Change in kinetic regime of protein aggregation with temperature increase. Thermal aggregation of rabbit muscle creatine kinase.
Creatine kinase thermal aggregation kinetics has been studied in 30 mM Hepes-NaOH buffer, pH 8.0, at two temperatures: 50.6 and 60 degrees 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 degrees C, but at temperature 60 degrees 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 degrees 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 degrees 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 degrees C, change in the kinetic regime of thermal creatine kinase aggregation took place: at 50.6 degrees C aggregation rate is limited by the stage of protein molecule denaturation, but at 60 degrees 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.
Fedurkina
N V
NV
Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow. fedurkina@inbi.ras.ru
Belousova
L V
LV
Mitskevich
L G
LG
Zhou
H-M
HM
Chang
Z
Z
Kurganov
B I
BI
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D018833:Chaperonins / Q000737:chemistry / Q000378:metabolism; D003402:Creatine Kinase / Q000737:chemistry / Q000378:metabolism*; D004795:Enzyme Stability; D006358:Hot Temperature; D018482:Muscle, Skeletal / Q000201:enzymology*; D011489:Protein Denaturation; D011817:Rabbits; D013816:Thermodynamics
D001426:Bacterial Proteins; C431133:Hsp16.3 protein, Mycobacterium tuberculosis; D003402:Creatine Kinase; D018833:Chaperonins