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.
A dispersive liquid-liquid microextraction method based on the dispersion of 1,2-dichlorobenzene as an extraction solvent into an aqueous phase in the presence of ethanol as a dispersive solvent for the preconcentration of Co 2+ and Ni 2+ ions is discussed. 1-Nitroso 2-naphtol was used as a chelating agent prior to the extraction and the preconcentrated analyte was determined by flame atomic absorption spectrometry. The effect of various experimental parameters including the extraction and dispersive solvent type and volume, pH, amount of the chelating agent, etc. on the microextraction and complex formation was investigated for finding the optimum conditions. The enhancement factors were about 61.9 and 51.8, the calibration graphs were linear in the range of 10-150 mgL -1 and 10-250 mgL -1 with detection limits of 2.42 mgL -1 and 1.59 mgL -1 , and RSD (n = 5) of 3.08% and 2.17% for cobalt and nickel, respectively. The method was successfully applied to the determination of Co and Ni in water and vitamin B 12 .
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