N-Homocysteinyl-Proteins - Homocysteine in Protein Structure/Function and Human Disease - page 76

SH

ALBUMIN

Albumin-Cys 34 -SH

S-S-Cys

ALBUMIN

Albumin-Cys 34 -S-S-Cys

S-S-Hcy

ALBUMIN

Albumin-Cys 34 -S-S-Hcy

NH 2

H
N

SH

ALBUMIN

HS C

O

N -(Hcy-SH)-Albumin-Cys 34 -SH

NH 2

H
N SH

ALBUMIN

Cys-S-S C

O

N -(Hcy-S-S-Cys)-Albumin-Cys 34 -SH

NH 2

H
N

S-S-Cys

ALBUMIN

Cys-S-S C

O

N -(Hcy-S-S-Cys)-Albumin-Cys 34 -S-S-Cys

Fig. 5.7 Structures of the different forms of human serum albumin. Reproduced from [96]

albumin is chromatographically separated from unmodified albumin by anion
exchange HPLC (Fig. 5.8 ). The different susceptibilities of albumin-Cys 34 -
S-S-Cys and albumin-Cys 34 -SH to the modification by Hcy-thiolactone are
consistent with a structural transition in albumin dependent on the status of the
Cys 34 residue [324].
The reactions of albumin-Cys 34 -S-S-Cys and albumin-Cys 34 -SH with
Hcy-thiolactone yield two different primary products, N-(Hcy-SH)-albumin-
Cys 34 -S-S-Cys (Reaction 5.3 ) and N-(Hcy-SH)-albumin-Cys 34 -SH (Reaction 5.4 ),
respectively (Fig. 5.9 ). However, these primary products are not observed due to
fast thiol-disulfide exchange reactions that result in the formation of a single
product, N-(Hcy-S-S-Cys)-albumin-Cys 34 -SH (Fig. 5.9 ), which is observed on an
anion exchange column (Fig. 5.8 ).
The thiol-disulfide exchange reactions occur in trans between different
molecules of N-(Hcy-SH)-albumin-Cys 34 -S-S-Cys or between N-(Hcy-SH)-
albumin-Cys 34 -SH and albumin-Cys 34 -S-S-Cys. The equilibrium is strongly shifted
toward N-(Hcy-S-S-Cys)-albumin-Cys 34 -SH because the Cys-34 thiolate anion has
unusually low pK a of ~5 [323] and thus is more thermodynamically stable that Hcy
thiolate anion (pK a of ~10) [191, 192]. The low pK a of the Cys-34 thiolate also
makes the thiol-disulfide exchange of N-(Hcy-SH)-albumin-Cys 34 -SH with
albumin-Cys 34 -S-S-Cys thermodynamically more favored than with cystine.