Change in kinetic regime of protein aggregation with temperature increase. Thermal aggregation of rabbit muscle creatine kinase

Abstract

Creatine kinase thermal aggregation kinetics has been studied in 30 mM Hepes-NaOH buffer, pH 8.0, at two temperatures: 50.6 and 60°C. Aggregation kinetics was analyzed by measuring the growth of apparent absorption (A) at 400 nm. It was found that the limiting value of apparent absorption (A _lim) is proportional to protein concentration at both temperatures. The first order rate constant (k _I) does not depend on protein concentration in the range 0.05–0.2 mg/ml at temperature 50.6°C, but at temperature 60°C it increases with the growth of protein concentration in the range 0.1–0.4 mg/ml. Kinetic curves, shown in coordinates {A/A _lim; t}, in experiments at 50.6°C fuse to a common curve, which coincides with the theoretical curve of creatine kinase denaturation calculated using the denaturation rate constant determined from differential scanning calorimetry. At temperature 60°C, half-transformation time t _1/2 = ln2/k _I decreases when protein concentration grows. We conclude that when temperature increased from 50.6 to 60°C, change in the kinetic regime of thermal creatine kinase aggregation took place: at 50.6°C aggregation rate is limited by the stage of protein molecule denaturation, but at 60°C it is limited by the stage of protein aggregate growth, which proceeds as a reaction of pseudo-first order. Small heat shock protein Hsp 16.3 Mycobacterium tuberculosis suppresses the creatine kinase aggregation.