Elsevier

Analytical Biochemistry

Volume 321, Issue 1, 1 October 2003, Pages 125-130
Analytical Biochemistry

An enzymatic electrochemiluminescence assay for the lethal factor of anthrax☆

https://doi.org/10.1016/S0003-2697(03)00424-XGet rights and content

Abstract

The lethal factor (LF) of anthrax toxin is the toxic component of the exotoxin (lethal toxin) secreted by toxic strains of Bacillus anthracis. The lethal factor is a zinc-dependent metalloprotease that specifically cleaves the mitogen-activated protein kinase kinase (MAPKK) family of enzymes. We took advantage of this substrate specificity to develop an electrochemiluminescence (ECL) peptide cleavage assay. The ECL assay uses the stable ruthenium (Ru) metal chelate that, in the presence of tripropylamine, generates a light reaction triggered by the application of an electric potential. The Ru label is specifically incorporated into the C-terminal CYS residue of a synthetic peptide (23mer) containing the MAPKK2 cleavage sequence of LF. Streptavidin-coated paramagnetic beads were the solid phase and facilitated separation and characterization of the enzymatic reaction products based upon N-terminal biotinylation of the peptide substrate. Intact peptide bound via the biotin moiety generated high signal due to the Ru label, whereas binding of the cleaved peptide fragment devoid of Ru label reduced the ECL signal. The proposed assay provides a novel opportunity for the screening of potential therapeutics against anthrax.

Section snippets

Lethal factor

LF of B. anthracis was purchased from List Biological Laboratories Inc. (Campbell, CA) and resuspended at 1 mg/ml in 5 mM Hepes containing 50 mM NaCl, pH 7.5, with 1 mg/ml bovine serum albumin to enhance stability. The solution was divided into 100-μl aliquots and stored frozen until used. Once opened, the remaining fraction was discarded.

Peptide synthesis

The biotinylated LF peptide (23mer) containing the MAPKK2 cleavage sequence was prepared and purified at the University of Texas Health Science Center (San

Confirmation of labeled substrate viability

The enzymatic ECL assay relies on the ability of the N-terminal biotin moiety of the peptide to be captured by the paramagnetic streptavidin-coated beads and the resulting reduction of the ECL signal after LF cleaves the C-terminal portion of the peptide. We synthesized a 23mer peptide based on the sequence of MAPKK-2 (Fig. 1) representing a 15-amino acid region of the native protein kinase with some modifications. The N terminus containing the biotin moiety was extended by the addition of

Discussion

The LF component of anthrax toxin has been implicated in the early stages of anthrax and therefore drugs designed to block the enzymatic activity of LF could be of therapeutic benefit. For that objective, we developed an enzymatic ECL assay (96-well format) that takes advantage of the metalloproteolytic activity of LF on the MAPKK family of enzymes. The assay uses a modified peptide based on the relevant target for the enzymatic activity of the LF. Modifications, such as the N-terminal addition

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Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army.

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