Interactive intermediates are formed during the urea unfolding of rhodanese.
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.
Horowitz
P M
PM
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
Butler
M
M
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
D003432:Cross-Linking Reagents; D013438:Sulfhydryl Compounds; D004120:Dimethyl Suberimidate; D014508:Urea; D013884:Thiosulfate Sulfurtransferase