ACID–BASE PROPERTIES - Oxidation of Amino Acids, Peptides, and Proteins: Kinetics and Mechanism - page 53

acids/peptides (i.e., speciation) can be calculated by applying their protonation/
dissociation constants. The next section in this chapter presents the procedure
to determine speciation. Cysteine was chosen as an example because of its
importance in the oxidative chemistry of proteins. Generally, cysteine was
found the most reactive amino acid in proteins with reactive species (Chapter
4). Protonation of amino acids depends on temperature, ionic strength, and
polarity of solvent (see the next section) and thus the speciation. The interac-
tions between amino acids and solvent with different dielectric constants
provide information on the chemistry of in vivo processes [3, 4].
In the biological environment, rates of the reactions depend on the confor-
mational dynamics of proteins. The degree of exposure of proteins to solvent
media (e.g., H 2 O) may therefore affect the oxidation of amino acid side chains
by reactive species. The dynamics of proteins has been studied extensively
using the hydrogen/deuterium exchange approach [5, 6]. The use of the hydro-
gen exchange (HX) phenomenon in protein measurements has a long history,
which was first described in 1950 to measure deuteration using density gradi-
ents [5, 7]. Hydrogen atoms of S-H, O-H, and N-H groups in proteins can
exchange with the surrounding water. The degree of deuteration in continuous-
labeling experiments is monitored as a function of time. The slow hydrogen/
deuterium exchange (HDX) occurs if sites in the protein are involved in stable
hydrogen bonds or exist inside the protein core. The details and mechanisms
of the exchange of hydrogen between solvent and protein have been reviewed
[8-11]. The exchange of deuterium for hydrogen can be followed by different
spectroscopic techniques. Of the several spectroscopic tools, nuclear magnetic
resonance (NMR) has been extensively used for the analysis of HX in proteins
[9, 12]. Mass spectrometry (MS)-based HDX measurements are also appropri-
ate because the mass of deuterium is almost twice the mass of hydrogen [10,
11, 13, 14]. In these measurements, only a miniscule amount of the protein is
needed. As little as 500-1000 pmol is needed for an entire experiment, which
includes the analysis of 10 points of exchange. Moreover, protein concentra-
tion can be as low as 0.1 μM, which is at least an order of magnitude smaller
than those of many other techniques used in studying the conformation and
dynamics of a protein. In the HX-MS approach, samples having multiple pro-
teins and ligands can be analyzed [10].
The hydrogen at the peptide backbone amide linkages in proteins can be
easily measured. Many hydrogen atoms on the side chains of proteins exchange
so rapidly that the measurement of them by MS is difficult. Hydrogens bonded
to carbons in proteins almost never exchange. The advantage of measuring
amide hydrogens is that they hold secondary structure elements (β-sheets, α-
helices) together. Moreover, every amino acid except proline has an amide
hydrogen located on the backbone. The exchange rates of NHs in folded pro-
teins are approximately eight orders of magnitude slower than those in unfolded
proteins. Thus, hydrogen bonding and solvent exposure determine the exchange
between the two interwoven proteins. Fast exchange rates are suggestive of
solvent exposure and/or no hydrogen bonding, while the reverse is true for