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Prevention of thermal aggregationof antibodies in aqueous solutionswas achieved by noncovalent association with hydrophobically modifiedpoly(acrylate) copolymers. Using a polyclonal immunoglobin G (IgG)as a model system for antibodies, we have studied the mechanisms bywhich this multidomain protein interacts with polyanions when incubatedat physiological pH and at temperatures below and above the proteinunfolding/denaturation temperature, in salt-free solutions and in0.1 M NaCl solutions. The polyanions selected were sodium poly(acrylates),random copolymers of sodium acrylate and N-n-octadecylacrylamide (3 mol %), and a random copolymerof sodium acrylate, N-n-octylacrylamide(25 mol %), and N-isopropylacrylamide (40 mol %).They were derived from two poly(acrylic acid) parent chains of Mw5000 and 150000 g·mol–1. The IgG/polyanion interactions were monitored by static and dynamiclight scattering, fluorescence correlation spectroscopy, capillaryzone electrophoresis, and high sensitivity differential scanning calorimetry.In salt-free solutions, the hydrophilic PAA chains form complexeswith IgG upon thermal unfolding of the protein (1:1 w/w IgG/PAA),but they do not interact with native IgG. The complexes exhibit aremarkable protective effect against IgG aggregation and maintainlow aggregation numbers (average degree of oligomerization <12at a temperature up to 85 °C). These interactions are screenedin 0.1 M NaCl and, consequently, PAAs lose their protective effect.Amphiphilic PAA derivatives (1:1 w/w IgG/polymer) are able to preventthermal aggregation (preserving IgG monomers) or retard aggregationof IgG (formation of oligomers and slow growth), revealing the importanceof both hydrophobic interactions and modulation of the Coulomb interactionswith or without NaCl present. This study leads the way toward thedesign of new formulations of therapeutic proteins using noncovalent1:1 polymer/protein association that are transient and require a markedlylower additive concentration compared to conventional osmolyte protectingagents. They do not modify IgG permanently, which is an asset forapplications in therapeutic protein formulations since the in vivoefficacy of the protein should not be affected. [ABSTRACT FROM AUTHOR]

Bacteriorhodopsin (BR) is a retinal protein that functions as a light-driven proton pump. In this study, six novel mutants including K41E and D102K, were obtained to verify or rule out the possibility that residues Lys41 and Asp102 are determinants of the time order of proton release and uptake, because we found that the order was reversed in another retinal protein archaerhodopsin 4 (AR4), which had different 41th and 102th residues. Our results rule out that possibility and confirm that the pK(a) of the proton release complex (PRC) determines the time order. Nevertheless, mutations, especially D102K, were found to affect the kinetics of proton uptake substantially and the pK(a) of Asp96. Compared to the wild-type BR (BR-WT), the decay of the M intermediate and proton uptake in the photocycle was slowed about 3-fold in D102K. Hence those residues might be involved in proton uptake and delivery to the internal proton donor.

This work utilizes a photoelectrochemical approach to study the pH dependence of proton release and uptake in the photocycles of two retinal proteins, bacteriorhodopsin (BR) and archaerhodopsin 4 (AR4). By detecting photoinduced potentials that originate from the proton concentration changes (DeltaH(+)) generated by proteins near the indium tin oxide (ITO) electrode, we show that the kinetics of release and uptake can be followed in a broad pH range, and the pK(a) of the proton release complex (PRC) can be easily determined under different conditions. Nonoriented protein films were deposited on the electrode, and photovoltage in an electrochemical cell was detected after illumination with a green flash. The kinetics of proton release and uptake could be measured as light-induced decreases and increases of the photopotential. A kinetic analysis was performed, and a formula describing proton fluxes of wild-type BR and AR4 and D96N mutant of BR was derived. Three componentsfast proton release, slow proton release, and proton uptakewere found in the wild-type retinal proteins; two components, fast and slow proton releases, were found in the D96N mutant. The pH dependence of the fraction of fast release over the whole release was used to determine the pK(a) for proton release in the photocycles of these retinal proteins. Measurements were also performed in conventional buffer solutions and crown ether. The presence of buffer in 10-50 mM concentration did not abolish the light-induced signals, indicating that the electrode response is much less sensitive to buffers than pH-sensitive dyes in a suspension due to a higher protein/buffer ratio near the electrode. This feature enables us to study effects of chemicals with high buffer capacity, and significant effects of buffers and crown ether on proton pumping behaviors of retinal proteins were revealed. In comparison with the classic pH-sensitive dye approach, the photoelectrochemical approach is convenient and efficient for measurements of transient proton concentration changes (DeltaH(+)) generated by a proton pump and thus might be utilized as a powerful tool for the investigation of light-driven proton pumping mechanisms in a wide pH range.