Dynamic light scattering study on phase separation of a protein-water mixture: Application on cold cataract development in the ocular lens (original) (raw)
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2007
We report a dynamic light scattering study on protein suspensions of bovine lens homogenates at conditions (pH and ionic strength) similar to the physiological ones. Light scattering data were collected at two temperatures, 20 o C and 37 o C, over a wide range of concentrations from the very dilute limit up to the dense regime approaching to the physiological lens concentration. A comparison with experimental data from intact bovine lenses was advanced revealing differences between dispersions and lenses at similar concentrations. In the dilute regime two scattering entities were detected and identified with the long-time, self-diffusion modes of α-crystallins and their aggregates, which naturally exist in lens nucleus. Upon increasing protein concentration significant changes in time correlation function were observed starting at ~75 mg ml-1 where a new mode originating from collective diffusive motions becomes visible. Self-diffusion coefficients are temperature insensitive, whereas the collective diffusion coefficient depends strongly on temperature revealing a reduction of the net repulsive interparticle forces with lowering temperature. While there are no rigorous theoretical approaches on particle diffusion properties for multi-component, nonideal hard-sphere, polydispersed systems, as the suspensions studied here, a discussion of the volume fraction dependence of the long-time, self-diffusion coefficient in the context of existing theoretical approaches was undertaken. This study is purported to provide some insight into the complex light scattering pattern of intact lenses and the interactions between the constituent proteins that are responsible for lens transparency. This would lead to understand basic mechanisms of specific protein interactions that lead to lens opacification (cataract) under pathological conditions.
Cold cataracts: a naturally occurring aqueous two-phase system
Journal of Chromatography B: Biomedical Sciences and Applications, 2000
The cytoplasm of the eye lenses shows a liquid-liquid phase transition similar to the one observed in aqueous two-phase systems. This phenomenon is known as cold cataracts. We have studied the solution behavior of the main protein fractions that constitute the lenses' cytoplasm using small-angle neutron scattering and dynamic light scattering. Our results provide evidence that an intricate balance of forces underlines the physical phenomena responsible for the optical properties of the lenses and for the phase transition that is observed as the temperature is lowered below some critical value. These forces include solvent-mediated forces besides the more conventional Coulombic and dispersion forces. This study suggests that solvent mediated forces must be included to successfully model liquid-liquid phase transitions like the ones observed in cold cataracts or in aqueous two-phase systems.
Statistical-thermodynamic model for light scattering from eye lens protein mixtures
The Journal of Chemical Physics, 2017
We model light-scattering cross sections of concentrated aqueous mixtures of the bovine eye lens proteins γBand α-crystallin by adapting a statistical-thermodynamic model of mixtures of spheres with short-range attractions. The model reproduces measured static light scattering cross sections, or Rayleigh ratios, of γB-α mixtures from dilute concentrations where light scattering intensity depends on molecular weights and virial coefficients, to realistically high concentration protein mixtures like those of the lens. The model relates γB-γB and γB-α attraction strengths and the γB-α size ratio to the free energy curvatures that set light scattering efficiency in tandem with protein refractive index increments. The model includes (i) hard-sphere α-α interactions, which create short-range order and transparency at high protein concentrations, (ii) short-range attractive plus hard-core γ-γ interactions, which produce intense light scattering and liquid-liquid phase separation in aqueous γ-crystallin solutions, and (iii) short-range attractive plus hard-core γ-α interactions, which strongly influence highly non-additive light scattering and phase separation in concentrated γ-α mixtures. The model reveals a new lens transparency mechanism, that prominent equilibrium composition fluctuations can be perpendicular to the refractive index gradient. The model reproduces the concave-up dependence of the Rayleigh ratio on α/γ composition at high concentrations, its concave-down nature at intermediate concentrations, non-monotonic dependence of light scattering on γ-α attraction strength, and more intricate, temperature-dependent features. We analytically compute the mixed virial series for light scattering efficiency through third order for the sticky-sphere mixture, and find that the full model represents the available light scattering data at concentrations several times those where the second and third mixed virial contributions fail. The model indicates that increased γ-γ attraction can raise γ-α mixture light scattering far more than it does for solutions of γ-crystallin alone, and can produce marked turbidity tens of degrees celsius above liquid-liquid separation.
Biophysical Journal, 1983
The bovine eye-lens protein, aL-crystallin, has been studied with photon correlation spectroscopy to obtain the mutual diffusion coefficient, Di, with fluorescence correlation spectroscopy to determine the tracer diffusion coefficient, DT, and with light scattering to get the isothermal osmotic compressibility (&r/bc)PT. The concentration dependence of D., DT, and (tr/bc)PT up to a volume fraction 4 of the protein of 2.5 x 10-2 has been interpreted on the basis of four different interaction potentials: (a) an extended hard-sphere potential; (b) a shielded Coulomb potential; (c) a shielded Coulomb interaction where the effect of counterions is included; (d) a simple mixed potential. The three parameters D., DT, and (ir/bc)PT have also been combined in the generalized Stokes-Einstein equation, Dm = [(ir/bc)P,T -(1 -0) -(DT)]/(kB-T). Our results indicate that, in the case that photon correlation spectroscopy gives the mutual diffusion coefficient Dm, the applicability of the Stokes-Einstein equation can be questioned; or that, when one assumes the Stokes-Einstein equation to be valid, there is significant discrepancy between the result of photon correlation spectroscopy and D..
Dynamic light scattering evidence of a `fragile' character of protein aqueous solutions
Chemical Physics Letters, 1999
Dynamic light scattering has been used to measure the mutual diffusion coefficient D of bovine serum albumin as a m function of temperature at different protein concentrations. A deviation from the Arrhenius behaviour is observed, at each protein concentration investigated, corresponding to a characteristic temperature T , whose value is found to be dependent A on the protein concentration itself. In the whole temperature range, the dependence of D on T is well represented by a m Vogel-Fulcher-Tamman law. The results are discussed in the framework of the so-called strongr fragile classification scheme for glass-forming liquids, connected to the peculiar properties of the water-protein system. q 1999 Elsevier Science B.V. All rights reserved.
Relaxometry of lens homogenates. II. temperature dependence and comparison with other proteins
Magnetic Resonance in Medicine, 1989
Magn. Reson. Med. 8,45 (1988)) on the magnetic field dependence of 1 / T , (NMRD profiles) of calf lens nuclear homogenates, at 25"C, to 5'C, and to other protein systems as well. These include concentrated solutions of myoglobin and bovine serum albumin, both globular proteins, the first compact and roughly spherical, the other extended, flexible, and with weak internal bonding; chicken lens homogenate, for which the dominant crystallins (lens proteins) are -70% a-helical compared with calf crystallins, which are essentially all &sheet; and hen egg white, both native and heatdenatured. Our earlier conjectures regarding a reversible change in protein organization of the calf lens crystallins as a function of solute protein concentration is given added support. Our findings suggest that cytoplasmic homogenate can be characterized as a heterogeneous and polymorphic solution of crystallins. At high concentrations the NH moieties of the protein backbone become accessible to solvent with water (not NH proton) exchange rates > lo4 s -I . This conclusion is based on two aspects ofthe observed NMRD profiles. At low crystallin concentration, the profiles of calf and chicken lens homogenates are similar in form to those of myoglobin and native hen egg white, a form that has been studied previously for a range of diamagnetic globular proteins and has been demonstrated to arise from the rotational thermal motion of the solute molecules. At high crystallin concentrations, the NMRD profiles of the lens homogenates develop a monotonic background (high rates at low fields), much like that of the heat-denatured egg-white sample and those of most tissues. In addition, there is a set of peaks in the central part of the profiles of the concentrated crystallins, seen also in the denatured egg white and some tissues but not in the myoglobin sample, which is known to arise from cross-relaxation interactions between the water protons and (through the intermediary of the NH proton) the I4N quadrupolar levels. The magnitude of these peaks, which is larger by an order of magnitude for native calf lens homogenates than for any tissue, requires that the majority of the NH moieties be accessible to water. Finally, going to 5°C for the native calf lens homogenate takes the sample below the temperature of reversible phase separation, and it becomes opaque. However, there is no change in the profile that can be attributed to this transition and its associated opacity. o
Photochemistry and Photobiology, 2008
The role of UVA radiation in the formation of human nuclear cataract is not well understood. We have previously shown that exposing guinea pigs for 5 months to a chronic low level of UVA light produces increased lens nuclear light scattering and elevated levels of protein disulfide. Here we have used the technique of dynamic light scattering (DLS) to investigate lens protein aggregation in vivo in the guinea pig/UVA model. DLS size distribution analysis conducted at the same location in the lens nucleus of control and UVA-irradiated animals showed a 28% reduction in intensity of small diameter proteins in experimental lenses compared with controls (P < 0.05). In addition, large diameter proteins in UVA-exposed lens nuclei increased five-fold in intensity compared to controls (P < 0.05). The UVA-induced increase in apparent size of lens nuclear small diameter proteins was three-fold (P < 0.01), and the size of large diameter aggregates was more than four-fold in experimental lenses compared with controls. The diameter of crystallin aggregates in the UVA-irradiated lens nucleus was estimated to be 350 nm, a size able to scatter light. No significant changes in protein size were detected in the anterior cortex of UVA-irradiated lenses. It is presumed that the presence of a UVA chromophore in the guinea pig lens (NADPH bound to zeta crystallin), as well as traces of oxygen, contributed to UVA-induced crystallin aggregation. The results indicate a potentially harmful role for UVA light in the lens nucleus. A similar process of UVA-irradiated protein aggregation may take place in the older human lens nucleus, accelerating the formation of human nuclear cataract.
Experimental Eye Research, 2001
The non-invasive technique of dynamic light scattering (DLS) was used to quantitatively characterize vitreous and lens structure on a molecular level by measuring the sizes of the predominant particles and mapping the three-dimensional topographic distribution of these structural macromolecules in three spatial dimensions. The results of DLS measurements in ®ve fresh adult bovine eyes were compared to DLS measurements in model solutions of hyaluronan (HA) and collagen (Coll). In the bovine eyes DLS measurements were obtained from excised samples of gel and liquid vitreous and compared to the model solutions. Measurements in whole vitreous were obtained at multiple points posterior to the lens to generate a three-dimensional`map' of molecular structure. The macromolecule distribution in bovine lens was similarly characterized.
Photochemistry and Photobiology, 2008
The role of UVA radiation in the formation of human nuclear cataract is not well understood. We have previously shown that exposing guinea pigs for 5 months to a chronic low level of UVA light produces increased lens nuclear light scattering and elevated levels of protein disulfide. Here we have used the technique of dynamic light scattering (DLS) to investigate lens protein aggregation in vivo in the guinea pig/UVA model. DLS size distribution analysis conducted at the same location in the lens nucleus of control and UVA-irradiated animals showed a 28% reduction in intensity of small diameter proteins in experimental lenses compared with controls (P < 0.05). In addition, large diameter proteins in UVA-exposed lens nuclei increased five-fold in intensity compared to controls (P < 0.05). The UVA-induced increase in apparent size of lens nuclear small diameter proteins was threefold (P < 0.01), and the size of large diameter aggregates was more than four-fold in experimental lenses compared with controls. The diameter of crystallin aggregates in the UVAirradiated lens nucleus was estimated to be 350 nm, a size able to scatter light. No significant changes in protein size were detected in the anterior cortex of UVA-irradiated lenses. It is presumed that the presence of a UVA chromophore in the guinea pig lens (NADPH bound to zeta crystallin), as well as traces of oxygen, contributed to UVA-induced crystallin aggregation. The results indicate a potentially harmful role for UVA light in the lens nucleus. A similar process of UVA-irradiated protein aggregation may take place in the older human lens nucleus, accelerating the formation of human nuclear cataract.