 derivatives with 2-aminoacridone electrophoresed using the Tris-borate buffer system and viewed using a cooled CCD imaging system.
  • the numbers in the figure include pairs of lanes with similar loadings, and the lanes are as follows: lanes 1, 0.8 pmol; lanes 2, 0.4 pmol; lanes 3, 0.2 pmol; lanes 4, 0.1 pmol; lanes 5, sample buffer alone. As little as 0.2 pmol was detected.
  • NeuSGl N-glycolylneuraminic acid 40 Neu5Ae N-acetylneuraminic acid 41 Neu5Ac-(2-3)- ⁇ -D-Gal-(1-4)-D-Glc N-acetylneuraminlactose42 Neu5Ac ⁇ 2-3Gal ⁇ 1-3(Fuc ⁇ 1-4)GlcNac ⁇ 1-3Ga ⁇ 1-dGlc monosialyl, monofucosyllacto- N-tetraose
  • FIGs 5a and 5b are shown CCD images of fluorescent electrophoretograms were made of gels using the Tris-borate buffer system, of a variety of mono- and oligosaccharides labelled with 2-aminoacridone.
  • the gels were viewed for 30 seconds for each image section.
  • the samples run of the gels corresponds to the number of each of the saccharides shown in Table 1.
  • the lanes labelled "W" are controls and contained no saccharide, in the labelling reaction.
  • the arrows show the positions of artif actual bands. 20 pmol of each of the saccharides analyzed was visible as a bright yellowish band.
  • Faint bluish bands were also present in all the samples, including the water blank at the positions indicated in Figures 5a and b and Figure 7. These bands were easily distinguishable by eye from the yellow saccharide bands when gels were viewed on a UV light box and moved faster than all of the neutral saccharides tested. More of these bands are visible in Figure 7 than in Figure 5 since a larger proportion of the reaction mixture was analyzed in the gel shown in Figure 7. A bright yellow band occurred at the cathodic and of each gel lane owing to the unreacted 2-aminoacridone which was uncharged at the pH of the electrophoresis. Most of this was removed by rinsing the sample wells prior to both CCD imaging and before disassembling the gel cassette for photography. In the latter case, any fluorophore which had diffused unto the gel was removed by cutting of the top 1-2 mm.
  • Figure 7 is a CCD image of a fluorescent electrophoretogram showing the positions of artifactual bands and various neutral saccharide derivatives.
  • the gel was viewed for 60 seconds.
  • Each of lanes 1, 2 and 3 contain a proportion of a standard reaction mixture which contains no saccharide.
  • the reaction mixture was dissolved in 100 ⁇ l of 6M-urea, and the lanes are as follows: lane 1, 0.5 ⁇ l; lane 2, 1.0 ⁇ l; lane 3, 2.0 ⁇ l.
  • the arrows labelled A, B, C and D show the positions of the major artifactual bands which appeared bluish when viewed on a UV light box.
  • the remaining lanes show four neutral reducing saccharides: lane 4, 3-O-methlygluco ⁇ e; lane 5, mannose; lane 6, glucose; lane 7, galactose. 10 nmol of each saccharide derivative was dissolved in 500 ul of 6M-urea and 20 pmol (1 ⁇ l) loaded per lane.
  • the mobilities of the labelled saccharides were dependent partly on the size of each saccharide molecule but were also strongly influenced by various molecular structures.
  • the effect of the size of a saccharide can be seen from examining the mobilities of glucose and all its straight chain alpha 1-4 linked oligomers from maltose to maltoheptaose ( Figure 5a, lane 16, and Figure 5b, lanes 23, 27, 29, 30 and 31) which showed a decreasing mobility with increasing degree of polymerization.
  • Figure 5a, lane 16, and Figure 5b lanes 23, 27, 29, 30 and 31
  • corresponding 2 and 6 deoxy derivatives of galactose and glucose were well separated and the six dimers of glucose were well separated into 3 pairs of anomers, nigerose and laminaribiose (1-3 linked), maltose and cellobiose (1-4 linked), and isomaltose and gentiobiose (1-6 linked).
  • the corresponding anomers in each pair had small mobility differences and this was also the case for maltotriose and cellotriose.
  • the beta-linked molecules had slightly higher mobilities than those were alpha-linked.
  • the electrophoretic mobilities of the acidic saccharides in the Tris-borate buffer system was also affected by small structural differences; for instance, galactose-6-sulphate and glucose-6-sulphate were separated. In general they had higher mobilities than the neutral saccharides.
  • the three mono-saccharides which contained a carboxyl group, glucuronic acid, N- glycolylneuraminic acid and N-acetylneuraminic acid each showed three separate bands. The reasons for this phenomenon is at present unknown. It was not see with N- acetylneuraminlactose or the sialylated complex oligosaccharides 42 and 43 (see Table 1). Electrophoresis is Tris-HCl/Tris-Glyeine
  • Glucuronic acid, N-glycolylneuraminic acid, and N-acetylneuraminic acid, but not N-acetylneuraminlactose appeared as three bands which were fainter than the other saccharides.
  • PAGE i.e., polyacrylamide gel electrophoresis
  • PAGE has been used previously for the separation of oligosaccharides which are charged naturally (references 5, 6, 7, 8) and also for the separation of uncharged oligosaccharides as borate ion complexes (references 2, 9, 10).
  • Numerous methods for the covalent labelling of reducing saccharides with fluorophores have been described (references 11 to 17) although PAGE has been used in only two recent reports (references 4, 18, 17) for the analysis of such derivatives.
  • reaction conditions were based on those used previously for the quantitative derivatization of reducing saccharides by an aromatic primary amine, 8- aminonaphthalene-1-3-6-trisulphonic acid (ANTS) (reference 4) and the conditions chosen gave quantitative derivatization for the 2- aminoacridone.
  • the method described shows high sensitivity using either photography or electronic imaging with the cooled CCD.
  • the latter system is approximately three times more sensitive and there is considerable potential for increasing its sensitivity by increasing the power of the light source, by matching the filters more accurately to the excitation and emission spectra of the fluorophore, by using a CCD camera cooled to a lower temperature, and by using a wider aperture lens.
  • the sensitivity of detection may be limited by the proportion of the reaction mixture which can be loaded into the sample wells. When using the system describe, this volume is 10 ⁇ l. Since the minimum volume of the 6M-urea sample solution required to dissolve the reaction mixture material is 50 ⁇ l, only one fifth of the available saccharide derivative can be loaded.
  • the 2-aminoacridone based PAGE analysis of reducing saccharides described here is in principle similar to that in which ANTS has been used and reported previously (reference 4).
  • the ANTS enabled all the reducing saccharides tested to be visualized in a single gel with high resolution without using borate ions in the electrophoresis buffer.
  • the fluorescent saccharide bands are considerably sharper in the ANTS system than those obtained using 2-aminoacridone and the electrophoretic mobilities of the ANTS derivatives were influenced strongly by the size of each molecule.
  • all of the alpha 1-4 linked straight chain polymers of glucose from maltose to (alpha-D-Glc-(1-4)) 25 alpha-D-Glc were separated in a single gel.
  • the anolyte was made by diluting 0.375 ml of cone. H 2 SO 4 in 250 ml of degassed H 2 O.
  • the catholyte was 20mM NaOH made by diluting 0.5 ml stock 10M NaOH in 250 ml degassed H 2 O.
  • the IEF gel was overlayed with approx. 30 ul of ampholyte solution (Pharmacia pH range 3-10 (5% v/v)).
  • the samples were dissolved in 6M-urea and layered under the ampholyte solution in the gel tube. Usually 0.5 to 2 ul was loaded. It was ensured that no air bubbles occurred in the system. Bromophenol blue in solution in 6M-urea was also loaded to act as a marker.
  • Saccharide derivatives were focused at 100V for 30 min. and then at 1000V for 60 min. In the later stages of the focusing the bromophenol blue, which turned yellow, appeared as an immobile band. Gels were removed from their tubes by gentle water pressure and placed across the top of a second vertical slab gel. Two gels were placed end to end on each second dimensional gel.
  • the second dimension was a uniform concentration (20% w/v) polyacrylamide gel with the dimensions 70mm (H) ⁇ 140 mm (W) ⁇ 0.75 mm (T).
  • the buffer system was based on that of Neville (reference 1).
  • the gel solution contained 20% (w/v) a ⁇ rylamide, 0.67% bis, 0.1% w/v ammonium persulphate, 0.01% v/v TEMED, ).042M Tris HCl buffer pH 9.18.
  • the anolyte was 0.42M tris HCl buffer pH 9.18 and the catholyte was 0.041M Trisbase, 0.04M boric acid, pH 8.64.
  • the IEF-gels were sealed in place with approx. 3 mm of 1.0% w/v agarose solution buffered with the stacking gel buffer for the electrophoretic system used (reference 1), that is 0.054M Tris base, 0.027M H 2 SO 4 , pH 6.1.
  • the samples were electrophoresed at 100V for 30 min., 200V for 30 min. and 500V for 60 min.
  • the voltages were held constant and the gel cooled by surrounding anolyte at approximately +7oC.
  • the electrophoresis was stopped when the bromophenol blue from the first dimension gel reached approximately 5 mm from the anodic end of the gel.
  • the gel was rinsed and either viewed using the Astromed CCD 2200 imaging system in vitro or removed from its cassette and photographed as described in example 1. Photographs of CCD images of typical two- dimensional fluorescent electrophoretograms are shown in Figures 8 and 9.
  • Figure 8 shows two IEF gels indicated at 1 and 2, each having the acid end at the left hand side ash shown in the Figure, placed side to side (i.e., end to end) on the second dimension gel which was run is a downwards direction as shown in the Figure.
  • the bottom of the second dimension gel is shown at 3.
  • the central spots towards the upper end of the second dimension gels are of oligosaccharide 56/51, with the spot in gel 1 being diminished compared with the spot on gel 2.
  • the uncharged reaction product of the digestion of oligosaccharide 56/51 by neuraminidase is not seen on the gel.
  • Other spots are of unknown identity or are artifacts.
  • Figure 9 is similar to Figure 8, with the left hand gel showing results for urine and neuraminidase and the right hand gel showing results for urine.
  • the diagonal line running across the two gels is a photographic artefact.
  • Samples were electrophoresed in an acidic gel buffer system which operates at approx. pH 2.71 for the first dimension. All the neutral saccharide derivatives and some of the acidic saccharide derivatives will be positively charged at this pH.
  • the second dimension was on a continuous buffer system containing borate ions at pH 8.3. All the saccharide derivatives will be negatively charged in this system.
  • Figure 10 is a photograph of a CCD image of the results obtained for a urine separation by this method.
  • Samples were electrophoreses in a buffer system based on that of Laemmli (reference 3), but with SDS omitted from the first dimension. This is a discontinuous system operating at alkaline pH and contains no borate ions.
  • the second dimension buffer system was based on that of Neville (reference 1) but the SDS omitted throughout. This system contains borate ions.
  • the gel pattern depends on the differential interaction of saccharide derivatives with borate ions.
  • Figures 11a and 11b are photographs of CCD images of results obtained for a complex mixture of 40 saccharides (Figure 11a) and also for urine ( Figure 11b). All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

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  • Spectroscopy & Molecular Physics (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Other In-Based Heterocyclic Compounds (AREA)
  • Saccharide Compounds (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)

Abstract

L'invention se rapporte à l'utilisation du marquage fluorescent 2-aminoacridone s'utilisant dans la séparation de mélanges de glucides et l'analyse de la structure de glucides. Les glucides à analyser peuvent être marqués par la 2-aminoacridone et ensuite, séparés les uns des autres par électrophorèse. L'électrophorèse peut se présenter sous une ou deux dimensions. On peut visualiser la bande produite par l'électrophorèse et la quantifier directement au moyen d'un éclairage par ultra-violets ou d'un dispositif à couplage de charge pour effectuer une détection photo-électrique. On peut également utiliser le marquage des glucides par la 2-aminoacridone pour analyser la structure des glucides en dédoublant (ou ajoutant) différents glucides marqués par la 2-aminoacridone avec des enzymes de modification des glucides à spécificité connue et en séparant ensuite les glucides par électrophorèse sous une ou deux dimensions. L'invention se rapporte également à des kits servant à effectuer le marquage par la 2-aminoacridone et l'électrophorèse.
PCT/US1991/009727 1990-12-22 1991-12-19 Analyse de glucides au moyen de la 2-aminoacridone Ceased WO1992011531A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB9027990.2 1990-12-22
GB909027990A GB9027990D0 (en) 1990-12-22 1990-12-22 Analysis of carbohydrates
GB9104412.3 1991-03-01
GB919104412A GB9104412D0 (en) 1991-03-01 1991-03-01 Improvements in or relating to electrophoresis

Publications (1)

Publication Number Publication Date
WO1992011531A1 true WO1992011531A1 (fr) 1992-07-09

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Application Number Title Priority Date Filing Date
PCT/US1991/009727 Ceased WO1992011531A1 (fr) 1990-12-22 1991-12-19 Analyse de glucides au moyen de la 2-aminoacridone

Country Status (5)

Country Link
EP (1) EP0516844A4 (fr)
JP (1) JPH05504363A (fr)
AU (1) AU1425892A (fr)
GB (1) GB2254851B (fr)
WO (1) WO1992011531A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5308460A (en) * 1992-10-30 1994-05-03 Glyko, Incorporated Rapid synthesis and analysis of carbohydrates
US5340453A (en) * 1989-09-27 1994-08-23 Astroscan, Ltd. Analysis of carbohydrates
US5472582A (en) * 1993-04-23 1995-12-05 Astromed Limited Analysis of carbohydrates using 2-aminoacridone
US6007691A (en) * 1991-05-07 1999-12-28 Glyko, Inc. Fluorophore assisted carbohydrate electrophoresis diagnosis
US8034558B2 (en) 2001-06-04 2011-10-11 Ge Healthcare Uk Limited Acridone derivatives as labels for fluorescence detection of target materials
WO2020151799A1 (fr) * 2019-01-21 2020-07-30 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Méthodes avancées d'identification haute performance automatisée de glucides et de motifs de composition de mélange de glucides et systèmes correspondants, ainsi que méthodes d'étalonnage de systèmes de détection de fluorescence à longueurs d'onde multiples correspondantes, fondées sur de nouveaux colorants fluorescents
WO2020151804A1 (fr) * 2019-01-21 2020-07-30 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e. V. Colorants 2(7)-aminoacridone et 1-aminopyrène sulfonés et leur utilisation comme marqueurs fluorescents, en particulier pour l'analyse des hydrates de carbone

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GB8714270D0 (en) * 1987-06-18 1987-07-22 Williams G R Analysis of carbohydrates
EP0641438A1 (fr) * 1991-11-21 1995-03-08 Glyko, Inc. Controle de medicaments assiste par fluorogene

Non-Patent Citations (4)

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Title
P. JACKSON et al., "Polyacrylamide gel electrophoresis of reducing saccharides labelled with the fluorophore 8-aminoaphthalene-1,3,6-trisulphonic acid: Application to enzymological structural analysis of oligo-saccharides", Electrophoresis 1991, 12, 94 -96. *
P. JACKSON, "Polyacrylamide gel electrophoresis of reducing saccharides labelled with the flurophore 2-aminoacridone: subpicomolar detection using an imaging system based on a cooled charge-coupled device", Anal. Biochem. 196(2), 238-44. *
P. JACKSON, "The use of polyacrylamide-gel electrophoresis for the high-resolution separation of reducing saccharides labelled with the flurophore 8-aminoaphthalene-1,3,6-trisulphonic acid", Biochem. J. (1990) 270, 705-713. *
See also references of EP0516844A4 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5340453A (en) * 1989-09-27 1994-08-23 Astroscan, Ltd. Analysis of carbohydrates
US6007691A (en) * 1991-05-07 1999-12-28 Glyko, Inc. Fluorophore assisted carbohydrate electrophoresis diagnosis
US5308460A (en) * 1992-10-30 1994-05-03 Glyko, Incorporated Rapid synthesis and analysis of carbohydrates
US5472582A (en) * 1993-04-23 1995-12-05 Astromed Limited Analysis of carbohydrates using 2-aminoacridone
US8034558B2 (en) 2001-06-04 2011-10-11 Ge Healthcare Uk Limited Acridone derivatives as labels for fluorescence detection of target materials
WO2020151799A1 (fr) * 2019-01-21 2020-07-30 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Méthodes avancées d'identification haute performance automatisée de glucides et de motifs de composition de mélange de glucides et systèmes correspondants, ainsi que méthodes d'étalonnage de systèmes de détection de fluorescence à longueurs d'onde multiples correspondantes, fondées sur de nouveaux colorants fluorescents
WO2020151804A1 (fr) * 2019-01-21 2020-07-30 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e. V. Colorants 2(7)-aminoacridone et 1-aminopyrène sulfonés et leur utilisation comme marqueurs fluorescents, en particulier pour l'analyse des hydrates de carbone
CN113646636A (zh) * 2019-01-21 2021-11-12 马克斯·普朗克科学促进学会 基于新型荧光染料的用于自动高性能鉴别碳水化合物和碳水化合物混合物组成模式的先进方法和系统以及用于校准多波长荧光检测系统的方法
US12174197B2 (en) 2019-01-21 2024-12-24 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E. V. Sulfonated 2(7)-aminoacridone and 1-aminopyrene dyes and their use as fluorescent tags, in particular for carbohydrate analysis

Also Published As

Publication number Publication date
EP0516844A1 (fr) 1992-12-09
GB9127091D0 (en) 1992-02-19
AU1425892A (en) 1992-07-22
GB2254851B (en) 1995-06-07
JPH05504363A (ja) 1993-07-08
GB2254851A (en) 1992-10-21
EP0516844A4 (en) 1993-08-25

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