Discoveries of Protein S- and N-Homocysteinylation - Homocysteine in Protein Structure/Function and Human Disease - page 54

Fig. 4.1 Reactions of Hcy-thiolactone and Hcy in human serum. (a) Kinetics of protein
N-homocysteinylation (filled circles) and protein S-homocysteinylation (filled squares) in the
presence of 12

M[ 35 S]Hcy-thiolactone. (b) Kinetics of protein S-homocysteinylation (filled

μ

M[ 35 S]Hcy. At the point indicated by an arrow, 10 mm DTT was added
(Reproduced from [81])

squares) with 12

μ

increasing Hcy concentration (Fig. 3.7 ) and decreases with increasing levels of
folic acid (which lowers Hcy levels) and HDL (which hydrolyzes Hcy-thiolactone)
(Table 3.9 ) [74].
Subsequent studies have revealed that the incubation of human serum with
[ 35 S]Hcy-thiolactone results in a progressive incorporation of the [ 35 S] radiolabel
into protein (Fig. 4.1a ). At 3 h, most of the [ 35 S] becomes protein bound and is
precipitable by trichloroacetic acid. Treatment with dithiothreitol (DTT) of the
[ 35 S]Hcy-thiolactone-modified serum protein releases only

30 % of the
incorporated [ 35 S] as free [ 35 S]Hcy, which suggests two modes of Hcy binding
to protein [78, 81]. [ 35 S]Hcy in the DTT-resistant fraction of the [ 35 S]-protein
adducts is bound to a side chain amino groups of protein lysine residues [78, 96].
Similar fractions of N-[ 35 S]Hcy-protein and S-[ 35 S]Hcy-protein adducts are
obtained with [ 35 S]Hcy-thiolactone concentrations ranging from 10 nM to 1 mM
[78].
Control experiments, with separately prepared S-[ 35 S]Hcy-protein, confirm
that DTT treatment releases all disulfide-bound Hcy from the protein.
For example, incubation of exogenous [ 35 S]Hcy with human serum results in a
progressive formation of S-[ 35 S]Hcy-protein adducts that are precipitable with
trichloroacetic acid. The treatment with DTT renders essentially all [ 35 S]Hcy
from the S-[ 35 S]Hcy-protein adducts trichloroacetic acid soluble (Fig. 4.1b ).
Polyacrylamide gel electrophoresis under nonreducing conditions demonstrates
that S-[ 35 S]Hcy-albumin represents most (

95 %) of S-[ 35 S]Hcy-protein in

>

human serum [81].