EFFECT OF PHENOLIC ACIDS ON PANCREATIC LIPASE STRUCTURE BY FLUORESCENCE SPECTROPHOTOMETRY

Pancreatic lipase is the primary enzyme responsible for dietary lipid hydrolysis, making its inhibition a target for anti-obesity therapies. Because synthetic inhibitors, like Orlistar, have been associated to unwanted side effects, there is growing interest in looking at natural polyphenols from plant sources, such as phenolic acids, as potential alternatives. The aim of this work is to analyze the possible interactions between phenolic acids (p-coumaric, caffeic, chlorogenic, and gallic acid) and porcine pancreatic lipase, and their effects on its conformation.

Porcine pancreatic lipase solution was prepared (20 mM Tris buffer; pH 7.2) at two concentrations. Intrinsic fluorescence was measured with a microplate reader using excitation and emission wavelengths of 290 and 320 nm, respectively. The assay was carried out in the absence and presence of different phenolic acid concentrations (10-400 µM), alone and in binary combinations. The Stern-Volmer equation and a modified double-logarithmic model were used to determine the quenching constant (K_sv), the bimolecular quenching rate constant (k_q), the binding Affinity constant (K_a), and the number of binding sites (n).

p-Coumaric, caffeic, and chlorogenic acids quenched the enzyme's intrinsic fluorescence in a concentration-dependent manner. Gallic acid exhibited a different behavior; its data points were scattered and non-linear, suggesting that it does not form a stable complex with the enzyme. The calculated k_q values ranged from 1.73-6.52×10¹² M⁻¹s⁻¹, approximately two orders of magnitude above the diffusion-controlled collision limit (~2.0 × 10¹⁰ M⁻¹s⁻¹). These values corresponded to static quenching mechanisms, indicating that the phenolic acids may form non-fluorescent complexes with the enzyme in its ground state.

Whereas at the higher enzyme concentration (2 mg mL^-1), p-coumaric acid exhibited the highest binding affinity (K_a=2.48±0.01×10³ M⁻¹; n=0.98±0.03), consistent with simple 1:1 binding at a single site. At a lower enzyme concentration (0.2 mg mL-1), caffeic acid became the strongest binder (Ka = 5.13±0.12×10³ M⁻¹; n = 0.92±0.01). This was attributed to fewer protein-protein interactions, which may allow more binding, such as hydrogen bonds, within its catechol group. 

When the phenolic acids were tested together in binary mixtures, caffeic and chlorogenic acids produced the strongest combined quenching effect (higher K_a). However, this mixture, for example, exhibited a lower binding-site calculated value (n=0.21±0.02). This was related to competition between them for the same or overlapping regions on the enzyme rather than binding independently.

Overall, these results indicate that hydroxycinnamic acids can interact with and induce structural changes in pancreatic lipase through static quenching, supporting their potential role as natural, dietary-derived anti-obesity agents.

The authors acknowledged UACJ for all the support.