Figure 7. Carbonyl adducts are increased in RBC ghosts post COCl₂ exposure.

Mice were exposed to air or COCl₂ (20 ppm, 10min). RBCs were separated from the plasma and hemolyzed with 20 mM hypotonic HEPES Buffer 24 hours later. The mixture was centrifuged at 14000 xg for 20 min and the resulting RBC pellet was dissolved in RIPA buffer. The protein was quantified by the BCA method and the presence of protein carbonyl groups was assessed using the OxyBlot Protein Oxidation Detection Kit according to the manufacturer’s protocol. Significant oxidation was noted in a protein around 95 KDa. Oxidation of high molecular weight proteins (around 250 kDa; presumably ankyrin and spectrin) was also observed (A). Each lane represents a different mouse. Additionally equal amounts of proteins were loaded into a 4-20% gradient gel and proteins were separated and stained with Amido Black (data not shown). There was no difference in protein levels between the air and the COCl₂ exposed mice. Panel B shows the quantification of carbonyl adducts. Values are means ± 1 SEM; Student’s t-test.