S-Homocysteinylated Proteins - Homocysteine in Protein Structure/Function and Human Disease - page 118

7.1 Plasma S-Homocysteinyl-Proteins

7.1.1 S-Hcy-Albumin

Two major forms of serum albumin exist in the circulation: albumin-Cys 34 -SH,
known as mercaptoalbumin, and albumin-Cys 34 -S-S-Cys (Fig. 5.7 ), accounting for
about two-thirds (0.45 mM) and one-third (0.15 mM), respectively, of total plasma
albumin [323]. Two minor forms, accounting for 1-2 % of total albumin, also exist
in the circulation: albumin-Cys 34 -S-S-Hcy [91, 103] and N-Hcy-albumin (Fig. 5.7 )
[79]. Albumin carries 80 % of plasma Hcy [103] and Cys [296]. The in vivo

ability of albumin to form a disulfide with Hcy has been recapitulated in vitro [78,
81, 91].
Bound Hcy and Cys form a disulfide bond with the conserved residue Cys 34 of
albumin, which is located in a 10- ˚ -wide hydrophobic pocket. The Cys 34 residue
has an unusually low pK a of 5 and thus exists as a thiolate anion at physiological
pH. The oxidation status of Cys 34 governs the local structure: buried for the reduced
thiolate form and exposed for the disulfide form [410]. These two structural forms
of albumin can be separated on a conventional [323] or HPLC anion exchange
columns [96] (Fig. 5.8 ). Albumin also carries S-linked GSH and CysGly (Fig. 7.1 )
[106, 408]. S-linked Hcy, Cys, GSH, and CysGly are also carried on globulin
(Fig. 7.1 ), HDL, and

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α 1 -acid glycoprotein, but not on transferrin (which does not
possess a cysteine residue with a free thiol) [106]. It is not known whether S-
thiolation occurs in the plasma or intracellularly during biogenesis of these proteins.
In vitro studies suggest that albumin-Cys 34 -S-S-Hcy can form in three
thiol-disulfide exchange reactions: (1) between Hcy and albumin-Cys 34 -S-S-Cys,
(2) between mercaptoalbumin (Alb-SH) and Hcy-S-S-Hcy, and (3) between Alb-
SH and Hcy-S-S-Cys [411, 412]. The physiological role of the redox transitions in
albumin is not understood.
In the presence of excess Hcy, S-linked Cys and CysGly are released from S-
thiolated albumin with a half-life of 30 min, and there is a corresponding increase in
S-Hcy-albumin. Under the same conditions, release of S-linked Cys and CysGly
from α 1 -acid glycoprotein is much slower with a half-life of 24 h. Half-maximal

increase in Hcy S-linked to α 1 -acid glycoprotein requires several hours [106].

Exchange reactions with excess GSH show more rapid displacement of Cys,
Hcy, and CysGly from albumin than from

α 1 -acid glycoprotein. However,
exchange reaction with GSH is faster: half-lives are 10 min for the displacement
of Cys and CysGly and 10 min for the displacement of Hcy from albumin and

about 3 h for thiol displacement from

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α 1 -acid glycoprotein [106]. These findings
suggest that GSH is more effective than Hcy in thiol-disulfide exchange reactions.