The role of ammonium sulfate precipitation in the removal of substances which interfere with enzymatic hydrolysis of conjugated estrogens in pregnancy urine

Abstract

Precipitation of the conjugated estrogens from pregnancy urine by 70% (w/v) (NH4)2SO4 was found to leave urinary substances in the supernatant which interfere with the assay following hydrolysis with enzymes (Ketodase and Glusulase). This removal of enzyme inhibitors is associated with a decrease in optimal pH for enzymic hydrolysis as well as with a reduction in the minimum amount of enzyme required to effect a maximum hydrolysis. One of the inhibitors removed is saccharolactone that may be present. An additional removal of enzyme inhibitors, with a further reduction in optimal pH, is effected by extraction of a solution of the (NH4)2SO4 precipitate by the technique of Kellie. These purification procedures result in an optimal pH for enzymatic hydrolysis equivalent to that reported for pure conjugates, and a 5- to 10-fold reduction in the minimum amount of enzyme required to effect a maximum hydrolysis. Propylene glycol, unlike its effect on phenolphthalein and pregnanediol glucuronides, has an inhibitory effect on the enzymic hydrolysis of estriol glucuronide.