Quantification of anti-aggregation activity of chaperones.

Anti-aggregation potential of the cell is determined by chaperones of a proteinaceous nature (mainly by small heat shock proteins) and low-molecular-weight chemical chaperones. To characterize the anti-aggregation activity of chaperones in vitro, appropriate test systems based, for example, on thermal or dithiothreitol-induced aggregation of model proteins can be used. Aggregation assays usually follow increment in the light scattering intensity or apparent optical absorbance. The initial parts of the dependences of the light scattering intensity (I) on time (t) can be described by quadratic equation: I=[KLS(t-t0)]2, where KLS is a parameter characterizing the initial rate of aggregation and t0 is the duration of lag phase. Based on the dependence of KLS on the initial concentration of the protein [P]0, the power coefficient a in the equation KLS=const [P]0a is determined. The (KLS/KLS,0)1/a versus chaperone concentration plot is used for analysis of the protective action of chaperones. The anti-aggregation activity of protein chaperones is expressed as an adsorption capacity of the chaperone with respect to target protein. The anti-aggregation activity of chemical chaperones is expressed as a semi-saturation concentration of the chaperone, i.e., the concentration of chaperone at which (KLS/KLS,0)1/a=0.5.