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Bioluminescent N-Acetyl Transferase-2 Enzyme Assay Using a Novel Luciferin Derivative...

Bioluminescent N-Acetyl Transferase-2 Enzyme Assay Using a Novel Luciferin Derivative as a Substrate

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Abstract

This is part of a series of seven articles describing new bioluminescent substrates to be used for drug screening and assaying enzymatic activity.

Michael P. Valley, Carolyn Woodroofe and James J. Cali

Promega Corporation
Publication Date: 2008

Introduction

The N-acetyltransferases are phase II biotransformation enzymes that acetylate aromatic amines and help determine the clinical efficacy and toxicity of drugs and other xenobiotic compounds (1) (2) . NAT2 is the variant commonly associated with the slow acetylator phenotype observed in humans. Luciferin-NAT2 is a specific substrate for this enzyme and shows little to no activity with NAT1. Luciferin-NAT2 is used in a luminogenic assay that correlates light output with NAT2 enzyme activity (Figure 1).

Schematic of Luciferin-NAT2 reaction.Figure 1. Schematic of Luciferin-NAT2 reaction.

The acetylation of Luciferin-NAT2 by NAT2 makes the compound a better substrate for the light-generating enzyme in the Luciferin Detection Reagent.

 

Luminogenic enzyme assays use probe substrates that are prosubstrates for the light-generating reaction of firefly luciferase. These prosubstrates are substantially quenched as light-generating luciferase substrates but are converted by a non-luciferase enzyme of interest (e.g., NAT2) to a more active luciferin substrate that is detected in a second reaction with a luciferin detection reagent (LDR). LDR is a firefly luciferase enzyme reaction mix that is devoid of luciferin. When combined with an enzyme reaction mixture that contains a luciferin derivative as the enzyme substrate, LDR generates light in proportion to the activity of the enzyme of interest (3) .

NAT2 Enzyme Assay

A 1mM stock solution of Luciferin-NAT2 (Cat.# P1721) was made in a 50/50 solution of water and methanol containing 2mM HCl. Higher concentrations of substrate can be made, but the compound is not soluble above 1.5mM. The NAT2 enzyme used to characterize this substrate was purchased from Sigma (Cat.# N 9160; 2.5mg/ml, 400U/mg). Experiments were performed in opaque white 96-well plates (Costar Cat.# 3917), and measured with the GloMax®-Multi Detection System (Cat.# E7031). All luminescence values are background subtracted using a no-enzyme control. Values are expressed in relative luminescent units (RLU).

The Km of Luciferin-NAT2 with NAT2 is 3.8 ± 0.5 µMFigure 2. The Km of Luciferin-NAT2 with NAT2 is 3.8 ± 0.5 µM.

To determine the Km of this substrate with NAT2, 25µl of enzyme (0.25U in 100mM HEPES [pH 8]) was mixed with 25µl of substrate (400µM Acetyl-CoA, variable concentration of Luciferin-NAT2 in 100mM HEPES [pH 8]). After incubating 1 hour at room temperature, 50µl of Luciferin Detection Reagent (LDR; Cat.# V8920, V8921) was added, and the luminescence was measured 20 minutes later. Since NAT2 is inhibited by methanol, a constant amount (1%) was maintained at all substrate concentrations in this experiment.

 

The luminescent signal is linearly dependent on the amount of NAT2 in the reaction.Figure 3. The luminescent signal is linearly dependent on the amount of NAT2 in the reaction.

To examine the enzyme-concentration dependence of the NAT2 reaction with luciferin-NAT2, the experiment described for Figure 2 was repeated with 4µM Luciferin-NAT2 and 0.01–1 units of NAT2.

The luminescent signal is linearly dependent on the time of the NAT2 reaction.Figure 4. The luminescent signal is linearly dependent on the time of the NAT2 reaction.

To examine the time dependence of the NAT2 reaction with luciferin-NAT2, the experiment described for Figure 2 was repeated with 4µM Luciferin-NAT2 and 0.25U of NAT2, and incubated for 0–60 minutes prior to adding LDR.

Conclusion

Luciferin-NAT2, a novel luciferin derivative, is a luminogenic substrate for the NAT2 enzyme. Using this substrate with a luminogenic enzyme assay approach harnesses the exquisite sensitivity, selectivity and simplicity of bioluminescence, and provides for a simple, rapid, multiwell plate-based NAT2 enzyme assay.

References

  1. Parkinson, A. (2001) Biotransformation of Xenobiotics, in Toxicology: The Basic Science of Poisons, Klassen, C.D., ed., McGraw-Hill. 133–224.
  2. Dupret, J.M. et al. (1994) Structure-function studies of human arylamine N-acetyltransferases NAT1 and NAT2. Functional analysis of recombinant NAT1/NAT2 chimeras expressed in Escherichia coli. J. Biol. Chem. 269, 26830–5.
  3. Cali, J.J. et al. (2006) Luminogenic cytochrome P450 assays. Exp. Op. Drug Metab. Toxicol. 2, 629–45.

How to Cite This Article

Valley, M. P., Woodroofe, C. and Cali, J. J. Bioluminescent N-Acetyl Transferase-2 Enzyme Assay Using a Novel Luciferin Derivative as a Substrate. [Internet] 2008. [cited: year, month, date]. Available from: http://www.promega.es/resources/pubhub/enotes/bioluminescent-n-acetyl-transferase-2-enzyme-assay-using-a-novel-luciferin-derivative-as-a-substrate/

Valley, M. P., Woodroofe, C. and Cali, J. J. Bioluminescent N-Acetyl Transferase-2 Enzyme Assay Using a Novel Luciferin Derivative as a Substrate. Promega Corporation Web site. http://www.promega.es/resources/pubhub/enotes/bioluminescent-n-acetyl-transferase-2-enzyme-assay-using-a-novel-luciferin-derivative-as-a-substrate/ Updated 2008. Accessed Month Day, Year.

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Figures

Schematic of Luciferin-NAT2 reaction.Figure 1. Schematic of Luciferin-NAT2 reaction.

The acetylation of Luciferin-NAT2 by NAT2 makes the compound a better substrate for the light-generating enzyme in the Luciferin Detection Reagent.

The Km of Luciferin-NAT2 with NAT2 is 3.8 ± 0.5 µMFigure 2. The Km of Luciferin-NAT2 with NAT2 is 3.8 ± 0.5 µM.

To determine the Km of this substrate with NAT2, 25µl of enzyme (0.25U in 100mM HEPES [pH 8]) was mixed with 25µl of substrate (400µM Acetyl-CoA, variable concentration of Luciferin-NAT2 in 100mM HEPES [pH 8]). After incubating 1 hour at room temperature, 50µl of Luciferin Detection Reagent (LDR; Cat.# V8920, V8921) was added, and the luminescence was measured 20 minutes later. Since NAT2 is inhibited by methanol, a constant amount (1%) was maintained at all substrate concentrations in this experiment.

The luminescent signal is linearly dependent on the amount of NAT2 in the reaction.Figure 3. The luminescent signal is linearly dependent on the amount of NAT2 in the reaction.

To examine the enzyme-concentration dependence of the NAT2 reaction with luciferin-NAT2, the experiment described for Figure 2 was repeated with 4µM Luciferin-NAT2 and 0.01–1 units of NAT2.

The luminescent signal is linearly dependent on the time of the NAT2 reaction.Figure 4. The luminescent signal is linearly dependent on the time of the NAT2 reaction.

To examine the time dependence of the NAT2 reaction with luciferin-NAT2, the experiment described for Figure 2 was repeated with 4µM Luciferin-NAT2 and 0.25U of NAT2, and incubated for 0–60 minutes prior to adding LDR.

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