Kinetic substrate quantification by fitting the enzyme reaction curve to the integrated Michaelis—Menten equation

Fei Liao; Kao-Cong Tian; Xiao Yang; Qi-Xin Zhou; Zhao-Chun Zeng; Yu-Ping Zuo
March 2003
Analytical & Bioanalytical Chemistry;Mar2003, Vol. 375 Issue 6, p756
Academic Journal
The reliability of kinetic substrate quantification by nonlinear fitting of the enzyme reaction curve to the integrated Michaelis-Menten equation was investigated by both simulation and preliminary experimentation. For simulation, product absorptivity c was 3.00 mmol-1 Lcm-1 and Km was 0.10 mmol L-1, and uniform absorbance error a was randomly inserted into the error-free reaction curve of product absorbance A1 versus reaction time t1 calculated according to the integrated Michaelis-Menten equation In [Am/ (Am - Ai)] + Ai/ (e × Km) = (Vm/Km) × ti. The experimental reaction curve of arylesterase acting on phenyl acetate was monitored by phenol absorbance at 270 nm. Maximal product absorbance Am was predicted by nonlinear fitting of the reaction curve to Eq. (1) with Km as constant. There were unique Am for best fitting of both the simulated and experimental reaction curves. Neither the error in reaction origin nor the variation of enzyme activity changed the background-corrected value of Am. But the range of data under analysis, the background absorbance, and absorbance error a had an effect. By simulation, Am from 0.150 to 3.600 was predicted with reliability and linear response to substrate concentration when there was 80% consumption of substrate at a of 0.001. Restriction of absorbance to 0.700 enabled Am up to 1.800 to be predicted at a of 0.001. Detection limit reached Am of 0.090 at a of 0.001. By experimentation, the reproducibility was 4.6% at substrate concentration twice the Km and Am linearly responded to phenyl acetate with consistent absorptivity for phenol, and upper limit about twice the maximum of experimental absorbance. These results supported the reliability of this new kinetic method for enzymatic analysis with enhanced upper limit and precision.


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