Assay of heat-labile enterotoxins by their ADP-ribosyltransferase activities.
Simple enzymatic assays to detect heat-labile enterotoxins whose modes of action are similar to that of cholera toxin were evaluated. The assays are performed by using an artificial substrate, diethylamino benzylidine-aminoguanidine, which is an ADP-ribose acceptor. The product, formed in the presence of NAD+, can be quantitated by spectrofluorometric, spectrophotometric, or high-performance liquid chromatographic (HPLC) methods. As little as 25 ng (spectrofluorometry) or 125 ng (spectrophotometry or HPLC) of cholera toxin can be detected in an assay volume of 250 microliters. The detection limit for heat-labile enterotoxin by either the spectrophotometric or HPLC methods was 125 ng/250 microliters. Because the results are quantitative, the enzymatic methods can be used for medium development, determination of factors that influence toxin production, and other applications that heretofore could be accomplished only with difficulty. The enzymatic methods add a new dimension to the assay of toxins that ribosylate arginine residues of proteins. Sensitivities of the assays might be improved by developing better synthetic substrates, and applications could be broadened by the development of artificial substrates containing other functional groups.
Narayanan
J
J
Department of Biochemistry and Biophysics, Iowa State University, Ames 50011.
Hartman
P A
PA
Graves
D J
DJ
D000818:Animals; D001419:Bacteria / Q000201:enzymology / Q000378:metabolism*; D001427:Bacterial Toxins; D002772:Cholera Toxin / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D004768:Enterotoxins / Q000032:analysis*; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D029968:Escherichia coli Proteins; D006801:Humans; D011065:Poly(ADP-ribose) Polymerases / Q000032:analysis*; D013050:Spectrometry, Fluorescence; D014734:Vibrio cholerae / Q000201:enzymology / Q000378:metabolism
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D002772:Cholera Toxin; C027538:heat-labile enterotoxin, E coli; D011065:Poly(ADP-ribose) Polymerases