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-butylhydroperoxide (t-BOOH) and 1.6 μM 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 μM 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 μM. 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.