Comparative Analysis of the Effects of α-Crystallin and GroEL on the Kinetics of Thermal Aggregation of Rabbit Muscle Glyceraldehyde-3-Phosphate Dehydrogenase

Abstract

Effects of alpha-crystallin and GroEL on the kinetics of thermal aggregation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been studied using dynamic light scattering and analytical ultracentrifugation. The analysis of the initial parts of the dependences of the hydrodynamic radius of protein aggregates on time shows that in the presence of alpha-crystallin or GroEL the kinetic regime of GAPDH aggregation is changed from the regime of diffusion-limited cluster-cluster aggregation to the regime of reaction-limited cluster-cluster aggregation, wherein the sticking probability for the colliding particles becomes lower the unity. In contrast to alpha-crystallin, GroEL does not interfere with formation of the start aggregates which include denatured GAPDH molecules. On the basis of the analytical ultracentrifugation data the conclusion has been made that the products of dissociation of GAPDH and alpha-crystallin or GroEL play an important role in the interactions of GAPDH and chaperones at elevated temperatures.

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

“…This fact indicates that GAPDH aggregation proceeds in the DLCA regime. The obtained result agrees with the data obtained by us earlier [28,32]. The initial part of the dependence of R h on time obtained in the absence of HP-␤-CD is linear (line 1 in Fig.…”

“…Parameter K LS may be considered as a measure of the rate of aggregation. Earlier we showed that thermal aggregation of GAPDH proceeded in the regime of diffusion-limited cluster-cluster aggregation (DLCA) [26,28,32], and above a definite value of time (t > t*) the dependence of the hydrodynamic radius of the protein aggregates on time followed the power law:…”

“…Naletova et al [24] have demonstrated that GroEL does not prevent thermal denaturation of GAPDH, but effectively binds the thermally denatured enzyme, thus preventing the aggregation of the denatured molecules. Earlier we studied the effect of ␣-crystallin and GroEL on thermal aggregation of GAPDH [25][26][27][28]. Furthermore, mechanisms of protective action of ␣-crystallin and GroEL were compared [28].…”

“…Earlier we studied the effect of ␣-crystallin and GroEL on thermal aggregation of GAPDH [25][26][27][28]. Furthermore, mechanisms of protective action of ␣-crystallin and GroEL were compared [28]. It has been shown that the products of dissociation of GAPDH, on the one hand, and the products of dissociation of ␣-crystallin or GroEL, on the other hand, play an important role in the interaction of GAPDH and chaperones at elevated temperatures.…”

Ultraviolet Spectroscopy as a Tool in Therapeutic Protein Development

“…47,66 It should be noted that a good correlation between the intensity of static light scattering and hydrodynamic size measured with dynamic light scattering is usually observed. 67 Percent inhibition of aggregation (%IA) is typically quantitatively estimated according to 68 : (7) where OD 350(sample) and OD 350(ref ) are the OD values of the protein at a certain wavelength above 320 nm (350 nm in this case) in the presence and absence of a given stress variable over similar incubation times, respectively. In addition to kinetic measurements, thermal aggregation of therapeutic proteins is often probed by this approach in screening for stabilizing excipients and/or formulations.…”