REACTIVE SPECIES - Oxidation of Amino Acids, Peptides, and Proteins: Kinetics and Mechanism - page 10

information on proteins [96]. This technique has limitations because partially
unfolded proteins do not crystallize, and hence, it is not always clear whether
conformations in the solid state are identical to the bulk solution phase
structures [97]. Time-resolved X-ray techniques may shed more light on
protein dynamics [96]. Nuclear magnetic resonance (NMR) spectroscopy can
provide insight on the structure and dynamics of proteins in solution [98-100].
Modern instruments have eliminated the limitations of high concentrations
of proteins using traditional NMR spectroscopy when collecting data.
However, determining structural information for proteins larger than 40-
50 kDa remains a challenge for NMR spectroscopy. Progress in the mapping
of proteins is summarized below, which includes the application of oxidative
labeling of proteins.
1.2.1 Oxidative Labeling
1.2.1.1 Carbonyl Labeling. Proteins containing carbonyls are produced by
reacting them with 2,4-dinitrophenylhydrazine (DNPH), followed by separa-
tion using gel electrophoresis [90]. The identification and quantification of
derivatized protein carbonyls can also be carried out by ratiometric Raman
spectroscopy [101]. Another approach of protein carbonyl labeling is by the
reaction with biotin hydrazide under mild pH conditions [102]. One drawback
of labeling is that both approaches cannot distinguish primary and secondary
carbonyls as well as carbonyls from glycation [103, 104]. It is also possible that
both reactants may not react with all types of oxidized amino acids [105].
1.2.1.2 Cysteine Residue Labeling. Labeling of the free SH group by a
reagent based on iodoacetamide, maleimide, and 5,5′-dithiobis-(2-nitrobenzoic
acid) allows indirect assessment of the oxidation of Cys [106-108]. Increased
Cys oxidation causes a decrease in the amount of labeling. One other approach
uses the blocking of reduced thiols present in solution before the reduction
of oxidized thiols with dithiothreitol (DTT), followed by labeling with
iodoacetamidofluorescein for separation using two-dimensional gel electro-
phoresis [109]. Thiol-specific biotin-HPDP (N-[6-(Biotinamido)hexyl]-3′-(2′-
pyridyldithio)propionamide) labeling has also been demonstrated for proteins
containing reversibly oxidized cysteines [110].
1.2.1.3 Reactive Species Labeling. Oxidized forms of chlorine, bromine,
and iodine readily react with Cys, Met, and aromatic amino acids to modify
the side chains of proteins (Fig. 1.4) [111, 112]. Aside from the side chains
shown in Figure 1.4, cystine also reacts to yield N -dischlorocystine. The stoi-
chiometric amounts of oxidizing agents used in the reaction mixtures deter-
mine the extent of oxidation of Cys, Met, and cystine. The reaction between
halide anions and oxidizers such as H 2 O 2 and • OH may also form oxidized
halide intermediates. Significantly, intermediates may react with water to form