Rapid detection of DPP-IV activity in porcine serum: A fluorospectrometric assay

• Published in: Analytical biochemistry Vol. 592; p. 113557
• Main Authors: Divya, K., Vivek, H.K., Priya, B.S., Nanjunda Swamy, S.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-03-2020
• Subjects: Animals; Bovine serum; Cattle; Dipeptidyl Peptidase 4 - blood; Diprotin A; DPP-IV; Gly-Pro-AMC; Human serum; Humans; Nanotechnology - methods; Porcine serum; Spectrometry, Fluorescence - methods; Swine; RFU; Human serum; DPP-IV; GLP-1; Porcine serum; AMC; Bovine serum; Diprotin A; Gly-Pro-pNA; Gly-Pro-AMC
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2019.113557

Dipeptidyl peptidase IV (DPP-IV) is an aminopeptidase that cleaves the N-terminal dipeptide from peptides bearing proline or alanine residues. Currently, DPP-IV activity is quantified by spectrophotometric or fluorometric methods, which employ Gly-Pro-pNA and Gly-Pro-AMC respectively, as substrate. However, these methods require high enzyme and substrate concentrations. In this study, we adapted the DPP-IV fluorospectrometric assay using NanoDrop 3300, which requires only nanogram levels of the enzyme (30 ng crude DPP-IV) and considerably low substrate concentrations (100 μM). Fluorescence measurement required a reaction mixture of only 2 μL, thus eliminating the need for microtiter plates or cuvettes.We employed this assay to demonstrate DPP-IV activity in porcine serum for the first time. The enzymatic activity peaked at pH 8.0 in porcine (84 nM/min), human (87 nM/min) and bovine (89.1 nM/min) sera, with the optimum temperature of 37 °C. The enzyme showed maximum activity upon incubation for 40 min at 37 °C. In contrast, activity in the porcine serum was the highest after incubation for 30 min at the same optimized parameters. The IC50 values of diprotin A against DPP-IV from human, porcine, and bovine sera were 7.83, 8.62, 9.17 μM, respectively. The present assay procedure is a convenient, sensitive, accurate and high-throughput method suitable for primary screening of DPP-IV inhibitors.
Fluoro spectrometric assay of DPP-IV by NanoDrop. [Display omitted]
•DPP-IV assay was adapted to fluorospectrometric method using NanoDrop3300.•The assay required low concentrations of enzyme (30 ng) and substrate (0.1 mM).•Only 2 μL of reaction mixture is required for the measurement of fluorescence.•DPP-IV activity in porcine serum is reported in this work for the first time.•A comparison of DPP-IV activity in human, porcine and bovine sera is presented.