Low concentrations of guanidinium chloride expose apolar surfaces and cause differential perturbation in catalytic intermediates of rhodanese.
The conformations of sulfur-free and sulfur-containing rhodanese were followed with and without the detergent lauryl maltoside after guanidinium chloride (GdmCl) addition to 5 M to study the apparent irreversibility of denaturation. Without lauryl maltoside, sulfur-containing rhodanese denatured in a transition giving, at approximately 2.3 M GdmCl, 50% of the total denaturation induced change observed by activity, CD, or intrinsic fluorescence. Sulfur-free rhodanese gave more complex behavior by intrinsic fluorescence and CD. CD showed loss of secondary structure in a broad, complex, and apparently biphasic transition extending from 0.5 to 3 M GdmCl. The interpretation of the transition was complicated by time-dependent aggregation due to noncovalent interactions. Results with the apolar fluorescence probe 2-anilinonaphthalene-8-sulfonic acid, implicated apolar exposure in aggregation. Sulfhydryl reactivity indicated that low GdmCl concentrations induced intermediates affecting the active site conformation. Lauryl maltoside prevented aggregation with no effect on activity or any conformational parameter of native enzyme. Transitions induced by GdmCl were still observed and consistent with several phases. Even in lauryl maltoside, an increase in apolar exposure was detected by 2-anilinonaphthalene-8-sulfonic acid, and by protein adsorption to octyl-Sepharose well below the major unfolding transitions. These results are interpreted with a model in which apolar interdomain interactions are disrupted, thereby increasing active site accessibility, before the intradomain interactions.
Horowitz
P
P
Criscimagna
N L
NL
D000817:Anilino Naphthalenesulfonates; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002417:Cattle; D002942:Circular Dichroism; D005456:Fluorescent Dyes; D005960:Glucosides / Q000494:pharmacology; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology*; D046911:Macromolecular Substances; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D013455:Sulfur; D013466:Sulfurtransferases; D013884:Thiosulfate Sulfurtransferase
C050774:2-anilinonaphthalene-8-sulfonic acid; D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D005960:Glucosides; D006146:Guanidines; D046911:Macromolecular Substances; C040358:dodecyl maltoside; D013455:Sulfur; D013466:Sulfurtransferases; D013884:Thiosulfate Sulfurtransferase; D019791:Guanidine