title: Dynamic structure/function relationships in the alpha-chymotrypsin deactivation process by heat and pH.
abstract: The activity decay of alpha-chymotrypsin due to temperature and pH has been related to the associated structural changes of the protein that could be studied with the help of melting temperature which was determined by using ultraviolet absorption spectroscopy, and fluorescence spectra measurements. The kinetic behaviour in activity loss of alpha-chymotrypsin followed a two-step deactivation model, involving an intermediate state. The increase in temperature and pH showed a clear deactivation effect, reducing exponentially the half-life of the enzyme. At pH 7.0, this two-step deactivation process was also observed with both the maximum fluorescence intensity (I(max)) and emission wavelength (lambda(max)). The series-type kinetic model allowed to establish a clear correlation between the activity and the fluorescence spectral normalized data: the intermediate state of the enzyme occurred at an identical deactivation denaturation level (alpha1), and a proportionality between the decay rate constants was observed. As a function of the incubation temperature, another correlation was observed between the alpha1 profile, initial lambda(max) and thermal unfolding transition, allowing to identify the intermediate state of the kinetic model as that obtained at the melting temperature (43.9 degrees C).
authors.affiliation: Departmento de Bioquímica y Biología Molecular B e Immunología, Facultad de Química, Universidad de Murcia, Spain.
authors.forename: P
authors.initials: P
authors.lastname: Lozano
authors.forename: T
authors.initials: T
authors.lastname: De Diego
authors.forename: J L
authors.initials: JL
authors.lastname: Iborra
mesh_terms: D000818:Animals; D002417:Cattle; D002918:Chymotrypsin / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008956:Models, Chemical; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
chemical_list: D002918:Chymotrypsin