title: Biophysical analysis of phaseolin denaturation induced by urea, guanidinium chloride, pH, and temperature.
abstract: The structural stability of phaseolin was determined by using absorbance, circular dichroism (CD), fluorescence emission, and fluorescence polarization anisotropy to monitor denaturation induced by urea, guanidinium chloride (GdmCl), pH changes, increasing temperature, or a combination thereof. Initial results indicated that phaseolin remained folded to a similar extent in the presence or absence of 6.0 M urea or GdmCl at room temperature. In 6.0 M GdmCl, phaseolin denatures at approximately 65 degrees C when probed with absorbance, CD, and fluorescence polarization anisotropy. The transition occurs at lower temperatures by decreasing pH. Kinetic measurements of denaturation using CD indicated that the denaturation is slow below 55 degrees C and is associated with an activation energy of 52 kcal/mol in 6.0 M GdmCl. In addition, kinetic measurement using fluorescence emission indicated that the single tryptophan residue was sensitive to at least two steps of the denaturation process. The fluorescence emission appeared to reflect some other structural perturbation than protein denaturation, as fluorescence inflection occurred approximately 5 degrees C prior to the changes observed in absorbance, CD, and fluorescence polarization anisotropy.
authors.affiliation: Department of Biochemistry, Louisiana State University, Baton Rouge 70803.
authors.forename: J M
authors.initials: JM
authors.lastname: Dyer
authors.forename: J W
authors.initials: JW
authors.lastname: Nelson
authors.forename: N
authors.initials: N
authors.lastname: Murai
mesh_terms: D002942:Circular Dichroism; D005454:Fluorescence Polarization; D019791:Guanidine; D006146:Guanidines / Q000737:chemistry*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010940:Plant Proteins / Q000737:chemistry*; D011489:Protein Denaturation; D014508:Urea / Q000737:chemistry*
chemical_list: D006146:Guanidines; D010940:Plant Proteins; C048826:phaseolin protein, Phaseolus vulgaris; D014508:Urea; D019791:Guanidine