Alpha-crystallin assisted refolding of enzyme substrates: optimization of external parameters.
alpha-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 alpha-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 alpha-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 alpha-crystallin mediated reactivation of above stated enzymes. We found that nucleotides and electrolytes had little effect on the refolding ability of alpha-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 alpha-crystallin reveals that hydrophobicity had stronger influence on the refolding capacity of alpha-crystallin than its oligomeric size.
Biswas
A
A
Protein Chemistry Laboratory, Department of Chemistry, Bose Institute, 93/1 A.P.C. Road, Kolkata 700 009, India.
Das
K P
KP
D000818:Animals; D002256:Carbonic Anhydrases / Q000737:chemistry; D002417:Cattle; D004798:Enzymes / Q000737:chemistry*; D007908:Lens, Crystalline / Q000378:metabolism; D008027:Light; D008291:Malate Dehydrogenase / Q000737:chemistry; D018832:Molecular Chaperones / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D012542:Scattering, Radiation; D013379:Substrate Specificity; D038201:alpha-Crystallins / Q000737:chemistry*
D004798:Enzymes; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D038201:alpha-Crystallins; D008291:Malate Dehydrogenase; D002256:Carbonic Anhydrases