Guanidine hydrochloride-induced denaturation of Pseudomonas cepacia lipase.
The guanidine hydrochloride-induced denaturation of Pseudomonas cepacia lipase (PCL) was studied at pH 7 by monitoring the changes in the fluorescence and circular dichroism of the enzyme. The denaturation was irreversible as a whole, and the addition of Ca2+ ions decreased the velocity of the denaturation. The denaturation process was well explained consistently by a two-step mechanism, as follows: [see equation in text] where N is the native state of PCL, D(I) an intermediate denatured-state which can be refolded into the native state, and D(F) the final denatured-state that can not be renatured. Ethanol (10%) increased the denaturation velocity by decreasing the refolding step, D(I) + Ca2+ --> N x Ca2+, which would be caused by the stabilization of D(I) by ethanol.
Tanaka
A
A
Faculty of Bioresources, Mie University, Tsu, Mie, 514-8507, Japan. akiyoshi@bio.mie-u.ac.jp
Okuda
K
K
Senoo
K
K
Obata
H
H
Inouye
K
K
D016956:Burkholderia cepacia / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D000431:Ethanol / Q000494:pharmacology; D019791:Guanidine / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D008049:Lipase / Q000737:chemistry* / Q000187:drug effects*; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors
D007477:Ions; D000431:Ethanol; D008049:Lipase; D019791:Guanidine; D002118:Calcium