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Laura Giurgola, Claudio Gatto, Jana D'Amato Tothova; Extraction of crystalline proteins from porcine eye lens and evaluation of their stability and relation with oxidative stress. Invest. Ophthalmol. Vis. Sci. 2020;61(7):5008.
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To extract crystalline proteins from porcine eye lenses and to evaluate their stability and relation with oxidative stress.
10 lenses were extracted from porcine eyes. The lenses were washed with phosphate-buffered saline (PBS) and homogenized in extraction buffer (20 mM Tris HCl, 5 mM EDTA, 2.6 ml/g of lenses) using Polytron homogenizer. The water-insoluble residues were eliminated from the homogenate by subsequent centrifugations at 12000 rpm for 10 minutes. Crystalline proteins concentration of the fresh extract was assessed with Bradford assay using Bradford reagent (Sigma Aldrich) and HPLC using Jupiter 5 µm C18 300 Å column. The stability was assessed by HPLC after storage of the extract at -20°C, +4°C, +25°C and +40°C for 5 months.The reactive oxygen species (ROS) probe Dehydrorodamine-123 (DHR123, Sigma Aldrich) was added to the lens homogenate solution containing 3 mg/ml of crystalline proteins and irradiated by UV light (254 nm) for 0, 5, 10, 15 minutes. Irradiated samples were analyzed both by HPLC for protein characterization and fluorimetry recording emission fluorescence spectra from 510 to 700 nm, using a Perkin Elmer luminescence spectrophotometer with excitation at 505 nm to determine ROS production.
HPLC analysis showed the presence of a specific peak pattern of crystalline proteins in the porcine eye lens extract corresponding to 21% of α, 66% of β, and 13% of γ crystalline proteins. The concentration of each crystalline protein decreased after 15 minutes of UV irradiation.The emission fluorescence spectra showed a peak at 527 nm corresponding to the presence of Rhodamine 123, as a result of the oxidation of DHR123 probe induced by the presence of ROS. After 5 months of storage at +40°C, α and γ crystallines showed concentration decrease of 13% and 66%, respectively. No changes in crystalline concentrations were observed at -20°C, +4°C, +25°C after 5 months of storage.
α, β and γ crystalline proteins were extracted from porcine eye lens and quantified. UV irradiation of crystalline proteins solution induced their degradation that could be related to ROS production. The extract showed excellent stability at -20°C, +4°C and +25°C for 5 months of storage. Additional studies are necessary to evaluate the oxidative stress mechanism that induces crystalline proteins degradation
This is a 2020 ARVO Annual Meeting abstract.
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