Specific adsorption of phosphate ions on proteins evidenced by capillary electrophoresis and reversed-phase high-performance liquid chromatography.
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 (alpha-lactalbumin). The theoretical electrophoretic mobility of globular proteins lysozyme and alpha-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 (pI=8-9), the electrophoretic mobility (mu) was constant and negative, highlighting a charge reversal due to phosphate adsorption. * For alpha-lactalbumin (holo form) experimental mu 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.
Rabiller-Baudry
M
M
Laboratoire des Procédés de Séparation, UC 991 INRA-Université Rennes 1, France. murielle.rabiller-baudry@univ-rennes1.fr
Chaufer
B
B
D000327:Adsorption; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D007768:Lactalbumin / Q000737:chemistry*; D009113:Muramidase / Q000737:chemistry*; D010710:Phosphates / Q000737:chemistry*
D010710:Phosphates; D007768:Lactalbumin; D009113:Muramidase