Mechanism of aggregation of UV-irradiated ß(L)-crystallin.

Thermal denaturation and aggregation of UV-irradiated ß(L)-crystallin from eye lenses of steers have been studied. The data on size-exclusion chromatography and SDS-PAGE indicated that UV irradiation of ß(L)-crystallin at 10 °Ð¡ resulted in fragmentation of the protein molecule and formation of cross-linked aggregates. Fluorescence data showed that tryptophan fluorescence in the irradiated protein decreased exponentially with the UV dose. Decrease in tryptophan fluorescence is a result of photochemical destruction, but not of conformational changes of protein, because there is no red shift in the fluorescence maximum. The differential scanning calorimetry (DSC) profiles of the samples of UV-irradiated and wild type ß(L)-crystallin were registered. The area under curves, which is proportional to the amount of the native protein, decreased exponentially with increasing the irradiation dose. The shape of the DSC profiles for the samples of UV-irradiated ß(L)-crystallin was identical to that for wild type ß(L)-crystallin. The DSC data allowed estimating the portion of UV-denatured ß(L)-crystallin, which is not registered by DSC, and the portion of the combined fraction consisting of native and UV-damaged molecules retaining the native structure. A conclusion has been made that UV-induced denaturation of ß(L)-crystallin follows the one-hit model. The study of the kinetics of thermal aggregation of UV-irradiated ß(L)-crystallin at 37 °Ð¡ using dynamic light scattering showed that the initial stage of aggregation was that of formation of the start aggregates with the hydrodynamic radius of 20 nm. Further sticking of the start aggregates proceeded in the regime of reaction-limited cluster-cluster aggregation. Splitting of the aggregate population into two components occurred above a definite point in time.

Effect of alpha-crystallin on thermal denaturation and aggregation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase.

The study of thermal denaturation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the presence of alpha-crystallin by differential scanning calorimetry (DSC) showed that the position of the maximum on the DSC profile (T(max)) was shifted toward lower temperatures with increasing alpha-crystallin concentration. The diminishing GAPDH stability in the presence of alpha-crystallin has been explained assuming that heating of GAPDH induces dissociation of the tetrameric form of the enzyme into dimers interacting with alpha-crystallin. The dissociation of the enzyme tetramer was shown by sedimentation velocity at 45 degrees C. Suppression of thermal aggregation of GAPDH by alpha-crystallin was studied by dynamic light scattering under the conditions wherein temperature was elevated at a constant rate. The construction of the light scattering intensity versus the hydrodynamic radius (R(h)) plots enabled estimating the hydrodynamic radius of the start aggregates (R(h,0)). When aggregation of GAPDH was studied in the presence of alpha-crystallin, the start aggregates of lesser size were observed.

Mechanism of chaperone-like activity. Suppression of thermal aggregation of betaL-crystallin by alpha-crystallin.

Thermal denaturation and aggregation of beta(L)-crystallin from bovine lens have been studied using differential scanning calorimetry (DSC) and dynamic light scattering (DLS). According to the DLS data, the distribution of the beta(L)-crystallin aggregates by their hydrodynamic radius (R(h)) remains monomodal to the point of precipitating aggregates (sodium phosphate, pH 6.8; 100 mM NaCl; 60 degrees C). The size of the start aggregates (R(h,0)) and duration of the latent stage (t(0)) leading to the formation of the start aggregates have been determined from the light scattering intensity versus the hydrodynamic radius plots and the dependences of R(h) on time. The R(h,0) value remains constant at variation of the beta(L)-crystallin concentration, whereas the t(0) value increases with diminishing beta(L)-crystallin concentration. The suppression of beta(L)-crystallin aggregation by alpha-crystallin is connected with the decrease in the R(h,0) value and increase in the t(0) value. In the presence of alpha-crystallin the aggregate population is split into two components. The first component is represented by stable aggregates whose size remains constant in time. The aggregates of the other kind grow until they reach the size characteristic of aggregates prone to precipitation. The DSC data show that alpha-crystallin has no appreciable influence on thermal denaturation of beta(L)-crystallin.