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The function of the enzyme human aldehyde oxidase (AOX1) is uncertain; however, recent studies have implicated significant biochemical involvement in humans. AOX1 has also rapidly become an important drug-metabolizing enzyme. Until now, quantitation of AOX1 in complex matrices such as tissue has not been achieved. Herein, we developed and employed a trypsin digest and subsequent liquid chromatography-tandem mass spectrometry analysis to determine absolute amounts of AOX1 in human liver. E. coli expressed human purified AOX1 was used to validate the linearity, sensitivity, and selectivity of the method. Overall, the method is highly efficient and sensitive for determination of AOX1 in cytosolic liver fractions. Using this method, we observed substantial batch-to-batch variation in AOX1 content (21-40 pmol AOX1/mg total protein) between various pooled human liver cytosol preparations. We also observed interbatch variation in Vmax (3.3-4.9 nmol min(-1) mg(-1)) and a modest correlation between enzyme concentration and activity. In addition, we measured a large difference in kcat/Km, between purified (kcat/Km of 1.4) and human liver cytosol (kcat/Km of 15-20) indicating cytosol to be 11-14 times more efficient in the turnover of DACA than the E. coli expressed purified enzyme. Finally, we discussed the future impact of this method for the development of drug metabolism models and understanding the biochemical role of this enzyme.

Absolute Quantification of Aldehyde Oxidase Protein in Human Liver Using Liquid Chromatography–Tandem Mass Spectrometry