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In Situ Measurements of pH Changes in β-Lactoglobulin Solutions under High Hydrostatic Pressure

Orlien, V., Olsen, K., Skibsted, L.H.
Journal of agricultural and food chemistry 2007 v.55 no.11 pp. 4422-4428
milk, food processing, high pressure treatment, pH, acidity, lactoglobulins, new methods, spectral analysis, aqueous solutions, measurement, dissociation, denaturation, optical properties, absorbance, dyes, spectrophotometers, validity, beta-lactoglobulin
A novel in situ method, in which the spectral changes of aqueous solutions under pressure are measured using optical pH indicators in a high-pressure spectrophotometer, has been developed in order to provide a quantitative description of the pressure dependence of acid/base equilibria of proteins. The self-consistent method, insensitive to compressibility, was developed for measurement of changes in pH with pressure based on α-naphthyl red and neutral red as these indicators were found to have pressure insensitive pK'(a) values. The method was validated for up to 500 MPa by measurement of the pressure-dependence of the weak acid buffers acetic acid/acetate and imidazolium/imidazole from which volumes of dissociation of (delta)V° = -11.2 and 3.7 mL/mol, respectively, were established. Succinic acid/hydrogensuccinate was surprisingly insensitive to pressure with (delta)V° = -0.9 mL/mol. For β-lactoglobulin B in an unbuffered aqueous solution with ionic strength of 0.05 M and pH 4, pressure up to 300 MPa increased pH up to 1.5 units depending on concentration (up to 5 mg/mL investigated), followed by a decrease to the initial pH 4 for pressure up to 500 MPa. The surprising increase in pH at pressure up to 300 MPa is suggested to be caused by an increase in the effective pK(a) values of aspartic acid and glutamic acid side chain in hydrophobic compartments of the protein created by pressure denaturation, leading to a binding of water protons and an increase in free hydroxide ions. For higher pressure the carboxylic side chains in the fully denatured protein again becomes exposed to the solvent, and pH decreases to the initial pH of the aqueous system.