Specific adsorption of phosphate ions on proteins evidenced by capillary electrophoresis and reversed-phase high-performance liquid chromatography

Abstract

Specific adsorption of phosphate ions at pH=7.0 was studied on different proteins, either counter-ions of phosphate (lysozyme, lactoferrin) or co-ion of phosphate (α-lactalbumin). The theoretical electrophoretic mobility of globular proteins lysozyme and α-lactalbumin (apo and holo (+1 calcium per molecule) forms) was compared with those measured by capillary electrophoresis in phosphate at pH 7.0, versus the ionic strength (I) in the range 0–0.775 mol L −1. The specific adsorption of phosphate ions was evidenced by difference. From the experimental charge number ( Z eff) of protein in phosphate medium, a phosphate content per protein molecule was determined at pH=7.0. • For lactoferrin (p I=8–9), the electrophoretic mobility ( μ) was constant and negative, highlighting a charge reversal due to phosphate adsorption. • For α-lactalbumin (holo form) experimental μ was roughly constant and more negative than predicted. Z eff increased continuously from −4 to −11 in the ionic strength range from 0.005 to 0.775 mol l −1, respectively. Accordingly, one to six phosphates were bound per molecule, respectively. • For lysozyme, experimental electrophoretic mobility was positive but lower than predicted. Z eff was only discrete values +5 for I in the range 0.001–0.020 mol l −1 and about +3 in the range 0.050–0.500 mol l −1, whereas the theoretical Z value was +7 at pH=7.0. Lysozyme bounds one phosphate at low ionic strength and about two — three at higher ionic strength. Reversed-phase HPLC confirms that adsorption of phosphate is different for the three proteins.