REACTIVE OXYGEN SPECIES - Oxidation of Amino Acids, Peptides, and Proteins: Kinetics and Mechanism - page 156

the sulfhydryl group (SH) to the thiolate ion (S − ) of cysteine. Thus, it could be
concluded that the greater reactivity of O 3 with the thiolate ion was mostly
responsible for the increase in the reaction rate with an increase in pH. The
estimated rate constants were 2.0 × 10 4 /M/s and 2.5 × 10 10 /M/s for reactions of
O 3 with cys(SH) and cys(S − ), respectively. The reactivity of O 3 with another
sulfur-containing amino acid, Met, was also high [223].
Hydrocarbon-like amino acids (glu and Asp) and amides (Asn, gln, and
Arg) have side chains that showed slow reactivity with O 3 . Two hydroxyamino
acids, Ser and Thr, also reacted slowly with O 3 . Ala and Val contain alkyl groups
and reacted slower than that of gly, which has no alkyl group. Butylamine
reacted much slower with O 3 than gly and also showed the influence of the
alkyl group on the rates (Fig. 4.14). Pro is a secondary amine and appears to
react faster than primary amines, such as gly (Fig. 4.14). It appears that the
imidazole ring in His was responsible for its high reactivity with O 3 . The aro-
matic amino acids, Phe and Trp, also had high reactivities with O 3 (Fig. 4.14).
It seems that the aromatic ring and the benzylic hydrogens influenced the
reactivity with O 3 .
The rate constants at pH 8.0 are provided in Table 4.10. Amount of ozone
required (i.e., ozone demand) for the oxidation of several amino acids were
determined experimentally [230], which aided in the determination of the rate
constants based on the decay rate of ozone, k (O 3 ). The calculated values of
k (AA) and half-lives are also provided in Table 4.10. The half-lives of most of
the reactions of amino acids with O 3 were determined to be in tens of seconds.
However, the half-lives for the oxidation of Met and Trp by O 3 were deter-
mined in milliseconds (Table 4.10).
Several studies on the products of the ozonation of amino acids, peptides,
and proteins in aqueous solution have been performed using spectrophotomet-
ric and chromatographic techniques, and now ESI and MALDI methods [224,
231-250]. Ozonation of gly by O 3 in the presence of HCO − ions produced
nitrate, oxamic, formic, and oxalic acids [251]. Ozonation of Met, cys, and glu-
tathionine formed sulfoxide and sulfone [236, 239, 252, 253]. Sulfoxide was the
main oxidation product of Met (Fig. 4.16) [225, 236]. However, a mass analysis
of the products showed both sulfoxide and sulfone as primary products of the
oxidation reaction [239, 254]. cys was oxidized as a first step to cystine (cySScy)
(Fig. 4.16), but a further oxidation reaction by ozone yielded cysteic acid [236,
255]. Recently, an interfacial ozonolysis of cys and gSH was carried out using
online thermospray ionization MS [252, 253]. When the surface of the aqueous
cys microdroplets was exposed to O 3 (g) for less than 1 millisecond, sulfenate
(cysSO − ), sulfinate ( CysSO − ), and sulfonate ( CysSO − ) were simultaneously
detected [252]. In similar experiments with gSH, sulfonates ( GSO /GSO
3

−

2

− )

were formed [253].
The oxidation of Trp, Tyr, and His alone and as part of peptide/proteins
residues by O 3 were carried out using the ESI-MS analysis [237, 239]. An
earlier study suggested Pro was the oxidized product of His [225], but a later
study reported an aspartyl residue, through a 2-oxohistidine intermediate, as