Studies on Synthetic and Naturally Occurring Enzyme Metabolites

In the General Introduction there is included a brief description of the biology and the chemical structure of starch followed by an account of contemporary opinions regarding the metabolism of starch. Carbohydrate is translocated in higher plants as sucrose which must be regarded as both the initial and the end product of starch metabolism. The process whereby it is converted to amylose and amylopectin and then in due season the carbohydrate rermobilized as sucrose are examined in detail. It is shown that all of the enzymic activities invoked are not only possible but have actually been demonstrated either in the higher plants or else closely associated with starch metabolism in other organisms. In the remainder of the Thesis an examination of the free sugars and the sugar phosphates which can be extracted from potato tubers in association with starch granules is described in detail. Potato starch granules were leached with aqueous methanol and the extract concentrated by vacuum distillation. The neutral sugars and the sugar phosphates present in this extract were then separated from each other using an anion exchange resin. The free sugars were identified by a variety of techniques but principally paper chromatography, and a further separation of the mono- and disaccharides was effected by charcoal column chromatography. The concentrations of the various sugars were determined colorimetrically, especial care being taken to adopt methods which gave a specific result for the sugar being examined and avoided interference from the other sugars present. Of the sugars identified, sucrose, glucose, fructose, maltose and ribose, sucrose was by far the most abundant (208mum/g. of starch extracted) . Glucose and fructose were present in almost equal amounts (66mum/g. of starch) whilst there was less ribose (26mum/g.) and maltose (12.5mum/g.). In a separate analysis myoinositol was also identified (2. 6mum/g.). The sugar phosphates proved to be more difficult to isolate and analyse. After considerable preliminary exploration it was decided merely to separate by anion exchange chromatography those esters which form a complex with borate from those which do not do so. The phosphate groups were then removed by enzymatic hydrolysis following which the carbohydrate moieties were identified and quantified. Extensive use was made of gas liquid chromatography at this stage. In a typical experiment only of the phosphate associated with starch granules was extracted. Of this extractable phosphate 78% was inorganic whilst of the organic phosphate (120mum/g. of starch) only 9.8% was ascribable to identified carbohydrate phosphates. Of these glycerophosphate was the most abundant (3.25mum/g.) followed by glucose-6-phosphate (2.77mum/g.), myo-inositol monophosphate (2.51mum/g.) and sucrose phosphate (2.13mum/g.). Glucose-1-phosphate and fructose-6-phosphate were also present (0.54 and 0.52mum/g. of starch respectively). In a final section is described the application of a selection of these techniques to starch granules freshly extracted from potatoes of known history. Qualitatively there appeared to be little difference between this starch and starch prepared and purchased commercially except that the presence of free ribose could not be confirmed. However with the exception of inorganic phosphate all of the identified metabolites were extracted in higher concentrations from fresh starch that from commercial starch