Dungan J M, Horowitz P M
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
J Protein Chem. 1993 Jun;12(3):311-21. doi: 10.1007/BF01028193.
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.
在30至40摄氏度之间，硫氰酸酶会发生荧光检测到的结构转变，导致酶失活和聚集，而这种现象会随着蛋白质浓度的增加而异常减少。8微克/毫升的硫氰酸酶在40摄氏度下孵育50分钟后会失活，但随着其浓度升高，它受到保护，以至于在200微克/毫升以上，至少70分钟内只有轻微失活。月桂基麦芽糖苷或低浓度的2-巯基乙醇会增加失活。该酶受到高浓度的2-巯基乙醇或底物硫代硫酸盐的保护。1,8-苯胺基萘磺酸盐的荧光显示在30至40摄氏度之间出现疏水位点。40摄氏度下的光散射动力学显示出三个阶段：初始延迟、相对快速增加，然后是更逐渐的增加。在几种情况下光散射会降低；在蛋白质浓度增加时；在高浓度的2-巯基乙醇存在时；在月桂基麦芽糖苷存在时；或在硫代硫酸盐存在时。聚集的酶无活性，尽管酶可以在没有明显聚集的情况下失活。戊二醛交联表明硫氰酸酶可以形成二聚体，并且在40摄氏度下会形成更高分子量的物种，而在23摄氏度下则不会。在40摄氏度下形成的沉淀物包含具有二硫键的单体、二聚体和多聚体。我们提出，受热扰动的硫氰酸酶增加了疏水暴露，并且它可以：(a) 在限速失活后聚集；或(b) 可逆地二聚化并保护自身免于失活和形成大聚集体。
