Interactions of lysozyme with hydrophilic and hydrophobic polymethacrylate stationary phases in reversed phase chromatography (RPC)

Abstract

Two silicas, one with a mean pore diameter of 30 nm and the other non-porous, were coated with polymethacrylates of increasing hydrophobicity in the sequence: poly-2-hydroxyethylmethacrylate (P2HEMA)1 polyethylmethacrylate (PEMA) and poly-n-octylmethacrylate (POMA). Association constants, Kass, between lysozyme and the coated silicas were determined by means of frontal analysis, and the apparent heats of adsorption, delta Happ, by means of microcalorimetry. Using Kass and delta Happ the changes in the apparent free energy, delta Gapp, and in the apparent entropy, delta Sapp, were calculated at a concentration of lysozyme < 10 mumol/l. The association between the lysozyme and the coated silica gave negative delta Happ and delta Sapp values. The delta Sapp values calculated from the experimental data in the absence of an added electrolyte and with 0.1 M sodium perchlorate present to be -12 (-96) J/mol deg on the P2HEMA silica, -27 (-51) J/mol deg on the PEMA silica, -98 (-186) J/mol deg on the POMA silica, respectively. The high delta Sapp value of the lysozyme on the POMA silica reflects a kind of stabilisation effect due to the conformational changes of lysozyme on the most hydrophobic POMA silica. As evidenced by RPC, lysozyme elutes on the POMA column in its totally unfolded form enabling a more disordered conformation with respect to entropy than in the native form.