Change in Kinetic Regime of Protein Aggregation with Temperature Increase. Thermal Aggregation of Rabbit Muscle Creatine Kinase

---

N. V. Fedurkina^1*, L. V. Belousova², L. G. Mitskevich¹, H.-M. Zhou³, Z. Chang⁴, and B. I. Kurganov¹

¹Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky pr. 33, 119071 Moscow, Russia; fax: (7-495) 954-2732; E-mail: fedurkina@inbi.ras.ru

²Department of Biochemistry, Biological Faculty, Lomonosov Moscow State University, 119992 Moscow, Russia; E-mail: anton@protein.bio.msu.ru

³Department of Biological Science and Biotechnology, Tsinghua University, Beijing 100084, P. R. China; E-mail: zhm-dbs@mail.tsinghua.edu.cn

⁴Shool of Life Science, Peking University, Beijing, 100871, P. R. China; E-mail: chang@pku.edu.cn

^* To whom correspondence should be addressed.

Received April 8, 2005; Revision received May 24, 2005

---

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.

---

KEY WORDS: creatine kinase, thermal aggregation, kinetics, small heat shock protein Hsp 16.3

DOI: 10.1134/S000629790603014X

---