Homocysteine-Thiolactone - Homocysteine in Protein Structure/Function and Human Disease - page 21

Table 3.1 Physical-chemical properties of L -Hcy-thiolactone and L -Hcy [68]

N H 2

L -Hcy-thiolactone L -Hcy

N H 3 +

O

Property

COO -

HS

S

Chemical character Aminoacyl-thioester Mercapto amino acid

UV spectrum Yes, λ ¼ 240 nm, No significant absorption at

λ >

max

5,000 M 1 cm 1

ε ¼ 220 nm

Stability at
37 C, t 0.5
Phosphate-saline

~30 h ~2 h

Human serum ~1 h ~2 h

6.67 a 9.04, 9.71 b

9.02, 9.69 (thiol group) b

pK a of amino
group

Chemical reactivity Acylates amino groups of

protein lysine residues c

Condenses to form
Hcy-thiolactone

Reacts with aldehydes to
afford tetrahydrothiazines a
Resistant to oxidation
Base-hydrolyzed to Hcy

Reacts with aldehydes
to afford
tetrahydrothiazines a
Oxidized to disulfides
Reacts with nitric oxide
to afford S-nitroso-Hcy d

a [84]
b [463]
c [73, 78]
d [75-77]

Fig. 3.2 (continued) (Sigma-Aldrich) were incubated in 1-ml ampoules (Wheaton) at 128 C.
Reaction mixtures were lyophilized and dissolved in 0.15 mL of 0.15 M K 2 HPO 4 , and Hcy-
thiolactone was extracted with 5 volumes of a chloroform/methanol (2:1, v/v) mixture. Hcy-
thiolactone, recovered from the organic phase (0.6 mL) by re-extraction with 0.15 mL of 0.1 N
HCl, was lyophilized and dissolved in 80 μL of water. An 8-μl aliquot of each sample was buffered
with 3 μL of 0.5 M Na 2 HPO 4 and treated with bleomycin hydrolase (1 μL of 100 μM solution) for
30 min at 25 C. Bleomycin hydrolase-treated and bleomycin hydrolase-untreated samples were
diluted with 0.5 mL of water, and Hcy-thiolactone was quantified by measuring absorbance at
235 nm (filled circle). L -Hcy-thiolactone (open circle) and D -Hcy-thiolactone (multiplication sign)
stereoisomers were distinguished by their susceptibility and resistance, respectively, to enzymatic
hydrolysis by bleomycin hydrolase. (b-e) two-dimensional thin-layer chromatography analyses
of the L -[ 35 S]Met/hydriodic acid digestion mixtures. (b) L -[ 35 S]Met before digestion. (c) Crude
D , L -[ 35 S]Hcy-thiolactone obtained by digestion of L -[ 35 S]Met (4 h, 128 C). (d) Purified D , L -[ 35 S]
Hcy-thiolactone preparation. (e) Treatment with bleomycin hydrolase resolves D , L -[ 35 S]Hcy-
thiolactone to L -[ 35 S]Hcy and D -[ 35 S]Hcy-thiolactone. Autoradiograms of the two-dimensional
thin-layer chromatography plates (6.7 cm 5.0 cm) are shown. Origin (marked with multiplication

sign) is at the lower left corner, the first dimension is from bottom to top, and the second dimension
is from left to right (Reproduced from [188])