[Frontiers in Bioscience 6, a17-24, August 1, 2001]
REACTIVE CARBONYL FORMATION BY OXIDATIVE AND NON-OXIDATIVE PATHWAYS
Sarah Adams, Pattie Green, Renee Claxton, Sabrina Simcox, Michelle V. Williams, Katherine Walsh and Christiaan
Biochemistry of Aging Laboratory, University of Florida, Box 118206, Gainesville, FL 32611
TABLE OF CONTENTS
3. Materials and Methods
Materials, reagents, and isolation of proteins
3.2. Metal-catalyzed protein oxidation
3.3. Protein modification by aldehydes
3.4. Protein oxidation by hypochlorous acid and peroxynitrite
3.5. Determination of reactive carbonyls in proteins
4.1. Metal-catalyzed oxidation
4.2. Carbonyl formation by non-oxidative mechanisms
4.3. Carbonyl formation by the strong oxidants hypochlorous acid and peroxynitrite
4.4. Hemoglobin, myoglobin, and cytochrome c have a similar absorbance spectrum
The spectrophotometric protein carbonyl assay is
used as an indicator of protein damage by free radical
and in a variety of pathologies.
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
albumin exposed to oxidative scenarios, such as
hypochlorous acid, peroxynitrite, and metal-catalyzed
oxidation exhibited variable, but increased levels of
Other non-oxidative modification systems, in
which proteins are incubated with various aldehydes, such
as malondialdehyde, acrolein, glycolaldehyde, and glyoxal
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
Oxygen free radicals are produced as byproducts
of many endogenous and exogenous sources including UV
light, radiation, neutrophil activity, and metabolism (1-4).
Oxidative stress often leads to lipid, nucleic acid,
carbohydrate, and protein modifications.
and modification have been shown to increase during aging
and exacerbate many pathological processes, such as
atherosclerosis and human cataracts (1, 3-5).
proteins can lead to the formation of oxidized amino acids,
such as dityrosine, 3-nitrotyrosine, 3-chlorotyrosine,
oxohistidine, and altered amino acid side chains containing
reactive carbonyls (2, 6-12).
Oxidation of proteins can
result in the loss of catalytic function, increased sensitivity