STUDIES ON PEANUT PROTEIN (ARACHIN) WITH SPECIAL REFERENCE TO SUBUNITS AND MOLECULAR SPECIES

Abstract

Naturally occurring proteins may exist either as a single polypeptide chain or several polypeptide chains are intervened 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 physiochemical 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. Peanut has attracted attention towards itself being one of the largest cultivated economic crop in India. It contains an average of 45-50% oil, 25-30% protein, 5-12%o carbohydrates, about 3% crude fibre, 2.5%. ash and 10-12%. non-protein nitrogen. Peanut contains two main storage proteins arachin and conarachin. Since the fractionation of original globulin (in 1880 by Ritthausen) into arachin and conarachin by the salting out technique many fundamental investigations on the nature of these proteins have revealed an increasing degree of complexity with increasing sophistication of techniques employed. The existing literature suggested that there was a need to investigate the homogeneity, behaviour and characteristics of molecular species of arachin : arachin I and arachin II, the different states of aggregation occurring with changes in the chemical nature, pH and ionic strength of the solutions. The (0 studies were also carried out to investigate the association-dissociation phenomenon of the complex protein, arachin. The present thesis comprises of six Chapters. The first Chapter General Introduction is an introductory one dealing with an introduction to the nature, structure and importance of proteins. In addition, it briefly reviews the work reported in the literature on the chemistry of peanut proteins and different techniques used to investigate the nature of arachin. Second Chapter Materials and Equipment gives the details of materials and equipment regarding their make, purity and other specifications. Third Chapter Molecular Species of Arachin describes the isolation, purification, molecular weight determination and weight ratios of arachin molecular species : arachin I and arachin II using different chromatographic techniques such as gel filtration, polyacrylamide gel electrophoresis and reversed-phase high performance liquid chromatography. Associationdissociation behaviour of these molecular species was also studied using polyacrylamide gel electrophoresis. Peanut meal was defatted and extracted with 10%NaCl solution. Arachin, the high molecular weight protein, was obtained by fractional precipitation with ammonium sulfate using upto 40% saturation. The protein was purified by reprecipitation and repeated dialysis. Fourth Chapter The Subunits gives detail account of studies on the number and kinds of subunits of arachin, arachin I and arachin II. Studies were carried out under different experimental conditions using slab gel electrophoresis, size-exclusion chromatography and reversed-phase high performance liquid (ii) chromatography to separate subunits of arachin, arachin I and arachin II. Seralose-6B, Sephadex G-100 and Sephadex G-150 were used to separate arachin subunits by size-exclusion chromatography. Gel electrophoresis was done under varying concentrations of gel 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 Arachin I Subunits deals with the reconstitution of arachin I subunits (SI to S6) to parent arachin I using SDSPAGE. Arachin I was allowed to reconstitute from the isolated subunits (SI to S6) by removing SDS. Seven combinations were tried for studying the reconstitution pattern. For example, subunits (SI to S5) were mixed in their weight ratio and codialysed against tris-HCl buffer. Similarly S6 was dialysed separately which was later on mixed in its weight ratio with the mixture (SI to S5). The combined subunits (SI to 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 subunits as above. Each time they were mixed in their weight ratios. In another set of experiment all the six subunits were dialysed together in the similar manner. Thus, seven 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. (iii) Sixth Chapter Synthesis and Liquid Chromatography of PTH-Amino Acids deals with synthesis, characterization and separation of PTH-amino acids under different liquid chromatographic conditions. Some new TLC, RP-TLC and RP-HPLC methods for the separation and identification of certain PTH-amino acids were systematically developed, and are presented in the thesis. These were also used for the identification of N-terminal residues obtained from the Edman degradation of each of the six subunits of arachin I.