Bovine Eyes Research Articles

Bovine posterior limbus: an evaluation of an alternative source for corneal endothelial and trabecular meshwork stem/progenitor cells.

A growing body of evidence has revealed that stem-like cells in the posterior limbus of the eye between the corneal endothelium (CE) and trabecular meshwork (TM) may be able to rejuvenate these tissues in disease. However, these cells have not been clearly defined and we have named them PET cells (progenitor cells of the endothelium and trabeculum). A good and inexpensive animal model for PET cells is lacking, so we investigated bovine eyes as an effective large tissue source. We showed the presence of stem/progenitor cells in the bovine CE, transition zone, and TM in situ. Floating spheres cultured from the CE and TM showed similar stem cell marker expression patterns. Both the CE and TM spheres were bipotent and highly proliferative, but with limited secondary sphere-forming capability. They were highly prone to differentiate back into the cell type of their tissue of origin. It is speculated that the PET cells become more tissue-specific as they migrate away from their niche. Here, we showed that PET cells are present in the posterior limbus of bovine eyes and that they can be successfully cultured and expanded. PET cells represent an attractive target for developing new treatments to regenerate both the CE and TM, thereby reducing the requirement for donor tissue for corneal transplant and invasive treatments for glaucomatous patients.

The effect of varying glucose levels on the ex vivo crystalline lens: implications for hyperglycaemia-induced refractive changes.

Refractive changes in diabetic eyes have long been reported but with equivocal results. The lens has been a more recent focus as the source of any change but it is possible that multiple sources of variation have made it difficult to demonstrate a systematic change clinically. The aim of this study was therefore to use a bovine lens model to investigate the optical changes in hyperglycaemia and when lenses are returned to normal glucose levels as would occur following commencement of treatment. Bovine eyes were obtained and their lenses excised under sterile conditions before placing them in culture medium within an incubator using standard tissue culture techniques. In the first experiment, lenses were transferred into culture medium containing 5mm (n=12), 15mm (n=12) and 30mm (n=12) glucose. Measurements were made of the change in back vertex focusing distance with equatorial lens diameter using the ScanTox(™) measurement system. From these measurements, the back vertex focal length and primary longitudinal spherical aberration were derived. In a second experiment, lenses maintained at 30mm glucose (n=7) were stepped down to 5mm glucose to simulate starting diabetic therapy and measured in the same way. Changes over time were assessed with a linear regression model. A trend towards myopia was observed with increasing hyperglycaemia, this was not statistically significant. When lenses were stepped-down from hyperglycaemia to normal physiological levels of glucose, a hyperopic shift was observed in line with published clinical studies that again failed to reach statistical significance. High variability in the measurement on longitudinal spherical aberration prevented any significant trends being measured. Our results suggest that there are no consistent crystalline lens-induced refractive changes following exposure to hyperglycaemia for time-periods up to 5days used in the current study. It is possible that bovine lenses are able to offset the raised osmotic pressure from high glucose levels in the short-term by a process of osmoregulation and that repeated osmotic stress or longer term exposure may be required to induce the changes in refraction that are seen clinically.

Ocular disease in cattle

This article looks at common medical condition affecting the bovine eye including corneal ulceration, stromal abscesses, infectious bovine keratoconjunctivitis, squamous cell carcinoma, and bovine iritis. It also covers anatomical features, first aid procedures and examination techniques.

Endothelial glycocalyx layer in the aqueous outflow pathway of bovine and human eyes

The glycocalyx layer on the vascular endothelium is known to have an important role as a transport barrier and in the mechanotransduction of fluid shear stress. The detailed structure and distribution of the glycocalyx in the bovine and human aqueous humor outflow pathways has not yet been reported. The purpose of this study was to determine whether this layer exists in the bovine and human aqueous outflow pathways and to compare the distribution and thickness therein. Enucleated bovine (N = 4) and human (N = 4) eyes were fixed using Alcian Blue to preserve the glycocalyx. The glycocalyx distribution and thickness (in regions where it was seen) were measured on the trabecular beams (TM), Schlemm's canal (SC)/aqueous plexus (AP), and collector channels (CC). The glycocalyx, which appears as a layer of hair-like brushes, coats the surface of the endothelium non-uniformly in the bovine and human aqueous outflow pathways with a thickness in bovine eyes of 68–122 nm and in human eyes of 52–166 nm (25th to 75th percentiles). The distribution of the glycocalyx in different regions of the outflow pathway is not the same between bovine and human eyes. In both species, the glycocalyx was most uniform in the CCs. Less coverage of glycocalyx was found in the AP than the TM in bovine eyes, while more coverage was found in SC than the TM in human eyes. Most interestingly, glycocalyx was also found filling most pores of the endothelium of AP/SC in both bovine and human eyes. Glycocalyx was usually not found coating the inner membranes of the giant vacuoles (GVs); however, in GVs with a visible pore, glycocalyx was frequently observed on the inner membranes of the GVs. Based on our findings and those from the vascular endothelium, it is likely that the glycocalyx in SC plays a role in transduction of shear stress and perhaps regulation of outflow resistance.

Internal Energy of Thermometer Ions Formed by Femtosecond Laser Desorption: Implications for Mass Spectrometric Imaging

Mass spectrometry (MS) imaging of biological samples would greatly benefit from improved lateral resolution and depth profiling which may be possible with femtosecond (fs), near-IR laser desorption microprobes to form ions directly in laser desorption ionization (fs-LDI) or via vacuum ultraviolet postionization (fs-LDPI). The use of fs-LDI-MS or fs-LDPI-MS for imaging, however, requires a determination of the magnitude of internal energy imparted by the ultrashort desorption pulse because the amount of internal energy determines the extent of ion fragmentation, and extensive fragmentation degrades MS imaging by complicating the mass spectra. This paper estimates internal energies imparted to 4-chlorobenzylpyridinium (CBP) thermometer ions desorbed from both simulated bacterial biofilms and bovine eye lens tissue samples by ∼75 fs, 800 nm laser pulses. Both direct ions and photoions formed by 10.5 eV single photon ionization of desorbed neutrals are analyzed by time-of-flight MS. Survival yields (SYs) of CBP varied from 0.2 to 0.8 and depended upon desorption laser fluence, overlap between desorption laser pulses, and whether direct ions or photoions were detected. SYs for photoions additionally depended on time delay between desorption and photoionization laser pulses, with the highest SYs seen at longer delay times. CBP internal energies were estimated using previously published computational results and compared with those from several other common MS imaging ion sources. The results are discussed in terms of their implications for MS imaging by fs-LDI and fs-LDPI.

Draft Genome Sequence of Moraxella bovoculi Strain 237T (ATCC BAA-1259T) Isolated from a Calf with Infectious Bovine Keratoconjunctivitis.

Moraxella bovoculi is a recently identified species, recovered from the bovine eye, which is under investigation as an etiological agent of infectious bovine keratoconjunctivitis. A draft genome sequence of the Moraxella bovoculi type strain 237T has been determined to identify features that may be important during host colonization.

New device for creating a continuous curvilinear capsulorhexis

To describe the evolution of a new device to facilitate continuous curvilinear capsulorhexis (CCC) creation. Department of Ophthalmology, University of Colorado School of Medicine, Aurora, Colorado, USA. Experimental study. Bench-side exvivo testing of unique prototypes for guidance and assistance of CCC in bovine and human eyes was performed. Five designs were sequentially tested as follows: a flexible circular blade of nickel-titanium alloy (nitinol), a flexible nitinol guide wire, a flexible elastomeric suction device, a combination approach of a nitinol guide wire and flexible silicone ring, and a freestanding micropatterned silicone ring. The first 3 designs were not amenable to insertion through a sub-2.4 mm corneal incision and failed to maintain adequate downward force to cut the capsule and/or prevent radial tears. The fourth design was successfully inserted through a 2.4 mm incision and maintained adequate downward pressure and contact to guide a manual CCC without radial tears. The final design was insertable through a 2.4 mm incision and exhibited self-adhesive characteristics after placement on the anterior capsule of an ophthalmic viscosurgical device-filled anterior chamber. Given the steep learning curve of manual capsulorhexis and the high cost of capsulotomy-assistive devices, such as the femtosecond laser, an alternative approach for creating a CCC is desirable. Performance of a highly precise manual CCC through a small incision using a medical-grade silicone device with an adhesive micropatterned design is a viable and cost-effective option for use in cataract surgery across a wide range of user experience. All authors are named as the inventors in a patent filed by the University of Colorado covering the details in this report.

Morphological and Hydrodynamic Correlations with Increasing Outflow Facility by Rho-Kinase Inhibitor Y-27632

Abstract Rho-kinase inhibitors affect actomyosin cytoskeletal networks and have been shown to significantly increase outflow facility and lower intraocular pressure in various animal models and human eyes. This article summarizes common morphological changes in the trabecular meshwork induced by Rho-kinase inhibitors and specifically compares the morphological and hydrodynamic correlations with increased outflow facility by Rho-kinase inhibitor, Y-27632, in bovine, monkey, and human eyes under similar experimental conditions. Interspecies comparison has shown that morphological changes in the juxtacanalicular connective tissue (JCT) of these 3 species were different. However, these different morphological changes in the JCT, no matter if it's separation between the JCT and inner wall in bovine eyes, or separation between the JCT cells or between the JCT cells and their matrix in monkey eyes, or even no separation between the inner wall and the JCT but a more subtle expansion of the JCT in human eyes, appear to correlate with the increased percent change of outflow facility. More importantly, these different morphological changes all resulted in an increase in effective filtration area, which was positively correlated with increased outflow facility in all 3 species. These results suggest a link among changes in outflow facility, tissue architecture, and aqueous outflow pattern. Y-27632 increases outflow facility by redistributing aqueous outflow through a looser and larger area in the JCT.

A technique for drug surrogate diffusion coefficient measurement by intravitreal injection

The aim of this work is to develop a technique to accurately measure the diffusion coefficient of drugs and drug surrogates in the vitreous humor using MRI. Fresh bovine eyes were used for drug diffusion study in the vitreous, and Gd-DTPA was chosen as a drug surrogate/contrast agent to visualize the diffusion process by MRI. Experiments were conducted by injecting 30μl of the Gd-DTPA with a concentration of 20mM/l in 0.9% saline in the vitreous of the whole bovine eye. MRI images were acquired at regular intervals for 1.5–2h, and Gd-DTPA concentration was determined from the MRI signal intensity. At each time point, concentration contours were constructed and a least-squares best fit to the corresponding theoretical contours, based on a cylindrical bolus model, was performed. The best fit at different time points resulted in fairly consistent diffusion coefficient values. With the surrogate injection technique perfected, highly-symmetric distribution of Gd-DTPA was observed, allowing for spherically symmetric mathematical models, with adjustments for ellipticity as needed, for the diffusion coefficient analysis. The analysis yielded an average diffusion coefficient value of (3.040±0.274)×10−6cm2/s. The 3-D MRI visualization together with careful symmetric injection of the surrogate/contrast agent has provided a quantitative tool for the accurate measurement of the diffusion coefficient. With symmetric injection and corresponding diffusion theory based on the point-source model with adjustments for nonzero bolus size and asymmetry, strong agreement of theory with experiments has been achieved.

Identification of a Novel Lipofuscin Pigment (iisoA2E) in Retina and Its Effects in the Retinal Pigment Epithelial Cells

Lipofuscin accumulation in retinal pigment epithelial (RPE) cells of the eye implicates the etiologies of Stargardt disease and age-related macular degeneration, a leading cause of blindness in the elderly. Here, we have identified a previously unknown RPE lipofuscin component. By one- and two-dimensional NMR techniques and mass spectrometry, we confirmed that this compound is a new type of pyridinium bisretinoid presenting an unusual structure, in which two polyenic side chains are attached to adjacent carbons of a pyridinium ring. This pigment is a light-induced isomer of isoA2E, rather than A2E, referred to as iisoA2E. This pigment is a fluorescent lipofuscin compound with absorbance maxima at ∼430 and 352 nm detected in human, pig, mouse, and bovine eyes. Formation of iisoA2E was found in reaction mixtures of all-trans-retinal and ethanolamine. Excess intracellular accumulation of this adduct in RPE cells in vitro leads to a significant loss of cell viability and caused membrane damage. Phospholipase D-mediated phosphodiester cleavage of the A2PE series generated isoA2E and iisoA2E, in addition to A2E, thus corroborating the presence of isoA2PE and iisoA2PE that may serve as biosynthetic precursors of isoA2E and iisoA2E.

Measuring The Intravitreal Mobility of Nanomedicines with Single-Particle Tracking Microscopy

To develop a robust assay to evaluate and compare the intravitreal mobility of nanoparticles in the intact vitreous body. Excised bovine eyes were prepared to preserve the fragile structure of the vitreous humor, while permitting high-resolution fluorescence microscopy and single-particle tracking analysis of intravitreally injected nanoparticles. This assay was validated by analyzing polystyrene beads and further employed to evaluate gene nanomedicines composed of poly(amido amine)s and plasmid DNA. The assay was able to distinguish immobilized cationic nanoparticles from mobile PEGylated nanoparticles. PEGylation of the polyplexes resulted in a drastic improvement of their mobility. An ex vivo eye model is presented for studying nanoparticle mobility in intact vitreous humor by single-particle tracking microscopy. These results give important guidelines for developing gene- and drug-delivery nanomedicines that are compatible with intravitreal administration.

Successful Transplantation of In Vitro Expanded Human Cadaver Corneal Endothelial Precursor Cells On to a Cadaver Bovine’s Eye Using a Nanocomposite Gel Sheet

Purpose: In vitro expansion of human corneal endothelial precursor (HCEP) cells has been reported via production of cell aggregated spheres. However, to translate this procedure in human patients warrants maintaining the position of the eyeballs facing down for 36 h, which is not feasible. In this study, we report a method using a nanocomposite (NC) gel sheet to accomplish the integration of HCEP cells to the endothelium of cadaver bovine’s eyes.Materials and Methods: HCEP cells were isolated from the corneal endothelium of a cadaver human eye and then expanded using a thermoreversible gelation polymer (TGP) as reported earlier. For the study, three cadaver bovine eyes were used. The NC gel sheets were inserted into the bovine eyes’, aligned and suture-fixed in position under the host endothelium. HCEP cells previously expanded in the TGP were harvested and injected using a 26-gauge syringe between the endothelium and the NC gel sheet. The eyes were left undisturbed for three hours following which the NC gel sheets were gently removed. The corneas were harvested and subjected to histopathological studies.Results: Histopathological studies showed that all the three corneas used for NC gel sheet implantation showed the presence of engrafted HCEP cells, seen as multi-layered cells over the native endothelium of the bovine cornea. Examination of the NC gel sheets used for implantation showed that only very few corneal endothelial cells remained on the sheets amounting to what could be considered negligible.Conclusion: The use of the NC gel sheet makes HCEP cell transplantation feasible for human patients. Further in vitro basic studies followed by translational studies are necessary to bring this method for clinical application in appropriate indications.

Bovine rod rhodopsin: 2. Bleaching in vitro upon 12C ions irradiation as source of effects as light flash for patients and for humans in space

Purpose: In a previous paper, we showed that chemiluminescence from radical recombination (initiated by lipid peroxidation and propagated by polyunsaturated fatty acids [PUFA]) has a bleaching effect comparable to that caused by light on the rhodopsin of retinal rod outer segment (RdOS) prepared from bovine eyes. Photons generated by radical recombination were suggested to be the origin of phosphenes perceived as light flashes by the human eye. Irradiation with 12C carbon ions was used in this study to stimulate radical production, propagation and recombination leading to photoluminescence.Materials and methods: 12C radiation bleached RdOS rhodopsin, but structural damage increasing with the radiation dose was also observed. For this reason, only the effects on rhodopsin at doses producing next to negligible biodamage and permitting regeneration have been considered as bleaching effects.Results: 12C irradiation bleached RdOS rhodopsin, but increasing structural damage with radiation dose was also observed. For the measure of bleaching and to reveal dose response effects on rhodopsin that were able to be regenerated only results from doses producing nearly negligible biodamage have been considered.Conclusions: Recombination of radicals appears responsible for the release of photons with subsequent bleaching of rhodopsin. This effect could have an important role in the generation of the anomalous visual effects (phosphenes) experienced by patients during hadrotherapy or by astronauts in space.

Aldose Reductase Inhibitors of Plant Origin

Diabetic complications are attributed to hyperglycaemic condition which is in turn associated with the polyol pathway and advanced glycation end products. Aldose reductase (AR) is the principal enzyme of polyol pathway which plays a vital role in the development of diabetic complications. AR inhibitory activity can be screened by both in vitro and in vivo methods. In vitro assays for AR enzyme are further classified on the basis of the source of enzyme such as rat lens, rat kidney, cataracted human eye lens, bovine eyes and human recombinant AR enzymes, whereas the in vivo model is based on the determination of lens galactitol levels. A number of synthetic AR inhibitors (ARIs) including tolrestat and sorbinil have been developed, but all of these suffer from drawbacks such as poor permeation and safety issues. Therefore, pharmaceutical companies and many researchers have been carrying out research to find new, potent and safe ARIs from natural sources. Thus, many naturally occurring compounds have been reported to have AR inhibitory activity. The present review attempts to highlight phytochemicals and plant extracts with potential AR inhibitory activity. It also summarizes the classes of compounds which have proven AR inhibitory activity. Phytochemicals such as quercetin, kaempferol and ellagic acid are found to be the most promising ARIs. The exhaustive literature presented in this article clearly indicates the role of plant extracts and phytochemicals as potential ARIs.

Reversible changes in aqueous outflow facility, hydrodynamics, and morphology following acute intraocular pressure variation in bovine eyes

Background Elevated intraocular pressure (IOP) is primarily due to increased aqueous outflow resistance, but how aqueous outflow resistance is generated and regulated are still not fully understood. The aim of this study is to determine whether changes in outflow facility, outflow pattern, and morphology following acute IOP elevation were reversible when the IOP was returned to a normal level in bovine eyes using a two-color tracer technique to label outflow patterns within the same eye. Methods Twelve fresh enucleated bovine eyes were perfused with Dulbecco's phosphate buffer saline (PBS) containing 5.5 mmol/L glucose (DBG) at 30 mmHg first to establish the baseline outflow facility followed by a fixed volume of red fluorescent microspheres (0.5 μm, 0.002% v/v). After the red tracer being replaced with DBG in the anterior chamber, perfusion was continued at 7 mmHg with the same volume of green tracer, followed by a fixative. In two control groups, the eyes were constantly perfused at either 30 mmHg (n=6) or 7 mmHg (n=6) using the same methods. The outflow facility (C, μl·min·-1mmHg-1), was continuously recorded. Confocal images were taken along the inner wall (IW) of the aqueous plexus (AP) in frontal sections. The percent of the effective filtration length (PEFL, PEFL=IW length exhibiting tracer labeling/total length of IW) was measured. Sections with AP were processed and examined by light microscopy. The total length of IW and the length exhibiting separation (SL) in the juxtacanalicular connective tissue (JCT) were measured. A minimum of eight collector channel (CC) ostia per eye were analyzed for herniations. Results In the experimental (30-7 mmHg) group, the outflow facility was significantly higher at 7 mmHg ((4.81±1.33) μl·min·-1mmHg-1) than that at 30 mmHg ((0.99±0.15) μl·min·-1mmHg-1, P=0.002), corresponding to a significant increase in the PEFL (P=0.0003). The percent of CC ostia exhibiting herniations in the experimental group ((67.40±8.90) μl·min·-1mmHg-1) decreased significantly compared to that in the control at 30 mmHg ((94.44±3.33) μl·min·-1mmHg-1, P=0.03), but higher than that in the control at 7 mmHg ((29.43±4.60) μl·min·-1mmHg-1, P=0.01). Washout-associated separation between the IW and JCT was found by light microscopy and percent separation length (PSL, PSL=SL/total length of IW) was decreased in the control at 30 mmHg compared to that in the experimental group and control at 7 mmHg. Conclusions The pressure-induced morphological and hydrodynamic changes were reversible. Changes (collapse of AP, separation between the JCT and IW, and herniation into CC ostia) influence the effective filtration area that regulates outflow facility.

Effects of Pressure and Electrical Charge on Macromolecular Transport Across Bovine Lens Basement Membrane

Molecular transport through the basement membrane is important for a number of physiological functions, and dysregulation of basement membrane architecture can have serious pathological consequences. The structure-function relationships that govern molecular transport in basement membranes are not fully understood. The basement membrane from the lens capsule of the eye is a collagen IV-rich matrix that can easily be extracted and manipulated in vitro. As such, it provides a convenient model for studying the functional relationships that govern molecular transport in basement membranes. Here we investigate the effects of increased transmembrane pressure and solute electrical charge on the transport properties of the lens basement membrane (LBM) from the bovine eye. Pressure-permeability relationships in LBM transport were governed primarily by changes in diffusive and convective contributions to solute flux and not by pressure-dependent changes in intrinsic membrane properties. The solute electrical charge had a minimal but statistically significant effect on solute transport through the LBM that was opposite of the expected electrokinetic behavior. The observed transport characteristics of the LBM are discussed in the context of established membrane transport modeling and previous work on the effects of pressure and electrical charge in other basement membrane systems.

The Mechanical Properties of Ex Vivo Bovine and Porcine Crystalline Lenses: Age-Related Changes and Location-Dependent Variations

The mechanical properties of ex vivo animal lenses from three groups were evaluated: old bovine (25–30 mo old, n = 4), young bovine (6 mo old, n = 4) and young porcine (6 mo old, n = 4) eye globes. We measured the dynamics of laser-induced microbubbles created at different locations within the crystalline lenses. An impulsive acoustic radiation force was applied to the microbubble, and the microbubble displacements were measured using a custom-built high pulse repetition frequency ultrasound system. Based on the measured dynamics of the microbubbles, Young's moduli of bovine and porcine lens tissue in the vicinity of the microbubbles were reconstructed. Age-related changes and location-dependent variations in the Young's modulus of the lenses were observed. Near the center, the old bovine lenses had a Young's modulus approximately fivefold higher than that of young bovine and porcine lenses. The gradient of Young's modulus with respect to radial distance was observed in the lenses from three groups.

Properties of membranes derived from the total lipids extracted from the human lens cortex and nucleus

Human lens lipid membranes prepared using a rapid solvent exchange method from the total lipids extracted from the clear lens cortex and nucleus of 41- to 60-year-old donors were investigated using electron paramagnetic resonance spin-labeling. Profiles of the phospholipid alkyl-chain order, fluidity, oxygen transport parameter, and hydrophobicity were assessed across coexisting membrane domains. Membranes prepared from the lens cortex and nucleus were found to contain two distinct lipid environments, the bulk phospholipid–cholesterol domain and the cholesterol bilayer domain (CBD). The alkyl chains of phospholipids were strongly ordered at all depths, indicating that the amplitude of the wobbling motion of alkyl chains was small. However, profiles of the membrane fluidity, which explicitly contain time (expressed as the spin-lattice relaxation rate) and depend on the rotational motion of spin labels, show relatively high fluidity of alkyl chains close to the membrane center. Profiles of the oxygen transport parameter and hydrophobicity have a rectangular shape and also indicate a high fluidity and hydrophobicity of the membrane center. The amount of CBD was greater in nuclear membranes than in cortical membranes. The presence of the CBD in lens lipid membranes, which at 37°C showed a permeability coefficient for oxygen about 60% smaller than across a water layer of the same thickness, would be expected to raise the barrier for oxygen transport across the fiber cell membrane. Properties of human membranes are compared with those obtained for membranes made of lipids extracted from cortex and nucleus of porcine and bovine eye lenses.

Signaling cross-talk between cannabinoid and muscarinic systems actives Rho-kinase and increases the contractile responses of the bovine ciliary muscle

The aim of the present study was to evaluate the role of a possible interaction between cannabinoid and muscarinic systems, both widely expressed in the ocular structure and involved in the control of bovine ciliary muscle contractility and intraocular pressure modulation. The ciliary muscle strips isolated by bovine eyes were exposed cumulatively to anandamide in the presence and in the absence of carbachol (5nM), in a miograph system for isometric recording. The experiments were also conducted in the presence of AM251 (100nM), 4-DAMP (100nM), Pertussis toxin (500ng/ml), U73122 (0.1 and 1μM), chelerythrine (1 and 10μM) and Y27632 (1 and 10μM). Contractile responses were expressed as the percentage of 10μM carbachol-induced contraction. The anandamide-induced contraction on bovine ciliary muscle strips was enhanced by the previous stimulation of Gq-protein-coupled muscarinic M3 receptors with carbachol. The contractile response to anandamide plus carbachol was affected by different inhibitors such as Pertussis toxin, phospholipase C, protein kinase C and Rho-kinase. The key results of the present study show that sequential activation of muscarinic M3 receptors and cannabinoid CB1 receptors produce synergistic contractile effects of the bovine ciliary muscle by involving the activation of Rho-kinase and protein kinase C.

Chemical Modification of Lysozyme, Glucose 6-Phosphate Dehydrogenase, and Bovine Eye Lens Proteins Induced by Peroxyl Radicals: Role of Oxidizable Amino Acid Residues

Chemical and structural alterations to lysozyme (LYSO), glucose 6-phosphate dehydrogenase (G6PD), and bovine eye lens proteins (BLP) promoted by peroxyl radicals generated by the thermal decomposition of 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH) under aerobic conditions were investigated. SDS-PAGE analysis of the AAPH-treated proteins revealed the occurrence of protein aggregation, cross-linking, and fragmentation; BLP, which are naturally organized in globular assemblies, were the most affected proteins. Transmission electron microscopy (TEM) analysis of BLP shows the formation of complex protein aggregates after treatment with AAPH. These structural modifications were accompanied by the formation of protein carbonyl groups and protein hydroperoxides. The yield of carbonyls was lower than that for protein hydroperoxide generation and was unrelated to protein fragmentation. The oxidized proteins were also characterized by significant oxidation of Met, Trp, and Tyr (but not other) residues, and low levels of dityrosine. As the dityrosine yield is too low to account for the observed cross-linking, we propose that aggregation is associated with tryptophan oxidation and Trp-derived cross-links. It is also proposed that Trp oxidation products play a fundamental role in nonrandom fragmentation and carbonyl group formation particularly for LYSO and G6PD. These data point to a complex mechanism of peroxyl-radical mediated modification of proteins with monomeric (LYSO), dimeric (G6PD), and multimeric (BLP) structural organization, which not only results in oxidation of protein side chains but also gives rise to radical-mediated protein cross-links and fragmentation, with Trp species being critical intermediates.