N-Homocysteinyl-Proteins - Homocysteine in Protein Structure/Function and Human Disease - page 77

a b c

Alb-Cys-S-S-Cys

250

30

N

-(Hcy-S-S-Cys)-Alb-Cys-SH

Alb-Cys-S-S-Cys

20

200

N -(Hcy-S-S-Cys)-Alb-Cys-SH

10

150

Alb-Cys-S-S-Cys

N- (Hcy-S-S-Cys) -

Alb-Cys-S-S-Cys

0

10

100

N- (Hcy-S-S-Cys)-

N -([ 35 S]Hcy-S-S-Cys)-Alb-Cys-SH

3,000

A lb-Cys-SH

N -(Hcy-S-S-Cys)- Alb-Cys-SH

50

2,000

5

1,000

0

0

0

12 13 14

12 13 14 15 11 12 13 14 15

Minutes

Minutes Minutes

Fig. 5.8 Anion exchange HPLC analysis of Hcy-thiolactone-modified albumin-Cys 34 -S-S-Cys.
Albumin-Cys 34 -S-S-Cys was modified with Hcy-thiolactone or [ 35 S]Hcy-thiolactone at 37 C and
analyzed by anion exchange HPLC. Panel (a), protein profiles after 0-h (top trace), 4-h (middle
trace), and 22-h (bottom trace) modification with Hcy-thiolactone. Panel (b) shows protein profiles
of the 10-h reaction with Hcy-thiolactone after an overnight incubation without (dotted line) and
with a twofold molar excess of cysteine (solid line). Panel (c), protein (upper panel) and 35 S(lower
panel) profiles after 4 h of modification with [ 35 S]Hcy-thiolactone (Reproduced from [96])

Hcy-thiolactone + albumin-Cys 34 -S-S-Cys

→

→ N -(Hcy-SH)-albumin-Cys 34 -S-S-Cys ↔ N -(Hcy-S-S-Cys)-albumin-Cys 34 -SH

Reaction 5.3 Mechanism of N-homocysteinylation of albumin-Cys 34 -S-S-Cys

→ N -(Hcy-SH)-albumin-Cys 34 -SH

(a) Hcy-thiolactone + albumin-Cys 34 - -SH

N -Hcy-SH-albumin-Cys 34 -SH + albumin-Cys 34 -S-S-Cys

↔

↔ N -(Hcy-S-S-Cys)-albumin-Cys 34 -SH + albumin-Cys 34 -SH

(b)

Reaction 5.4 Mechanism of N-homocysteinylation of mercaptoalbumin (albumin-Cys 34 -SH)

The formation of a mixed disulfide bond between Cys and N-linked Hcy induces
substantial structural changes that lead to increased sensitivity of N-(Hcy-S-S-Cys)-
albumin-Cys 34 -SH, compared with N-(Hcy-SH)-albumin-Cys 34 -SH, to proteolysis
by trypsin or chymotrypsin [96].
Other plasma N-Hcy-proteins also undergo facile thiol-disulfide exchange with
albumin-Cys 34 -S-S-Cys. For example, when equimolar amounts of N-Hcy-trans-
ferrin and albumin-Cys 34 -S-S-Cys are incubated together, albumin-Cys 34 -S-S-Cys
is quantitatively converted to albumin-Cys 34 -SH (Reaction 5.5 ). Unmodified trans-
ferrin does not induce this reaction. N-homocysteinylated fibrinogen, antitrypsin,
hemoglobin, myoglobin, and cytochrome c, but not unmodified native proteins, also
removed cysteine from the albumin-Cys 34 -S-S-Cys disulfide with the liberation of
albumin-Cys 34 -SH. The equilibrium of those reactions is strongly shifted toward
N-(Hcy-S-S-Cys)-protein and albumin-Cys 34 -SH [96].