[Catalytic properties and thermostability of horseradish peroxidase covalently bound with Sepharose by carbohydrate residues of the enzyme].
Authors: Berezin, Ugarova, Fel'dman
Journal: Biokhimiia (Moscow, Russia)
Summary
# Editorial Summary Horseradish peroxidase (HRP) has long been exploited in equine diagnostics and research applications, yet maintaining enzyme stability and catalytic efficiency during storage and use remains challenging. Berezin and colleagues developed a method to covalently bind HRP to Sepharose matrices via the enzyme's carbohydrate residues (oxidised with periodate), then compared catalytic kinetics and thermal stability of the native, soluble oxidised, and immobilised forms using standard peroxidase assays with o-dianisidine substrate. Whilst oxidising the carbohydrate residues halved the catalytic turnover rate (kcat) in soluble enzyme without affecting substrate affinity (Km), immobilisation produced a striking 20-fold improvement in o-dianisidine affinity alongside unchanged hydrogen peroxide Km—suggesting the matrix environment fundamentally altered substrate accessibility. The immobilised preparation demonstrated superior thermostability, with the degree and type of protein modification significantly influencing enzyme longevity. For equine professionals developing point-of-care diagnostics or enzyme-based assays, this work underscores how matrix selection and binding chemistry can substantially improve both shelf-life and detection sensitivity, though practitioners should anticipate reduced reaction rates unless substrate concentration is carefully optimised.
Read the full abstract on PubMed
Practical Takeaways
- •Not applicable to equine practice - this is a fundamental biochemistry study on enzyme immobilization techniques with no direct clinical relevance
Key Findings
- •Horseradish peroxidase can be stably immobilized on Sepharose 4B through covalent binding via oxidized carbohydrate residues
- •Oxidation of carbohydrate residues decreased catalytic rate (kcat) approximately 2-fold for soluble peroxidase but did not affect Km for H2O2
- •Immobilized peroxidase showed 20-fold decrease in Km for o-dianizidine substrate compared to native enzyme
- •Matrix chemical structure and protein functional group modification significantly affect immobilized enzyme stability