A fluorogenic substrate for detection of organophosphatase activity.
A new fluorogenic substrate for the specific detection of organophosphatase (OPase) activity has been designed and evaluated. Our results indicate that 7-diethylphospho-6,8-difluor-4-methylumbelliferyl (DEPFMU) is hydrolyzed specifically by the OPases, mammalian serum paraoxonase and bacterial organophosphorus hydrolase (OPH). The apparent K(m) of DEPFMU is 29 microM for OPH and 91 and 200 microM for the PON1 L(55)R(192) and PON1 L(55)Q(192) isoforms of human paraoxonase, respectively. DEPFMU-based assay systems are 10-100 times more sensitive for OPH and mammalian paraoxonase detection than existing methods. Importantly, DEPFMU is poorly hydrolyzed by both serum and cellular phosphatases and, therefore, may be used as part of a robust and sensitive assay for detecting not only purified, but also highly impure, preparations of OPase such as blood samples. The superior sensitivity of DEPFMU makes it potentially useful in the search for new enzymes that may hydrolyze nerve poisons such as sarin, soman, and VX, monitoring the decontamination of organophosphates (OPs) by OPH and determining serum paraoxonase activity which appears to be important for protection against atherosclerosis, sepsis, and OP toxicity.
Soukharev
Serguei
S
Plasma Derivatives Department, Holland Laboratory, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855, USA. soukharevs@usa.redcross.org
Hammond
David J
DJ
D000818:Animals; D043303:Aryldialkylphosphatase / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D006868:Hydrolysis; D051379:Mice; D015394:Molecular Structure; D010755:Organophosphates / Q000737:chemistry* / Q000378:metabolism; D010261:Paraoxon / Q000032:analysis; D011817:Rabbits; D014162:Transfection; D014468:Umbelliferones / Q000737:chemistry* / Q000378:metabolism
C492761:7-diethylphospho-6,8-difluoro-4-methylumbelliferyl; D005452:Fluoresceins; D005456:Fluorescent Dyes; D010755:Organophosphates; D014468:Umbelliferones; C492762:di-diethylphosphofluorescein; D043303:Aryldialkylphosphatase; D010261:Paraoxon