ARTICLE pubs.acs.org/Biomac
Mobility of Molecules and Ions Solubilized in Protein Gels: Diffusion in the Thick Fraction of Hen Egg White Keith M. Krise and Bratoljub H. Milosavljevic* Department of Chemistry, The Pennsylvania State University, 104 Chemistry Research Building, University Park, Pennsylvania 16802, United States ABSTRACT: The thick fraction of hen egg white is a protein hydrogel with an immeasurably high viscosity composed of ∼90% water that can serve as a model system for mammalian mucous membrane. Measurements of the rate constants of diffusion-controlled reactions occurring within the gel (and corresponding activation energies) and electric conductivity revealed that the thick fraction of egg white can be envisioned as a 3D network comprising hydrated protein molecules (held by intermolecular SS bridges) surrounded by water pools and channels (of nonuniform diameters) that have a microviscosity that is very similar to that of bulk water. This was corroborated by differential scanning calorimetry measurements that revealed that 16% of water is bound to proteins. The melting kinetics of ice crystallites (produced from the freezable water) indicates nonhomogeneous water pool size.
’ INTRODUCTION The use of radiation and heat (hyperthermia therapy) to treat cancer and the use of ultrasound to break up kidney stones (lithotripsy) can adversely affect healthy tissue and cells in the treatment area by disrupting the 3D structure of proteins.13 The epithelial cells that comprise these luminal surfaces are covered by a protective, viscous mucous membrane that consists of heavily glycosylated and cysteine-rich proteins, namely, mucin glycoproteins that contain several hundred oligosaccharide chains in O-glycosidic linkages to amino acids serine and threonine of the polypeptide chain.46 Disulfide bonds, between the cysteine residues of mucin monomers as well as noncovalent intermolecular interactions between carbohydrate moieties give the mucous membrane viscous and gel properties. The proteins in the mucous membranes are well-hydrated, with the mucous membrane typically consisting of about 9095% water.4 To understand better the molecular level effects of radiation, heat, and ultrasound on proteins in biomolecular gels, the thick fraction of egg white could serve as a convenient and economical model system for the luminal mammalian mucous membrane. Like the mammalian mucous, the thick fraction of egg white contains highly glycosylated, cysteine-rich, and hydrated proteins.710 In our previous research,11 we characterized the thick fraction of egg white and determined that the thick fraction of egg white is a hydrated, protein gel composed of an ovomucin ovomucoid protein network connected by disulfide bonds with ∼90% water (similar to the water content in mucous membranes). Also within the thick fraction of egg white are protein monomers, agglomerates, and conglomerates including the egg white proteins ovalbumin and conablumin. An understanding of the state of water in protein hydrogels is needed for studies of radiation-, heat-, and ultrasound-induced r 2011 American Chemical Society
protein denaturation mechanisms in such media. In particular, the G values (concentration of species produced or reacted per unit dose absorbed) and mobility of radicals produced by ionizing radiation and sonication of hydrogels depend on microviscosity and will impact the extent to which proteins are denatured. To assess the state of water in the thick fraction of egg white, we compared the rheological properties of it on the microscale to those of pure water. This work expands on our previous work with simple aqueous polymer solutions in which we showed that (despite high macroviscosity) the physiochemical properties of water in poly(vinyl alcohol) aqueous solutions (up to 10% (w/w)) were the same as those of pure water.12 Here using diffusion-controlled reactions and electric conductivity involving small reactants and ions, we investigated the mobility of molecules and ions solubilized within the thick fraction of egg white to characterize its microscale rheological properties. Differential scanning calorimetry (DSC) was used to gain further information about the amount of bound water in the hydrogel as well as water pool and channel sizes within the thick fraction. The data obtained are used to envision the thick fraction of egg white at the molecular level, providing the necessary (missing) data so that it could be used as a fully characterized model system in radiation research.
’ EXPERIMENTAL SECTION Materials. All chemicals used, tris(2,20 -bipyridine)ruthenium(II) chloride, magnesium chloride, methyl viologen dichloride salt, Received: March 27, 2011 Revised: May 4, 2011 Published: May 06, 2011 2351
dx.doi.org/10.1021/bm200417t | Biomacromolecules 2011, 12, 2351–2356
Biomacromolecules potassium chloride, and sodium chloride, were of the highest purity. All chemicals passed spectrophotometric analysis tests before use and were used as received from Sigma-Aldrich Chemical. Egg white thick and thin fractions were isolated from fresh hen eggs from a local farm. Steady-State and Time-Resolved Studies. The concentration of Ru(bpy)32þ used in both steady-state and time-resolved experiments was 50 μM. Optical absorption spectra were obtained using a Varian UVvis spectrophotometer (model Cary 4000), and optical emission spectra were obtained using a Horiba Scientific Fluorolog spectrofluorimeter (model FL3-1/IHR320 IR). Steady-state fluorescence anisotropy measurements were obtained by placing HOYA 62.0s (PL) linear polarizers in the excitation and emission light paths in the Horiba Scientific Fluorolog spectrofluorimeter. Luminescence decays were obtained using a laser photolysis setup consisting of a laser from Stanford Research Systems (model NL-100, λem= 337.1 nm, pulse width = 3.5 ns), a photodiode from Electro-Optics Technology (model 23-2618A, rise time = 0.5 ns), a (600 ( 10) nm optical interference filter, and a Tektronix 200 MHz storage oscilloscope (model TDS 2022B). Laser photolysis measurements were taken in the temperature range of 1540 °C, and for these temperature-resolved measurements, the cuvette was immersed in a temperature-controlled water bath. Because of extensive foaming, thick and thin fraction of egg white samples cannot be deoxygenated by purging with inert gases. To avoid this problem, laser photolysis measurements in the absence of oxygen were performed in a custom-made quartz cuvette fused with a 500 mL Erlenmeyer flask. A glass stopper was made with two glass tubings with valves, thus allowing oxygen-free work by saturating the gaseous atmosphere above the solution with nitrogen gas. To prevent water loss (which was found to be negligible), the Erlenmeyer flask atmosphere was saturated with nitrogen, and the valves were closed. The solution was stirred gently and slowly to facilitate oxygen release from the solution into the flask atmosphere above the solution. This process was repeated (five times) until gas chromatography indicated 25% of ice was melted in this interval because, as has been previously shown, the enthalpy of melting depends on crystallite size.38
’ CONCLUSIONS The photophysics of Ru(bpy)32þ along with the kinetics of its excited state quenching by methyl viologen in the thick fraction, indicate that organometallic and aromatic cations diffuse via subsequent adsorption and desorption steps due to interactions with proteins. Electric conductivity measurements show that the mobility of small monatomic ions in both the thick and thin fraction of egg white is not affected by the presence of protein molecules. Experimentally obtained second-order quenching rate constant for Ru(bpy)32þ quenching by oxygen in the thick fraction of egg white is (1.1 ( 0.2) 109 M1 s1 at room temperature, which is comparable to quenching rate constants obtained in pure water. The activation energy for excited Ru(bpy)32þ by oxygen quenching reaction in the thick fraction of egg white was determined to be ∼1.5 times larger than that in 2355
dx.doi.org/10.1021/bm200417t |Biomacromolecules 2011, 12, 2351–2356
Biomacromolecules water, which (taken together with all previously mentioned data) indicates that oxygen diffuses at the same rate as that in bulk water. DSC experiments reveal that 16% of the present water is bound to protein molecules, whereas the melting kinetics of ice produced from free water indicate nonuniform crystallite sizes, which in turn can be attributed to nonuniform water pool sizes. The data obtained suggest that the thick fraction of egg white can be envisioned as a 3D network comprising hydrated protein molecules (held by intermolecular SS bridges) surrounded by water pools and channels (of nonuniform sizes) that have a microviscosity very similar to that of bulk water.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]. Fax: þ (814) 863-6195.
’ ACKNOWLEDGMENT We thank the Pennsylvania State University Department of Chemistry for support.
ARTICLE
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