40mM EDTA in (non-metallo)protease assay buffer - (Mar/05/2014 )
Does anyone know the reason for the extremely high concentrations of EDTA in casein based protease assays for papain? The AOAC/FCC/ US & European Pharmacopeia all give 14g/L (example below). To me it looks like a decimal point typo that has been duplicated for decades but maybe it is something to do with interference from cystein.
”Dissolve 3.55 g Na2HPO4 in 400 mL H2O in 500 mL volumetric flask. Add 7.0 g 2Na.2H2O.EDTA and 3.05 g cysteine.HCl.H2O. Adjust to pH 6.0 ± 0.1 with 1M HCl and dilute to volume with H2O. Prepare fresh daily.”
this is just a guess (and probably wrong, but...), casein is highly phosphorylated and will bind metals. a high concentration of edta should keep metals away from the casein.
also, the original investigator may have been using poorly deionized (or not deionized) water.
Ahh yes. Thanks.
I think you are correct; it would compete for the Ca and stop the casein forming micelles.