Inhibition assays of horseradish peroxidase by hexavalent chromium and other heavy metals

Abstract

ABSTRACT In this study, individual and combinations of heavy metals were tested at different pH for their inhibition potential by horseradish peroxidase (HRP). At pH of 6.4, maximum velocities for a single substrate concentration of hydrogen peroxide was observed. The double reciprocal of Lineweaver-Burk plot gave the same values of Km and decreased Vmax represents the non-competitive type of inhibition by hexavalent chromium metal ions. The IC10 value of Cr6+ ion for free HRP was achieved at 0.55ppm, while inhibition for 1ppm of substrate concentration was 24.5%. For other tested heavy metals, no significant inhibition properties were observed except for Cd2+ and Mg2+ metal ions. However, when combined with Cr6+ ions, substantial changes were detected. Based on IC10 values, the sensitivity order for free HRP for individual metal ions was observed as Cd2+ > Cr6+ > Mg2+ > Cu2+ > Cr3+ > Mn2+ while the combination with Cr6+ gave the order as Cd2+ > Cu2+ > Cr3+ > Cr6+ > Mg2+ > Mn2+. The order of inhibition byquaternary combinations of metal ion were in the order (Cr6+ + Cd2+ + Cu2+ + Cr3+) > (Cr6+ + Cu2+ + Mg2+ + Cd 2+) ≥ (Cr6+ + Cd2+ + Mg2+ + Cr3+) ≥ (Cr6+ + Cu2+ + Mg2+ + Cr3+), while the quinary combination (Cr6+ + Cr3+ + Cu2+ + Mg2+ + Cd2+) was the most effective grouping. The studies shown excellent selectivity towards Cr6+ and suggest further use of HRP enzymes in the design and selection of biosensors for heavy metals and other pollutants.