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

摘要

Kinetics of urease denaturation by anionic surfactant (sodium n-dodecyl sulphate, SDS) at concentrations below the critical micelle concentration (CMQ 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) (k1)->(<- k-1) Intermediate (I)(k1 ->)(<- k-2) 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 x 10(-3), k(2) = 5.173 x 10(-8) x 10(-5), k(-1) = 0.09432 3.6 x 10(-4) and k(-2) = 2.079 x 10(-3) 5.6 x 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. (c) 2006 Elsevier B.V. All rights reserved.