EFFECT OF DENATURANTS ON THE STRUCTURE AND ACTIVITY OF DDT-DEHYDROCHLORINASER. LATHA, I.M. MANDAPPA, M.S. THAKUR AND H.K. MANONMANI
Urea, sodium dodecyl sulfate (SDS) and guanidine hydrochloride (GdmCl) are often used as excellent denaturing or unfolding agents. We studied the effect of these denaturing agents on the structural changes of DDT- enzyme of Psuedomonas putida T5. There was a progressive loss in catalytic activity of DDT- dehydrochlorinase with increasing concentrations of denaturants, namely urea, SDS and GdmCl. At 10 M urea, 5 % SDS and 1 M GdmCl, the extent of loss in enzyme activity was 98, 78 and 100% respectively. The emission spectrum of urea denatured enzyme did not show shift, but that of SDS and GdmCl treated enzyme showed very marginal shift. The secondary structure analysis of the enzyme by CD spectrum suggested a predominance of b-structure in the untreated enzyme. Urea denatured enzyme revealed complete loss of a-helix and there was substantial reduction in b sheets. SDS treated enzyme showed increase in a-helix, turns and random structure. However, there was a marginal loss in b sheets. GdmCl treated enzyme showed complete absence of a-helix compared to the control enzyme. The b-sheets were also reduced and the number of turns increased. The effects of GdmCl were almost similar to that of urea as observed in CD spectrum. The urea polyacrylamide gel showed only one band whereas SDSpolyacrylamide gel showed closely moving two bands.
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