Kinetics of protein aggregation. Quantitative estimation of the chaperone-like activity in test-systems based on suppression of protein aggregation.
The experimental data on the kinetics of irreversible aggregation of proteins caused by exposure to elevated temperatures or the action of denaturing agents (guanidine hydrochloride, urea) have been analyzed. It was shown that the terminal phase of aggregation followed, as a rule, first order kinetics. For the kinetic curves registered by an increase in the apparent absorbance (A) in time (t) the methods of estimation of the corresponding kinetic parameters A(lim) and kI (A(lim) is the limiting value of A at t --> infinity and kI is the rate constant of the first order) have been proposed. Cases are revealed when the reaction rate constant kI calculated from the kinetic curve of aggregation of the enzymes coincides with the rate constant for enzyme inactivation. Such a situation is interpreted as a case when the rate of aggregation is limited by the stage of denaturation of the enzyme. A conclusion has been made that, in order to establish the mechanism of protein aggregation, the kinetic investigations of aggregation should be carried out over a wide range of protein concentrations. The refolding experiments after denaturation of proteins by guanidine hydrochloride or urea have been also analyzed. It was shown that aggregation accompanying refolding follows first order kinetics at the final phase of the process. The model of protein refolding explaining such a kinetic regularity has been proposed. When aggregation of protein substrate follows first order kinetics, parameters A(lim) and kI may be used for the quantitative characterization of the chaperone-like activity in the test-systems based on suppression of protein aggregation.
Kurganov
B I
BI
Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, 117071, Russia. kurganov@gagarinclub.ru
D000818:Animals; D019791:Guanidine / Q000737:chemistry; D006361:Heating; D007700:Kinetics; D018832:Molecular Chaperones / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*; D014508:Urea / Q000737:chemistry
D018832:Molecular Chaperones; D011506:Proteins; D014508:Urea; D019791:Guanidine