title: A Numerical Approach for Kinetic Analysis of the Nonexponential Thermoinactivation Process of Uricase.
abstract: Prior to the exponential decrease of activity of a uricase from Candida sp. during storage at 37 °C, there was a plateau period of about 4 days at pH 7.4, 12 days at pH 9.2, and about 22 days in the presence of 30 μM oxonate at pH 7.4 or 9.2, but no degradation of polypeptides and no activity of resolved homodimers. To reveal determinants of the plateau period, a dissociation model involving a serial of conformation intermediates of homotetramer were proposed for kinetic analysis of the thermoinactivation process. In the dissociation model, the roles of interior noncovalent interactions essential for homotetramer integrity were reflected by an equivalent number of the artificial weakest noncovalent interaction; to avoid covariance among parameters, the rate constant for disrupting the artificial weakest noncovalent interaction was fixed at the minimum for physical significance of other parameters; among thermoinactivation curves simulated by numerical integration with different sets of parameters, the one for least-squares fitting to an experimental one gave the solution. Results found that the equivalent number of the artificial weakest noncovalent interaction primarily determined the plateau period; kinetics rather than thermodynamics for homotetramer dissociation determined the thermoinactivation process. These findings facilitated designing thermostable uricase mutants.
authors.affiliation: Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Clinical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Wu
authors.affiliation: Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Clinical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, China.
authors.forename: Xiaolan
authors.initials: X
authors.lastname: Yang
authors.affiliation: Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Clinical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, China.
authors.forename: Deqiang
authors.initials: D
authors.lastname: Wang
authors.affiliation: Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Clinical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, China.
authors.forename: Xiaolei
authors.initials: X
authors.lastname: Hu
authors.affiliation: Central Laboratory, Yongchuan Hospital, Chongqing Medical University, No.439, Xuanhua Road, Yongchuan, Chongqing, 402160, China.
authors.forename: Juan
authors.initials: J
authors.lastname: Liao
authors.affiliation: Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Clinical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, China.
authors.forename: JingJing
authors.initials: J
authors.lastname: Rao
authors.affiliation: Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Clinical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Pu
authors.affiliation: Molecular Modeling and Biopharmaceutical Center and Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY, 40536, USA.
authors.forename: Chang-Guo
authors.initials: CG
authors.lastname: Zhan
authors.affiliation: Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Clinical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, China. liaofeish@yeah.net.
authors.forename: Fei
authors.initials: F
authors.lastname: Liao
mesh_terms: D002175:Candida / Q000737:chemistry / Q000201:enzymology*; D019281:Dimerization; D057075:Enzyme Assays; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000302:isolation & purification; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015233:Models, Statistical; D011487:Protein Conformation; D013816:Thermodynamics; D014503:Urate Oxidase / Q000737:chemistry* / Q000302:isolation & purification
chemical_list: D005656:Fungal Proteins; D014503:Urate Oxidase
keywords: A plateau period; Dissociation kinetics; Oxonate; Thermostability; Uricase