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An aggregation test system based on the aggregation of UV-irradiated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from rabbit skeletal muscle has been proposed. On the basis of the measurements of the enzyme activity and differential scanning calorimetry data a conclusion has been made that UV radiation results in formation of damaged protein molecules with lower thermostability. It was shown that the order of aggregation rate for UV-irradiated GAPDH with respect to the protein was close to 2. This means that such a test system allows detecting the effect of various agents exclusively on the stage of aggregation of unfolded protein molecules. The influence of α-crystallin and 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) on aggregation of UV-irradiated GAPDH was studied. Despite the fact that HP-β-CD accelerates thermal aggregation of non-irradiated GAPDH, in the case of aggregation of UV-irradiated GAPDH HP-β-CD reveals a purely protective effect., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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.

It has been shown that the relatively low concentrations of proline (0.1 M) have a slight accelerating effect on thermal aggregation of glycogen phosphorylase b (Phb) from rabbit skeletal muscle registered by the accumulaton of the aggregated protein. The suppression of Phb aggregation at high proline concentrations is mainly due to the protective action of proline on the stage of unfolding of the Phb molecule. The enhancement of Phb stability in the presence of the high concentrations of proline was demonstrated by the data on differential scanning calorimetry, analytical ultracentrifugation and thermoinactivation kinetics. The construction of the protein aggregate size versus time plots allowed the acceleration of the stage of Phb aggregation in the presence of high concentrations of proline to be demonstrated. The obtained results are consistent with the predictions of the crowding theory.

Ca(2+)- and Mg(2+)-induced association of phosphorylase kinase (PhK) from rabbit skeletal muscle has been studied at the magnitudes of the ionic strength close to the physiological values (40 mM Hepes, pH 6.8, containing 0.1 M NaCl, 0.1 mM Ca(2+), 10 mM Mg(2+); 25 degrees C) and under the molecular crowding conditions produced by high concentrations (1 M) of the natural osmolyte, trimethylamine N-oxide (TMAO). In the presence of 0.1 M NaCl two forms of PhK were registered, namely the "basic form" and "highly associated form", suggesting that PhK association may be treated as an example of cooperative association. According to the data on dynamic light scattering the average hydrodynamic radii of these forms were 16 and 144 nm. The addition of 1 M TMAO produces the time dependent increase in the light scattering intensity caused by the conversion of the basic form into the highly associated form. According to the data of the sedimentation analysis the basic form of PhK comprises a hexadecamer (M(r)=1320 kDa) and its small associates. The removal of Ca(2+) by addition of EGTA results in the reverse conversion of the highly associated form into the basic form suggesting reversibility of self-association of PhK. FAD, the ligand that is specifically bound to PhK, blocks the conversion of the basic form of PhK into the highly associated form.