The role of metallic cations in the inhibitory activity of doxycycline. (A) Zn²⁺ or Cu²⁺ decreased doxycycline inhibitory activity. Doxycycline (5 mmol/L) was treated with ZnCl₂ (•), CuSO₄ (□), or CaCl₂ (▪) at designated molar concentration ratios before it was added at 1/10 (vol/vol) dilution to the proteinase. Proteinase Inhibition (%) = (Original Activity − Residual Activity)/(Original Activity − Residual Activity After Incubation With Doxycycline Not Treated With Cations) × 100. (B) Restoration of proteinase activity by metallic cations. The plasma proteinase was treated with 1 mmol/L EDTA (♦) or 1 mmol/L doxycycline (□). CaCl₂ or ZnCl₂ was then added at the designated concentrations. The proteinase activity was plotted against the cation concentrations.