STUDIES ON RAPESEED (Brassica campestris L.) HIGH MOLECULAR WEIGHT PROTEIN

Abstract

Proteins are among the most important nitrogeneous components of the living cells in animals and plants. Their functions range from catalysts (enzymes) to regulators to structural components. A significant proportion of an animal is in the form of protein. They are the principal constituents of skin, muscle, blood, cartilage, hair, nerves, antibodies, enzymes and many hormones. All animals require a constant supply of new proteins, both for replacement of old protein ard for growth. The building blocks of proteins are about twenty (X-amino acids [NH2CH(R)COOH] of the reconfiguration, all differing in the structure of R, linked together by peptide bonds (-CO-NH-) in chains that may consist of a few dozen to 1000 amino acid residues. The protein molecule may contain one long polypeptide chain or many such chains linked together by different kinds of bonds. These polypeptide chains are referred to as subunits. The sequence determines conformation of the protein which regulates its functional specificity. The sequence determination becomes important because it is useful in the study of gene structure and evolution and provides a link between genetic message in DNA and three dimensional structure. Further, the amino acid sequence of a protein can be related through the genetic code to the nucleotide sequence of the gene that directed its 11 synthesis, and small medicinally useful polypeptides can be synthesized chemically. The determination of primary structure of proteins, that is the arrangement of various amino acid residues along the polypeptide chain is a formidable task. The progress in sequence analysis over the last few decades has been made possible by the introduction of stepwise degradation of peptides and proteins. Edman degradation of peptides and proteins by phenylisothiocyanate virtually lies at the core of all modern sequencing strategies. An increasing interest in Brassicaceae and particularly in rapeseed (Brassica species) proteins as a consequence of their being one of the most important oilseed crops in the world has allowed the characterization and elucidation of their gene nucleotide sequences. Rapeseed contains two major types of storage proteins, one is a 12S neutral globulin and the others are 1.7S small basic albumins. The main function of the storage proteins is to provide an adequate nitrogen supply to the germinating seedling and they are also of interest in clinical research. 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 (HMW) protein have been carried out. Ill Rapeseed (Brassica campestris L.) high molecular weight (HMW) protein contains six different subunits. The present studies include the reconstitution of the HMW protein from isolated subunits, the complete primary structural determination of one of the six subunits of the protein, the hydrogen ion titration of the protein and development of rapid TLC systems for the identification of unknown PTHamino acids obtained during the sequence determination by manual Edman degradation. The thesis comprises of the following six chapters: Chapter 1 General Introduction Chapter 2 Materials and Equipment Chapter 3 Studies on the Reconstitution of the HMW Protein Chapter 4 Amino Acid Sequence of a Subunit of the HMW Protein Chapter 5 Hydrogen Ion Titration of the HMW Protein Chapter 6 TLC of PTH-Amino Acids Chapter 1 is an introduction to the nature, structure and importance of proteins and sequencing studies. In addition, it briefly reviews the work reported in the literature on the chemistry of rapeseed proteins. IV Chapter 2 gives the details of materials and equipment regarding their make, purity and other specifications. Chapter 3 describes the isolation, purification, molecular weight determination, subunit composition of the rapeseed protein, molecular weights and weight ratios of different subunits, first two N-terminal amino acid residues of each subunit, amino acid composition of the native protein and the different subunits and the results of the reconstitution of the rapeseed HMW protein from isolated subunits. The high molecular weight protein was isolated from a 10% sodium chloride extract of the defatted rapeseed meal, by ammonium sulphate fractionation. 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 dissociated into six different subunits in the presence of SDS. Molecular weights of the intact protein and the individual subunits were determined by SDS-PAGE. SDS-PAGE in presence and/or absence of 2-mercaptoethanol indicated that there were no S-S linkages in the protein. 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 calculated. The HMW protein was allowed to reconstitute from the isolated subunits by removing SDS. The six isolated 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 and DEAEcellulose chromatography and some PAGE photographs showing the dissociation of rapeseed HMW protein in the presence of SDS, 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 lowest molecular weight (15600) was chosen for the sequence determination. It was subjected to N-terminal analysis and treated with cyanogen bromide, and digested with the 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 VI 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 125 amino acid residues was established from the individual sequences of the polypeptide fragments, by overlapping technique. Chapter 5 consists of the results of the hydrogen ion titration of the rapeseed high molecular weight protein. Hydrogen ion titration was performed on the pure rapeseed protein at 30°C in 0.5 M KC1 solution (u -0.15). The titration curve was obtained by plotting the number of hydrogen ions dissociated against pH of the solution. The titration curve was analyzed to estimate the number of side chain carboxyl, imidazole and e-amino groups. The information derived from the titration curve was correlated with the amino acid assay of the protein and found to be in good agreement with it. The hydrogen ion titration curve of the protein is also presented. Chapter 6 deals with the development of new TLC systems for identification and resolution fo PTH-amino acids. The reliable identification of PTH-amino acids obtained from each Edman degradation step by two complementary techniques is needed. TLC was chosen as one of the methods Vll for the identification of PTH-amino acids because it is an inexpensive, simple, reliable, rapid and sensitive analytical technique. During the studies on sequence determination the need to identify/resolve certain combinations of PTH-amino acids arose and literature also did not reveal any solution to these. Therefore, simple TLC systems were developed to identify several of PTH-amino acids, of which the solvent systems chloroform-acetic acid (9:1, v/v) and chloroform-ethanol (15:2, v/v) were found to be very effective in resolving and identifying PTH-asp and PTH-glu. The solvent systems developed were actually employed during the course of studies on sequencing of the subunit of the HMW protein.