REACTIVE OXYGEN SPECIES - Oxidation of Amino Acids, Peptides, and Proteins: Kinetics and Mechanism - page 159

modeling, and molecular dynamics [257]. Results indicate pathways of the
reaction mechanism involve hydrogen bonding and close-ring structure and
therefore ozonolysis was controlled by entropy effects. calculated energies
and entropies of activation allowed to proposed mechanisms for different
pathways of ozonolysis.
There are few studies on the oxidation of proteins by O 3 [225, 237]. The
oxidation of band 3 protein, cytochrome c , BSA, and human serum albumin
(HSA) by O 3 has been carried out to examine the oxidation susceptibility of
Trp residues present in different regions of proteins [225]. Isolation of band 3
protein from the ozone-treated ghosts followed by subjection of it to trypsin
digestion yielded water-soluble peptides from the cytoplasmic N-terminal tail
and the interhelical loops. Fluorescent peptides include WMEAAR from the
N-terminal cytoplasmic tail and gWVIHPLgLR from the outer loop between
helices 7 and 8. Trp residues in band 3 were monitored in three different envi-
ronments: the N-terminal cytoplasmic tail, the transmembrane helices, and in
one of the interhelical loops external to the lipid bilayer [225]. cytochrome c
contained the gITWK peptide in which Trp was at position 59 interacting with
the heme moiety. Digestion by trypsin of BSA produced two fluorescent pep-
tides (FWgK and AWSVAR) and two Trp residues, which were in different
regions of the protein. HSA contained the AWAVAR peptide with one Trp
residue within it.
The fluorescence of control- and O 3 -treated proteins at pH 7.5 were mea-
sured (Fig. 4.17). As shown in Figure 4.17a,b, the peptides in band 3 and cyto-
chrome c were not oxidized by O 3 . The Trp in N-terminal cytoplasmic tail of
band 3 is expected to be resistant to oxidation by O 3 because many proteins
of the glycolytic sequence of the tail possibly react first before ozone could
reach to Trp on the N-terminal cytoplasmic tail. A protection of Trp in the
interhelical loop from the peripheral proteins on the outside of the membrane
may be another cause of no oxidation observed in band 3. HPLc and amino
acid analysis of O 3 -treated cytochrome c demonstrated a modification of the
protein and oxidation of Met. However, the Trp residue did not change, which
suggests its protection by the porphyrin ring [225]. The results shown in Figure
4.17c,d clearly indicate the peptides in both BSA and HSA were oxidized by
O 3 . Trp residues in these proteins are involved in structures of membrane and
hence are not protected from the attack of O 3 . It is likely that the two Trp
residues of BSA reacted differently with O 3 due to their positions in the three-
dimensional structure. Overall, ozonolysis of tryptophans in studied proteins
was affected by the position of the Trp residues within the protein.
The oxidation of five different proteins (invertase, pectinase, papain, trypsin,
and gelatin) by O 3 has been examined to obtain clues on the action of ozone
on more complex biological species such as virus and bacteria [237]. Measure-
ments of spectroscopy and polarimetry were used to monitor the ozonolysis
of the proteins. The results in Figure 4.18 showed two spectral maxima, one at
222 nm, attributed to the n → π* transition of the amide bond, and the other
at 275 nm, attributed to the aromatic acids, which are mainlyTrp (ε 277nm = 5500/M/