Chemistry: electrical and wave energy – Processes and products – Electrostatic field or electrical discharge
Patent
1989-02-14
1992-04-14
Niebling, John F.
Chemistry: electrical and wave energy
Processes and products
Electrostatic field or electrical discharge
204299R, 356344, B01D 6142, C25D 1300, C01N 2100
Patent
active
051045087
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to analysis of carbohydrates.
BACKGROUND TO THE INVENTION
A large number of different carbohydrates exist, including many compounds of importance in biological processes. Carbohydrate structures can occur alone, but sometimes also occur in association with biomolecules such as lipids (glycolipids, glycosphingolipids), proteins (glycoproteins, proteoglycans), or other molecular structures. Carbohydrates are made up of a variety of different monosaccharide units or building blocks, which may occur along, e.g. as glucose or fructose, or which may be linked by a range of different glycosidic bonds to form larger molecules such as disaccharides, e.g. lactose and sucrose, oligosaccharides and polysaccharides, e.g. cellulose and starch. Larger units may be linear or they may have a branched structure, e.g. as shown in FIG. 1, where Mn represents monosaccharide type "n".
Given two monosaccharide units (say M1 and M2) linked in a particular way it is often possible to find reagents or groups of reagents (such as the enzymes known as glycosidases which cleave glycosidic bonds between monosaccharide units) which will selectively cleave the bond between M1 and M2 or between a monosaccharide and the molecular structure to which the monosaccharide is attached.
Given a suitable set of reagents e.g. enzymes, it is possible to cleave a carbohydrate structure in many possible ways, and to obtain a collection of different fragments which can be analysed by various separation methods such as by chromatography. From a knowledge of the resulting fragments it is possible to make various deductions concerning the structure of the original carbohydrate. This type of approach is described in, e.g., the paper by Tomiya et al. in Analytical Biochemistry, 163, 484-499 (1987).
However there are limitations to the known techniques, and the present invention aims to provide improved methods, and apparatus.
SUMMARY OF THE INVENTION
According to one aspect of the present invention there is provided a method of analysing a carbohydrate, comprising decomposing a carbohydrate structure into various constituent fragments, separating the fragments, and viewing light from the separated fragments using a charge coupled device (CCD).
In a further aspect the present invention provides apparatus for analysing a carbohydrate, comprising means for decomposing a carbohydrate structure into various constituent fragments, means for separating the fragments, and a CCD for detecting a pattern of light from the separated fragments.
Use of a CCD enables results to be analysed more conveniently and potentially with greater sensitivity than has hitherto been possible.
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 2000 Imaging System produced by Astromed Limited, Cambridge, United Kingdom. The CCD is preferably cooled to at least as low as -25.degree. C., with sensitivity being significantly increased by further cooling down as far as -160.degree. C. Typical operation temperatures are in the range -40.degree. C. to -120.degree. C.
Prior to viewing the separated fragments, some sort of treatment or processing may be required, either before or after separation, in order to make the fragments more conducive to visualisation.
For example, the structure prior to treatment of the resulting fragments may be labelled in known manner with a detectable labelling reagent, e.g. a radioactive, fluorescent or luminescent compound. As a further possibility, treatment may be effected with a reagent or reagents to produce a detectable colourimetric change.
The labeling reagent may be attached to sites on the carbohydrate units, after release if necessary from an attached biomolecule. Alternatively, the biomolecule may be modified in known way to enable incorporation of a suitable labeling reagent.
The carbohydrate structure may be decomposed by known chemical techniques, e.g. as disclosed in the paper referred to above.
The collection of fragments may
REFERENCES:
Analytical Biochemistry 115, pp. 170-176 (1981) (Poretz et al.).
Analytical Biochemistry 97, pp. 438-449 (1979) (Weitzman et al.).
J. Biochem., vol. 85, No. 4, pp. 989-994 (1979) (Hase et al.).
"Heterogeneity of Lipopolysaccharides, Analysis of Polysaccharide Chain Lengths by Sodium Dodecylsulfate-Polyacrylamide Gel Electrophoresis", Barbara Jann, Konrad Reske, and Klaus Jann, Eur. J. Biochem., vol. 60, pp. 239-246 (1975).
Jackson Peter
Williams Gwynfor R.
Astroscan Ltd.
Koestner Caroline
Niebling John F.
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