title: An enzymatic electrochemiluminescence assay for the lethal factor of anthrax.
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.
authors.affiliation: Toxinology and Aerobiology Division, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702, USA. victor.rivera@det.amedd.army.mil
authors.forename: Victor R
authors.initials: VR
authors.lastname: Rivera
authors.forename: Gerald A
authors.initials: GA
authors.lastname: Merrill
authors.forename: Jill A
authors.initials: JA
authors.lastname: White
authors.forename: Mark A
authors.initials: MA
authors.lastname: Poli
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial; D001408:Bacillus anthracis / Q000201:enzymology*; D001427:Bacterial Toxins; D001710:Biotin; D002352:Carrier Proteins / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004563:Electrochemistry / Q000379:methods*; D008163:Luminescent Measurements; D048370:MAP Kinase Kinase 2; D045726:Metalloproteases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011480:Protease Inhibitors / Q000032:analysis; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011817:Rabbits; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D002352:Carrier Proteins; D011480:Protease Inhibitors; C030325:anthrax toxin; D001710:Biotin; D011505:Protein-Tyrosine Kinases; D048370:MAP Kinase Kinase 2; D020929:Mitogen-Activated Protein Kinase Kinases; D045726:Metalloproteases