17E-5 |
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H. H. KSHIRSAGAR, M. Venkatachalam, and S. K. Sathe. Dept. of Nutrition, Food & Exercise Sciences, Florida State Univ., 402 Sandels Bldg., Mail code 1493, Tallahassee, FL 32306-1493 Most investigations concerned with soybean protein chemical denaturation typically use a large molar excess of chemical denaturant(s) and often, crude proteins. The purpose of the present study was to use purified soybean proteins and appropriate denaturant concentrations that would permit evaluation of subtle protein structural changes. Soybean (Century cultivar) 11S (glycinin) and 7S (b-conglycinin) were prepared using the procedure described by Nagano et al [JAFC 40(6):941-944, 1992]. Stock protein solutions (50 mg/ml) were prepared in sodium phosphate buffer (0.02 M, pH 7.5). Protein structural changes caused by urea (0-6 M), guanidine-HCl (0-6 M), and SDS (0-10 mM) were determined by monitoring intrinsic fluorescence (quenching studies) and circular dichroic (CD) spectroscopy. In the 11S control, 73% and 26% tryptophan residues were accessible to acrylamide and iodide quenching, respectively while the corresponding values for 7S were 91% and 65%. A low Stern-Volmer quenching constant (KSV=2.13) further suggested significant number of 11S tryptophan residues to be buried in the hydrophobic region of molecule. At 2 M guanidine HCl a sharp rise in KSV (increase range 3-4 units) coupled with an increased accessibility to quenchers (accessibility ranges were 77-100% for acrylamide and 47-100% for iodide), indicated substantial denaturation of 11S. At urea concentrations >1 M, 11S and 7S denaturation occurred in a one-step pattern. As expected, at > 2 mM SDS, both proteins registered maximum tryptophan exposure due to complete protein denaturation. At 0-0.2 mM SDS, steady increase in shoulder at 222 nm indicated a gradual increase in helicity while at 0.5-2 mM SDS, increased signal intensity at 198 nm suggesting increase in random coil. Quenching results indicated acrylamide to be a more effective quencher than iodide. The results suggest that further studies should be completed to fully understand the stages involved in 11S and 7S denaturation.
Session 17E, Food Chemistry: Proteins
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