REACTION OF OZONE WITH PROTEIN TRYPTOPHANS - BAND-III, SERUM-ALBUMIN,AND CYTOCHROME-C

Treatment of red cell ghosts with ozone inhibited both AChE (marking t he outside of the membrane) and G3PDH (marking the inside of the membr ane). There was no change in tryptophan fluorescence of the ghosts aft er the ozone treatment. Band 3 protein was isolated fi om the ozone-tr eated ghosts. The protein was digested with trypsin to obtain water so luble peptides from the cytoplasmic N-terminal tail and the interhelic al loops. Fluorescent peptides included GWV-IHPLGLR from the outer loo p between helices 7 and 8, and peptide WMEAAR from the N-terminal cyto plasmic tail. Neither one of these peptides was oxidized by ozone. Thi s was true whether or not the ghosts were sealed. We conclude that the position of these tryptophans either in the membrane structure, or be cause of binding to other proteins in the cytoplasmic tail, protects t hem from oxidation by ozone. Treatment of horse heart cytochrome c wit h ozone did not change the absorbance spectrum in the heme region or t he 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 t he ozone-treated cytochrome c with trypsin gave peptides which demonst rated normal fluorescence. (Cytochrome c has abnormally low fluorescen ce, which is not changed by ozone exposure.) The peptides were separat ed by HPLC. The fluorescence of the tryptophan-containing peptide (GIT WK) was not decreased by treatment of the cytochrome c by ozone. Amino acid analysis of the ozone-treated cytochrome c indicated that methio nine was oxidized. We conclude that tryptophan in cytochrome c is prot ected hom oxidation by ozone because of the interaction with the porph yrin ring. Bovine serum albumin and human serum albumin were treated w ith ozone. There was a monotonic decrease in tryptophan fluorescence i n both cases. Digestion of BSA with trypsin produced two fluorescent p eptides. The peptide FWGK was identified by coelution with the authent ic peptide. The putative peptide AWSVAR was not the same as the chemic ally synthesized peptide. The peptide sequences FWGK and ''AWSVAR'' we re both oxidized in ozone-treated bovine serum albumin, with no detect able discrimination. Tryptic digestion of the ozone-treated human seru m albumin produced a single fluorescent peptide, which was oxidized by ozone. The putative peptide AWAVAR in the tryptic digest of HSA was d istinct from chemically synthesized peptide. The oxidation of tryptoph ans in proteins by ozone is markedly influenced by position in tertiar y structure, position in membrane structure, and by chemical interacti ons within the protein. (C) 1997 Academic Press.