title: Reaction of ozone with protein tryptophans: band III, serum albumin, and cytochrome C.
abstract: Treatment of red cell ghosts with ozone inhibited both AChE (marking the outside of the membrane) and G3PDH (marking the inside of the membrane). There was no change in tryptophan fluorescence of the ghosts after the ozone treatment. Band 3 protein was isolated from the ozone-treated ghosts. The protein was digested with trypsin to obtain water soluble peptides from the cytoplasmic N-terminal tail and the interhelical loops. Fluorescent peptides included GWVIHPLGLR from the outer loop between helices 7 and 8, and peptide WMEAAR from the N-terminal cytoplasmic tail. Neither one of these peptides was oxidized by ozone. This was true whether or not the ghosts were sealed. We conclude that the position of these tryptophans either in the membrane structure, or because of binding to other proteins in the cytoplasmic tail, protects them from oxidation by ozone. Treatment of horse heart cytochrome c with ozone did not change the absorbance spectrum in the heme region or the tryptophan absorbing region. HPLC of the ozone-treated cytochrome c showed that cytochrome c was being modified, indicated by a change in the elution time. Treatment of cytochrome c with ozone did not change the activity in the NADH-cytochrome c reductase assay. Digestion of the ozone-treated cytochrome c with trypsin gave peptides which demonstrated normal fluorescence. (Cytochrome c has abnormally low fluorescence, which is not changed by ozone exposure.) The peptides were separated by HPLC. The fluorescence of the tryptophan-containing peptide (GITWK) was not decreased by treatment of the cytochrome c by ozone. Amino acid analysis of the ozone-treated cytochrome c indicated that methionine was oxidized. We conclude that tryptophan in cytochrome c is protected from oxidation by ozone because of the interaction with the porphyrin ring. Bovine serum albumin and human serum albumin were treated with ozone. There was a monotonic decrease in tryptophan fluorescence in both cases. Digestion of BSA with trypsin produced two fluorescent peptides. The peptide FWGK was identified by coelution with the authentic peptide. The putative peptide AWSVAR was not the same as the chemically synthesized peptide. The peptide sequences FWGK and "AWSVAR" were both oxidized in ozone-treated bovine serum albumin, with no detectable discrimination. Tryptic digestion of the ozone-treated human serum albumin produced a single fluorescent peptide, which was oxidized by ozone. The putative peptide AWAVAR in the tryptic digest of HSA was distinct from chemically synthesized peptide. The oxidation of tryptophans in proteins by ozone is markedly influenced by position in tertiary structure, position in membrane structure, and by chemical interactions within the protein.
authors.affiliation: Department of Botany and Statewide Air Pollution Research Center, University of California at Riverside, Riverside, California, 92521, USA.
authors.forename: J B
authors.initials: JB
authors.lastname: Mudd
authors.forename: P J
authors.initials: PJ
authors.lastname: Dawson
authors.forename: S
authors.initials: S
authors.lastname: Tseng
authors.forename: F P
authors.initials: FP
authors.lastname: Liu
mesh_terms: D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry*; D002417:Cattle; D003574:Cytochrome c Group / Q000737:chemistry*; D004910:Erythrocyte Membrane / Q000737:chemistry; D006736:Horses; D006801:Humans; D010084:Oxidation-Reduction; D010126:Ozone / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D012709:Serum Albumin / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D003574:Cytochrome c Group; D010455:Peptides; D012709:Serum Albumin; D010126:Ozone; D014364:Tryptophan