Suppression effect of guanidine hydrochloride on α-cyclodextrin-assisted refolding of denatured α-amylase

It is well established that alpha-cyclodextrin (α-CD) is an effective refolding aid for some proteins in dilution additive mode. On the other hand, it has recently been shown that guanidine hydrochloride (GuHCl), the well known protein denaturant, acts, at well defined mild concentrational ranges, as a stabilizer of the “molten globule” intermediates with the final renaturation as well as aggregation of the denatured protein under investigation. In the present study, the effect of GuHCl on α-CD efficacy on protein renaturation was investigated. The unassisted and α-CD-assisted refolding of chemically denatured α-amylase in the presence of various α-CD and GuHCl concentrations were evaluated, using activity assay, far-UV circular dichroism (CD) spectroscopy, turbidimetric analysis and hydrophobic probe 8-anilinonaphtalene-1-sulfonic acid (ANS) fluorescence techniques. The recovered activity, average helical structure and the extent of dilution-induced aggregation of refolded α-amylase were affected with increase of final GuHCl concentration in dilution buffer, especially in α-CD-assisted refolded samples. The refolding yield and protein helicity decreased, while apparent turbidity due to protein aggregation (Alim) and its rate were increased. The results clearly indicated that α-cyclodextrin effectively suppressed aggregation, especially at lower GuHCl concentrations in the refolding buffer. Based on these results, application of low dilution factors in α-CD-dependent protein renaturation systems should be avoided due to inhibitory effects of high final denaturant concentration on α-CD efficacy besides its direct influence on the production of aggregation-prone intermediate(s) of protein molecules.