Colorimetrical rate assay for urinary dipeptidyl peptidase IV (DPPIV) activity using a new substrate

We synthesized a new substrate glycyl-L-proline 3,5-dibromo-4-hydroxyanilide (Gly-Pro-DBAP), for dipeptidyl peptidase IV (DPPIV). Its hydrolysis by DPPIV resulted in the formation of a chromophore, 2,6-dibromophenol-indo-p-xylenol, and its maximal absorption wavelength (600 nm) was longer than that of p-nitroaniline (415 nm) released from conventional substrate, glycyl-L-proline p-nitroanilide (Gly-Pro-pNA). We also established the rate assay for urinary DPPIV activity using Gly-Pro-DBAP. The optimum pH was between 8.5 and 9.0. The apparent Km was 1.1 mmol/1. The detectable range was 2.5-350 U/l. No changes in blank values occurred throughout the enzyme reaction in the optimum pH. Its value was also much lower than Gly-Pro-pNA. CVs for within-run and between-run were 1.1% (n = 10) and 3.0% (n = 10), respectively. Among tested peptidases, only DPPIV could hydrolyze Gly-Pro-DBAP. Among the protease inhibitors, only two, diprotin-A and phenylmethylsulfonyl fluoride (PMSA), could inhibit DPPIV activity. The present method did not interfere with urinary ingredients such as hemoglobin. The correlation between the present (y) and conventional (x) methods is presented by the equation y = 1.121x + 0.096 (r = 0.993). Thus the present method provides practical advantages over the conventional method for routine laboratory use.