Reactive carbonyl formation by oxidative and non-oxidative pathways.
The spectrophotometric protein carbonyl assay is used as an indicator of protein damage by free radical reactions in vitro and in a variety of pathologies. We investigated model proteins and a variety of oxidative and non-oxidative reactions, as well as what effects hemoglobin, myoglobin, and cytochrome c might have on levels of protein carbonyls. We show that oxidative as well as non-oxidative mechanisms introduce carbonyl groups into proteins, providing a moiety for quantification with 2,4-dinitrophenylhydrazine (DNPH). Bovine serum albumin exposed to oxidative scenarios, such as hypochlorous acid, peroxynitrite, and metal-catalyzed oxidation exhibited variable, but increased levels of carbonyls. Other non-oxidative modification systems, in which proteins are incubated with various aldehydes, such as malondialdehyde, acrolein, glycolaldehyde, and glyoxal also generated significant amounts of carbonyls. Furthermore, purified myoglobin, hemoglobin, and cytochrome c show high absorbance at the same wavelengths as DNPH. The high levels observed are due to the innate absorbance of hemoglobin, myoglobin, and cytochrome c near the assay spectra of DNPH. These studies show that carbonyl content could be due to oxidative as well as non-oxidative mechanisms and that heme-containing compounds may effect carbonyl quantification.
Adams
S
S
Biochemistry of Aging Laboratory, University of Florida, Box 118206, Gainesville, FL 32611, USA.
Green
P
P
Claxton
R
R
Simcox
S
S
Williams
M V
MV
Walsh
K
K
Leeuwenburgh
C
C
D000447:Aldehydes / Q000494:pharmacology; D000818:Animals; D003574:Cytochrome c Group / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry; D017665:Hydroxyl Radical / Q000737:chemistry; D006997:Hypochlorous Acid / Q000494:pharmacology; D008670:Metals / Q000494:pharmacology; D009211:Myoglobin / Q000737:chemistry; D010084:Oxidation-Reduction; D030421:Peroxynitrous Acid / Q000494:pharmacology; D010659:Phenylhydrazines / Q000737:chemistry; D011506:Proteins / Q000032:analysis*; D013053:Spectrophotometry / Q000379:methods*
D000447:Aldehydes; D003574:Cytochrome c Group; D006454:Hemoglobins; D008670:Metals; D009211:Myoglobin; D010659:Phenylhydrazines; D011506:Proteins; D030421:Peroxynitrous Acid; C004787:2,4-dinitrophenylhydrazine; D017665:Hydroxyl Radical; D006997:Hypochlorous Acid