A new multi-wavelength model-based method for determination of enzyme kinetic parameters

Journal of biosciences

Mohammad-Reza Rashidi3¹  Jalal Hanaee¹  Mohammad-Hossein Sorouraddin²  Javad Vallipour²  Kaveh Amini²  Abdolhossein Naseri² 

[1] Department of Medicinal Chemistry, School of Pharmacy, Tabriz University of Medical Sciences, 51664-14766 Tabriz, Iran$$;Department of Analytical Chemistry, Faculty of Chemistry, University of Tabriz, Tabriz, Iran$$

关键词: Enzyme kinetics;    Lineweaver–Burk plot analysis;    Michaelis–Menten constants;    multi-wavelength model-based method;    spectrophotometry;   

DOI  :  

来源: Indian Academy of Sciences

【 摘 要 】

Lineweaver–Burk plot analysis is the most widely used method to determine enzyme kinetic parameters. In the spectrophotometric determination of enzyme activity using the Lineweaver–Burk plot, it is necessary to find a wavelength at which only the substrate or the product has absorbance without any spectroscopic interference of the other reaction components. Moreover, in this method, different initial concentrations of the substrate should be used to obtain the initial velocities required for Lineweaver–Burk plot analysis. In the present work, a multi-wavelength model-based method has been developed and validated to determine Michaelis–Menten constants for some enzyme reactions. In this method, a selective wavelength region and several experiments with different initial concentrations of the substrate are not required. The absorbance data of the kinetic assays are fitted by non-linear regression coupled to the numeric integration of the related differential equation. To indicate the applicability of the proposed method, the Michaelis–Menten constants for the oxidation of phenanthridine, 6-deoxypenciclovir and xanthine by molybdenum hydroxylases were determined using only a single initial concentration of the substrate, regardless of any spectral overlap.

【 授权许可】

Unknown