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

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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.

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