High-throughput micro-plate fluorescence quenching for screening the binding of mycotoxins to albumins

Abstract

Filamentous fungi, which commonly contaminate food and animal feed, produce toxic metabolites called mycotoxins. Legal limits on their maximum levels in food require food producers to carry out highly specific analytical tests for mycotoxins, such as Ochratoxin A (OTA), patulin (PAT) and zearalenone (ZEN). Serum albumins are known to bind some mycotoxins, presenting an opportunity for the development of novel capture and clean-up strategies. The aim of this study was to validate a fluorescence quenching micro-plate methodology for high-throughput screening of the binding constants of OTA, PAT and ZEN to albumins. Herein, we present measurements of equilibrium binding constants of these mycotoxins to several albumins using fluorescence quenching, measured in 96-well micro-plates. In addition, commercial native human serum albumin (HSA) was compared to recombinant HSA produced by Pichia pastoris KM71H transformed with the pPICZ9ssHSAH6 vector. Both OTA and ZEN showed slightly higher affinity for recombinant versus native HSA, whereas the difference was negligible for PAT. The order of affinity for native HSA is OTA>ZEN>PAT, with binding constants, Ksv (L/mol), of 6.22 x 105, 2.97 x 104 and 9.50 x 103, respectively. The use of different buffers (pH 7.0-7.4) was also tested, where the affinity of ZEN for HSA decreased slightly in PBS compared to 10 mM Tris-HCl and 20 mM PB, whereas PAT binds more strongly to HSA in PB. The affinity of mycotoxins to bovine- and rat-serum albumins (BSA, RSA) was also tested and compared to HSA. For ZEN the order of affinity was found to be RSA>HSA>BSA, and HSA> BSA>RSA for OTA and PAT. The results will aid in the development of novel protein-based extraction columns for analytical control