Cytochrome p450 enzymes are a large family of membrane heme proteins involved in eliminating drugs as well as synthesis or elimination of steroids, eicosanoids, and vitamins. Most of the P450 enzymes in humans are known and characterized based on their structure and functions. Many of them have orthologous protein in other species. We are interested in characterizing the drug metabolism cytochrome P450 enzymes in canines because canines have notable differences in the metabolism of chocolate, caffeine, and many drugs when compared to humans. This has implications in the treatment of dogs in veterinary medicine, but also in drug metabolism studies which are sometimes conducted in dogs. CYP1A2 metabolizes many substrates like phenacetin, caffeine, acetaminophen, and theobromine (from chocolate) which makes it likely that there are differences between the activity of the canine and human CYP1A2 enzyme. The human CYP1A2 protein is well characterized and has a crystal structure available. Less studies have been conducted to look at the biochemistry of the purified canine CYP1A2.<br/><br/>Synthesis of the canine CYP1A2 gene, followed by cloning into the pCW plasmid, was done by Twist Bioscience. The canine enzyme was grown in <i>E. coli</i> JM109 cells, isolated by breaking the cells open through sonication, solubilized with the detergent CHAPS, and purified with nickel affinity and ion exchange chromatography. The purity of the protein after purification was measured by gel electrophoresis and UV-vis spectrophotometry. The purified protein was used to determine the metabolism of phenacetin and caffeine, two substances known to be metabolized by human CYP1A2.

Canine cytochrome P450 1A2 purification and metabolism of phenacetin and caffeine

ACS Spring Meeting 2021

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