N-Homocysteinyl-Proteins - Homocysteine in Protein Structure/Function and Human Disease - page 93

50 to 160 mM in 10 mM sodium monophosphate for 6-min and 3-min re-equilibra-
tion time and a flow rate of 0.5 mL/min. The effluent is monitored in UV at 240 nm,
λ max for Hcy-thiolactone [68].
Using this assay, N-linked Hcy has been discovered in individual human blood
proteins, with highest amounts found in albumin and hemoglobin. Lower amounts
of protein N-linked Hcy are found in human fibrinogen, transferrin, antitrypsin,
IgG, LDL, and HDL. N-linked Hcy is also present in albumins from other species,
including sheep, pig, rabbit, chicken, rat, and mouse. This study also demonstrated
for the first time that protein N-linked Hcy is a significant component of human
plasma. The plasma concentrations of N-Hcy-protein vary between different
individuals from 0.5 to 13 M, which represent from 0.3 to 23 % of plasma tHcy.

Plasma N-Hcy-protein is positively correlated with plasma tHcy [79].

μ

5.5.2 High-Performance Liquid Chromatography Assays
with Fluorescence Detection

5.5.2.1 Derivatization with OPA

The original protein N-linked Hcy assay has been modified to increase the sensitiv-
ity and allow analyses of much smaller samples [297]. The new assay requires only
5 L plasma or 0.1 mg purified protein and uses post-column derivatization with

alkaline OPA followed by fluorescence detection.
After removal of free and disulfide-bound Hcy by treatments with DTT and
ultrafiltration on Millipore 10-kDa cutoff devices, protein is hydrolyzed with 6 M
HCl, 25 mM DTT. The hydrolysates are lyophilized, dissolved in 10

μ

L water
containing 20,000 cpm [ 35 S]Hcy-thiolactone (40,000 Ci/mol) as a tracer to monitor
recovery, and the liberated Hcy-thiolactone is purified by two-dimensional TLC on
cellulose or silica gel plates (5 cm

μ

4 cm). The [ 35 S]Hcy-thiolactone spot is
localized by autoradiography and scraped off the plate, and Hcy-thiolactone is
recovered by elution with 60 L cold 2 mM HCl.

The recovered Hcy-thiolactone is quantified by HPLC on a cation exchange
PSEA column (2 mm

μ

35 mm) eluted isocratically with 10 mM sodium phos-
phate, pH 6.6, and 20 mM NaCl at a flow rate of 0.36 mL/min for 5 min. The
effluent is mixed in a three-way tee with 2.5 mM OPA and 0.25 MNaOH, delivered
at a flow rate of 0.18 mL/min. The mixture passes through Teflon tubing reaction
coil (0.3 mm i.d. 3 m) and is monitored with a Jasco 1,520 fluorescence detector

using excitation at 370 nm and emission at 480 nm.
The assay is calibrated with in vitro-prepared N-Hcy-albumin standard
containing 0.027 mol N-linked Hcy/mol protein (Fig. 5.14 ). The sensitivity of this
assay allows detection of as little as 0.00006 mol Hcy/mol protein. With this assay,
N-linked Hcy in individual proteins was found to vary from as high as
0.470-0.515 mol/mol protein for human and equine ferritins to as low as
0.00006 mol/mol protein for chicken lysozyme. An example of HPLC