On the dinitrophenylation of bovine pancreatic ribonuclease A kinetics of the reaction in water and 8 M urea

Abstract

Activity measurements and chromatographic analyses of reaction products were used to determine the kinetics of substitution by FDNB of reactive amino groups in bovine pancreatic RNase A. The inactivation process obeyed second-order kinetics over the concentration range allowed by the solubility of the reagent. The pH-independent second-order rate constants for substitution at the α-amino group and for the ϵ-amino group in position 41 were determined to be ~0.8 and 26.4 m −1 min −1 at 15 °. These values are of the same order of magnitude as those for the corresponding rate-constants for substitution in model peptides (~1 and 14 m −1 min −1, respectively). Reaction at position 41, which inactivates the enzyme, is governed by the dissociation of a group with a p K app of 8.8 ± 0.1, an observation which in large part accounts for the exceptional reactivity of this side-chain. Evidence was obtained that showed the amino group of lysine 7, unavailable to reaction in the native enzyme, but exposed when substitution at position 41 has occurred, has a reactivity equivalent to that of the amino group at position 41. The reactivity at position 41 was partially abolished when dinitrophenylation occurred in 8 m urea solution, or when a carboxymethyl group modified the imidazole side-chain at position 119.