N-Homocysteinyl-Proteins - Homocysteine in Protein Structure/Function and Human Disease - page 97

treated with cysteamine HS-CH 2 CH 2 -NH 2 . This generates an adduct (N-
Hcy-S-S-CH 2 CH 2 -NH 2 )-ApoAI, in which cysteamine is bound via a disulfide
bond to the N-linked Hcy residue and introduces a free amino group into the N-
homocysteinylated protein. The additional amino group changes the protein's
isoelectric point, which allows separation of the more basic (N-
Hcy-S-S-CH 2 CH 2 -NH 2 )-ApoAI adduct from unmodified ApoAI by isoelectric
focusing on polyacrylamide gels. Separated modified and unmodified forms of
ApoAI are detected by Western blotting and staining with an antihuman ApoAI
polyclonal antibody (goat). The stained bands are quantified by digital scanning and
the ratio of N-Hcy-ApoAI/total ApoAI is calculated. The results are corrected for an
occasional nonspecific band by subtracting the ratio obtained without cysteamine
treatment from that with cysteamine treatment [303].
The reproducibility of this semiquantitative method is 19.1 %. In a population of
27 healthy subjects, the concentration of N-Hcy-ApoAI varies from 1.2 to 14.5 mg/
dL, whereas the ratio of N-Hcy-ApoAI/total ApoAI varies from 1 to 7.4 % (ApoAI
concentration varies from 110 to 200 mg/dL). N-Hcy-ApoAI was significantly
correlated with total ApoAI (r ¼

0.001). However, N-Hcy-ApoAI is
not correlated with tHcy, which varied in the normal range from 5 to15

0.673, p ¼

μ M.

M
N-Hcy-ApoAI in plasma. However, such high N-Hcy-ApoAI concentrations are
unrealistic because total N-Hcy-protein levels in plasma are known to be about
0.5

The N-Hcy-ApoA1 values of 1.2 to 14.5 mg/dL correspond to 0.57 μ Mto6.9 μ

μ M, most of which is due to N-Hcy-albumin [78, 115, 300, 358].

5.5.5 Detection of Protein -Homocysteinylation by Selective

Reactions with Aldehydes

N

Hcy and Hcy-thiolactone are known to react with aldehydes to yield 1,3-
tetrahydrothiazine-4-carboxylic acid derivatives (Table 3.1 ) (Reaction 3.5 ) [84].
These reactions are being explored for the detection of free Hcy in biological
samples [227, 359, 360] and N-Hcy-proteins [214].
The condensation reaction between an aldehyde tag and -amino

groups of N-linked Hcy residue in N-Hcy-protein yields 1,3-thiazine adduct
(Reaction 5.6 ). Under acidic conditions (pH

γ -thiol and α

3), the reaction of the aldehyde
with N-linked protein Hcy is irreversible, while a possible competing Schiff base
formation with the protein amino groups is reversible and favors the protonated free
amines (Reaction 5.6 ). For example, rhodamine aldehyde selectively reacts with N-
Hcy-myoglobin (containing 0.4 N-Hcy/mol protein) to form a fluorescent 1,3-
thiazine adduct [214]. Mass spectrometric analyses show that N-linked Hcy in
myoglobin is quantitatively converted to a corresponding 1,3-thiazine derivative
and that the reaction is completed in 3 h. Selectivity is demonstrated by
LC-ESI-MS, which shows no detectable formation of a Schiff base under acidic
(pH

¼

¼ 3) conditions. Quantification of the fluorescence associated with the band of