Salicylic acid has long been recognized for its ability to inhibit polyphenol oxidase (PPO) activity and suppress enzymatic browning in fruits and vegetables. Traditionally, this inhibition is attributed to the structural similarity between salicylic acid and phenolic substrates, enabling competitive binding at the active site of PPO. However, the potential contribution of salicylic acid’s acidification effect—its capacity to lower pH and thereby alter enzyme conformation—has often been overlooked. This study systematically investigated the dual inhibitory mechanisms of salicylic acid by manipulating buffer conditions to isolate and evaluate the roles of acidification and molecular binding.
Experiments were conducted using media with varying buffering capacities: ultrapure water, and phosphate-buffered saline (PBS) at 5, 10, and 20 mmol/L at pH 6.8. As buffer concentration increased, the extent of pH reduction caused by salicylic acid diminished, indicating a decreased acidification effect. In ultrapure water, as little as 0.05 mmol/L salicylic acid reduced PPO activity nearly to zero, accompanied by a pH drop to 4.3. In contrast, under high-buffering conditions, higher concentrations of salicylic acid (up to 3 mmol/L) were required for equivalent inhibition. The half-maximal inhibitory concentration (IC₅₀) increased from 0.02 mmol/L in ultrapure water to 2.6 mmol/L in 20 mmol/L PBS, confirming that buffering mitigates acidification-driven inhibition.
To assess the binding effect independently, pH was restored to 6.8 after salicylic acid treatment. Under these controlled conditions, PPO activity recovered significantly, particularly in low-buffer systems. At high salicylic acid concentrations (9 mmol/L), residual activity remained around 46% even after pH adjustment, suggesting a strong binding component. Kinetic analysis revealed a linear Lineweaver-Burk plot with unchanged Vmax but increasing Km, indicating competitive inhibition. The inhibition constant (Ki) was determined as 0.56 mmol/L, consistent with salicylic acid acting as a substrate mimic.
Circular dichroism (CD) spectroscopy showed that salicylic acid induced conformational changes in PPO, reducing α-helix content from 34.18378-89-7 Biological Activity 4% to 20.cIAP2 Antibody custom synthesis 7% and increasing β-sheet content.PMID:35179218 These structural alterations suggest a reversible destabilization of the enzyme’s native fold, which may impair catalytic efficiency.
Molecular docking simulations revealed salicylic acid binds primarily via hydrogen bonds with HIS61, HIS85, HIS259, HIS263, and hydrophobic interactions with VAL283. At acidic pH (5.0–5.5), additional electrostatic interactions and shorter hydrogen bond distances emerged, lowering binding energy and enhancing stability. This synergistic effect confirms that acidic pH not only reduces PPO activity directly but also strengthens salicylic acid binding.
In conclusion, both acidification and binding effects contribute to salicylic acid’s inhibition of PPO. In weakly buffered systems, acidification dominates; in stronger buffers, binding becomes more significant. The cooperative action of pH reduction and molecular interaction enhances overall efficacy. These findings highlight the importance of considering food matrix pH and buffering capacity when applying salicylic acid for browning control, offering a refined strategy for effective postharvest preservation.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
