The effect of guanidine hydrochloride on the conformation of bovine pancreatic DNAase I as measured with circular dichroism

Abstract The denaturation of bovine pancreatic DNAase I (EC 3.1.21.1) by guanidine hydrochloride (GdnHCI) has been investigated with circular dichroism in the presence and absence of 1 mM Ca 2+ at the wavelength region of 210–240 nm at 12, 25 and 36°C. The change of the molar ellipticity at 220 nm by GdnHCl titration showed cooperative transition at each temperature and the midpoints of the titrations occurred near 2 M GdnHCl. At each temperature, the denaturation of DNAase I in the presence of 1 mM Ca 2+ occurred a little slowly as compared with that in the absence of Ca 2+ . This suggests that 1 mM Ca 2+ can to some extent stabilize the secondary structure of DNAase I against GdnHCl denaturation. The apparent free energy for the denaturation of DNAaseI obtained by GdnHCl titration was calculated as 9.3±0.3 kcal/mol and 8.9±0.2 kcal/mol at 25°C in the presence and absence of I mM Ca 2+ , respectively. The possible regions for the α-helix and β-structure of DNAase I were predicted from the amino acid sequence by probability calculation of Chou, P.Y. and Fasman, G.D., Adv. Enzymol. 47, 45–148. The characteristic feature is that the NH 2 -terminal half of DNAase I is rich in β-structure and the COOH-terminal half contains mainly α-helix.