Effect of Denaturants on the Structure and Activity of DDT Dehydrochlorinase.

Urea, sodium dodecyl sulfate (50s) 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 pulido. TS. There was a progressive loss in catalytic
activity of DDT- dehydrochlorinase with increasing concentrations of denaturants, namely urea, 50s and
GdmQ. At 10 M urea, 5 % 50s 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 50s and
GdmCl treated enzyme showed vel)' marginal shift. The secondary structure analysis of the enzyme by CD
spectrum suggested a predominance of l1-structure in the untreated enzyme. Urea denatured enzyme
revealed complete loss of «-helix and there was substantial reduction in 11 sheets. SDS treated enzyme
showed increase in a-helix, turns and random structure. However, there was a marginal loss in 11 sheets.
GdmCl treated enzyme showed complete absence of a-helix compared to the control enzyme. The l1-sheets
were also reduced and the number of turns increased. The effects of GdmCI were almost similar to that of
urea as observed in CO spectrum. The urea polyacrylamide gel showed only one band whereas 505-
polyacrylamide gel showed closely moving two bands.