Methods of synthesizing a series of chemically-defined AMP, ADP, ATP, adenylyl imidodiphosphate and pyrophosphate derivatives suitable for affinity chromatography are extensively described. Each derivative has a single primary amino group at the end of a hexamethylene ;spacer' chain for attachment to CNBr-activated agarose. The synthesis of the derivative where the ;spacer' arm is attached directly to the 8 position of the adenine ring to produce 8-(6-aminohexyl)amino-AMP involves the direct bromination of AMP in the 8 position followed by displacement of the halogen by 1,6-diaminohexane. This monophosphate derivative can then be converted into the corresponding di- or triphosphate forms by direct phosphate condensation with carbonyl di-imidazole. A second series of adenosine phosphate derivatives with the phosphate moieties unsubstituted has been similarly prepared from N(6)-(6-aminohexyl)-AMP (Guilford et al., 1972). A third type of ligand has been synthesized by condensing the phosphoryl imidazolide of AMP with 6-aminohex-1-yl phosphate. This compound, P(1)-(6-aminohex-1-yl) P(2)-(5'-adenosyl) pyrophosphate, has an unsubstituted adenine ring. The synthesis of a fourth type of ligand, 6-aminohex-1-yl pyrophosphate, was done by heating 6-aminohexan-1-ol with crystalline pyrophosphoric acid under reduced pressure. The structures of the synthesized compounds were confirmed by chemical, electrophoretic and chromatographic methods and by u.v. spectrometry. The general applicability of the synthetic methods used is discussed in relation to the preparation of other affinity adsorbents. Examples are given where these derivatives have been successful in reversibly binding dehydrogenases, kinases and myosin and its proteolytic subfragments. The partial purification of rat liver glucokinase on an ADP derivative is shown.
Three samples of field-grown winter wheat (Triticum arstivum cv. Atou) with different protein contents were produced by late application of urea as a nitrogenous fertiliser. Baking tests (a conventional fermentation procedure) indicated that the breadmaking quality of the flours increased as protein content was raised from the lowest to the intermediate level, but that the flours of intermediate and highest protein content were of equivalent breadmaking quality. To compare gluten baking quality independently of protein quantity, loaves were also baked from 'flours' reconstituted to equivalent protein levels using the isolated glutens. The flours of low and intermediate protein content yielded glutens of similar baking quality. The gluten derived from the flour of highest protein content gave a lower loaf volume and texture score: subsequent biochemical investigations suggested that this was due to an effect of the relative levels of nitrogen and sulphur available to the plants grown on this particular soil. Analysis of the flours and glutens indicated that the ratio of su1phur:nitrogen fell as grain protein content increased and this correlated with a lower proportion of the sulphur amino acids cyst(e)ine and methionine. Gel electrophoresis studies revealed, in particular, an increase in the proportion of the sulphur-deficient, w-gliadin species as grain protein content increased. Agarose gel filtration chromatography of the flour and gluten proteins also suggested a correlation between the extent of aggregation of their glutenin components (mediated by disulphide bonds involving cystine residues) and their functional properties. The results of this study suggest that for wheat grown on this particular soil late application of high levels of a nitrogenous fertiliser in the absence of sulphur fertilisation led to a change in the balance between available nitrogen and available sulphur, such that the available sulphur levels became insufficient for 'normal' grain development. Nevertheless, the results indicated that considerable alteration in the biochemical characteristics of the flour proteins occurred before gluten baking quality was noticeably affected.
Actin and myosin were immobilized by coupling them to agarose matrices. Both immobilized G-actin and immobilized myosin retain most of the properties of the proteins in free solution and are reliable over long periods of time. Sepharose-F-actin, under the conditions used in this study, has proved unstable and variable in its properties. Sepharose-G-actin columns were used to bind heavy meromyosin and myosin subfragment 1 specifically and reversibly. The interaction involved is sensitive to variation in ionic strength, such that myosin itself is not retained by the columns at the high salt concentration required for its complete solubilization. Myosin, rendered soluble at low ionic strength by polyalanylation, will interact successfully with the immobilized actin. The latter can distinguish between active and inactive fractions of the proteolytic and polyalanyl myosin derivatives, and was used in the preparation of these molecules. The complexes formed between the myosin derivatives and Sepharose-G-actin can be dissociated by low concentrations of ATP, ADP and pyrophosphate in both the presence and the absence of Mg2+. The G-actin columns were used to evaluate the results of chemical modifications of myosin subfragments on their interactions with actin. F-Actin in free solution is bound specifically and reversibly to columns of insolubilized myosin. Thus, with elution by either ATP or pyrophosphate, actin has been purified in one step from extracts of acetone-dried muscle powder.
The application of buffers containing the anionic detergent sodium dodecyl sulphate (SDS) to the study of wheat flour proteins is described. SDS-containing buffers are more effective than alternative solvents, including those which contain a cationic detergent, e.g. cetyltrimethylammonium bromide, in terms both of their ability to solubilise wheat proteins and of their suitability as buffers for column chromatography.Thus, a solvent containing 0 . 0 6 9~ (2%) SDS solubilises a high proportion (95 %) of flour protein, and gel filtration chromatography on columns of Sepharose CL-4B in the presence of 3.47 x l o -3~ (0.1 %) SDS, particularly when used in conjunction with polyacrylamide gel electrophoresis, is a powerful tool for the detailed analysis of flour proteins. This technique enables flour proteins to be resolved into three major groups which have been identified as glutenins, gliadins and albumins plus globulins. It can be used to monitor increases in the molecular size of the glutenin fraction during the preparation of gluten from flour, and preliminary data suggests that there may be a correlation between the molecular size of this protein fraction and the breadmaking quality of the flour.
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