The Refolding Of Denatured Carbonic Anhydrase Assisted By Additives

The role of various additives on the refolding of denatured bovine carbonic anhydrase Ⅱ(CAB) was investigated. The effects of additive concentration in two different GdmCl concentrations,pH of the solution and temperature were studied. The kinetics of CAB refolding assisted by amides were analyzed, the principles of CAB refolding assisted by acetamide discussed and the possibility of other amides as additives was discussed and experimented. It was indicated that the denatured CAB could be properly refolded in the presence of acetamide, urea, thiourea, acetone, β-CD, CTAB+β-CD and Tween 40+β-CD. The refolding yield of CAB was improved as the concentration of β-CD, CTAB+β-CD and Tween 40+β-CD increased, while there was an optimal additive concentration for CAB reach its highest yield when acetamide, urea, thiourea were used. The yield can be improved at a wide pH range when acetamide, urea, thiourea and acetone existed. The optimal temperature were 37, 37 and 45 ℃for aetamide, urea, thiourea respectively. The kinetic model was in good agreement with the experimental results. The mechanism of CAB refolding assisted by amides were analogous to that of GdmCl. The kN, bigger than that of control, decreases with increasing of additive concentration, while there existed an optimal additive concentration minimizing the aggregating rate constant(kA) and maximum kN /kAwhich leads to the maximum recovery of CAB. When lowering the concentration of GdmCl to 0.0625 mol·L-1, the refolding yield could be the same as that in higher GdmCl concentration (0.125 mol·L-1) if the acetamide or urea concentration in the refolding buffer were a little increased. However, thiourea should always be used in high GdmCl concentration due to the unsufficient depressing effect of thiourea on the CAB intermolecular interactions.The possibility of acetamide as a denaturant was tested, it was found that acetamide in high concentration lead to the unconvertible denaturation of CAB although it was a good additive.CAB renatuaration could also be assisted by other amides, such as formamide, propamide, etc, with which the change of CAB refolding yield with time, pH of the buffer and temperature were similar to that of acetamide.