Thesis (M.S.E.C.E.)--University of Massachusetts Amherst, 2008. / Includes bibliographical references (p. 42-43).

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The degree of biochemical variability is, in many cases, thought to be related to environmental stability. Intraspecific protein variation was examined in Euphausia pacifica using starch gel electrophoresis. Specimens were collected from eight oceanic and three neritic stations over a wide geographic area. The interspecific aspect of this study examined the general protein patterns of five species of North Pacific euphausiids. Temperature, salinity and oxygen were monitored, at the time of biological collections, in order to distinguish between water types. Two major protein patterns characterize E. pacifica from the different regions. The pattern possessing the greatest number of bands occurs in highest frequency in areas of greatest environmental instability (as characterized by temperature, salinity, and oxygen); the highest frequency occurrence of the pattern with the lowest number of bands is in environmentally stable regions. The expression of each pattern appears related to the degree of fluctuation of the physical parameters monitored, but these parameters are not suggested as the direct cause of which pattern is expressed. The physical parameters serve only as a tool for characterizing the stability of a region. Analysis of the electrophoretic protein patterns from five species of euphausiids, Euphausia pacifica, E. gibboides, Thysanoessa spinifera, T. inspinata and Nematoscelis difficilis, revealed that each species possesses a unique protein pattern. The interspecific and intergeneric similarities of protein patterns parallel the existing morphological taxonomy. / Science, Faculty of / Zoology, Department of / Graduate

In this research experiments were carried out to estimate hydrophobic and electrostatic interactions in soluble proteins. Five proteins, lysozyme, lactalbumin, ovalbumin, myoglobin and ribonuclease-A were chromatographed isocratically on a HIC column at several molalities (0.3-1.3m) of each of three different neutral salts, ferrous sulfate, ammonium sulfate and sodium sulfate. The calculated retention coefficients were then fitted to a recently developed chromatographic model in two ways. a) Multiple regression analysis was conducted to estimate C values according to the non-linear model (log k = A + B log m + C m) . b) Simple regression analysis was conducted to estimate C′ values according to the linear model (log k = A′ + C′m) at higher salt concentrations (above 0.3m). Results indicated that C′ values better estimate the hydrophobic interactions than C values, in experiments conducted only at higher salt concentrations. The comparison of C and C′ values with ANS, CPA, and Bigelow's average hydrophobicity indices showed no clear correlations. But, omission of ovalbumin improved the correlation coefficient of C′ with ANS. Both parameters indicated straight line relationships with molal surface tension increment of salts. Further, the same model was used to estimate the hydrophobic and electrostatic interactions in protein-protein interactions. Lysozyme and avidin were chromatographed on a lysozyme immobilized affinity column. Lysozyme-lysozyme interaction showed more affinity for hydrophobic interactions at low pH values. Avidin-lysozyme interaction showed both hydrophophobic and electrostatic interactions. Both interactions showed a greater change in the strength of hydrophobic interaction rather than the surface area of interaction, to changing pH. / Land and Food Systems, Faculty of / Graduate

Rapeseed meal was extracted successively with water, hydrochloric acid at pH 2 and sodium hydroxide at pH 10 according to the three stage method of Kodagoda et al. (40); with some modifications. The prior extraction of meal with organic solvents was discontinued to decrease the loss of protein during preparation of protein isolates. The oxalic acid treatment used by Kodagoda et al. for lowering the high ash content in the acid-extracted protein fraction (IA₁) was not essential and was eliminated from the procedure. Decolorization of the protein isolates was attempted using hydrogen peroxide as an oxidizing agent and sulfur dioxide as a reducing agent. At the concentrations of 0.02, 0.2 and 2%, hydrogen peroxide significantly improved the colour of the acid-extracted and the base-extracted protein isolates. The effect of hydrogen peroxide on the amino acid composition of the protein isolates with and without added cysteine was studied under the different conditions of pH and temperature. The hydrogen peroxide treatment decreased the methionine and cystine contents of the protein isolates except that the added cysteine protected cystine in the protein, whereas, the sulfur dioxide treatment was effective for colour improvement without damaging the amino acid composition of the protein isolates. The water-extracted protein isolate (IW) was passed through an activated charcoal column to eliminate glucosinolates according to the method of Woyewoda et al. (76). Amino acid analysis showed a slight decrease in phenylalanine and tyrosine of the protein isolate. Some functional properties of the protein isolates were studied. The base-extracted protein isolate (IB) revealed the best emulsification capacity and stability, whereas the second acid-extracted protein isolate (IA₂; the first and second protein isolates were prepared from the acid extract by precipitating at pH 3.8 and 7.0 respectively) showed the greatest foamability. However, the hydrogen peroxide treatment slightly deteriorated the emulsification properties of protein isolate IB and improved the whipping ability of protein isolate IA₂. It is assumed that conformational changes in protein molecules caused by the hydrogen peroxide treatment affected the functional properties of these protein isolates. The total phospholipid content of the protein isolates was determined and a significant correlation with the emulsification capacity of the protein isolates was observed. / Land and Food Systems, Faculty of / Graduate

This thesis deals with studies on the hydrophobicity of proteins and enzymes and is divided in four chapters summarized separately below. (1) Traditional methods of coagulating milk for the manufacture of cheese suffer from two major drawbacks, namely, the high cost of the enzyme and the fact that they are batch systems. An obvious solution to both problems would be the use of immobilized enzymes to coagulate milk. Hydrophobic adsorption offers certain potential advantages over other techniques of enzyme immobilization. The objective of this part of the thesis was to immobilize the milk clotting enzymes chymosin and pepsin on various hydrophobic carriers and to assess their suitability for continuous coagulation of skimmilk. All enzyme-carrier preparations exhibited high initial activity on exposure to milk. However, the deactivation rates were very high. The main reason for this rapid deactivation appeared to be the loss of enzyme from the carriers, since soluble activity was detected in all enzyme preparations. The enzyme loss was due to the physical desorption of enzyme from the carriers as well as to the relatively rapid leakage of the ligand from the carrier (phenoxyacetyl cellulose). The best enzyme preparation was obtained with phenoxyacetyl cellulose. However, a study indicated that the continuous coagulation of skimmilk with proteases immobilized on the hydrophobic supports used in this study was not economically feasible. (2) The fat binding capacity (FBC) of food proteins is an essential functional property. However, fat binding as determined by existing methods has been mainly attributed to physical entrapment of the oil rather than to the binding with proteins. A simple turbidimetric method, thus, was developed for determining the FBC of various proteins. The turbidity was dependent on wavelength, blending time and volume of oil. The regression equation for predicting FBC was: FBC (%) = 30.271 + 1.381 S[sub=o] - 0.014 S[sub=o] x s where S[sub=o] and s are surface hydrophobicity and solubility index, respectively. A highly significant correlation (R² = 0.802, P < 0.01) was found between S[sub=o], S[sub=o] x s, and FBC of 11 food proteins tested. Advantages of the method developed include the small amount of sample required and the fact that the measured values would reflect the true fat binding capacity of proteins by minimizing the fat-entrapping effects. (3) The objectives of this part of the thesis were to determine the effects of heating on the emulsifying properties of selected food proteins, and, to assess the value of Sq as a predictor of these properties. The results obtained indicated that the emulsifying properties of the proteins studied were differently affected by heating, and that heat-denaturation was not always accompanied by loss of functionality, but, on the contrary, resulted in great improvement. The emulsifying properties could well be predicted solely on the basis of S[sub=o] level but not on the basis of solubility level, which indicated that S[sub=o] is a very important property determining protein functionality. However, the emulsion activity, emulsion stability and fat binding of the proteins studied could be well explained and more accurately predicted by S[sub=o] and solubility together. (4) The objectives of this part were to evaluate the thermal properties (thickening, coagulation and gelation) of selected food proteins and to assess the value of hydrophobicity as their predictor. The results obtained indicated that the average (S) and not the surface hydrophobicity was important for these properties. The thermal properties studied could not be explained by either the average hydrophobicity or sulfhydryls alone. Instead, they could well be predicted using average hydrophobicity and sulfhydryls together. / Land and Food Systems, Faculty of / Graduate

A notable feature of this century has been the introduction of medical science to large, undeveloped areas throughout the world. Backward, mainly tropical, regions have become fertile fields for study, particularly of nutritional and parasitic disorders, and the application of the modern biochemical methods have show that many inhabitants of these countries lack certain of the biochemical "normal" values seen in Europeans. One of the most striking differences is that of the serum proteins. Although by no means confined to this continent, these differences are commonly found in Africans.

Due to the complex nature of the protein molecule no adequate system of classification and separation of the simple proteins has yet been devised . Present methods of separation have many disadvantages. Because of these disadvantages new methods of separation were investigated. Chromatographic adsorption which makes use of the difference in adsorption affinities of substances for an adsorbent seemed to offer possibilities of an accurate and simple method for protein separation. Aluminum oxide was chosen as the most suitable adsorbent from such substances as fuller's earth, activated carbon, silica gel, and bauxite. To a column of alumina a solution or egg albumin was added. The fluid issuing from the column was caught in ml. portions and checked qualitatively with biuret solution for the presence of protein. Quantitative measuremetts of the protein content of each ml. fraction was accomplished by the use of biuret solution and a colorimeter. The alumina and egg albumin chromatogram was extruded and the presence of adsorbed zones on the column investigated by biuret solution. A zone 2.5 to 3 cm. long starting at the top of the column was formed. Efforts to elute this zone with water, salt solutions, slightly acidic and basic solutions, and ethanol-water mixtures failed. The fluid issuing from the column on development gave positive tests for protein content. This experimsnt showed that egg albumin was separated into two components by adsorbing it on alumina. Quantitative examination of the protein content of the fluid issuing from the columns showed that there was no variation of the ml. fractions from the alumina column, the silica gel column, and the calcium carbonate column. However, variations occurred in the ml. fractions taken from the activated carbon columns and the fuller' s earth column. One-dimensional and two-dimensional paper chromatography were tried. The paper was cut in strips for one-dimensional chromatography and left in sheets for two-dimensional chromatography. Acetate buffers of various pH's were used as developing solvents. No separation of the egg albumin by these methods was detected.

A Thesis Submitted to the Faculty of the School of Informatics, Indiana University, Indianapolis By Narmada Shenoy In Partial Fulfillment of the Requirements for the Degree of Master of Science August 2005 / Microtubules have many roles in the cytoskeletal infrastructure. This infrastructure underlies vital processes of cellular life such as motility, division, morphology, and intracellular organization and transport. These different roles are carried out by the creation of different microtubule (MT) systems (such as basal bodies, centrioles, flagellum, kinetochores, and mitotic spindles). The changing roles require the cytoskeleton to be both dynamic and static in nature. Guiding these processes are a network of proteins that direct cellular behavior through their ability to bind microtubules (MTs) in a spatial- and temporal-specific manner. The identification and characterization of the suite of microtubule binding and associated proteins (MAPs) involved in MT systems is important for the understanding of the biological form and function of each MT system. This research involved the analysis and annotation of four MAPs – Ensconsin in Humans, Hook (homolog 3) in Humans, Protein Regulator of Cytokinesis 1 (PRC1) in Humans and Anaphase Spindle Elongation protein (ASE1) in yeast. A bioinformatics approach was used for the annotation and analysis. A protocol for analysis and annotation of MAPs was developed. During the process, some limitations in using bioinformatics tools and procedures were encountered. These limitations were overcome, the initial protocol was improved on and a modified protocol of analysis was developed. A database was designed and built to hold annotated information on the MAPs. We seek to disseminate this database and its functionalities as a web resource to the scientific community. It will provide an excellent forum for researchers to obtain relevant information on MT binding and associated proteins (MAPs). Infection by parasitic protozoa causes incalculable morbidity and mortality to humans and agricultural animals. In this research, we have also focused on MAPs in parasitic organisms of the Apicomplexan and Trypanosomatid genera. The protocol for analysis incorporates steps to analyze MAPs from these organisms as well. Malaria (a potentially life threatening disease) is caused by Plasmodium, an Apicomplexan parasite. This parasite is transmitted to people by the female Anopheles mosquito, which feeds on human blood. African Sleeping Sickness is an acute disease 8 caused by Trypanosoma brucei that typically leads to death within weeks or months if not treated. Microtubule-associated proteins (MAPs) and their alteration of the unique microtubule (MT) systems play major roles in these organisms throughout their life cycle and are required for their pathogenic mechanisms. Each parasite contains unique MT systems that will test our annotation process as well as prepare the DB for addition of other novel MT systems, such as those contained with plants. Additionally, these single cell organisms have a multistage life cycle that provide similar annotation challenges to those encountered when one considers multi-cellular organisms. Therefore, a researcher working on any MT system within the database will find useful information regardless of the organism that they are studying. This will leave us with a sub-set of MAPs from parasitic organisms in our database that are potential drug-targets.