A concentrate containing up to 73% protein N was recovered from cheese whey by using cellulose acetate ultrafiltration membrances designed to reject solutes larger than 30,000 molecular weight by a continuous washing procedure. Conditions necessary for increasing the ultrafiltration process for cheese whey are reported. Variables include pressure, membrane porosity, feed rate, clarification, temperature and pH. The objective was to prepare whey products with a minimum concentration of monovalent salts and maximum concentration of protein while still maintaining a high flux rate. As expected pH adjustment to 7.0 and clarification at 2000 X g for 5 min were critical in increasing flux rate. However, membrane blockage occurred and gel electrophoresis indicated that (β-casein and αs-casein were the major components responsible yet salts and lactose may also be implicated to a lesser degree. Flux rate increased with temperature but was not affected by pressure. Results indicate that concentrating 3-4X would be practical but higher levels would be uneconomical due to the accumulation of viscous materials on the membrane. Gel filtration showed that whey proteins are retained almost quantitatively in the concentrate while low molecular weight nitrogen containing material pass the membrane into the permeate. / Land and Food Systems, Faculty of / Graduate

A diafiltration technique was used to study drug-protein interactions. Fraction V human serum albumin and plasma and two drugs (phenylbutazone and bishydroxycoumarin) with a high affinity for these substances were used in this investigation. Preliminary experiments were carried out to check for release of foreign substances and for binding of drug to the Amicon diafiltration apparatus. A binding experiment, in the absence of drug, revealed release of a protein-like, ultraviolet absorbing substance from Fraction V human serum albumin. The most suitable method of purification for albumin was by diafiltration with Tris buffer. Binding curves for bishydroxycoumarin - human serum albumin, phenylbutazone - human serum albumin, and bishydroxycoumarin - plasma interactions were obtained. The r and r/Df [subscript omitted] values were calculated and binding parameters estimated by both graphical extrapolation and by a computer non-linear least squares fit analysis. Binding curves were not independent of human serum albumin concentration, but the cause of this effect was not fully resolved. Results showed the diafiltration technique can yield precise data, can be used over a wide macromolecule concentration range and produces a binding curve, from one experiment, over a wide range of molar binding ratios. Use of the Amicon diafiltration apparatus in desorption (washout) experiments and equilibrium or direct experiments was also investigated. Attempts were made to obtain binding data by centrifugation (ultrafiltration) and by a gel filtration technique (Sephadex G-25 batch method). These methods yielded unsatisfactory results which could not be compared with those obtained by diafiltration. This abstract represents the true contents of the thesis submitted. / Pharmaceutical Sciences, Faculty of / Graduate

The histidine specific reagent 2-phenyl-l,4-dibromoacetoin (PDA) has been applied to αSI-casein B. Reaction of αSI-casein with PDA for 26 hours resulted in the loss of about two residues each of histidine and methionine per αSI-casein monomer (molecular weight = 27,000). The modified protein was 90% soluble in 8 mM calcium chloride and precipitated quantitatively at 13 mM calcium chloride. The control was precipitated quantitatively at 8 mM. The calcium binding capacity of the modified αSI-casein was reduced to about 4.5 calcium ions per PDA αsi-casein monomer from 12.4 calcium ions per αSl-casein monomer. Reaction of αSI-casein with PDA resulted in the production of aggregated material which remained at the origin on polyacrylamide gel electrophoresis in the presence of urea and 2-mercaptoethanol and which was eluted at the void volume of a Sephadex G-200 column. While following the time course of the reaction of PDA with αSI-casein, it was found by N-bromosuccinimide (NBS) spectrophotometric titration (6 M urea-acetate-formate buffer, pH 4.0) that two residues of tryptophan per monomer αSI-casein could be detected at zero reaction time but 3.1 residues could be detected after 30 hours. Comparison of the results obtained with αSI-casein, PDA αSl-casein, β-lactoglobulin and α-chymotrypsinogen using both the NBS titration procedure and the p-dimethylaminobenzaldehyde method of Spies and Chambers (46) suggest the presence of a "buried" tryptophan residue in αSI-casein. PDA has a negligible effect on the NBS titration procedure so the residue "exposed" cannot be an artifact generated by PDA bound to the protein. Increasing the urea concentration to 10 M did not expose the third tryptophan residue to NBS. / Land and Food Systems, Faculty of / Graduate

No description available.

Ion exchange chromatography methods which have been developed in recent years appear to offer a sensitive technique that can be utilized very advantageously in studies on various proteins and their properties. The application of such a procedure for the successful fractionation of bovine sarcoplasmic proteins should stimulate interest and research in characterizing the changes occurring in beef muscle during the post-mortem aging period. The research described herein pertains to the development and application of a DEAE-cellulose ion exchange chromatography procedure for the fractionation of bovine sarcoplasmic proteins. Results of preliminary experiments indicated that columns packed under pressure possessed superior fractionational qualities than did columns packed without pressure. Also in the preliminary experiments, a Tris buffer system (a starting buffer of 0.04 M Tris phosphate, pH 9.0, and a limiting buffer of 0.5 M Tris H₂PO₄, pH 3.6) and a concave gradient elution procedure were developed which were found to separate the sarcoplasmic proteins satisfactorily. At least 16 components were recognized to be fractionated in the chromatography of beef sarcoplasmic proteins extracted two hours post-mortem. Duplication of the chromatographic results was found to be quite good. Some changes that had occurred in the sarcoplasmic proteins during a post-mortem aging period of 10 days were detected by the chromatographic technique. The changes observed were the appearance of new components, and disappearance of some fractions while others diminished. Data obtained from experiments on the pre-chromatographic treatment of the samples showed that any deviation in procedure always resulted in chromatographic differences. Hence, strict uniformity must be maintained throughout the chromatographic procedure in order to obtain reproducible results. Although the experimental evidence obtained in this study indicates that further research must be completed on the chromatographic procedure, the method does offer a sensitive technique for gaining new information on the properties of the sarcoplasmic proteins. / Graduation date: 1965

Lu, Yao. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

No description available.

The benefits of fortification of poor quality food proteins such as wheat gluten with limiting amino acids depend on the biological availability of the added amino acids and their stability with respect to processing and storage. Although simple addition of amino acids in free form is convenient, the potential improvement in nutritional quality by this method of fortification may not materialize due to possible losses during processing steps such as washing, susceptibility to degradative reactions, and different rates of absorption and utilization compared to protein-bound amino acids. In this study, covalent attachment of lysine and threonine to wheat gluten was investigated using the chemical carbodiimide reaction and the enzymatic plastein reaction. The nutritional quality of enriched products was evaluated by in vitro and microbiological tests, and susceptibility to destruction by heating in the presence of a reducing sugar was investigated. Covalent attachment of lysine ethyl ester or threonine to gluten by the plastein reaction utilizing the enzyme papain was not successful. Although lysine and threonine contents in the products were increased, these results were attributed to selective enzymatic release of other amino acids. The amino acid compositions of undialyzable "plastein" products were markedly different from the original gluten substrate, and product yields were low. The results suggest the formation of many dialyzable peptides and free amino acids, and inability for protein re-synthesis from these low molecular weight compounds. Covalent lysine and threonine contents were increased using the carbodiimide reaction. In general, the reaction was most influenced by pH, reactant concentration and type of reactant. Products enriched via primarily peptide bonds as well as products enriched via peptide and isopeptide bonds could be prepared using various starting materials. Lysine, N -acetyl lysine, N -benzylidene lysine and threonine were coupled through amide bond formation to gluten, sodium stearate-solubilized gluten, acid-solubilized gluten or pepsin-solubilized gluten. Sodium stearate solubilization did not improve extent of amino acid incorporation. Pepsin hydrolysis of gluten enhanced amino acid attachment but decreased product yields. 4.0-fold and 6.5-fold increases in lysine content resulted by reaction of pepsin-solubilized gluten with N e-benzylidene lysine and N-acetyl lysine, respectively. However, yields of these products were low (47% and 58%). 1.6-fold, 2.0-fold and 2.5-fold increases were obtained by reaction of gluten with Ne-benzylidene lysine, lysine and N£-acetyl lysine, respectively, with product yields of 90 to 95%. At least 20-fold and 5-fold increases could be achieved by reaction of 0.5N HCI and 0.05N HCI solubilized glutens respectively with lysine, with product yields of 80 to 90%. 4-fold and 2-fold increases in threonine content resulted from reaction of threonine with pepsin-solubilized gluten and gluten, respectively. Simultaneous attachment of lysine or lysine derivative and threonine was not effective. In vitro evaluation of availability and digestibility of covalently enriched products was carried out by the DNBS reaction and pepsin pancreatin digestion tests. The results indicate the formation of isopeptide bonds involving the £-amino group of lysine unless N -substituents of lysine were used. Isopeptide bonds involving the Y_carboxyl groups were indicated when gluten had been solubilized by acid treatment. Peptide bond formation predominated when N -benzylidene or N -acetyl lysine was attached to pepsin-solubilized gluten or gluten. The high hi vitro availability and digestibility values for N £-benzylidene lysine enriched products suggest lability of the Schiff's base linkage of this N £-substituent, in contrast to the stability of the amide linkage in Ne -acetyl lysine. Microbiological evaluation by a Tetrahymena bioassay confirmed the nutritional improvement of gluten by covalent attachment of lysine, Ne-acetyl lysine or Ne-benzylidene lysine. Relative nutritive value of gluten was 54, whereas covalently and freely enriched glutens had relative nutritive values similar to that of the reference casein, assigned a value of 100. Covalently and freely lysine enriched glutens were compared for color and extent of lysine destruction after baking. In general, covalently enriched products had lighter color and higher percentages of total, DNBS-available and pepsin-pancreatin-digestible lysine contents than freely enriched products. N e-Benzylidene lysine enriched gluten was particularly stable, with relative nutritive value of 88 compared to 44 for baked gluten. It is concluded that covalently attached lysine is more stable than free lysine for enrichment of food proteins susceptible to Maillard reaction. / Land and Food Systems, Faculty of / Graduate

Male Fischer rats (n=43) were used in a diet-diet/ exercise design to investigate the apparent protein sparing effects of exercise. The animals were divided into five groups: INITIAL (baseline), SEDENTARY (control), DIET, DIET/EXERCISE, and EXERCISE. Carcasses were analyzed for body composition, the blood for plasma alanine concentration and the urine for urea nitrogen concentration. The results showed no significant differences between groups in urinary urea nitrogen, plasma alanine, body weight, or carcass weights. The EXERCISE group had a significant increase in percent protein and a significant decrease in percent fat and grams of fat when compared to all other groups (p <.05).

Abstract not available