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a
b
Human serum
120
Human serum
120
100
100
N -Ac-N
α
-Hcy-Lys
N -Hcy-N
α
-Ac-Lys
N -Hcy-Lys
N -Hcy-LysAla
N
α
-Ac-Lys
LysAla
Hcy
N -Hcy-Lys-Alb
N -Hcy-Lys-Hb
N -Hcy-Lys-Alb/IAA
N -Hcy-Lys-Hb/IAA
Alb
Hb
80
80
60
60
40
40
20
20
0
0
-1
-0.5
0
0.5
1
0
1
2
3
4
Log [competitor, mM]
Log [competitor, µg/mL]
c
d
Rabbit serum
Rabbit serum
120
120
100
100
N -Ac-N
α
-Hcy-Lys
N -Hcy-N
α
-Ac-Lys
N -Hcy-Lys
N -Hcy-LysAla
N -Hcy-LysLeu
Val-(N -Hcy-Lys)
N -Hcy-tertaLys
80
80
N -Hcy-Lys-Alb
N -Hcy-Lys-Hb
N -Hcy-Lys-LDL
Hb
Alb
60
60
40
40
20
20
0
0
-1
-0.5
0
0.5
1
0
1
2
3
4
Log [comeptitor, mM]
Log [competitor µg/mL]
Fig. 6.2 Specificity of binding to N-Hcy-hemoglobin of a human anti-N-Hcy-protein autoanti-
body (a, b) and a rabbit polyclonal anti-N-Hcy-protein antibody (c, d). Microtiter wells are coated
with 10
gmL
−
1
human N-Hcy-hemoglobin and incubated with a 40-fold dilution of a human
serum (a, b) or 200-fold dilution of rabbit antiserum (c, d) with or without indicated competitor.
Rabbit antiserum is obtained from animals inoculated with N-Hcy-keyhole limpet hemocyanin.
Results are expressed as B/B
o
, where B is the amount of IgG bound in the presence of competitor
and B
o
that without competitor (Reprinted from [172])
μ
a
b
0.14
0.14
0.12
0.12
R
2
=0.25
0.10
0.1
0.08
0.08
R
2
=0.02
0.06
0.06
0.04
0.04
0.02
0.02
0
0.00
5
15
25
35
100
200
300
400
500
tHcy (µM)
Cysteine (µM)
Fig. 6.3 Relationships between serum anti-N-Hcy-protein IgG and tHcy (a) or cysteine (b)in
healthy human subjects (Reprinted from [172])
cysteine or methionine [172]. The Hcy-dependent variation in anti-N-Hcy-protein
autoantibody titers is consistent with the Hcy-thiolactone hypothesis (Fig.
6.1
):
elevation in Hcy leads to inadvertent elevation in Hcy-thiolactone, observed
ex vivo in human fibroblasts [73] and endothelial cells [64, 74], and in vivo in
humans and mice [64, 93-95]. Protein modification by Hcy-thiolactone generates
neo-self antigens, Nε
-Hcy-Lys-protein.
Increased accumulation of neo-self
Nε
-Hcy-Lys epitopes in proteins, observed ex vivo in cultured human cells treated
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