title: Interactive intermediates are formed during the urea unfolding of rhodanese.
abstract: Structural transitions have been studied on the pathway for urea denaturation of rhodanese. Unlike guanidinium hydrochloride, urea gives no visible precipitation. Increasing urea concentrations cause a transition in which the enzyme activity is completely lost by 4.5 M urea, and there is a shift of the intrinsic fluorescence maximum from 335 nm for the native enzyme to 350 nm. There is a maximum exposure of organized hydrophobic surfaces at 4.5 M urea as reported by the fluorescence of 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid. Above 4.5 M urea, this probe reports the progressive loss of organized hydrophobic surfaces. The polarization of the intrinsic fluorescence falls with increasing urea concentrations in a complex transition showing that rhodanese flexibility increases in at least two phases. Rhodanese becomes increasingly susceptible to digestion by subtilisin between 3.5 and 4.5 M urea, giving rise to large fragments. At urea concentrations > 5 M, rhodanese is completely digested. There is a small increase in the rate of sulfhydryl accessibility between 3.5 and 4.5 M urea, but there is a large increase in the sulfhydryl accessibility above 4.5 M urea. Dimethyl suberimidate cross-linking shows the presence of associated species in 3-5 M urea, but there are few cross-linkable species at lower or higher urea concentrations. These results are consistent with a model in which urea unfolding of rhodanese is associated with the initial production of a species having organized regions of structure with exposed hydrophobic surfaces separated by flexible elements.
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
authors.forename: P M
authors.initials: PM
authors.lastname: Horowitz
authors.forename: M
authors.initials: M
authors.lastname: Butler
mesh_terms: D000818:Animals; D002417:Cattle; D003432:Cross-Linking Reagents / Q000737:chemistry; D004120:Dimethyl Suberimidate / Q000737:chemistry; D005454:Fluorescence Polarization; D008099:Liver / Q000201:enzymology; D011489:Protein Denaturation; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013884:Thiosulfate Sulfurtransferase / Q000737:chemistry* / Q000648:ultrastructure; D014508:Urea / Q000737:chemistry
chemical_list: D003432:Cross-Linking Reagents; D013438:Sulfhydryl Compounds; D004120:Dimethyl Suberimidate; D014508:Urea; D013884:Thiosulfate Sulfurtransferase