α-Crystallin Assisted Refolding of Enzyme Substrates: Optimization of External Parameters

α-Crystallin is known to act as a molecular chaperone by preventing the aggregation of partially unfolded substrate proteins. It is also known to assist the refolding of a number of denatured enzymes, but the activity yield is often less than 20%. In this paper, we have tried to tune the refolding ability of α-crystallin in vitro by optimizing various external parameters . We wanted to find out the best possible condition under which it can exhibit maximum refolding capacity. We found that under suitable condition in vitro α-crystallin can refold denatured malate dehydrogenase, carbonic anhydrase and lactate dehydrogenase to recover more than 40% activity. We also measured the effect of several external factors such as nucleotides, osmolytes, electrolytes, temperature etc. on the in vitro α-crystallin mediated reactivation of above stated enzymes. We found that nucleotides and electrolytes had little effect on the refolding ability of α-crystallin. However, in presence of different osmolytes, we found that its ability to reactivate denatured substrate proteins enhanced significantly. Refolding in presence of pre-incubated α-crystallin reveals that hydrophobicity had stronger influence on the refolding capacity of α-crystallin than its oligomeric size.