Measurement of environmental formylmethionyl-peptides.
Siegel PD; Ronk EA; Clark PR; Shahan TA; Castranova V
J Toxicol Environ Health 1994 Jul; 42(3):275-288
Experiments directed at developing an assay for determining formylmethionyl peptides (FMPs) in environmental samples were described. FMPs were naturally occurring bacterial ligands that can cause biological effects at nanomolar concentrations. Attempts were made to develop an FMP assay based on high performance liquid chromatography (HPLC) utilizing N-formylmethionyl-leucyl- phenylalanine (59880976) (fMLP) as the analyte. When samples were analyzed by a published HPLC method in which an ultraviolet detector set at 420 nanometers (nm) and a mobile phase consisting of 60 to 40 methanol/0.1 molar (M) acetic-acid was used, both fMLP and oxidized fMLP (oxyfMLP) could be detected. The detection limit, however, was only 850 nanograms (ng). Switching to a variable wavelength detector set at 190nm and a mobile phase consisting of 25 to 75 acetonitrile/0.1M phosphate buffer improved the detection limit to 6.5ng; however, three oxyfMLP peaks were detected. Stock solutions of fMLP standards and environmental samples such as cotton dust and sorghum stored at -20 degrees-C did not show good stability as peaks corresponding to oxyfMLP were detected. The modified HPLC method did not have sufficient sensitivity to detect fMLP in environmental air samples, which typically contained only a few milligrams of total dust. Deformylases were isolated from Escherichia-coli in an attempt to develop a competitive binding assay for detecting fMLP. Advantage was taken of the fact that deformylases liberated formic- acid. This project was unsuccessful due to the lack of specificity. Receptor binding studies on guinea-pig pulmonary macrophages, rabbit neutrophils, and human neutrophils utilizing tritium labeled fMLP were undertaken to investigate the possibility they could be used to develop a competitive binding assay. The receptor binding patterns indicated the presence of at least three fMLP receptors that would interfere with measuring fMLP in a competitive assay. The authors conclude that attempts to develop an assay for determining fMLP in environmental samples have thus far been unsuccessful.
NIOSH-Author; Analytical-methods; Proteins; Microorganisms; Natural-products; Chromatographic-analysis; Chemical-binding; Chemoreceptors; In-vitro-studies
P. D. Siegel, Ph.D., NIOSH/DRDS, 944 Chestnut Ridge Road, MS 211, Morgantown, WV 26505, USA
Journal of Toxicology and Environmental Health