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

Journal of Biosciences volume 35, pages 395–403 (2010)Cite this article

Abstract

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.

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Author notes

  1. Kaveh Amini

    Present address: Department of Chemistry, York University, Toronto, ON, M3J 1P3, Canada

Affiliations

  1. Department of Analytical Chemistry, Faculty of Chemistry, University of Tabriz, Tabriz, Iran

    Mohammad-Hossein Sorouraddin, Kaveh Amini, Abdolhossein Naseri & Javad Vallipour

  2. Department of Medicinal Chemistry, School of Pharmacy, Tabriz University of Medical Sciences, 51664-14766, Tabriz, Iran

    Jalal Hanaee & Mohammad-Reza Rashidi

Authors
  1. Mohammad-Hossein Sorouraddin
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  2. Kaveh Amini
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  3. Abdolhossein Naseri
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  4. Javad Vallipour
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  5. Jalal Hanaee
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  6. Mohammad-Reza Rashidi
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Correspondence to Mohammad-Reza Rashidi.

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Sorouraddin, MH., Amini, K., Naseri, A. et al. A new multi-wavelength model-based method for determination of enzyme kinetic parameters. J Biosci 35, 395–403 (2010). https://doi.org/10.1007/s12038-010-0045-z

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  • DOI: https://doi.org/10.1007/s12038-010-0045-z

Keywords

  • Enzyme kinetics
  • Lineweaver-Burk plot analysis
  • Michaelis-Menten constants
  • multi-wavelength model-based method
  • spectrophotometry