Thermal aggregation of bovine serum albumin (BSA) has been studied using dynamic light scattering, asymmetric flow field-flow fractionation and analytical ultracentrifugation. The studies were carried out at fixed temperatures (60°C, 65°C, 70°C and 80°C) in 0.1 M phosphate buffer, pH 7.0, at BSA concentration of 1 mg/ml. Thermal denaturation of the protein was studied by differential scanning calorimetry. Analysis of the experimental data shows that at 65°C the stage of protein unfolding and individual stages of protein aggregation are markedly separated in time. This circumstance allowed us to propose the following mechanism of thermal aggregation of BSA. Protein unfolding results in the formation of two forms of the non-native protein with different propensity to aggregation. One of the forms (highly reactive unfolded form, U_hr) is characterized by a high rate of aggregation. Aggregation of U_hr leads to the formation of primary aggregates with the hydrodynamic radius (R_h,1) of 10.3 nm. The second form (low reactive unfolded form, U_lr) participates in the aggregation process by its attachment to the primary aggregates produced by the U_hr form and possesses ability for self-aggregation with formation of stable small-sized aggregates (A_st). At complete exhaustion of U_lr, secondary aggregates with the hydrodynamic radius (R_h,2) of 12.8 nm are formed. At 60°C the rates of unfolding and aggregation are commensurate, at 70°C the rates of formation of the primary and secondary aggregates are commensurate, at 80°C the registration of the initial stages of aggregation is complicated by formation of large-sized aggregates.

Heat shock proteins (hsps) are involved in multiple cellular processes during normal and stress conditions, particularly in the folding of polypeptides. A newly recognized property of the members of the Hsp70 family is their ability to interact with lipids, opening ion conductance pathways in artificial membranes, and integrating into natural membranes. The formation of Hsp70 channels in biological membranes and their function is still elusive. In this study, we showed that Hsp70 and Hsc70 display a highly selective interaction with phosphatidylserine moieties on membranes, followed by rapid incorporation into the lipid bilayer. Addition of Hsp70 or Hsc70 into the extracellular medium resulted in a viability decrease of cells beading PS on the exterior surface, such as PC12 cells. This toxic effect is modulated by the presence of ATP or ADP and can be blocked by screening PS moieties with annexin 5. These observations suggest that the presence of Hsp70 in the extracellular medium may be an accelerator of apoptosis since the presence of PS on the surface is an early indicator of this process. These findings may also explain the toxicity observed in cells overexpressing Hsp70s and provide a rational for the tight regulation of Hsp70 expression.—Arispe, N., Doh, M., Simakova, O., Kurganov, B., De Maio, A. Hsc70 and Hsp70 interact with phosphatidylserine on the surface of PC12 cells resulting in a decrease of viabifity.FASEB J . 18, 1636–1645 (2004)