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.