POTENTIATION OF GLUCURONIDASE HYDROLYSIS OF SODIUM PREGNANEDIOL GLUCURONIDATE

Abstract

It has been observed that after long standing a reduction occurs in the activity of glucuronidase preparations of mammalian origin (e.g. Ketodase) on sodium pregnanediol glucuronidate (NaPG), either as a pure solution or in urine. This deterioration is characterized by increases in the optimum pH and in the amount of enzyme required to yield a complete hydrolysis of the NaPG. The fact that complete enzyme activity on pure NaPG solutions is restored by the addition of propylene glycol suggests that this deterioration might involve some glucuronyl transfer mechanism. Propylene glycol increases the activity of enzyme on pure NaPG solution by a factor of 6–15, reducing the amount of enzyme required to give a 95% hydrolysis in 4 hours from about 150 units/ml to 10 units/ml or by reducing the time required for 10 units/ml of enzyme to effect this hydrolysis from 24 to 4 hours. The presence of inhibitors in urine reduces this factor to 2, thus it requires half as much enzyme to effect a 95% or complete hydrolysis of the NaPG of urine in the presence of propylene glycol as in its absence. About two-thirds of the inhibitors toward the Ketodase hydrolysis of the NaPG in urine can be removed by simple (NH4)2SO4precipitation and extraction techniques. Thus 25 units/ml urine equivalent are capable of giving a complete hydrolysis of the NaPG in such extracts in the presence of propylene glycol, compared to 150 units/ml for the original urine under similar circumstances or 300–600 units/ml in the absence of propylene glycol.