A new fluorescence assay for dipeptidylpeptidase IV using tripeptide L-prolyl-L-prolyl-L-alanine as substrate

We have developed a new fluorescence assay for dipeptidylpeptidase IV using a tripeptide, L-prolyl-L-prolyl-L-alanine, which might be one of the potential natural substrates. The principle of the assay is based on the measurement of fluorescent adduct between alanine liberated from the tripeptide by enzymatic hydrolysis and o-phthaldialdehyde in the presence of 2-mercaptoethanol in aqueous alkaline medium. This new assay is sensitive enough to measure the enzyme activity in as little as 0.01 microliter of human serum and in crevicular fluid obtained from human gingival sulcus. The Km value for the tripeptide was 1.7 x 10(-5) M which is less than one-tenth of that obtained with other chromogenic or fluorogenic substrates. The interference by serum was overcome by simply incorporating the same amount of serum in the standards.