Effect of Mixed Crowding on Refolding of Human Muscle Creatine Kinase

Zhi-Min      Lin; Sen      Li

Abstract

The effects of mixed crowding agents containing both sucrose and dextran 70 on refolding process of human muscle creatine kinase (HCK) were studied by enzyme activity assay and aggregation measurements. The results showed that sucrose and dextran have opposite effect on parameters of HCK during refolding: reactivation yield, refolding rates and amount of aggregation, as they were both used in the mixed crowding agents. The exclusion volume effect of dextran and osmophobic effect of sucrose on HCK refolding can be counteracted by each other: sucrose bated the aggregation induced by dextran and increased the final reactivation yield and refolding rate of the slow track, while dextran inhibited the effect of sucrose to prevent aggregation and help correct folding. The effects of human cyclophilin 18 (hCyp18) and casein on folding of HCK were also studied in crowding conditions, and it was found that the chaperone function of hCyp18 was additive with sucrose but blocked by dextran, and that the aggregation-improving effect of casein was additive with dextran 70 but impaired by sucrose to certain extent. This study indicates osmolytes and macromolecule crowding agents play different roles in the physiological conditions and could lead to better understanding of protein folding in the intracellular environment.

Keywords: Dextran, folding, human muscle creatine kinase, macromolecule crowding, mixed crowding, sucrose, osmolytes, muscle, macromolecule, mixed, (HCK), (hCyp18), crowding agents, lysozyme, GroEL, Gibbs energy, kcat, KM, kcat/KM, BSA, (SDS-PAGE), GBC U/V spectrophotometer, semilogarithmic, kinetic, PPI