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First published on October 11, 2006; DOI: 10.1124/dmd.106.011601

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Received for publication June 23, 2006.
Revised October 2, 2006.
Accepted for publication October 4, 2006.

A Real-Time Fluorescence Assay for Measuring N-Dealkylation

Richard T. Mayer 1*, E. Kurt Dolence 2, Gabriele E. Mayer 3

1 USDA, ARS 2 Unviersity of Wyoming 3 University of Wyoming

* Address correspondence to: E-mail: dmayer{at}uwyo.edu

Abstract

A real-time fluorescence assay system utilizing a series of 9-N-(alkylamino)acridine derivatives (methyl, ethyl, n-propyl, n-butyl, n-pentyl, and benzyl) that are N-dealkylated to 9-aminoacridine (9AA) is described. The product, 9AA, is ca. 27-fold more fluorescent than the substrates using excitation and emission wavelengths of 405 and 455 nm, respectively. Tests using expressed CYPs 1A1, 1A2, 3A4, 3A5, 1B1, 2C9, 2C19, and 2D6 indicated that N-dealkylase activity is specific for CYP1A1 and CYP2D6. CYP2D6 N-dealkylated methyl, ethyl, n-propyl, and n-butyl substrates while CYP1A1 N-dealkylated these plus the n-pentyl derivative. Activities using 5 µM 9-N-(alkylamino)acridine substrates ranged from 0.1 - 0.9 pmole 9AA/min/pmole CYP. Kinetic constants for CYP1A1 N-dealkylation of the 9-N-(methylamino)acridine (MAA) and 9-N-(ethylamino)acridine (EAA) were Km 1.09 ± 0.68 and 0.35 ± 0.21 µM and the Vmax 61.9 ± 48.5 and 113.8 ± 8.4 pmol 9AA/min/pmol CYP1A1, respectively. Kinetic constants for CYP2D6 N-dealkylation of MAA and EAA were Km 7.9 ± 5.4 and 3.2 ± 1.6 µM and the Vmax 501 ± 35.4 and 702.7 ± 257 pmol 9AA/min/pmol CYP2D6, respectively. The experimental binding energies ({Delta}Gbind) were calculated for MAA with CYP1A1 and CYP2D6 to be -8.266 and -7.074 kcal/mole, respectively. The {Delta}Gbind values for EAA with CYP1A1 and CYP2D6 were -8.950 and -7.618 kcal/mole, respectively. The substrates were suitable for monitoring N-dealkylase activity in microsomal preparations (human, rat, and monkey hepatic preparations) and human hepatocellular carcinoma cell suspensions. Assays were conducted by monitoring reactions either in 96-well microtiter plates using a fluorescence plate reader or in cuvettes using a spectrofluorimeter.


Key words: CYP1A, CYP2D, cytochrome P450, cytochrome P450 catalyzed oxidations, hepatocytes, human CYP enzymes, microsomes, monooxygenases, P450 mechanism


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[Abstract] [Full Text] [PDF]


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