Denaturation of jack-bean urease by sodium n-dodecyl sulphate: A kinetic study below the critical micelle concentration

Abstract

Kinetics of urease denaturation by anionic surfactant (sodium n-dodecyl sulphate, SDS) at concentrations below the critical micelle concentration (CMC) is investigated spectrophotometrically at neutral pH and the corresponding two-phase kinetic parameters of the process are estimated from a three-state reversible process using a binomial exponential relation based on the relaxation time method as: Native ( N ) ⇌ k − 1 k 1 Intermediate ( I ) ⇌ k − 2 k 1 Denatured ( D ) Using a prepared computer program, the experimental data are properly fitted into a binomial exponential relation, considering a two-phase denaturation pathway including a kinetically stable folded intermediate formed at SDS concentration of 1.1 mM. Forward and backward rate constants are estimated as: k 1 = 0.2141 ± 4.5 × 10 −3, k 2 = 5.173 × 10 −3 ± 8.3 × 10 −5, k −1 = 0.09432 ± 3.6 × 10 −4 and k −2 = 2.079 × 10 −3 ± 5.6 × 10 −5 s −1 for the proposed mechanism. The rate-limiting step as well as the reaction coordinates in the denaturation mechanism are established. The mechanism involves formation of a kinetically stable folded native like intermediate through the electrostatic interactions. The intermediate was found to be more stable even than the native form (by about 9 kJ mol −1) and still hexamer, because no loss of amplitude was observed. Electrophoresis experiments on the native and surfactant/urease complexes indicated a higher mobility for the kinetically folded native like intermediate.