AMINO ACID SEQUENCING OF ARACHIN SUBUNIT AND STUDY OF STRUCTURAL CLASS OF ARACHIN

Abstract

Proteins are among the most important components of all living systems. They are of great nutritional importance and are directly involved in the chemical processes for the maintenance of life. Proteins primarily are composed of about twenty amino acids differing in the structure of its side chain R, linked together by peptide bonds in chains through various permutations and combinations of their arrangements that may consist of a few dozen to more than a thousand amino acids. Proteins are all functionally specific, and this specificity is due to the specific sequence in which the amino acids are bound. A single disturbance in the sequence of amino acids can lead to biological, pathological or physiological disorder. Due to biological activity dependence on the primary structure of the protein, it becomes important to know the amino acid sequence. Further, it is useful in the study of gene structure and evolution and provides a link between genetic message in DNA and three dimensional structure. Also, the amino acid sequence of a protein can be related through the genetic code to the nucleotide sequence of the gene that directed its synthesis, and small medicinally useful polypeptides can be synthesized chemically. The determination of the primary structure of polypeptides and proteins is a formidable task. It is accomplished generally by cleaving the larger chains into smaller and manageable segments and determining their sequence first. The method of stepwise degradation of the peptides by PITC devised by P. Edman virtually lies at the core of all modern day sequencing. i • 11 Peanut, member of the family Leguminosae, has attracted attention towards itself being one of the largest oil seed crop world wide, it is also a rich source of protein, vitamin B -B , nicotinic acid, vitamin E and vitamin B„. They contain an average of 45-50°/ oil, 25-30% protein, b 5-12°/. carbohydrates, about 3% of crude fibre, 2.5% of ash and 10-12% of non protein nitrogen. Peanuts contain two main storage proteins arachin and conarachin. Arachin, the major storage protein is obtained by ammonium sulphate saturation upto 40% and conarachin I and conarachin II by 60% and 80% saturation respectively. Plant proteins being easily available and having high nutritional value are of use in todays life. Since scanty attention was being paid towards structural aspects of plant proteins and due to increasing interest in peanut proteins, studies on structure of arachin have been carried out. Peanut protein arachin contains six different subunits. The present studies include reconstitution behaviour of arachin from its subunits, complete amino acid sequence of a subunit of arachin, the determination of structural class of arachin by correlation coefficient method. During these sequencing studies some new TLC systems for the identification of unknown PTH-amino acids obtained during the sequence determination by manual Edman degradation, were also developed and included here. The thesis comprises of the following five chapters: Chapter 1 General Introduction. Chapter 2 Materials and Equipment. m Chapter 3 A. Studies on the Reconstitution Behaviour of Arachin Subunits. B. Sequence Determination of the Subunit. Chapter 4 Study of Structural Class of Arachin. Chapter 5 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 peanut proteins. Chapter 2 gives the details of materials and equipment regarding their make, purity and other specifications. Chapter 3A describes the isolation, purification, characterisation, molecular weight determination, subunit composition of arachin subunit,' molecular weights and weight ratios of different subunits, amino acid composition of the different subunits and reconstitution behaviour of arachin and part B deals with complete amino acid sequence of one of arachins subunit. Arachin was isolated from the defatted peanut meal in 10% NaCl by ammonium sulphate fractionation. The protein was purified by dialysis and its purity ascertained by DEAE-cellulose chromatography and polyacrylamide gel electrophoresis (PAGE) under different gel concentrations. IV Arachin dissociated into six different subunits in the presence of SDS. The molecular weights of arachin and its six subunits was determined by SDS-PAGE. SDS-PAGE in the presence of 2-mercaptoethanol still showed six subunits thereby indicating the absence of any S-S linkages in arachin. The subunits were separated and isolated by Sephadex gel filtration and PAGE, and purified by dialysis and were lyophilized. Their concentration were determined by UV and Folin Lowry method. The amino acid analysis was performed for intact arachin as well for its different subunits. Arachin was allowed to reconstitute from the isolated subunits by removing SDS. The isolated six subunits of arachin were separately dissolved in phosphate buffer and were mixed in their weight ratios. The mixture of the subunits was dialyzed against sodium phosphate buffer at 20° 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 DEAE-cellulose chromatography and PAGE showing the dissociation and reconstituted proteins are also presented. For the complete amino acid sequence,one of the arachin subunit was subjected to direct Edman degradation and was digested with enzymes trypsin, Staphylococcal aureus V8 protease and chymotrypsin to effect specific cleavages. The various fragments obtained from these cleavages were separated and isolated. Manual Edman degradation on each fragment was carried out to determine the N-terminal sequence. The PTH-amino acids obtained were identified by UV 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 was established from the individual fragment sequences by overlapping technique. Chapter 4 deals with the study of structural class of arachin by the correlation coefficient method. The complete amino acid composition of intact arachin was established. Using this composition the amino acid frequencies in intact arachin was calculated. The correlation coefficients of arachin with the four standard structural classes were calculated and the structural class was predicted to be B based on maximum correlation coefficient. Chapter 5 deals with the development of new TLC systems for identification and resolution of PTH-amino acids. For the reliable identification of the PTH-amino acids TLC was chosen as one of the methods as it was sensitive, inexpensive, reliable and rapid. Therefore, simple TLC systems were developed to identify several of the PTH- amino acids, of which the solvent system acetone-dichloromethane (0.3:8) was the most effective in resolving many of the difficult combinations. The solvent systems developed were actually employed during the course of studies on sequencing of the arachin subunit.