A sensitive method for the assay of serum prolyl endopeptidase

A new fluorimetric assay for the determination of prolyl endopeptidase (EC 3.4.21.26) was developed. The synthetic substrate Z-glycyl-prolyl-4-methylcoumarinyl-7-amide (0.2 mmol/l; 5 microliters), K-phosphate buffer (100 mmol/l, pH 7.5; 100 microliters), and serum (10 microliters) are incubated for 120 min at 37 degrees C. The reaction is stopped with acetic acid (1.5 mol/l; 500 microliters) and the released 7-amino-4-methylcoumarin is measured fluorimetrically. The mean value of prolyl endopeptidase catalytic activity concentration in serum for 120 healthy volunteers was 0.455 (SD = 0.092) mumol of 7-amino-4-methylcoumarin released per litre of serum per minute. The proposed procedure is sensitive, robust, and economical.