STUDIES ON RAPESEED HIGH MOLECULAR WEIGHT PROTEIN

Abstract

Proteins are highly complex organic nitrogenous substances found in the cells of all animals and plants. They occupy a central position in the architecture and functioning of living matter and are intimately connected with all phases of chemical and physical activity that constitute the life of the cell, and their functions in biological systems include transport and storage, coordinated motion, mechanical support, immune protection, control of growth and differentiation. Proteins are quantitatively the main material of animal tissues for which they constitute approximately three-fourth of the dry substances. All proteins, regardless of their functions or biological activities, are built from the basic set of twenty a-amino acids [NH2CH(R)COOH] of the L-configuration which have been united through the loss of one H20 molecule to form peptide bonds (-CO-NH-). The backbone of the polypeptide chain contains hundreds of amino acid units in a single chain. Protein molecule may contain one long polypeptide chain or many such chains linked together by -S-S-, Hydrogen bonds and ionic forces which give the proteins its secondary structure. Finally, the chains are coiled and folded in a 3 D pattern called its tertiary structure. All proteins are functionally specific, and this specificity is due to the specific sequence in which the amino acids are bound. In order to understand the functional specificity of a protein molecule it becomes important to know the subunit structure and sequence of amino acids for each of the subunits. Due to biological activity dependence on the primary structure of the protein, it is necessary to know the amino acid sequence. The sequence analysis is further important as it can (i) be related through genetic code to the nucleotide sequence of the gene that directed its synthesis. Amino acid sequence provides a link between genetic message in DNA and three dimensional structure of a protein molecule. A single disturbance in the sequence of amino acids can lead to a biological, pathological or physiological disorder. Knowledge of amino acids sequence also reveals evolutionary history and provides a basis for molecular pathology. Agricultural archaeology is another area in which the importance of amino acid sequence information is now well established. The primary structure determination i.e. the sequence of amino acids in a given protein is still a challenging task. It is accomplished generally by cleaving the larger chains into smaller and manageable segments and then determining their sequence first. Edman degradation for sequence analysis of peptides and proteins by phenylisothiocynate virtually lies at the core of all modern sequencing techniques. Rapeseed is among the five largely cultivated oil seed crops in the world. An increasing interest in Brassicaceae and particularly in rapeseed (Brassica species) proteins, has allowed the characterization and elucidation of their gene nucleotide sequences. In view of the importance of plant proteins, their easy availability and high nutritional value and since scanty attention was paid to their structural aspects and due to the increasing interest in rapeseed proteins in particular, studies on certain aspects of rapeseed high molecular weight protein have been carried out. Rapeseed of the variety 'YID-1' grown at PUSA, India of 1990 harvest were obtained for the sequence analysis. There are two major types of storage proteins one is 12S (ii) neutral globulin and the other is a 1.7S small basic albumin. Rapeseed (Brassica campestris L.) contains six different subunits. The present studies include the reconstitution behaviour of HMW protein from its subunits, complete amino acid sequence of a subunit of rapeseed , the determination of structural class of rapeseed by correlation coefficient method. The thesis has been divided into the following six chapters: Chapter I General Introduction Chapter II Materials and Equipments Chapter III Studies on the Reconstitution Behaviour of Rapeseed HMW Protein. Chapter IV Sequence Determination of a Subunit of HMW Protein. Chapter V Study of Structural Class of Rapeseed Chapter I includes brief introduction about the nature, structure and importance of proteins. It reviews the work reported in the literature on the chemistry of rapeseed proteins. Chapter II describes the details of materials and equipments regarding their make, purity and other specifications. Chapter III presents the isolation, purification, molecular weight determination, subunit composition of the rapeseed protein, molecular weights, weight ratio of the different subunits, amino acid composition of the native protein, the different subunits, first two N-terminal amino acid residues of each subunit and the result of the reconstitution of (Hi) rapeseed HMW protein from isolated subunits. The high molecular weight protein was precipitated by ammonium sulphate fractionation from a 10% sodium chloride extract of the defatted rapeseed meal. The protein was purified by Sephadex gel filtration and dialysis. The homogeneity of the protein was ascertained by DEAE-cellulose chromatography and polyacrylamide gel electrophoresis (PAGE) under different gel concentrations. The HMW protein in the presence of SDS, dissociated into six different subunits. Molecular weights of the intact protein and the individual subunits were determined by SDS-PAGE. Absence of -S-S- linkages in the protein was indicated by SDS-PAGE in presence and/or absence of 2-mercaptoethanol. The six subunits were separated and isolated by gel filtration and PAGE. The isolated subunits were purified by dialysis and lyophilized. The concentration of all the subunits was determined by UV and Folin-Lowry method. Amino acid analysis was performed for the intact protein and individual subunits and the number of each amino acid residue was calculated. The hmw protein was allowed to reconstitute from the isolated subunits by removing sodium dodecyl sulphate. The isolated six subunits of the protein were separately dissolved in phosphate buffer and were mixed in their weight ratio. The mixture of the subunits was dialyzed against sodium phosphate buffer at 20°C for 48 hours with two changes of the buffer solution. It was then lyophilized and the percentage yield of reconstituted protein was calculated. Besides, elution profiles of the protein on Sephadex gel filtration, DEAE-cellulose chromatography and some PAGE photographs showing the dissociation of rapeseed HMW protein in the presence of (iv) SDS, and SDS plus 2-mercaptoethanol, individual subunits of the native protein and the reconstituted protein are also presented. Chapter 4 presents the complete amino acid sequence of one of the six subunits present in the rapeseed HMW protein. The subunit with the molecular weight (18,300) was chosen for the sequence determination. It was subjected to N-terminal analysis and treated with cyanogen bromide, and digested with enzymes, trypsin, Staphylococcal aureus V8 protease and chymotrypsin to effect specific cleavages. The various fragments produced from chemical and enzymatic cleavages were separated and isolated.The N-terminal sequence analysis by manual Edman method was carried out for each of these fragments. PTH-amino acids obtained during Edman degradation were identified by UV spectroscopy and TLC in different solvent systems. The amino acid composition of all the fragments were in complete agreement with their respective amino acid sequences and the complete amino acid sequence of the subunit containing 172 amino acid residues was established from the individual sequences of the polypeptide fragments, by overlapping technique. Chapter 5 consists of the results of the study of structural class of rapeseed by the correlation coefficient method. The complete amino acid composition of intact rapeseed was established. Using this composition the amino acid frequencies in intact rapeseed was calculated. The correlation coefficients of rapeseed with the four standard structural classes were calculated and the structure class was predicted to be based on maximum correlation coefficient.