Determination of antifungal proteins in soil by liquid chromatography

A liquid chromatography method was developed for the determination of antifungal/antimicrobial proteins Rs-AFP1 and Dm-AMP1 in sandy loam soils. The extraction of these highly basic proteins was achieved by mechanical shaking with aqueous Tris buffer pH 9 containing guanidinium thiocyanate salt (4.1 M), EDTA and nonionic polyoxyethylene 20 cetyl ether, Brij-58 detergent. The extracts were cleaned up on Oasis HLB polymer solid-phase extraction cartridges and quantified by liquid chromatography fluorescence detection based on the fluorescence properties of the tryptophan content of these proteins. The detector response was linear for 0.3-10 microg mL(-1). Procedural recoveries were tested in the range 10-100 mg kg(-1). The limit of quantification was 10 mg kg(-1 )protein in the soil sample representing the lowest validated fortification level. The antifungal proteins were found to be stable in soil extract tested up to 9 days when stored at 4 degrees C.