A Colorimetric 96-Well Microtiter Plate Assay for the Determination of Enzymatically Formed Citrulline

doi:10.1006/abio.2000.4805

Analytical Biochemistry

Volume 286, Issue 2, 15 November 2000, Pages 257-264

https://doi.org/10.1006/abio.2000.4805 Get rights and content

Abstract

$l$ -Citrulline constitutes a product of a number of enzymatic reactions. In the past a number of colorimetric methods for the determination of $l$ -citrulline, upon its chemical modification with diacetyl monoxime at 95°C, have been reported. However, all these methods are time- and material-consuming. In this work, using the same chemical reaction, a new method for the use in 96-well polystyrene microtiter plates was developed. The method is fast and requires substantially less material as the enzymatic reaction is performed in a volume of 60 μl. The applicability of this enzymatic assay was established using $l$ -N^ω,N^ω-dimethylarginine dimethylaminohydrolase, which generates $l$ -citrulline from side-chain methylated derivatives of $l$ -arginine. The detection limit for $l$ -citrulline is about 0.2 nmol. In addition, our studies show that most commonly used biochemical buffers and buffer additives do not affect the assay. This method may prove useful in the studies of other $l$ -citrulline producing enzymes including nitric oxide synthase.

References (38)

L.A. Knodler et al.
J. Biol. Chem.
(1998)
K. Sugawara et al.
Anal. Biochem.
(1998)
J.D. Gary et al.
Prog. Nucleic Acid Res. Mol. Biol.
(1998)
T. Ogawa et al.
Arch. Biochem. Biophys.
(1987)
T. Ogawa et al.
J. Biol. Chem.
(1989)
R.R. Giraldez et al.
Anal. Biochem.
(1998)
V.B. Kumar et al.
Anal. Biochem.
(1999)
T.P. Misko et al.
Anal. Biochem.
(1993)
T.R. Boyde et al.
Anal. Biochem.
(1980)
D.B. Shindler et al.
Anal. Biochem.
(1979)

B. Zarabian et al.

Anal. Biochem.

(1987)

D. Hunninghake et al.

Anal. Biochem.

(1966)

G. Ceriotti et al.

Clin. Chim. Acta

(1966)

G. Guthöhrlein et al.

Anal. Biochem.

(1968)

R.B. Moore et al.

Anal. Biochem.

(1970)

A. Prescott et al.

Anal. Biochem.

(1976)

Y. Yui et al.

J. Biol. Chem.

(1991)

W. Wang et al.

Anal. Biochem.

(1995)

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The anti-obesity and anti-diabetic effects of Cit in rats are sex-dependent, with Cit being more effective in female than male rats.
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Peptidyl arginine deiminase 4 (PAD4) is an important biocatalytic enzymes involved in the conversion of protein arginine to citrulline, its dysregulation has a great impact on many physiological processes. Recently, PAD4 has emerged as a potential therapeutic target for the treatment of various diseases including rheumatoid arthritis (RA). Traditional Chinese Medicines (TCMs), also known as herbal plants, have gained great attention by the scientific community due to their good therapeutic performance and far fewer side effects observed in the clinical treatment. However, limited researches have been reported to screen natural PAD4 inhibitors from herbal plants. The color developing reagent (COLDER) or fluorescence based methods have been widely used in PAD4 activity assay and inhibitor screening. However, both methods measure the overall absorbance or fluorescence in the reaction solution, which are easy to be affected by the background interference due to colorful extracts from herbal plants. In this study, a simple, and robust high-performance liquid chromatography ultraviolet-visible (HPLC-UV) based method was developed to determine PAD4 activity. The proposed strategy was established based on COLDER principle, while used hydrophilic l-arginine instead of hydrophobic N-benzoyl-l-arginine ethyl ester (BAEE) as a new substrate to determine PAD4 inhibition activity of herbal extracts. The herbal extracts and PAD4 generated hydrophobic l-citrulline were successfully separated by the HPLC, and the developed method was optimized and validated with a known PAD4 inhibitor (GSK484) in comparison with COLDER assay. The IC₅₀ value of GSK484 measured by HPLC-UV method was 153 nM, and the detection limit of the citrulline was 0.5 nmol, respectively, with a linear range of 0.5 nmol to 20 nmol. The IC₅₀ value of the HPLC-UV method was improved by nearly three times compared with COLDER assay (527 nM), and the results indicated the reliability of PAD4 inhibition via HPLC-UV method. The inhibitory effect against PAD4 were fast and accurately screened for the twenty-four extracts from eight herbs. Among them, Ephedra Herba extracts showed significant inhibitory activity against the PAD4 with the IC₅₀ values of three extracts (ethanol, ethyl acetate and water) ranging from 29.11 μg/mL to 41.36 μg/mL, which may help researchers to discover novel natural compounds holding high PAD4 inhibition activity.
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Dimethylarginine dimethylaminohydrolase (DDAH, EC 3.5.3.18) catalyzes the hydrolysis of asymmetric N^ω,N^ω-dimethyl-l-arginine (ADMA), an endogenous inhibitor of human nitric oxide synthases. The active-site cysteine residue has been proposed to serve as the catalytic nucleophile, forming an S-alkylthiourea reaction intermediate, and serving as a target for covalent inhibitors. Inhibition can lead to ADMA accumulation and downstream inhibition of nitric oxide production. Prior studies have provided experimental evidence for formation of this covalent adduct but have not characterized it kinetically. Here, rapid quench-flow is used with ADMA and the DDAH from Pseudomonas aeruginosa to determine the rate constants for formation (k₂ = 17 ± 2 s⁻¹) and decay (k₃ = 1.5 ± 0.1 s⁻¹) of the covalent S-alkylthiourea adduct. A minimal kinetic mechanism for DDAH is proposed that supports the kinetic competence of this species as a covalent reaction intermediate and assigns the rate-limiting step in substrate turnover as hydrolysis of this intermediate. This work helps elucidate the different reactivities of S-alkylthiourea intermediates found among the mechanistically diverse pentein superfamily of guanidine-modifying enzymes and provides information useful for inhibitor development.
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Citation Excerpt :
Using this technique, we incorporated citrulline in PAD4 at two known autocitrulllination sites, R372 and R374. Using benzoylarginine ethyl ester (BAEE, a small molecule substrate of PADs) and a traditional COLDER assay [56] to detect citrulline formation, we found that the R372Cit and R374Cit mutants are 181- and 9-fold less active than WT PAD4, indicating that citrullination can have profound effects on PAD4 activity. We also assessed the cellular activity of WT PAD4 and the R374Cit mutant and showed that citrullination of R374 leads to 6-fold less citrullinated histone H3, consistent with the in vitro data.
Citrullination is a post-translational modification (PTM) that converts peptidyl-arginine into peptidyl-citrulline; citrullination is catalyzed by the protein arginine deiminases (PADs). This PTM is associated with several physiological processes, including the epigenetic regulation of gene expression, neutrophil extracellular trap formation, and DNA-damage induced apoptosis. Notably, aberrant protein citrullination is relevant to several autoimmune and neurodegenerative diseases and certain forms of cancer. As such, the PADs are promising therapeutic targets. In this review, we discuss recent advances in the development of PAD inhibitors and activity-based probes, the development and use of citrulline-specific probes in chemoproteomic applications, and methods to site-specifically incorporate citrulline into proteins.

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^☆: This work was supported by Swiss National Science Foundation Grant 31-58858.99.

²: To whom correspondence should be addressed. Fax: +41-(0)1-63 5 59 05. E-mail: [email protected].

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Regular ArticleA Colorimetric 96-Well Microtiter Plate Assay for the Determination of Enzymatically Formed Citrulline☆

Abstract

J. Biol. Chem.

Anal. Biochem.

Prog. Nucleic Acid Res. Mol. Biol.

Arch. Biochem. Biophys.

J. Biol. Chem.

Anal. Biochem.

Anal. Biochem.

Anal. Biochem.

Anal. Biochem.

Anal. Biochem.

Anal. Biochem.

Anal. Biochem.

Clin. Chim. Acta

Anal. Biochem.

Anal. Biochem.

Anal. Biochem.

J. Biol. Chem.

Anal. Biochem.

Regular Article
A Colorimetric 96-Well Microtiter Plate Assay for the Determination of Enzymatically Formed Citrulline☆