Peroxynitrite-dependent chemiluminescence of amino acids, proteins, and intact cells.
Exposure of proteins to ONOO- (fatty acid-free bovine serum albumin (BSA) and histones, 10 mg/ml) was accompanied by light emission which could be detected using a photon counter. Light emission upon addition of ONOO- to either histones or BSA increased linearly with ONOO- concentration at a rate of 50 +/- 4 and 66 +/- 4 cps/(mg protein.mM ONOO-), respectively (averages+SE). Bicarbonate (25 mM) increased ONOO(-)-dependent BSA chemiluminescence approximately 3-fold above baseline (221 +/- 6 cps/(mg protein.mM ONOO-)). The peak of peroxynitrite-dependent light emission was around 40-fold higher than when 1 mM tert-butyl-hydroperoxide (t-BOOH) and 1.6 microM hemin were used as oxidants. Fatty acid-containing BSA (0.04-0.08%) emitted 3.4-fold more light than pure BSA. Chemiluminescence increased with pH, being 4.5-fold higher at pH 8.8 than at pH 6.0. However, the half-life of emissive species did not change with pH, suggesting that the process leading to the formation of electronically excited states is the same at all pHs. Tryptophan or N-acetyltyrosine oxidation by ONOO- was accompanied by chemiluminescence (130 +/- 10 and 14 +/- 3 cps/(mg amino acid.mM ONOO-), respectively). Exposure of DNA or isolated nucleotides to either t-BOOH/hemin or ONOO- was not accompanied by light emission. Leptomonas seymouri (an insect parasite used as a model of intact cells) exposed to ONOO- emitted 3700 +/- 400 cps/(mg protein.mM ONOO-), compared to 55 +/- 3 cps/(mg protein.mM peroxide) when t-BOOH was used as oxidant. While chemiluminescence of L. seymouri exposed to ONOO- increased measured at concentrations as low as 30 microM, carbonyl formation (from protein oxidation) and thiobarbituric acid-reactive substances (lipid peroxidation) could be measured only if cells were exposed to initial ONOO- larger than 700 microM. Spectral analysis suggests that excited carbonyls (emission wavelength 340-450 nm) are not produced in high proportions. A substantial amount of light is generated above 500 nm, part of which could come from triplet states of tryptophan and tyrosine.
Watts
B P
BP
Department of Biomedical Sciences, College of Allied Health Professions, University of South Alabama, Mobile 36688-0002.
Barnard
M
M
Turrens
J F
JF
D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D001639:Bicarbonates / Q000494:pharmacology; D002478:Cells, Cultured; D005227:Fatty Acids / Q000494:pharmacology; D006427:Hemin / Q000494:pharmacology; D006657:Histones / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008163:Luminescent Measurements; D009566:Nitrates / Q000494:pharmacology*; D009696:Nucleic Acids / Q000737:chemistry; D009711:Nucleotides / Q000737:chemistry; D010084:Oxidation-Reduction; D010545:Peroxides / Q000494:pharmacology; D011506:Proteins / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D013053:Spectrophotometry; D014351:Trypanosomatina / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry; D020122:tert-Butylhydroperoxide
D000596:Amino Acids; D001639:Bicarbonates; D005227:Fatty Acids; D006657:Histones; D009566:Nitrates; D009696:Nucleic Acids; D009711:Nucleotides; D010545:Peroxides; D011506:Proteins; C039642:peroxynitric acid; D012710:Serum Albumin, Bovine; D014443:Tyrosine; D006427:Hemin; D014364:Tryptophan; D020122:tert-Butylhydroperoxide