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.
Goossens
F
F
Laboratory for Medical Biochemistry, University of Antwerp, Wilrijk, Belgium.
De Meester
I
I
Vanhoof
G
G
Scharpé
S
S
D000293:Adolescent; D000328:Adult; D000368:Aged; D002021:Buffers; D003374:Coumarins; D005069:Evaluation Studies as Topic; D005470:Fluorometry / Q000379:methods* / Q000706:statistics & numerical data; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008875:Middle Aged; D000085822:Prolyl Oligopeptidases; D012680:Sensitivity and Specificity; D012697:Serine Endopeptidases / Q000097:blood*; D013379:Substrate Specificity
D002021:Buffers; D003374:Coumarins; C020988:N-carbobenzoxyglycyl-prolyl-4-methylcoumarinyl amide; D012697:Serine Endopeptidases; C000705287:PREPL protein, human; D000085822:Prolyl Oligopeptidases