The inhibition of polyphenol oxidase (PPO) is a critical target in preserving the quality of fresh fruits and vegetables. While salicylic acid has emerged as a potent natural inhibitor, its effectiveness varies significantly with environmental conditions such as pH. This study investigates how acidic pH enhances the inhibitory action of salicylic acid through both structural destabilization and strengthened molecular binding.
To isolate the influence of pH, salicylic acid solutions were adjusted to fixed pH values of 6.8, 6.5, 6.0, and 5.5 before incubation with PPO. At each pH, residual enzyme activity was measured after treatment with 3 and 9 mmol/L salicylic acid. Results showed a clear pH-dependent inhibition trend: at pH 6.8, residual activity was 73.4% and 45.9%, respectively, while at pH 5.5, it dropped to 18.1% and 5.6%. The IC₅₀ values decreased from 12.2 mmol/L at pH 6.8 to just 0.6 mmol/L at pH 5.5, indicating a dramatic increase in potency under acidic conditions.
Kinetic analysis confirmed that salicylic acid acts as a competitive inhibitor across all pH levels, with constant Vmax and increasing Km values, consistent with substrate competition at the active site. However, the magnitude of inhibition intensified with decreasing pH, suggesting additional mechanisms beyond simple binding.278779-30-9 Description
Circular dichroism (CD) spectra revealed progressive loss of α-helical structure in PPO with increasing acidity.531-95-3 site At pH 5.PMID:35094229 5, α-helix content declined by nearly 40% compared to native PPO, accompanied by a significant rise in β-sheet formation. These changes correlate with reduced enzymatic activity and indicate unfolding of the tertiary structure, likely exposing hydrophobic regions and facilitating inhibitor access.
Molecular docking simulations at different pH levels demonstrated a marked enhancement in binding affinity under acidic conditions. At pH 6.8, the lowest binding energy was −75.8 kcal/mol; at pH 5.5, it dropped to −138.6 kcal/mol. The improved stability arose from new electrostatic interactions between deprotonated carboxylate groups of salicylic acid and protonated histidine residues (HIS61, HIS85, HIS259, HIS263, HIS296). Additionally, hydrogen bonds shortened by up to 0.8 Å, strengthening their contribution to complex stability. At pH 5.0, further hydrogen bonding with ASN260 was observed, reinforcing the interaction network.
These findings demonstrate that low pH does not merely enhance the acidification effect but actively promotes the binding of salicylic acid to PPO by altering the enzyme’s conformation and charge distribution. The synergistic interplay between pH-induced unfolding and specific molecular interactions results in far greater inhibition than either mechanism alone.
In practical applications, this synergy suggests that combining salicylic acid with naturally acidic food matrices—such as citrus fruits or berries—can achieve superior browning control with lower inhibitor concentrations. It also underscores the need for precise pH management in preservation protocols. By leveraging both intrinsic acidity and effective inhibitors, food processors can develop more sustainable and efficient strategies to maintain freshness, color, and nutritional value in perishable produce.MedChemExpress (MCE) offers a wide range of high-quality research chemicals and biochemicals (novel life-science reagents, reference compounds and natural compounds) for scientific use. We have professionally experienced and friendly staff to meet your needs. We are a competent and trustworthy partner for your research and scientific projects.Related websites: https://www.medchemexpress.com
