Two new solvent systems, n-hexane + propionic acid (26:5, v/v) and chloroform + acetone (29:3, v/v), for the rapid resolution and identification of an 18-component mixture of phenylthiohydantoin amino acids are reported. Using these systems certain difficult combinations of phenylthiohydantoin amino acids are resolved. Two more solvent systems, viz chloroform + acetic acid (27:3, v/v) and chloroform + methanol (30:4, v/v), are developed to resolve phenylthiodantoin derivatives of aspartic and glutamic acids.
A subunit (M, 15 600) from the high molecular weight protein from rapeseed was separated and isolated; its purity and homogeneity were ascertained. The subunit was cleaved with cyanogen bromide, trypsin, chymotrypsin, and Staphylococcus aureus V8 protease. The fragments were separated and isolated by polyacrylamide gel electrophoresis, gel filtration, column chromatography on Dowex 1 x 2, and paper electrophoresis. The amino acid compositions of the intact subunit and different fragments obtained from enzymatic and chemical cleavages were determined. The subunit and its fragments were sequenced by manual Edman method. The phenylthiohydantoin amino acids obtained after each step were identified by thin-layer chromatography and ultraviolet spectroscopy. The complete amino acid sequence of the subunit consisting of 125 amino acid residues has been established by the overlapping method.
The high molecular weight protein was isolated from rapeseed and characterised. Six subunits were isolated in SDS (0.01%) solution on polyacrylamide gel electrophoresis and by gel filtration on Sephadex G‐100. Reassociation by removing SDS by co‐dialysis, against 10mM sodium phosphate buffer (pH 7.9) was done and the yield was about 90%. The reconstituted protein was indistinguishable from the intact protein in all respects. The subunits isolated from the native protein and the reconstituted protein were found to have identical molecular weights and N‐terminal amino acids. No disulphide bonds were observed in the subunit association. Amino acid analysis of the proteins and the six subunits was performed and the number of each amino acid residue calculated.
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