Refolding Increases the Chaperone-like Activity of αH-Crystallin and Reduces Its Hydrodynamic Diameter to That of α-Crystallin.

αH-Crystallin, a high molecular weight form of α-crystallin, is one of the major proteins in the lens nucleus. This high molecular weight aggregate (HMWA) plays an important role in the pathogenesis of cataracts. We have shown that the chaperone-like activity of HMWA is 40% of that of α-crystallin from the lens cortex. Refolding with urea significantly increased-up to 260%-the chaperone-like activity of α-crystallin and slightly reduced its hydrodynamic diameter (Dh). HMWA refolding resulted in an increase in chaperone-like activity up to 120% and a significant reduction of Dh of protein particles compared with that of α-crystallin. It was shown that the chaperone-like activity of HMWA, α-crystallin, and refolded α-crystallin but not refolded HMWA was strongly correlated with the denaturation enthalpy measured with differential scanning calorimetry (DSC). The DSC data demonstrated a significant increase in the native protein portion of refolded α-crystallin in comparison with authentic α-crystallin; however, the denaturation enthalpy of refolded HMWA was significantly decreased in comparison with authentic HMWA. The authors suggested that the increase in the chaperone-like activity of both α-crystallin and HMWA could be the result of the correction of misfolded proteins during renaturation and the rearrangement of protein supramolecular structures.

Oxidative damage to ßL-crystallin in vitro by iron compounds formed in physiological buffers.

ßL-crystallin aggregation due to oxidative damage in the presence of H2O2 and ferric chloride was studied in-vitro under conditions close to physiological. It was shown that the protein aggregation characterized by the nucleation time and the aggregation rate significantly depended on the composition of the isoosmotic buffers used, and decreased in the series HEPES buffer > Tris buffer > PBS. Ferric chloride at neutral pH was converted into water-insoluble iron hydroxide III (≡FeIIIOH). According to the data of scanning electron microscopy the ≡FeIIIOH particles formed in HEPES buffer, Tris buffer, and PBS practically did not differ in structure. However, the sizes of ≡FeIIIOH floating particles measured by dynamic light scattering differed significantly and were 44 ± 28 nm, 93 ± 66 nm, 433 ± 316 nm (Zaver ± SD) for HEPES buffer, Tris buffer, and PBS, respectively. It was found by the spin trap method that the ability of ≡FeIIIOH to decompose H2O2 with the formation of a •OH decreases in the series HEPES buffer, Tris buffer, and PBS. The authors suggest that the ability to generate •OH during the decomposition of H2O2 is determined by the total surface area of ≡FeIIIOH particles, which significantly depends on the composition of the buffer in which these particles are formed.