Homocysteine-Thiolactone - Homocysteine in Protein Structure/Function and Human Disease - page 28

3.3 Quantification Methods

3.3.1 Assays Based on Radiolabeling

The discovery that Hcy-thiolactone is formed biologically in living cells relied on
the use of radiolabeled precursors such as [ 35 S]sulfate [63], [ 35 S]cysteine [197],
[ 35 S]methionine [73, 138], and [ 35 S]homocysteine [73, 74] as tracers. After
incubating cultured cells with any of those precursors, the labeled media and cells
are collected separately. The labeled cells are extracted with formic acid. [ 35 S]Hcy-
thiolactone is separated from other [ 35 S]-labeled metabolites present in cell extracts
and culture media by two-dimensional thin-layer chromatography (TLC) using as
little as 5

4 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 the second-dimension solvent.
[ 35 S]Hcy-thiolactone is visualized by autoradiography using Kodak BioMax X-ray
film and quantified by scintillation counting. An illustration of the two-dimensional
TLC separation of the radiolabeled Hcy-thiolactone is shown in Fig. 3.1 . The
radiolabeling methods have an excellent reproducibility and sensitivity (allowing
detection of

μ L sample and cellulose or silica gel plates as small as 5 cm

100 femtomoles of Hcy-thiolactone) and have been used extensively
to elucidate Hcy-thiolactone metabolic pathways in microorganisms such as
Escherichia coli [193, 197] and the yeast Saccharomyces cerevisiae [63, 221],
plants [190], as well as in a variety of cultured human and rodent cells [73, 74, 138].

<

3.3.2 Assays Based on Direct UV Monitoring

Similar to other thioesters [189], Hcy-thiolactone absorbs ultraviolet light with the
maximum at 240 nm and 5,000 L mol 1 cm 1 in water [68]. Spectral monitor-

ing at 240 nm has been used for direct quantification of Hcy-thiolactone in bacterial
cultures [193, 194]. Because of its limited sensitivity (the limit of detection is
0.1 mM), the use of this method is limited to experimental systems in which high
concentrations of Hcy-thiolactone are generated.

ε ¼

3.3.3 High-Performance Liquid Chromatography-Based
Assays

Nonradioactive HPLC-based assays have subsequently been developed and are
available for Hcy-thiolactone quantification in biological samples, including
plasma, urine, and tissues. The assays are based on HPLC coupled with UV
absorbance or fluorescence detection.