Thermally perturbed rhodanese can be protected from inactivation by self-association.

A fluorescence-detected structural transition occurs in the enzyme rhodanese between 30-40 degrees C that leads to inactivation and aggregation, which anomalously decrease with increasing protein concentration. Rhodanese at 8 micrograms/ml is inactivated at 40 degrees C after 50 min of incubation, but it is protected as its concentration is raised, such that above 200 micrograms/ml, there is only slight inactivation for at least 70 min. Inactivation is increased by lauryl maltoside, or by low concentrations of 2-mercaptoethanol. The enzyme is protected by high concentrations of 2-mercaptoethanol or by the substrate, thiosulfate. The fluorescence of 1,8-anilinonaphthalene sulfonate reports the appearance of hydrophobic sites between 30-40 degrees C. Light scattering kinetics at 40 degrees C shows three phases: an initial lag, a relatively rapid increase, and then a more gradual increase. The light scattering decreases under several conditions; at increased protein concentration; at high concentrations of 2-mercaptoethanol; with lauryl maltoside; or with thiosulfate. Aggregated enzyme is inactive, although enzyme can inactivate without significant aggregation. Glutaraldehyde cross-linking shows that rhodanese can form dimers, and that higher molecular weight species are formed at 40 degrees C but not at 23 degrees C. Precipitates formed at 40 degrees C contain monomers with disulfide bonds, dimers, and multimers. We propose that thermally perturbed rhodanese has increased hydrophobic exposure, and it can either: (a) aggregate after a rate-limiting inactivation; or (b) reversibly dimerize and protect itself from inactivation and the formation of large aggregates.