Binding of Lysozyme to Unimolecular Micelles Formed from Hydrophobically-Modified Polyelectrolytes

Abstract

The binding of lysozyme, a basic protein with the isoelectric point (pI) of approximately 11.5, to unimolecular micelles (unimer micelles) formed from pyrene (Py)-labeled copolymers of sodium 2-(acrylamido)-2-methylpropanesulfonate and N-cyclododecylmethacrylamide was investigated by fluorescence spectroscopy, nonradiative energy transfer (NRET), quasielastic light scattering (QELS), and turbidimetry in 0.25 M NaCl aqueous solutions. The unimer micelle is a negatively charged particle with an apparent hydrodynamic diameter of approximately 11 nm, whose hydrophobic surface is partly exposed to the aqueous phase. When lysozyme was bound to the unimer micelles, Py fluorescence was observed upon excitation of tryptophan (Trp) residues at 290 nm due to NRET from singlet-excited Trp to Py labels, thus allowing us to monitor the lysozyme binding by NRET. Lysozyme was found to bind to unimer micelles even at pH 12.5 (>pI), although its net charge was negative. It is known that lysozyme forms an aggregate at this high pH. Immediately after lysozyme was mixed with unimer micelles in a 0.25 M NaCl aqueous solution, the aggregate of lysozyme binds to the unimer micelle mainly due to hydrophobic interaction. The bound lysozyme aggregate dissociates into lysozyme monomers (or dimers) on the surface of the unimer micelle over a period of approximately 12 h, leading to a complex with an apparent hydrodynamic diameter of approximately 20 nm. Effects of pH and ionic strength on the lysozyme binding indicated that even at a pH > pI electrostatic interaction was in effect conjointly with hydrophobic interaction for the binding of lysozyme to unimer micelles. Upon a decrease in the pH to approximately 8.5, the complexes form aggregates with sizes more than 100 times larger than the discrete complex of lysozyme and unimer micelles, leading to bulk-phase separation. This phase separation at pH ≤ 8.5 is due to charge neutralization between the positive charge of bound lysozyme and the negative charge of the unimer micelle.