The Detection, Analysis and Chemistry of Sugar Nucleotides

Sugar nucleotides are generally extracted with ethanol or with dilute perchloric or trichloroacetic acids, so that the extracts are essentially free of protein. Ethanol may extract a considerable amount of lipid, while the acids tend to cause appreciable losses of sugar nucleotides by hydrolysis. Trichloroacetic acid can be extracted with ether, while perchloric acid is best removed as its potassium salt. All extractions must be performed in the cold and extraction with ethanol is possible well below 0°C, though it fails to denature all pyrophosphatases and this can lead to losses. Saukkonen (1964) has reviewed a number of procedures for extraction and there is undoubtedly still some room for improvements. 

The separation of sugar nucleotides from such extracts is by no means easy, especially since it has to be as rapid as possible in order to minimise hydrolysis losses. Adsorption onto a basic ion-exchange resin is commonly used as a first step. It can be performed as a column method, as a batch extraction or, on a micro-scale, on sheets of ion-exchange materials. Considerable losses may occur on the resin. Elution can be by one of several different procedures, of which washing with ammonium chloride is one of the more useful (Recondo, Dankert and Passeron, 1965), since the salt is somewhat volatile. Other systems used include displacement by the chlorides of calcium, sodium or lithium, or by ammonium formate buffer. Hydrolysis losses are particularly likely with the last. 

Paper chromatography has been used to separate sugar nucleotides, both in acid extracts and in eluates from resins. In general, the former is feasible only where very small amounts of material are to be analysed. Solvent systems based on mixtures of ethanol and ammonium acetate buffer of about neutral pH have generally been preferred, as the buffer is volatile and hydrolysis is 

On a small scale desalting is possible and has been achieved by freezedrying, chromatography in organic solvents or by gel-filtration on thin layers. 

Because of the lability of sugar nucleotides, every effort should be made to minimise the number of procedures involved in their separation and characterisation and to make these as rapid and mild as possible. For this reason, thin layer procedures are greatly to be preferred. If initial cleaning-up and concentrating of extracts can be minimised or avoided, that too is advantageous. 

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