Intermolecular Interactions of ?-Carrageenan with Phytanic Acid and Ca++ at the Air-Water Interface Using X-Ray Reflectivity

Polysaccharides, especially sulfated polysaccharides extracted from red seaweed, find use in pharmaceutical, industrial, and numerous biological applications, such as anticoagulant, antithrombotic, antiviral, anti-tumor, and immunomodulatory activities. These applications all relate to their ability to bind to either solid, fluid interfaces (e.g. air/water surfaces or lipid biomembranes). These negatively charged polyelectrolytes are receiving additional recent attention because of their ability to constrain or completely eradicate the infectious capacity of coronaviruses by binding to the enveloping lipid membrane of the virus particle, indicating their potential to be used to reduce infection rates and motivating the study of their interaction with lipid bilayers membranes.

Here, we used X-ray reflectivity (XRR), and X-ray fluorescence (XRF) to study the binding at the air/water interface of the polysaccharide λ-carrageenan, present in the aqueous subphase, with an insoluble monolayer of phytanic acid at the interface. Phytanic acid, a fatty acid with a sixteen carbon backbone, and with methyl groups along the backbone, presents a relatively large area per molecule, similar to the phospholipids on bilayer membranes, and therefore constitutes a simple model for the biomembrane surface. At neutral pH, both carrageenan and the carboxyl group of phytanic acid are negatively charged, and we focus particularly on the role of calcium cations in the subphase as an electrostatic bridge for the complexation. Surface concentration, surface area, and molar volume of molecules present in every slab constituting the different monolayers were computed using electron density balance and molar volume balance equations. The calculations were carried out by using the reflectivity curves to fit values for the slab thickness, and electron density of every layer present at the air water interface. Additionally, these fitting parameters permitted to calculate the electron density profiles (EDP) of the molecules present at the interfacial region.

We find, in the absence of the calcium in the subphase, that λ-carrageenan molecules bind to the phytanic acid headgroups by hydrogen bonding despite the electrostatic repulsion, which produces a reduction of the surface concentration of the fatty acid (increase of the surface area). Langmuir trough measurements confirm this increase in area. Moreover, when both λ-carrageenan and Ca⁺⁺ are present in the subphase, the interactions with phytanic acid headgroups are stronger due to the divalent cation bridging the negative charges of the monolayer and the carrageenan. Quantitative measurements of the calcium cation concentration using X-ray fluorescence confirms a large concentration of the calcium cation in the surface layer. This calcium cation mediated interactions induce the recruitment of more λ-carrageenan molecules from the bulk to the interface and reduce the phytanic acid headgroup’s hydration, as also seen in the Langmuir trough measurements.