ACID–BASE PROPERTIES - Oxidation of Amino Acids, Peptides, and Proteins: Kinetics and Mechanism - page 55

Numerous experimental methods have been applied to determine dis-
sociation constants, which include liquid-liquid partitioning, high-pressure
liquid chromatography, capillary zone electrophoresis, matrix-assisted laser
desorption/ionization time-of-flight MS, and titration methods [24-31]. Poten-
tiometric titrations are frequently applied to determine dissociation constants
[32, 33]. However, when dissociation constants are particularly low or high, or
in cases where the substance has very low solubility, spectrophotometric
methods are used. Theoretical calculations using quantum theoretical tech-
niques have also been applied to predict the values of dissociation constants
[26, 34-36]. These calculations have shown to successfully predict the dissocia-
tion constants of amino acids and peptides in water [26].
The values of thermodynamic constants of amino acids and simple peptides
are presented in Tables 2.1 and 2.2 [28, 32, 37-45]. Examination of simple pep-
tides allowed understanding of reactions of various peptides with reactive
species [46, 47] (Chapters 5 and 6). Most of the amino acids have an α-
carboxylic acid group with p K a1 values of ∼2.0 to 2.5 and an α-amino group
with p K a2 values of ∼9 to 10. The values are lower than those of monofunctional
carboxylic acids and amines, respectively, due to the negative charge on the
carboxylate group at high pH and the positive charge on the amino group at
low pH, which stabilizes amino acids [48]. Both Asp and Glu have a carboxyl
group in the side chain, which have p K a values in the range of 3.5-4.5 when
incorporated into a protein. Arg, Lys, and His have basic side chains, and their
p K a values in proteins are in the range of 9.5-10.5, 12.0-13.0, and 6.0-7.0,
respectively. The resonance stabilization of Arg makes it a relatively strong
base. Because His has a p K a value close to the pH of a living cell, the pH deter-
mines the charge on a particular His in a protein. The side chains of Asn and
Gln have a resonance-stabilized unprotonated amide group and are not basic.
This is similar to the aromatic side chain of Trp. The p K a value of Cys is ∼9.
Proteins usually have uncharged thiols; however, ionization may take place in
some enzymes [48]. Cystine has a disulfide bridge and has four protonation
sites (Table 2.1).
The formation of a peptide involves condensation of the carboxylic group
of one amino acid with the amino group of the next amino acid to yield a
peptide bond (amide linkage). The amide group itself does not demonstrate
properties of either the amino group or the carboxylic group. Generally, a
simple peptide (e.g., diglycine and triglycine) has two p K a values, one from the
C-terminus and the other from the N-terminus (Table 2.2). Glutathione is a
tripeptide (γ-L-glutamyl-L-cysteinyl-glycine [GSH]) and has four p K a values.
The oxidized form of GSH is glutathione disulfide (GSSG) with four carbox-
ylic and two amino groups, producing six p K a values (Table 2.2). Metal binding
to GSH and GSSG thus varies with pH significantly. Moreover, the variation
of redox potential of the two GSH-GSSG pairs can be understood in depth
by knowing accurately the p K a values of GSH and GSSG [49].
As stated earlier, temperature influences dissociation of amino acids (i.e.,
speciation), which, in turn, affects the reaction rates of functional groups of