N-Homocysteinyl-Proteins - Homocysteine in Protein Structure/Function and Human Disease - page 92

and quantified by scintillation counting. The radiolabeling method has been used to
provide the first evidence that protein N-homocysteinylation occurs in cultured
human umbilical vein endothelial cells (Fig. 3.7 ) and normal human fibroblasts and
that the accumulation of N-Hcy-protein increases in CBS-deficient fibroblasts and
breast cancer cells [73, 74]. This method has also been instrumental in the demon-
stration that protein N-homocysteinylation is universal in mammalian cells and
occurs in another kingdom of life, the plants [190].
Subsequently, to facilitate studies of N-Hcy-protein in humans and experimental
animals, HPLC-based assays have been developed. Liquid chromatography/mass
spectrometry assays are now used for monitoring site-specific N-homocystei-
nylation. Immunoassays with rabbit polyclonal anti-N-Hcy-protein IgG antibodies
are also used for monitoring N-Hcy-protein. Methods are also being developed for
the detection of N-Hcy-protein by exploiting their selective reactions with
aldehydes.

5.5.1 High-Performance Liquid Chromatography Assay
with UV Detection

In the first HPLC assay developed for the determination of N-linked Hcy content in
proteins, N-Hcy-protein is subjected to acid hydrolysis under reducing conditions.
Under these conditions the liberated Hcy is converted to Hcy-thiolactone, which is
then quantified by cation exchange HPLC with UV detection [79].
The assay requires 200 L human plasma or 10 mg protein sample. Free and

disulfide-bound Hcy are removed by treatments with 5 mM dithiothreitol (DTT) in
phosphate-buffered saline and ultrafiltration on Millipore 10-kDa cutoff devices at
4 C (repeated six times). The completeness of the treatments is verified by assaying
Hcy in the last filtrate. Protein sample is then transferred from the ultrafiltration
device to a 1-mL glass ampoule, supplemented with DTT to 25 mM and HCl to
6 M, frozen at 80 C, sealed under vacuum, and hydrolyzed at 120 C for 1 h. The
hydrolysate is lyophilized, dissolved in 2 M ammonium bicarbonate, 1 M
dipotassium phosphate, supplemented with [ 35 S]Hcy-thiolactone (18,000 cpm,
40,000 Ci/mol) as a tracer to monitor recovery. Hcy-thiolactone is extracted with
chloroform/methanol (2:1, v/v) followed by re-extraction of the organic layer with
0.1 M HCl. The acid extracts are lyophilized, taken up in water, and further purified
by two-dimensional thin-layer chromatography on cellulose or silica gel plates
(6.7 cm

μ

5 cm). The first dimension solvent is butanol/acetic acid/water (4/1/1,
v/v), while isopropanol/ethyl acetate/water/ammonia (12/12/1/0.12, v/v) is used as
the second-dimension solvent. The [ 35 S]Hcy-thiolactone spot is visualized by
autoradiography using Kodak BioMax X-ray film, scraped off the plate, and Hcy-
thiolactone is eluted with cold 2 mM HCl (60

L, on ice). Final purification and
quantification is by cation exchange HPLC on a polysulfoethylaspartamide column
(PSEA, 2.1

μ

, 300 ˚ , from PolyLC, Inc.) using NaCl gradient from

200 mm, 5 μ