Stabilization of Diluted Aqueous Solutions of Horseradish Peroxidase

Effects of pH, enzyme concentration, and various supplements on the catalytic activity, temperature stability, and secondary structure of horseradish peroxidase (HRP) were studied in diluted aqueous solutions. In 5.0 mM citrate-phosphate buffer (pH 4.2) at 55°C and infinite dilution, HRP was inactivated with a rate constant of 2.86 × 10^–3 s^–1. CaCl₂, BSA, and glycerol caused protective effects, whereas KCl, LiCl, maltose, PEG-6000 (at a concentration above 3%), Triton X-100, ethanol, and Kathon CG had an opposite effect and altered the secondary structure of HRP. Two HRP-stabilizing media: the glycerol-based one containing 10% ethanol and 20% glycerol, or the protein-based one containing 0.1% Kathon CG and 0.2 mg/ml of BSA in 50.0 mM Tris-HCl buffer (pH 7.2) supplemented with 50 mM CaCl₂ were developed, and the stability of HRP (0.36 nM) and its immunoglobulin, cortisol, and progesterone conjugates were compared in these two media. The protein-based medium displayed a greater stabilizing effect particularly on HRP-steroid conjugates.