Chemical and Microstructural Characterization of pH and [Ca2+] Dependent Sol-Gel Transitions in Mucin Biopolymer

Abstract

Mucus is responsible for controlling transport and barrier function in biological systems, and its properties can be significantly affected by compositional and environmental changes. In this study, the impacts of pH and CaCl₂ were examined on the solution-to-gel transition of mucin, the primary structural component of mucus. Microscale structural changes were correlated with macroscale viscoelastic behavior as a function of pH and calcium addition using rheology, dynamic light scattering, zeta potential, surface tension, and FTIR spectroscopic characterization. Mucin solutions transitioned from solution to gel behavior between pH 4-5 and correspondingly displayed a more than ten-fold increase in viscoelastic moduli. Addition of CaCl₂ increased the sol-gel transition pH value to ca. 6, with a twofold increase in loss moduli at low frequencies and ten-fold increase in storage modulus. Changing the ionic conditions-specifically [H⁺] and [Ca²⁺] -modulated the sol-gel transition pH, isoelectric point, and viscoelastic properties due to reversible conformational changes with mucin forming a network structure via non-covalent cross-links between mucin chains.

Figure 1

Sol-gel transitions can take place in structured fluids in response to changes in environmental factors such as pH, ion concentration, inclusion/particle chemistry and size, temperature, and shear forces. Gels form when the self-affinity of solubilized material increases to form a network structure while remaining soluble. Green lines represent mucin protein cores and yellow lines represent oligosaccharide side chains. In addition to disulfide bonding at the ends of individual mucins (red and black dots), physical and chemical non-covalent cross-linking occurs between mucins in the gelled state (blue circles).

Figure 2

Flow sweeps over a shear rate of 0.001–100 s⁻¹ show samples at pH 2.1, 4.0, and 5.8 with 10 mM CaCl₂ display shear-thinning behavior with a more linear response than samples without CaCl₂.

Figure 3

Aqueous PGM solutions display pH-dependent sol-gel behavior and moduli, G′ (closed symbols) and G′′ (open symbols). Oscillating frequency sweep rheology data are shown over 0.01–10 rad/s for pH values of 2.1, 4.0, and 5.8.

Figure 4

Oscillating frequency sweep rheology data over 0.01–2 rad/s for aqueous PGM solutions at pH values of 2.1 (square), 4.0 (circle), and 5.8 (triangle), with 0.01 M calcium chloride added.

Figure 5

Storage (G′) and loss (G′′) moduli of PGM solutions at pH 2.1 are shown to decrease with the addition of 10 mM calcium chloride.

Figure 6

Storage (G′) and loss (G′′) moduli of PGM solutions at pH 4.0 increase with the addition of 10 mM calcium chloride.

Figure 7

Storage (G′) and loss (G′′) moduli of PGM solutions at pH 5.8 increase with the addition of 10 mM calcium chloride.

Figure 8

Average PGM hydrodynamic diameters determined by DLS reveal a sharp decrease in effective particle size at pH 4 and above, regardless of CaCl₂ addition of 10 mM.

Figure 9

PGM solutions at pH values below the pH_G (~ pH 4) exhibit significantly higher surface tension values. Addition of 10 mM CaCl₂ showed a negligible effect on the surface tension.

Figure 10

Zeta potential data show PGM solutions have a higher isoelectric point when 10 mM CaCl₂ is present.

Figure 11

Representative FTIR spectra of 10 mg/mL PGM solutions at various pH values, both with (dashed lines) and without (solid lines) 10 mM CaCl₂.