Please use this identifier to cite or link to this item: http://localhost:8081/xmlui/handle/123456789/1425
Authors: Arya, Upasana
Issue Date: 2006
Abstract: Proteins are the most important nitrogen containing biological molecules of all the living cells. Of all chemical compounds, proteins must almost certainly be ranked first, for they are the substance of life. They make up large part of the animal body including the principal material like skin, muscle, tendons, nerves, blood, enzymes, antibodies and many hormones. Chemically proteins are high polymers formed by the amino acids, the building blocks of proteins, which are linked together by peptide bonds. Naturally occurring proteins may exist either as a single polypeptide chain or several polypeptide chains are intertwined or chemically bonded leading to a complex molecular association of polypeptide chains. Several high molecular weight proteins, such as seed globulins, e.g., glutenin from wheat, soyprotein, peanut proteins, also exist in the form of different molecular species, which might exhibit distant chemical and physicochemical properties to each other. The biological activity of a protein is governed by its conformation which in turn depends upon sequence of amino acid residues. For the complex proteins, such as mentioned above, it becomes necessary to examine the molecular species, the polypeptide architecture of the protein and the subunits. For the research purpose soybean (Glycine max) is chosen as itcombines in one crop both the dominant world supply of edible vegetable oil, and the dominant supply of high-protein feed supplements for livestock. Other fractions and derivatives of the seed have substantial economic importance in a wide range of industrial, food, pharmaceutical, and agricultural products. Soybean has been a valuable resource for humans by providing both high quality protein (40%) and oil (20%). On an average, soybean contains about 40% proteins, 20% oil, 30% carbohydrates, 5% fibres and 5% ash. Soybeans proteins can be divided in albumins (10%), extracted by water, and globulins (90%) extracted by dilute salt solutions. Soybean storage globulins are composed of two major components, p-conglycinin and glycinin corresponding to 7S and US globulins, respectively. Glycinin, the most abundant storage protein of soybean, account for 40% of total seed protein and may represent as much as 10 to 15% of the dry weight of mature seed. The existing literature suggested that there was a need to investigate the homogeneity, behaviour and characteristics of glycinin and its molecular species: glycinin I and glycinin II. Therefore, the present work is aimed to investigate the different states of aggregation of glycinin occurring with changes in the chemical nature, pH and ionic strength of the solutions. The studies were also carried out to investigate the subunit structure, association-dissociation phenomenon and reconstitution behavior of the complex protein, glycinin. For the sake of convenience, simplicity and clarity, the work embodied in the thesis has been organized as follows: The first chapter, General Introduction is an introductory one and presents an up-todate review of all the literature on glycinin. It deals with the nature, structure and importance of proteins along with the classification of seed storage proteins. In addition, it reviews the work reported in literature on the biochemistry of soybean proteins including their ultracentrifuge sedimentation pattern, extraction procedures, amino acid composition, subunit composition and different techniques used to investigate the nature of glycinin. The problem elucidation of present research activities has also been posed in the context of cited work. Second Chapter, Materials and Equipment, gives the details of materials and equipment regarding their make, purity and other specifications. Third Chapter, Molecular Species of Glycinin, describes the isolation, purification, protein determination, molecular weight determination and weight ratios of glycinin and its molecular species: glycinin I and glycinin II using different chromatographic techniques such as gel filtration, polyacrylamide gel electrophoresis and reversed-phase high performance liquid chromatography. Soybean meal was defatted and extracted with tris buffer and was further purified on Sephadex G-150 column by gel filtration chromatography. Glycinin was resolved into its two molecular species, glycinin I and glycinin II by the above mentioned techniques using different buffer systems and their association-dissociation behaviour was also studied using polyacrylamide gel electrophoresis. Fourth Chapter, The Subunits, gives detail account ofstudies on the number, kinds and weight ratios of subunits of glycinin, glycinin I and glycinin II. Studies were carried out under different experimental conditions using tube gel, slab gel electrophoresis, size exclusion chromatography and reversed-phase high performance liquid chromatography to separate subunits of glycinin, glycinin I and glycinin II. Seralose-6B, Sepahdex G-100 and Sephadex G-150 were used to separate glycinin subunits by size exclusion chromatography. Gel electrophoresis was done under varying concentrations ofgel using continuous and discontinuous buffer systems for both tube and slab gel electrophoresis. In addition, the subunits were separated by reversed-phase HPLC under different experimental conditions using gradient program. Fifth Chapter, Reconstitution of Glycinin I Subunits, deals with the reconstitution of glycinin I subunits (SI to S6) to parent glycinin I using SDS-PAGE. Glycinin I was found to consist ofthree basic (SI, S2, S3) and three acidic (S4, S5, S6) subunits and was allowed to reconstitute from the isolated subunits (SI to S6) by removing SDS. Different combinations were tried for studying the reconstitution pattern. For example, subunits (SI - S5) were mixed in their weight ratio and codialyzed against phosphate buffer. Similarly S6 was dialyzed separately. The 6th subunit (S6) was later on mixed in its weight ratio with the mixture (SI to S5). The combined subunits (SI - S5 plus S6) were allowed to reconstitute. The experiment was repeated successively by taking S5, S4, S3, S2, and SI for independent dialysis followed by mixing with the remaining five in subunits as above. Each time they were mixed in their weight ratios. In another set of experiment all the six subunits were dialyzed together in the similar manner. Finally, efforts were also done to obtain pseudoglycinin made up of single acidic-basic subunit complex by codialyzing acidic and basic subunits in different combinations. Thus, different sets of reconstituted proteins were obtained and studied by polyacrylamide gel electrophoresis. A comparison was also made between Coomassie Brilliant Blue staining and silver staining for the detection of subunits on polyacrylamide gel slab.
Other Identifiers: Ph.D
Research Supervisor/ Guide: Bhushan, Ravi
metadata.dc.type: Doctoral Thesis
Appears in Collections:DOCTORAL THESES (chemistry)

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