Development of a Dihydropteroate Synthase-Based Fluorescence Polarization Assay for Detection of Sulfonamides and Studying Its Recognition Mechanism

Abstract

In this study, the dihydropteroate synthase of Staphylococcus aureus was obtained, and its recognition mechanisms for 31 sulfonamide drugs were studied. Results showed that their core structure matched well with the binding pocket of paraaminobenzoic acid, and all the sulfonamide side chains were out of the binding pocket. Hydrogen bonds and hydrophobic interactions were the main intermolecular forces, and the key amino acids were Gly171 and Lys203. The binding sites in sulfonamide molecules were mainly around the para-aminobenzenesulfonamide part. This enzyme was used to develop a fluorescence polarization assay for detection of these drugs in chicken muscles. The change trends of half of inhibition concentrations and crossreactivities for the 31 drugs were identical with the receptor−ligand affinities. The limits of detection were in the range of 2.0−38.5 ng/g, and one assay could be finished within several minutes. Therefore, this method could be used for multiscreening of sulfonamide residues in meat samples.

Production of a Natural Dihydropteroate Synthase and Development of a Signal-Amplified Pseudo-Immunoassay for the Determination of Sulfonamides in Pork

“…For a comparison to related methods, the main results of some previously reported conventional immunoassays and DHPS-based assays are summarized in Table . First, the operation steps of the present method were the same as those of the direct competitive methods ,,,, and fewer than those of the indirect competitive methods, ,,, but more than those of the fluorescence polarization methods. ,, Second, the assay time (from adding sample) of the present method was comparable to or shorter than − ,,,,,, that of the indirect competitive and direct competitive methods, but longer than the fluorescence polarization methods. ,, Third, the sensitivity of the present method was higher than all of those methods, even the fluorescence polarization methods and the chemiluminescence method. Fourth, the detection spectrum of the present method was broader than all of those methods.…”

“…For development of a signal-amplified ELISA, SZ was coupled with HRP, sHRP, and bHRP, individually, to prepare three enzyme-labeled conjugates (HRP-SZ, sHRP-SZ, and bHRP-SZ) by using the glutaraldehyde method. In our recent report, the molecular docking showed that DHPS could interact with the aromatic primary amine, phenyl ring, and −SO 2 N– part in SAs molecules . So this conjugate synthesis method would not influence the DHPS bindings for the three conjugates, even if the aromatic primary amine in the SZ molecule was occupied.…”

“…For development of an optimal SA-pELISA method, several important parameters were optimized with PABA as the representative (10 ng/mL). DHPPP is the first substrate of DHPS, and our recent report has shown that the binding of SAs with DHPS is actually the binding of SAs with DHPS-DHPPP complex, and the DHPS-SAs binding is weak if DHPPP is absent . Therefore, the concentrations of DHPS and DHPPP were optimized first.…”

“…The horseradish peroxidase (HRP), streptavidin-horseradish peroxidase (sHRP), and biotinylated horseradish peroxidase (bHRP) were from Shanghai Yuanye Biological Technology Co., Ltd. (Shanghai, China). Dihydropteridine pyrophosphate (DHPPP, the first substrate of DHPS) was synthesized in our recent report . Other common chemicals were from Shanghai Saen Chemical Company (Shanghai, China).…”

“…For an ELISA, the core material is the used recognition reagent. By now, various recognition reagents have been used to develop conventional ELISA and pseudo-ELISA for detection of SAs, such as polyclonal antibody, − monoclonal antibody, − recombinant antibody, , aptamer, molecularly imprinted polymer, and dihydropteroate synthase. − …”