J. Plant Biochemistry & BiotechnologyVol. 7,13-21, January 1998 Complete Amino Acid Sequence of a Subunit from Rapeseed Protei n Ravi Bhushan and Anil K Tyagi Department of Chemistry, University of Roorkee, Roorkee 247 667, India A subunit of molecular weight 18300 has been separated and isolated from seeds of Brasslca campesfr;s L. This subunlt was cleaved by using cyanogen bromide, trypsin, Staphylococcus aureus V8 protease and chymotrypsin; the fragments obtained from enzymatlc and chemical cleavages were separated and isolated by polyacrylamide gel electrophoresis and gel filtration. The amino acid analyses were carried out. The complete amino acid sequence of the subunit containing 172 amino acid residues has been established by manual Edman method. Key words: amino acid sequence, protein subunit, rapeseed. Rapeseed holds an important place among oilseeds as it contains substantially higher content of sulphur containing amino acids than soy protein as well as adequate amounts of other essential amino acids and due to high contents of Iys, met and cys it has a nutritive value as high as good animal protein and more than any other known vegetable protein (1). Because of their importance, rapeseed proteins have allowed their characterization and elucidation of their gene nucleotide sequences (2, 5). Rapeseed contains two major types of storage proteins, one is a 12S neutral globulin (6-8) and the others are 1 .7S small basic albumin (9). These storage proteins are of interest in clinical research and they also supply an adequate amount of nitrogen to germinating seedlings. Reports on the reconstitution of the rapeseed HMW protein from isolated subunits along with the amino acid composition of the native protein and the individual subunits (10) and hydrogen ion titration of the 12S rapeseed HMW (11), and complete amino acid sequence of one of the subunits (20), have .already been made from this laboratory. The amino acid composition (6,8,12- 14), effect of denaturants (15,16), subunit composition (17,18) and the secondary structure (19) of the proteins obtained from different rapeseed varieties have been reported. Sequence determination of a protein is a significant step towards the elucidation of the molecular basis of its biological activity. The amino acid sequence is the link between the genetic message in DNA and three dimensional structure is the basis of protein's biological function. Further, amino acid sequence of a protein reveals much about its evolutionary history. Literature shows that HMW protein from rapeseed variety sarson prain cultivar type 42 has six subunits (10), complete amino acid sequence of one of them of molecular weight 15,600 has been reported from this lab (20). Therefore, the complete amino acid sequence of a subunit of molecular weight 18,300 of the high molecular weight protein isolated from Brassica campestris L. variety YID-1 obtained from the Indian Agricultural Research Institute, New Delhi, has been established and presented here in this study. Materials and Methods Materials - Powdered rapeseed (500 g) of the mentioned variety was defatted by refluxing with n-hexane at 60°C and the defatted rapeseed meal was air dried. All the reagents and chemicals used were of analytical grade (Pierce, Sigma, SISCO and Fluka). Standard proteins were from Sigma. TPCK-treated trypsin (Sigma), S. aureus V8 protease (Miles), chymotrypsin and cyanogen bromide (SISCO Research laboratory, Bombay, India) were used. Standard phenylthiohydantoin (PTH) amino acids were synthesized (21) and their purity was ascertained by ultraviolet spectroscopy (21) and TLC (22- 24). The UV spectra were recorded on a Beckman DU- 6 UV spectrophotometer. Pharmacia gel electrophoresis apparatus model GE2I4LS was used. Waters amino acid analyser model 501 was used for amino acid analysis. Separation and recovery of subunits - The six different subunits of the protein were separated on PAGE using 10% polyacrylamide gels. Gly-NaOH buffer (pH 9.0) was employed as the electrode buffer. Samples of the protein pretreated with SOS (0.01%) in the presence and/or absence of 0.2 M 2-mercaptoethanol were applied to PAG-rods and a current of 2mAltube was applied for 90 min (11). The gels were fixed in 10% sulphosalicylic acid solution. The protein bands were located on the gel rods by staining with Commassie brilliant blue R-250 (0.1% in MeOH-HOAc-H 2 0, 45:10:45) followed by destaining with 7% acetic acid. The coloured bands, six in all,