Denaturation of jack-bean urease by sodium n-dodecyl sulphate: a kinetic study below the critical micelle concentration.
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: 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(-3)+/-8.3 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 kJmol(-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.
Nazari
K
K
Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran. nazarikh@ripi.ir
Mahmoudi
A
A
Esmaeili
N
N
Sadeghian
L
L
Moosavi-Movahedi
A A
AA
Khodafarin
R
R
D032449:Canavalia / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008823:Micelles; D011489:Protein Denaturation; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*; D013501:Surface-Active Agents / Q000737:chemistry*; D014510:Urease / Q000737:chemistry*
D008823:Micelles; D013501:Surface-Active Agents; D012967:Sodium Dodecyl Sulfate; D014510:Urease