Effects of Cu2+ on alkaline phosphatase from Macrobrachium rosenbergii

To gain insight into the effect of Cu2+ on the activity and structure of alkaline phosphatase (ALP) from Macrobrachium rosenbergii, the enzyme was purified using ammonium sulfate fractionation, Sephacryl S-200, and DEAE anion exchange chromatography. We studied Cu2+-mediated inhibition and aggregation of ALP, and found that Cu2+ significantly inactivated ALP activity with an IC50 of 1.47 +/- 0.02 mM. We further revealed that Cu2+ reversibly inhibited ALP in a mixed-type manner with K-i = 0.41 +/- 0.02 mM. Time-interval kinetics showed that the inhibition followed first-order reaction kinetics. This process was associated with conformational changes and significant transient free-energy change. Spectrofluorometry results showed that Cu2+ induced ALP tertiary structural changes, including the exposure of hydrophobic surfaces that directly induced ALP aggregation. The results provide new information regarding ALP from M. rosenbergii. (C) 2018 Elsevier B.V. All rights reserved.