Hello,
I'm trying to preform an esterase assay to test PETase enzyme, but the substrate (p-NPA) keeps turning yellow the moment it touches the buffer solution (Sodium phosphate pH 8).
From what I read online, the buffer should stay clear, and the pH of 8 should be ok for the pNPA.
what a I doing wrong?
Thanks
Hi there,
Your pNPA is already partially or totally hydrolyzed... I agree it should remain clear if 100% intact. To measure the degree of hydrolysis, measure OD at 410nm (with read OD within the linearity domain of the spec) and compare the deduced pNP concentration with the theoritical pNPA concentration.
p-Nitrophenyl acetate hydrolysis isn't just dependent upon the pH, its hydrolysis can be brought about by the presence of nucleophiles of the appropriate softness/hardness see
Coles, B. Effects of modifying structure on electrophilic reactions
with nucleophiles. Drug Metab. Rev. 1985, 15, 1307−1334.
So if your buffer contains any thiols (or other soft nucleophiles) I would expect the p-Nitrophenyl acetate to be liberate the yellow p-Nitrophenol rapidly.
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