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|Title:||STUDIES ON PEANUT (Arachis hypogea L) PROTEINS|
|Authors:||Reddy, K. Raveendra Nadha|
|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. Proteins make up a large part of the animal body. They are the principal material of skin, muscle, tendons, nerves, blood, enzymes, antibodies and many harmones. Chemically proteins are high polymers formed by the amino acids, the building blocks of proteins, which are linked together by peptide bonds. The protein may contain one long polypeptide chain or many such chains linked together by different kinds of bods. These polypeptide chains are generally referred as subunits. The conformation and shape of the subunits and their association to constitute protein molecule determines the overall shape, size and function of the protein. In order to understand the functional specificity of a protein molecule it is necessary to know the subunit structure and sequence of amino acids for each of the subunits (i.e. primary structure). The functional specificity of a protein rests in its conformation which in turn is determined by amino acid sequence. The sequence analysis is further important as it can be related, through genetic code, to the nucleotide sequence of the gene that directed its synthesis; sequence IV is a link between genetic message in DNA and 3 dimensional structure. Knowledge of amino acid sequence reveals evolutionary history and provides a basis for 'molecular pathology'. The determination of primary structure i.e. the sequence of amino acids in a given protein is still a challenging task. Edman degradation for sequence analysis lies virtually at the core of all modern sequencing strategies. Much work has been done on animal proteins, but plant proteins received only a little attention though they are easily available for structural studies. In view of these reasons peanuts have been selected for the present studies. Peanuts of the variety 'Type 25' grown in Uttar Pradesh state of India of 1985 harvest were used. Peanut (Arachis hypogea L.) contains two proteins viz arachin and conarachin. They are having 6 and 7 subunits respectively. The present studies include the complete primary structure determination of one of the subunits of arachin, reconstitution of conarachin from the isolated subunits and development of rapid TLC systems for the identification of unknown PTH-amino acids obtained during the sequence determination by manual Edman degradation. The thesis has been divided into the following five chapters: Chapter 1 Gemeral Introduction Chapter 2 Materials and Equipment Chapter 3 Amino Acid Sequence of a Subunit of Arachin Chapter 4 Studies on Conarachin II Chapter 5 TLC of PTH-Amino Acids. Chapter 1 presents briefly the introduction to proteins and their importance, and the detailed literature compilation on the chemistry of peanut proteins. Chapter 2 gives the details of materials and equipment regarding their make, purity and other specifications. Chapter 3 presents the introduction on arachin, the major peanut protein, and the primary structure of one of the subunits of arachin. Arachin was isolated from 10% sodium chloride extract of defatted peanut meal by adding ammonium sulphate to 40% saturation. The separated protein was purified and VI characterised by gel filtration, ion exchange chromatography, PAGE, molecular weight determination, amino acid analysis etc. Arachin was dissociated into six subunits in the presence of SDS. The six subunits were separated, isolated and characterised by PAGE, gel filtration, dialysis etc. One of the subunits with lowest molecular weight was choosen for the sequence determination. It was subjected to N-terminal analysis and CNBr, NBS and trypsin digestion. The various fragments from CNBr, trypsin and NBS digestion were separated and isolated, and amino acid analyses and N-terminal sequence analysis by manual Edman method were carried out for each of the fragments. PTH-amino acids obtained during Edman degradation were identified by UV and TLC in different solvent systems. Complete sequence of the same subunit was established, from the individual sequences of the above polypeptides, by overlapping technique. Chapter 4 reports the results of reconstitution of conarachin II, its amino acid composition, first two Nterminal residues of each subunit, their molecular weights, weight ratios of different subunits and the reconstitution yield of conarachin II. Besides these, some PAGE photographs are also presented showing the dissociation of conarachin II in the presence of SDS, SDS plus 2-mercaptoethanol, individual subunits of conarachin II and reconstituted ccnarachin II. VI1 Conarachin II was precipitated by ammonium sulphate fractionation from arachin and conarachin I free 10% sodium chloride extract of defatted peanut meal. The separated conarachin II was purified and the homogeneity was established by PAGE and DEAE-cellulose chromatography. Molecular weights of intact conarachin II and its subunits (seven in all) were determined by polyacrylamide gel electrophoresis. Conarachin II pretreated with SDS in the presence and/or absence of 2-mercaptoethanol was carried out to find out whether S-S linkages were present. The seven subunits separated by SDSPAGE were then isolated. The isolated subunits were dialysed to further purify them. The concentration of all the subunits was determined by UV and Folin-Lowry method. BSA was used as a standard for determining the concentration of unknown protein. Conarachin II was allowed to reconstitute from the isolated subunits by removing SDS. The seven isolated subunits of conarachin II were separately dissolved in phosphate buffer and were mixed in their weight ratio. The mixture of subunits was dialysed against sodium phosphate buffer at 8°C for 72 hours. The dialysed solution was lyophilised and the percentage yield of reconstituted conarachin II was calculated. The fifth chapter deals with TLC studies of PTHamino acids. The identification of PTH-amino acids, from Vlll Edman degradation, by two complementary techniques is necessary. TLC was choosen as one of the methods for their identification because it is inexpensive, easily available, reliable, rapid and sensitive. During the studies on sequence determination difficulties were encountered to identify/resolve certain combinations of PTH-amino acids and literature also did not reveal any solution to these. Therefore, simple TLC systems were developed to identify several of PTH-amino acids and were used during the course of studies on sequencing of arachin subunit.|
|Appears in Collections:||DOCTORAL THESES (chemistry)|
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