ABSTRACT
Effect of 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD) on thermal aggregation of creatine kinase from rabbit skeletal muscle (RMCK) at 48 degrees C has been studied using dynamic light scattering. An increase in the duration of the lag period on the kinetic curves of aggregation, registered as an increment of the light scattering intensity in time, has been observed in the presence of HP-beta-CD. It has been shown that the initial parts of the dependences of the hydrodynamic radius (R(h)) of the protein aggregates on time follow the exponential law. The reciprocal value of parameter t(2R) (t(2R) is the time interval over which the R(h) value is doubled) was used to characterize the rate of aggregation. A 10-fold decrease in the 1/t(2R) value was observed in the presence of 76mM HP-beta-CD. Judging from the data on the kinetics of RMCK inactivation and the data on differential scanning calorimetry of RMCK, HP-beta-CD does not affect the rate of RMCK unfolding.
Subject(s)
Creatine Kinase/chemistry , Creatine Kinase/metabolism , Muscle, Skeletal/enzymology , Temperature , beta-Cyclodextrins/pharmacology , 2-Hydroxypropyl-beta-cyclodextrin , Animals , Enzyme Activation/drug effects , Enzyme Stability/drug effects , Kinetics , Light , Particle Size , Protein Binding/drug effects , Protein Denaturation/drug effects , Rabbits , Scattering, Radiation , Spectrometry, FluorescenceABSTRACT
The study of thermal denaturation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the presence of alpha-crystallin by differential scanning calorimetry (DSC) showed that the position of the maximum on the DSC profile (T(max)) was shifted toward lower temperatures with increasing alpha-crystallin concentration. The diminishing GAPDH stability in the presence of alpha-crystallin has been explained assuming that heating of GAPDH induces dissociation of the tetrameric form of the enzyme into dimers interacting with alpha-crystallin. The dissociation of the enzyme tetramer was shown by sedimentation velocity at 45 degrees C. Suppression of thermal aggregation of GAPDH by alpha-crystallin was studied by dynamic light scattering under the conditions wherein temperature was elevated at a constant rate. The construction of the light scattering intensity versus the hydrodynamic radius (R(h)) plots enabled estimating the hydrodynamic radius of the start aggregates (R(h,0)). When aggregation of GAPDH was studied in the presence of alpha-crystallin, the start aggregates of lesser size were observed.
Subject(s)
Glyceraldehyde-3-Phosphate Dehydrogenases/chemistry , Muscles/enzymology , Protein Denaturation , alpha-Crystallins , Animals , Calorimetry, Differential Scanning , Dimerization , Enzyme Stability , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Protein Conformation , Rabbits , TemperatureABSTRACT
Thermal denaturation and aggregation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been studied using differential scanning calorimetry (DSC), dynamic light scattering (DLS), and analytical ultracentrifugation. The maximum of the protein thermal transition (T(m)) increased with increasing the protein concentration, suggesting that the denaturation process involves the stage of reversible dissociation of the enzyme tetramer into the oligomeric forms of lesser size. The dissociation of the enzyme tetramer was shown by sedimentation velocity at 45 degrees C. The DLS data support the mechanism of protein aggregation that involves a stage of the formation of the start aggregates followed by their sticking together. The hydrodynamic radius of the start aggregates remained constant in the temperature interval from 37 to 55 degrees C and was independent of the protein concentration (R(h,0) approximately 21 nm; 10 mM sodium phosphate, pH 7.5). A strict correlation between thermal aggregation of GAPDH registered by the increase in the light scattering intensity and protein denaturation characterized by DSC has been proved.
Subject(s)
Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Hot Temperature , Muscles/enzymology , Animals , Calorimetry, Differential Scanning , Glyceraldehyde-3-Phosphate Dehydrogenases/antagonists & inhibitors , Kinetics , Rabbits , UltracentrifugationABSTRACT
Thermal denaturation and aggregation of beta(L)-crystallin from bovine lens have been studied using differential scanning calorimetry (DSC) and dynamic light scattering (DLS). According to the DLS data, the distribution of the beta(L)-crystallin aggregates by their hydrodynamic radius (R(h)) remains monomodal to the point of precipitating aggregates (sodium phosphate, pH 6.8; 100 mM NaCl; 60 degrees C). The size of the start aggregates (R(h,0)) and duration of the latent stage (t(0)) leading to the formation of the start aggregates have been determined from the light scattering intensity versus the hydrodynamic radius plots and the dependences of R(h) on time. The R(h,0) value remains constant at variation of the beta(L)-crystallin concentration, whereas the t(0) value increases with diminishing beta(L)-crystallin concentration. The suppression of beta(L)-crystallin aggregation by alpha-crystallin is connected with the decrease in the R(h,0) value and increase in the t(0) value. In the presence of alpha-crystallin the aggregate population is split into two components. The first component is represented by stable aggregates whose size remains constant in time. The aggregates of the other kind grow until they reach the size characteristic of aggregates prone to precipitation. The DSC data show that alpha-crystallin has no appreciable influence on thermal denaturation of beta(L)-crystallin.