Near real-time determination of metabolic parameters for unquenched 6-mercaptopurine and xanthine oxidase samples using capillary electrophoresis

• CE injections stop the enzyme reaction without sample quenching or purification.
• Sequential sample injections lead to accurate progress curve generation.
• Initial velocities are accurately derived for the drug metabolism reaction.
• Precise kinetic constants including enzyme activity are determined in short times.
• Competitive inhibition constants for XO and two drugs are reported.

The enzyme activity of xanthine oxidase (XO) is influenced by several environmental factors including solution conditions, storage conditions, inhibitors, other enzymes, and activators. For instance, the metabolic reaction involving XO and the pro-drug 6-mercaptopurine, a drug used in the treatment maintenance of acute lymphatic leukemia, Crohn's disease, and ulcerative colitis, is often modified through the use of inhibitors, which varies the kinetic parameters associated with this reaction. Methods that provide fast and accurate determination of these kinetic constants can help in understanding the mechanism of these reactions. Herein, sequential and time-delayed electrokinetic injections of unpurified and unquenched samples containing xanthine oxidase, 6-mercaptopurine, and the inhibitor allopurinol are evaluated using capillary electrophoresis (CE). Using progress curve analysis, the Michaelis constant, apparent Michaelis constant, and inhibition constant are estimated to be 43.8 ± 2.0 μM, 143.0 ± 3.7 μM and 13.2 ± 1.4 μM, respectively. In addition, a turnover number of 7.9 ± 0.2 min−1 is quantified. These values are consistent with some previously published values but were obtained without user intervention for reaction monitoring. This unique application of CE enzyme assays offers substantial advantages over traditional methods by determining kinetic parameters for enzymatic reactions with minimal (nL) sample volumes, short (<30 min) reaction analysis times, without any sample quenching or purification, and minimal user intervention.

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