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.
Bolygo
Elek
E
Jealott's Hill International Research Centre, Syngenta, RG42 6EY, Bracknell, Berkshire, UK. elek.bolygo@syngenta.com
Ricketts
Dean
D
Moffatt
Frank
F
Jessop
Michael
M
Attenborough
Sheila
S
D000935:Antifungal Agents / Q000032:analysis / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D023082:Defensins; D010940:Plant Proteins / Q000032:analysis* / Q000737:chemistry; D012987:Soil / Q000032:analysis*
D000935:Antifungal Agents; D023082:Defensins; C099969:Dm-AMP1 protein, Dahlia merckii; D010940:Plant Proteins; D012987:Soil