INTERACTIONS OF LYSOZYME WITH HYDROPHILIC AND HYDROPHOBIC POLYMETHACRYLATE STATIONARY PHASES IN REVERSED-PHASE CHROMATOGRAPHY (RPC)

Two silicas, one with a mean pore diameter of 30 nm and the other non- porous, were coated with polymethacrylates of increasing hydrophobicit y in the sequence: poly-2-hydroxyethylmethacrylate (P2HEMA)(1) polyeth ylmethacrylate (PEMA) and poly-n-octylmethacrylate (POMA). Association constants, K-ass, between lysozyme and the coated silicas were determ ined by means of frontal analysis, and the apparent heast of adsorptio n, Delta H-app, by means of microcalorimetry. Using K-ass and Delta H- app the changes in the apparent free energy, Delta G(app), and in the apparent entropy, Delta S-app, were calculated at a concentration of l ysozyme < 10 mu mol/l. The association between the lysozyme and the co ated silica gave negative Delta H-app and Delta S-app values. The Delt a S-app values calculated from the experimental data in the app app ap p absence of an added electrolyte and with 0.1 M sodium perchlorate pr esent 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, respe ctively. The high Delta S-app value of the lysozyme on the POMA silica reflects a kind of stabilisation effect due to the conformational cha nges of lysozyme on the most hydrophobic POMA silica. As evidenced by RPC, lysozyme elutes on the POMA column in its totally unfolded form e nabling a more disordered conformation with respect to entropy than in the native form.