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

Abstract

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.

Modulation of physiological and pathological activities of lysozyme by biological membranes

“…The penetration of the specific HLH domain, embracing the amino acid residues Asp 87 -Arg 114 , into the lipid bilayers and subsequent disruption of membrane integrity have been demonstrated to be responsible for the bactericidal activity of Lz [24]. Furthermore, Lz antimicrobial action has been shown to be enhanced by modifying the charge and hydrophobicity of the protein [10,28]. In addition, Lz partially unfolded by heat treatment proved to exert catalytically independent antimicrobial action against both G+ and G-bacteria [24].…”

“…Discovered by Alexander Fleming in 1922, lysozyme was the first protein whose three-dimensional structure was characterized at atomic resolution [1]. Owing to its well-defined geometry and physicochemical properties Lz represents an attractive model in approaching a broad spectrum of fundamental problems including protein adsorption at interfaces [2], lipid-protein interactions [3][4][5], membrane fusion [6,7], protein folding, aggregation and amyloid fibrillogenesis [8][9][10]. A number of studies provide evidence for pronounced lipid-associating ability of lysozyme [11][12][13][14].…”

Binding of Polycarboxylic Acids to Cationic Mixed Micelles: Effects of Polymer Counterion Binding and Polyion Charge Distribution

“…Acrylic acid/ acrylamide, AA/AAm, (70/30) copolymer was purchased from General Science Co. (Tokyo), and dialyzed and freezedried before use. Poly(acrylamido-2-methylpropane sulfonate), PAMPS, and AMPS/AAm copolymers were prepared by polymerization of 2-(acrylamido)-2-methylpropanesulfonic acid with or without acrylamide as reported previously (41). Cetyltrimethylammonium chloride, CTAC, (Fluka) and Cetyltrimethylammonium bromide, CTAB, (Aldrich) were recrystallized twice from methanol.…”

Potentiometric studies on the interaction between superoxide dismutase and hyaluronic acid

“…The ''primary'' soluble complex consists of a number of proteins bound to a single polymer chain according to the previous report. 28 With the decrease of pH from 6.5 to 3.7, some SOD dimmers with a particle size of 20-30 nm occur in the free SOD solution, but the size of HA scarcely varies in the free HA solution. Therefore, a larger fluctuation of particle sizes directly indicates a variance of particle constituents for SOD-HA mixtures.…”

“…[23][24][25][26][27] Fluorometry was carried out to observe nonradiative energy transfer during the binding process. 28 Isothermal titration calorimetry was performed to measure some important thermodynamic parameters, such as a binding constant, stoichiometry, enthalpy, and entropy. 29 Microcalorimetry experiments performed on the protein-polyelectrolyte systems, such as hyaluronic acid (HA)/poly(L-lysine) and bovine serum albumin/poly(allylamine hydrochloride), showed that counter ions were released in the complexation process, and the binding between polyanions and polycations was mainly entropically driven.…”