Biochemical characterization of a new glycosylated protease from Euphorbia cf. lactea latex

Abstract

© 2015 Elsevier Masson SAS. A dimeric protease designated as EuP-82 was purified from Euphorbia cf. lactea latex. Since its proteolytic activity was inhibited by a serine protease specific inhibitor (PMSF), EuP-82 was classified as a serine protease. N-glycan deglycosylation tests revealed that EuP-82 was a glycosylated protein. MALDI-TOF MS showed that EuP-82 was a homodimer, which was its active form. The optimal conditions for fibrinogenolytic activity were at pH 11 and 35°C. EuP-82 enzyme had broad range of pH stability from 4 to 12. Moreover, the enzyme was still active in the presence of reducing agent (β-mercaptoethanol). EuP-82 was a proline-rich enzyme (about 20.69mol%). Increased proline production can be found in higher plants in response to both biotic and abiotic stresses, high proline in the molecule of EuP-82 might stabilize its activity, structure and folding. Based on the N-terminal amino acid sequences and peptide mass fingerprint (PMF) of EuP-82, the enzyme was identified as a new serine protease. The digested products from EuP-82 cleavage of human fibrinogen were analyzed by SDS-PAGE and PMF. The results confirmed that EuP-82 could digest all subunits of human fibrinogen. EuP-82 cleaved fibrinogen with a Michaelis constant (K<inf>m</inf>) of 3.30 ± 0.26 μM; a maximal velocity (V<inf>max</inf>) of 400.9 ± 0.85 ng min^-1; and a catalytic efficiency (V<inf>max</inf>/K<inf>m</inf>) of 121.5 ± 9.25 ng μM^-1min^-1. EuP-82 has potential for use in medicinal treatment, for example thrombosis, since the enzyme had fibrinogenolytic activity and high stability.