Effect of Protein Aggregation on the Binding of Lysozyme to Pyrene-Labeled Polyanions

The binding of lysozyme to pyrene (Py)-labeled homo- and copolymers of sodium 2-(acrylamido)-2-methylpropanesulfonate and acrylamide was investigated by a combination of scattering techniques (turbidimetry, quasielastic light scattering, and electrophoretic light scattering), potentiometry, and fluorescence techniques. Lysozyme, a basic protein with a high isoelectric point of 11.5, is known to exist in a dimer form at ∼5 < pH < ∼10 and in an aggregate form at pH > ∼10. Potentiometric titration indicates binding of lysozyme aggregates to Py-labeled polysulfonates in 0.2 M NaCl at pH as high as 12.3. Nonradiative energy transfer (NRET) from tryptophan residues of lysozyme to Py labels commences near pH 10.5 when pH is further decreased. This onset of NRET is accompanied by an onset of solution turbidity, arising from the formation of lysozyme−polymer complexes with diameters 10−100 times larger than those of the individual components. This pH for the onset of NRET and turbidity, which is independent of both ionic strength and polymer linear charge density, is close to the pH at which the lysozyme aggregate dissociates into dimers. With a further decrease in pH, macroscopic phase separation occurs due to the association of the complexes of the lysozyme dimers with the polymer; the pH at which the macroscopic phase separation commences to occur is dependent on ionic strength.