Effect of 2-hydroxypropyl-β-cyclodextrin on thermal inactivation, denaturation and aggregation of glyceraldehyde-3-phosphate dehydrogenase from rabbit skeletal muscle

Effect of 2‐hydroxypropyl‐β‐cyclodextrin on thermal stability and aggregation of glycogen phosphorylase b from rabbit skeletal muscle

“…It is interesting to note that the increase in the rate of thermal aggregation in the presence of HP-b-CD was observed also for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from rabbit skeletal muscles. 34,35 The molecule of GAPDH is a tetramer with a molecular mass of 144 kDa. Thermal denaturation of GAPDH proceeds according to the dissociative mechanism, i.e., includes the stage of reversible dissociation of the original tetrameric form into oligomeric forms of lesser size.…”

Selection of Test Systems for Estimation of Anti-aggregation Activity of Molecular Chaperones

“…To apply the kinetic data obtained by this means for elucidation of the aggregation mechanism and quantitative estimation of the effects of different agents on the aggregation rate, the relationship between increment in the light scattering intensity and the amount of the aggregated protein should be determined using the methods that allow the concentration of the aggregated protein to be directly registered. To characterize the initial rate of aggregation, the initial parts of kinetic curves registered by measurement of increment in the light scattering intensity were approximated by the quadratic function [8][9][10][11][12][13][14][15]: (2) In this empiric equation K LS is a constant that can be considered as a measure of the initial rate of aggregation and t 0 is the duration of the lag phase. To substantiate the propriety of using parameter K LS for characterization of the initial rate of aggregation, the comparison of the kinetic data obtained by measuring the light scattering intensity with direct determination of the amount of the aggregated protein should be carried out.…”

How to Quantify the Chaperone-Like (Anti-Aggregation) Activity?

“…Parameter K agg is a measure of the initial rate of aggregation. The applicability of equation 1for the description of the initial parts of the kinetic curves of protein aggregation was demonstrated for heat denaturation of glycogen phosphorylase b (Phb) [9][10][11][12], creatine kinase [13] and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from rabbit skeletal muscles [14,15] and dithiothreitol-induced aggregation of α-lactalbumin [16] and insulin [17]. The measurements of the hydrodynamic radius of protein aggregates using dynamic light scattering allow us to conclude that for the proteins under study the point in time t=t 0 corresponds to the appearance of start aggregates.…”

“…It has been just such protein substrates which allowed us to register the effect of chemical chaperones directly on the stage of aggregation of denatured protein molecules. In other test-systems, for example, in the testsystems based on heat-induced aggregation of proteins, the general effect of chemical chaperone involves the action of chaperone on the aggregation stage and the action of chaperone on the stages preceding the aggregation stage [10,[38][39][40].…”