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.

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The ability of synthetic polyanions to suppress thermo-aggregation of the oligomeric enzymes (glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and aspartate aminotransferase) has been established. The ability of the polyanions to reduce the thermo-aggregation increased in the order poly(methacrylic acid) < poly(acrylic acid) < sodium poly(styrene sulphonate), which agreed well with the increase, in the same order, of the charge density of the chains. The lengthening of the chains, as well as the rise in their relative content, resulted in an increase of the ability to reduce thermo-aggregation, mentioned above. Complete prevention of the enzyme aggregation was achieved when highly charged polyanions of a relatively high degree of polymerization were used in a concentration sufficient to solubilize the protein. Complexing with the polyanions prevented thermo-aggregation of the enzymes, but not their thermo-denaturation. The adverse effect of the complexing polyanions on the catalytic activity was reduced by the addition of a synthetic polycation, which resulted in a significant reactivation (up to 40%) of the enzyme. The possibility of preventing the thermo-aggregation of enzyme molecules and then partly restoring the enzyme activity, appears to be of particular interest when studying the aggregation mechanism of proteins that are prone to form the amyloid structures responsible for the development of neurodegenerative diseases like Alzheimer's disease, bovine spongiform encephalopathy and Huntington disease. This finding can also be considered as an important step in the creation of artificial chaperones.

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