Ethanol ethylacetate

Protein carbonyl content (PCC) was quantified as an indicator for protein damage in the retina. Samples and controls were each measured in duplicates. Supernatant (100 µl, prepared as described above for TBARS analysis) was reacted with 400 µl 10 mM 2,4-dinitrophenylhydrazine (DNPH, Aldrich) in 2M HCl (Frutarom), or 2M HCl alone (for controls). Reaction tubes were incubated for one hour at room temperature in the dark, while vortexing every 15 min. 500 µl 20% tri chloro-acetic acid (TCA, SIGMA) were added, followed by five min incubation on ice, after which tubes were centrifuged at 10,000 g for 10 min at 4°C. Supernatant was discarded; pellets were re-suspended in 1 ml 10% TCA, and again incubated and centrifuged as described above. Pellets were washed three times with ethanol:ethylacetate (1∶1, SIGMA) for 10 min, and centrifuged after each wash. The pellets were re-suspended in 500 µl 6 M guanidine-hydrochloride (BIO LAB) in 0.5 M K₃PO₄ (SIGMA) pH 2.5, and again centrifuged. Absorbance was measured using a spectrophotometer at λ = 370 nm. Protein carbonyl content was established using the corrected absorbance (CA) that was calculated by subtracting the average absorbance of the controls from that of the samples [28] –[30] .

Oxidative damage to proteins was assessed using the 2,4-dinitrophenylhydrazine (DNPH) method, suitable for the quantification of protein carbonyls [25 (link)]. One mL of the sperm lysate was subjected to trichloroacetic acid (TCA; 20% w/v; Sigma-Aldrich, St. Louis, MO, USA) treatment, subsequently mixed with 1 mL DNPH (10 mM in 2 N HCl; Sigma-Aldrich, St. Louis, MO, USA) and incubated at 37 °C for 1 h. The mixture was treated again with 1 mL TCA, incubated at 4 °C for 10 min and centrifuged (11,828× g, 10 min). The pellet was washed three times with 1 mL ethanol/ethyl acetate (1/1; v/v; Sigma-Aldrich, St. Louis, MO, USA) and finally resuspended in 1 mL 6 M guanidine HCl (Sigma-Aldrich, St. Louis, MO, USA). The absorbance was measured at 360 nm with the Cary 60 spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). guanidine HCl (6M) was used as a blank. The molar absorption coefficient of 22,000/M/cm was applied for the calculation of the protein carbonyl concentration in the samples. The extent of protein oxidation is expressed in nmol protein carbonyls/mg protein [23 (link)].