A chromogenic substrate for solid-phase detection of phospholipase A₂.
A method for solid-phase detection of phospholipase A₂ (PLA₂) was developed. The method uses 1-octanoyloxynaphthalene-3-sulfonic acid, which was found to be a good substrate of PLA₂. The substrate is hydrolyzed by PLA₂ into 1-naphthol-3-sulfonic acid, which is spontaneously coupled with coexisting diazonium salt to form a red-purple azo dye. Streptomyces and bovine pancreatic PLA₂ spotted on a nitrocellulose membrane could be detected by this method with considerable sensitivity. In addition, colonies of recombinant Escherichia coli producing bacterial PLA₂ were distinguishable from those producing an inactive mutant PLA₂, facilitating high-throughput screening in directed evolution of the enzyme.
Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Chisato
C
Eba
Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Aoi
A
Okano
Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Hideo
H
Nakano
Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. Electronic address: iwasaki@agr.nagoya-u.ac.jp.
Yugo
Y
Iwasaki
D000818:Animals; D002417:Cattle; D002863:Chromogenic Compounds / Q000378:metabolism*; D003101:Collodion / Q000737:chemistry; D057075:Enzyme Assays / Q000379:methods*; D006868:Hydrolysis; D008567:Membranes, Artificial; D009282:Naphthalenesulfonates / Q000378:metabolism*; D054467:Phospholipases A2 / Q000378:metabolism*; D013451:Sulfonic Acids / Q000378:metabolism*
D002863:Chromogenic Compounds; D008567:Membranes, Artificial; D009282:Naphthalenesulfonates; D013451:Sulfonic Acids; D003101:Collodion; D054467:Phospholipases A2
Artificial substrate; Diazo coupling; High-throughput screening; Phospholipase A(2); Solid-phase detection