Partial purification and characterisation of dipeptidyl peptidase II from porcine skeletal muscle

Abstract

The purification and study of biochemical properties of dipeptidyl peptidase II (DPP II; EC 3.4.14.2) from porcine skeletal muscle have been carried out in the present work. The purification included ammonium sulphate fractionation and two HPLC chromatographic separations using a Resource-Q anion exchange column. The enzyme was purified 1270 fold, with a 1.6% recovery and was completely separated from DPP IV activity. The pure enzyme displayed one main protein band with a Mr of 58 kDa on SDS-PAGE. Maximum activity was reached at pH 5.5 and 65°C. Those synthetic substrates containing Pro in N-penultimate position were the most efficiently hydrolysed, whereas in the case of peptides, DPP II efficiently hydrolysed both X-Pro- and X-Ala- peptides. The serine peptidase inhibitors PMSF and Pefabloc SC suppressed DPP II activity in a high degree, whereas 3, 4-DCI and cysteine peptidase inhibitors exerted little effect. Alkaline metal salts inhibited the enzyme activity according to the size of the cation, and among the assayed divalent cations, only Cu 2+, Fe 2+ and Hg 2+ showed significant inhibition of the activity. This is the first time that porcine muscle DPP II has been purified and its biochemical characteristics studied. So, these results contribute to improve the knowledge in relation with the proteolytic chain and the generation of flavour characteristics in meat products.