Quantitative assessment of the glyoxalase pathway in
Leishmania infantum as a therapeutic target by modelling
and computer simulation
Marta Sousa Silva
1
, Anto
´
nio E. N. Ferreira
1
, Ana Maria Toma
´
s
2,3
, Carlos Cordeiro
1
and Ana Ponces Freire
1
1 Centro de Quı
´
mica e Bioquı
´
mica, Departmento de Quı
´
mica e Bioquı
´
mica, Faculdade de Cie
ˆ
ncias da Universidade de Lisboa, Portugal
2 ICBAS – Instituto de Cie
ˆ
ncias Biome

e Bioquı
´
mica, Departmento de Quı
´
mica
e Bioquı
´
mica, Faculdade de Cie
ˆ
ncias
da Universidade de Lisboa, Edifı
´
cio C8,
Lisboa, Portugal
Fax: +351 217500088
Tel: +351 217500929
E-mail:
⁄ enzimol
Note
The mathematical model described here has
been submitted to the Online Cellular Sys-
tems Modelling Database and can be
accessed free of charge at chem.
sun.ac.za/database/silva/index.html
(Received 12 November 2004, revised 21
January 2005, accepted 28 February 2005)
doi:10.1111/j.1742-4658.2005.04632.x
The glyoxalase pathway of Leishmania infantum was kinetically character-
ized as a trypanothione-dependent system. Using time course analysis
based on parameter fitting with a genetic algorithm, kinetic parameters

glyoxal concentration.
Abbreviations
DHAP, dihydroxyacetone phosphate; GAP,
D-glyceraldehyde-3-phosphate; Glx I, glyoxalase I; Glx II, glyoxalase II; HTA, hemithioacetal;
MG, methylglyoxal; TFA, trifluoroacetic acid; T(SH)
2
, N
1
,N
8
-bis(glutathionyl)-spermidine; SDL-TSH, S-D-lactoyltrypanothione.
2388 FEBS Journal 272 (2005) 2388–2398 ª 2005 FEBS
conditions, these trioses readily undergo an irreversible
b-elimination reaction of the phosphate group from
their common 1,2-enediolate form, forming oxopro-
panal (methylglyoxal) [4]. Methylglyoxal is also formed
as a by-product of the triose phosphate isomerase cata-
lysed reaction [5] and in bacteria may be enzymatically
synthesized from dihydroxiacetone phosphate by meth-
ylglyoxal synthase (EC 4.1.99.11), an enzyme not
found in eukaryotic cells [6–8]. Once formed, methyl-
glyoxal irreversibly modifies amino groups in lipids,
nucleic acids and proteins, forming advanced glycation
end products [9]. It is therefore toxic, mutagenic and
an inhibitor of glycolytic enzymes [10]. The glutathi-
one-dependent glyoxalase pathway is the main detoxifi-
cation system for methylglyoxal [11]. It first reacts
nonenzymatically with glutathione, forming a hemithio-
acetal that is isomerized to the thiol ester S-d-lactoyl-
glutathione by glyoxalase I (Glx I; lactoylglutathione

Database and can be accessed at .
ac.za/database/silva/index.html free of charge.
Results and Discussion
The potential of the glyoxalase system as a possible
therapeutic target relies on its role as the main cata-
bolic pathway for methylglyoxal in eukaryotic cells. To
cause damage to Leishmania, or to any other trypano-
somatid, conditions must be sought that lead to an
increase of methylglyoxal concentration. A quantitative
analysis of the most critical parameters of the pathway
regarding this goal requires the knowledge of the intra-
cellular concentrations of all metabolites involved and
a kinetic model that accurately describes the glyoxalase
system in Leishmania.
Methylglyoxal was identified in Leishmania infantum
by HPLC and appears to be the only 2-oxoaldehyde
detected. This metabolite is present, in early stationary
phase cells, at a concentration of 9.67 pmol per 10
8
promastigotes. This low methylglyoxal concentration
suggests that its formation in L. infantum is nonenzy-
matic as observed in other cells [14,15]. To confirm this
hypothesis, methylglyoxal synthase activity was
assayed by measuring methylglyoxal formation from
dihydroxyacetone phosphate (DHAP). When compar-
ing the rates of methylglyoxal formation in the pres-
ence and in the absence of L. infantum extract, no
significant differences were found. DHAP forms
methylglyoxal at a rate of 0.17 lmÆmin
)1

trypanothione
-SH group
Hemithioacetal
Methylglyoxal
Dihydroxyacetone
phosphate
3-P-1,2-enediol
D-glyceraldehyde
-3-phosphate
O
3
POCH
2
O
H
OH
H
H
OH
O
3
POCH
2
OH
O
3
POCH
2
OH
O

3-phosphate (GAP) (Fig. 2) using the steady state
concentrations of these trioses as previously reported
[16]. Concerning the intracellular low molecular mass
thiols of L. infantum, at early stationary phase of
growth, HPLC analysis of monobromobimane deriva-
tives revealed the presence of GSH and T(SH)
2
at
retention times of 13.6 and 21.2 min, respectively
(Fig. 3B). T(SH)
2
was present at a concentration of
3.04 nmol per 10
8
promastigotes, while GSH concen-
tration was 0.50 nmol per 10
8
promastigotes, a much
lower value. Unidentified thiols (U marked peaks)
were also shown to be present in this parasite, at
retention times of 14.5 and 23.3 min (Fig. 3B). GSH
is present at a molar ratio of 1 : 6 relative to trypan-
othione, making T(SH)
2
a good candidate for repla-
cing GSH in the glyoxalase pathway in L. infantum,
as occurs in other enzymatic systems in trypanosom-
atids. Substrate dependence of the glyoxalase enzymes
was then evaluated in this parasite by initial rate ana-
lysis.

of 0.24 ± 0.04 mm and a
V of 0.19 ± 0.02 lmolÆmin
)1
Æmg
)1
using methyl-
glyoxal trypanothione hemithioacetal (Table 1). For
Fig. 2. The glyoxalase pathway in Leishmania infantum. Reactions
1 and 2 correspond to the nonenzymatic (n.e.) formation of MG
from dihydroxyacetone phosphate (DHAP) and
D-glyceraldehyde-3-
phosphate (GAP). Reactions 3 and 4 correspond to the reversible
reaction between MG and reduced trypanothione [T(SH)
2
]. Reac-
tions 5 and 6 are catalysed by Glx I and Glx II, respectively. Num-
bered reactions are described in Table 3.
A
B
Fig. 3. HPLC analysis of the glyoxalase pathway metabolites in
Leishmania infantum promastigotes. (A) Analysis of 2-oxoalde-
hydes, showing the presence of MG as 2-methylquinoxaline and
the internal standard (IS, 1 l
M 2,3-dimethylquinoxaline). Other
peaks are due to the reagent. (B) Thiol analysis, as monobromobi-
mane derivatives. Glutathione (GSH) and trypanothione (T(SH)
2
)
were identified. Peaks marked R are due to the derivatizing reagent
monobromobimane, while U marked peaks are unidentified thiols.

Michaelian enzymes [11,20]. When fitting a single-
enzyme model for glyoxalase I (single substrate
irreversible Michaelis–Menten) to time courses for
lactoyltrypanothione concentration, only a poor fit was
possible (Fig. 4A,A¢). Other rate laws were investigated
as possible alternatives and again no better fitting was
achieved (data not shown). As we could detect the
activity of both enzymes with trypanothione derived
substrates we next fitted a two-enzyme kinetic model
(single substrate irreversible Michaelis-Menten). In this
case an excellent fit was achieved (Fig. 4B,B¢) and the
kinetic parameters for both enzymes were determined
(Tables 1 and 2). This fit was obtained using only
two progress curves corresponding to 0.14 mm and
0.27 mm hemithioacetal. The analysis was also per-
formed with more than two curves and identical results
were obtained. For Glx I we determined an apparent
K
m
of 0.253 mm and an apparent V of 0.21 lmolÆ
min
)1
Æmg
)1
(Table 1) while for Glx II a K
m
of
0.098 mm and a V of 0.18 lmolÆmin
)1
Æmg

V
(lmolÆmin
)1
Æmg
)1
)
K
m
(mM)
V
(lmolÆmin
)1
Æmg
)1
)
Leishmania infantum GSH 1.85 ± 0.35 0.19 ± 0.02 – –
T(SH)
2
0.24 ± 0.04 0.19 ± 0.02 0.253 0.21
Plasmodium falciparum [19] GSH 0.77 ± 0.15 NC
a
––
Leishmania major [18] T(SH)
2
0.32 ± 0.03 NC
a
––
Saccharomyces cerevisiae [20] GSH 0.51 ± 0.06 NC
b
0.62 ± 0.18 NC

––
S. cerevisiae [20] SDL-GSH 0.32 ± 0.13 NC
b
0.09 ± 0.05 NC
b
a
NC, not comparable (data from recombinant enzyme).
b
NC, not comparable (data from permeabilized cells).
M. Sousa Silva et al. The glyoxalase pathway in Leishmania infantum
FEBS Journal 272 (2005) 2388–2398 ª 2005 FEBS 2391
possible post-translational modifications preserved, we
achieved a characterization of the glyoxalase system
sufficient to elaborate a minimal model of its global
kinetic behaviour (Fig. 2). A reference steady state was
defined by the experimentally determined enzyme
activities using time course analysis and the measured
intracellular trypanothione concentration. The rate of
methylglyoxal formation was calculated using the pre-
viously determined triose phosphate concentrations
[16] and rate constants [21].
When simulating the effects of changing glyoxalase
I or glyoxalase II activities on methylglyoxal steady-
state concentration, surprising results were obtained
(Fig. 5A,B). To increase methylglyoxal concentration
by about 50%, glyoxalase I activity must be
decreased to 10% of its reference value (Fig. 5A).
Varying glyoxalase II activity causes no noticeable
change on the concentration of methylglyoxal within
the tested range of variation (Fig. 5B). By contrast,

enzyme model (blue line, A), fitting a two-
enzyme model (red line, B) and fitting a
single-enzyme model with competitive prod-
uct inhibition (yellow line, C). The best fit for
each model was obtained by least squares
minimization using two time courses and a
genetic algorithm to search the parameter
space. Numerical solvers of ODE initial
value problems and the genetic algorithms
were implemented in the software package
AGEDO. For each model, plots of residuals
are shown in A¢,B¢ and C¢.
The glyoxalase pathway in Leishmania infantum M. Sousa Silva et al.
2392 FEBS Journal 272 (2005) 2388–2398 ª 2005 FEBS
possible for extreme modulations of enzyme activities.
In particular, in the combinations involving the
decrease of glyoxalase II activity the effect is equival-
ent to the modulation of the other parameters alone,
as shown in the combination involving glyoxalase I
and glyoxalase II (Fig. 6C).
The simulation results, based on experimentally
determined parameters and a kinetic model of the
AB
DC
Fig. 5. Sensitivity analysis of the glyoxalase
pathway in Leishmania infantum. The effects
of system parameters on the intracellular
steady-state concentration of methylglyoxal
were investigated by finite parameter chan-
ges (between 0.05- and three-fold) around

20
40
60
80
0.2
0.4
0.6
0.8
1.0
0.2
0.4
0.6
0.8
1.0
MG
GLX I
initial SH
0
20
40
60
80
0
20
40
60
80
0.2
0.4
0.6

40
60
80
AB
C
D
0.2
0.4
0.6
0.8
1.0
Fig. 6. Sensitivity analysis of the glyoxalase
pathway in Leishmania infantum, studying
the effects of two simultaneous system
parameters on the intracellular steady-state
concentration of methylglyoxal, by finite
parameter changes (between 0.05- and
onefold, except for MG input that was
between one- and 3.5-fold) around the
reference steady state. All values are fold
variations relative to the reference state
(normalized values). System parameters
were: initial trypanothione concentration and
methylglyoxal input (A), initial trypanothione
concentration and glyoxalase I activity (B),
glyoxalase II activity and glyoxalase I activity
(C), methylglyoxal input and glyoxalase I
activity (D).
M. Sousa Silva et al. The glyoxalase pathway in Leishmania infantum
FEBS Journal 272 (2005) 2388–2398 ª 2005 FEBS 2393

Again, in the work with yeast referred to above, the
most sensitive strain to methylglyoxal is the one lack-
ing glutathione synthase I, DGSH1, with a lower intra-
cellular GSH concentration [21]. In Trypanosoma
brucei, trypanothione depletion results in growth arrest
and increased sensitivity to oxidative stress [25]. Inhibi-
tion of trypanothione biosynthesis most likely impairs
several pathways vital to the survival of the parasite.
Moreover, resistance to carbonylic stress caused by
methylglyoxal will be compromised. From a practical
point of view, trypanothione depletion might be
achieved by inhibiting trypanothione synthetase the
enzyme that in T. brucei, T. cruzi and L. major was
shown to catalyse the formation of that thiol from
spermidine and glutathione [26–28]. This enzyme,
essential to T. brucei [29] and very likely to the other
trypanosomatids, is considered one of the most prom-
ising targets for chemotherapy.
In summary, research efforts in search for more
effective drugs against trypanosomatids have revealed
important aspects of these parasites’ biochemistry.
Effective therapies must rely on unique aspects such as
glycolysis compartimentation and thiol metabolism.
Trypanothione is essential for cell viability and plays a
major role in the defence against oxidative stress
caused by hydrogen peroxide and organic hydroper-
oxides. It is also the physiological substrate of the gly-
oxalase pathway, the main detoxification system for
methylglyoxal and other 2-oxoaldehydes, arising from
nonenzymatic reactions.

analytical grade and all solvents were of HPLC grade.
A Beckman DUÒ (Fullerton, CA, USA) 7400 diode array
spectrophotometer with a thermostated multicuvette holder,
with stirring, was used for the determination of protein con-
centration and to monitor enzyme activity. Centrifugations
were performed in a refrigerated Eppendorf (Hamburg,
Germany) 5804R centrifuge. Thiol determinations and
methylglyoxal (MG) quantifications were performed in a
Beckman Coulter HPLC coupled with a Jasco FP-2020 Plus
(Tokyo, Japan) fluorescence detector. In these assays, a
Merck LichroCART (Darmstadt, Germany) 250–4
(250 · 4 mm) column with stationary phase Merck LiChro-
spher
Ò
(Darmstadt, Germany) 100 RP-18 (5 lm) was used.
Preparation of metabolites
High-purity MG was prepared by acid hydrolysis of meth-
ylglyoxal dimethylacetal, in 10% (v ⁄ v) H
2
SO
4
, and purified
The glyoxalase pathway in Leishmania infantum M. Sousa Silva et al.
2394 FEBS Journal 272 (2005) 2388–2398 ª 2005 FEBS
by fractional distillation under reduced pressure in nitrogen
atmosphere [30]. The solution obtained was calibrated with
yeast Glx I and bovine liver Glx II.
Oxidized glutathione (GSSG) and oxidized trypanothione
(TS
2

and 35 l g ÆL
)1
streptomycin, at 25 °C [32].
Preparation of Leishmania infantum extracts
Promastigotes of L. infantum at early stationary phase of
growth (about 150 mL, containing approximately 10
9
cells)
were washed twice in NaCl ⁄ P
i
, and suspended in 1 mL
NaCl ⁄ P
i
. To prepare the protein extracts for enzyme assays,
cells were submitted to eight freeze–thaw cycles (on ice and
50 °C) and the supernatant was recovered after centrifuga-
tion at 10 500 g for 10 min. Protein concentration was quan-
tified according to Bradford using BSA as the standard [33].
For thiol identification and MG quantification, cells were
lysed and deproteinized with 0.5 m perchloric acid. The sus-
pension was kept on ice for 10 min, vortexed for 2 min and
centrifuged at 4 °C, 10 500 g, for 5 min. The recovered
supernatant was immediately analysed or stored at )80 °C
[14].
Thiol assay
Intracellular thiols were derivatized with the fluorescent
label monobromobimane and analysed by HPLC. The deri-
vatization procedure was based on the methods described
by Tang et al. [34] and by Ondarza et al. [35], with some
modifications. A 100 lL aliquot of the L. infantum extract

blocked with 5 mm N-ethylmaleimide for 20 min at 60 °C
before derivatization.
Methylglyoxal assay
Intracellular methylglyoxal was measured in L. infantum
(100 lL extract) with a specific HPLC-based assay, by deri-
vatization with 1,2-diaminobenzene and using 2,3-dimethyl-
quinoxaline as internal standard [36].
Methylglyoxal synthase activity was assayed by measuring
methylglyoxal formation from DHAP. The reaction
occurred in 1 mL reaction volume, in 0.1 m potassium phos-
phate buffer, pH 6.8, at 30 °C. DHAP was added to a 50-
and a 100-lL aliquot of the L. infantum extract, to a final
concentration of 1 mm. The reaction was stopped with the
addition of perchloric acid to 0.5 m final concentration.
Controls were performed without L. infantum extract and
the rates of methylglyoxal formation compared. Methylgly-
oxal was measured in all samples, at time zero and after
2.5 h of incubation, with the HPLC assay referred to above.
Enzyme kinetic assays
Enzyme activities were determined at 30 °C in a 2 mL reac-
tion volume, in 0.1 m potassium phosphate buffer, pH 6.8.
Magnetic stirring in the spectrophotometer cuvette was
used to maintain isotropic conditions.
The Glx I activity assay was based on the method des-
cribed by Martins et al. [20] with some modifications. Glx I
activity was assayed with GSH, with dithiothreitol reduced
GSSG, and with reduced trypanothione [T(SH)
2
], using
MG in excess (3.34 mm). Initial concentrations of GSH and

2
and MG using yeast glyoxalase I, as previ-
ously described. Concentrations of SDL-GSH between 0.5
and 4 mm were used and SDL-TSH concentrations between
0.05 and 0.10 mm were prepared. The reactions occurred in
the same conditions, and were started with the addition of
protein extract (15 lg of total protein). The hydrolysis of
both thiolesthers was followed at 240 nm. Glyoxalase II
activity with SDL-GSH was also assayed by following
GSH formation at 412 nm with 5,5¢-dithiobis(2-nitro-
benzoic acid) [20].
Determination of kinetic parameters
The kinetic parameters for glyoxalase I and II were deter-
mined using two different approaches, initial rate analysis
and time course analysis.
Initial rate data were fitted to irreversible single substrate
Michaelis–Menten models. Non-weighted hyperbolic regres-
sion by the method of least squares was performed with the
program HYPER (J. S. Easterby, University of Liverpool,
UK; />In time course analysis the parameters were determined
by minimization of the difference between experimental
time course data and the corresponding values predicted by
the solution of the differential equations derived from a
mathematical model of the kinetic assay. In this analysis,
different models were tested. In ‘single-enzyme model’, only
the reaction of glyoxalase I with an irreversible Michaelis–
Menten rate law was considered (Scheme 1).
HTA
SDL-TSH
[]

TSH-SDL
TSH-SDL
2
2
2
+
=
m
K
V
v
HTA
SDL-TSH
In ‘single-enzyme model with product inhibition’, only
the reaction of glyoxalase I was considered, with an irre-
versible Michaelis–Menten rate law with competitive prod-
uct inhibition (Scheme 3).
HTA
SDL-TSH
[]
[]
[]
HTA
TSH-SDL
1
HTA
1
1
1
1

i¼1
X
OBS
k
t
i
ðÞÀX
SIM
k
t
i
hðÞ
ÀÁ
2
Eqn ð1Þ
In this equation, p is the number of time courses used in
the analysis, n
k
is the number of points in time course k,
X
OBS
k
t
i
ðÞis the experimental value of the SDL-TSH for time
Table 3. Rate equations and kinetic parameters of the glyoxalase
pathway model. Rate equations are shown in Fig. 2. Kinetic models
for the two enzymes were experimentally validated by time course
analysis. Intracellular concentrations of methylglyoxal and trypano-
thione were calculated using an estimate of the L. infantum cell

4
+ v
6
Rate equations
v
1
¼ k
1
GAP
v
2
¼ k
2
DHAP
v
3
¼ k
3
MG T(SH)
2
v
4
¼ k
4
HTA
v
5
¼ V
5
HTA ⁄ (K

¼ 1.01 min
)1
V
5
¼ 2 · 3.042 mMÆmin
)1
V
6
¼ 2 · 2.653 mMÆmin
)1
K
m5
¼ 2 · 0.253 mM
K
m6
¼ 2 · 0.0980 mM
GAP ¼ 0.0072 mM
DHAP ¼ 0.16 mM
T(SH)
2
(at time zero) ¼ 2 x 0.45 mM
The glyoxalase pathway in Leishmania infantum M. Sousa Silva et al.
2396 FEBS Journal 272 (2005) 2388–2398 ª 2005 FEBS
course k at time t
i
, and X
SIM
k
t
i

from the Fundac¸ a
˜
o para a Cieˆ ncia e a Tecnologia,
Ministe
´
rio da Cieˆ ncia e Tecnologia, Portugal.
References
1 Muller S, Liebau E, Walter RD & Krauth-Siegel RL
(2003) Thiol-based redox metabolism of protozoan
parasites. Trends Parasitol 19, 320–328.
2 Hannaert V, Saavedra E, Duffieux F, Szikora JP, Rig-
den DJ, Michels PA & Opperdoes FR (2003) Plant-like
traits associated with metabolism of Trypanosoma para-
sites. Proc Natl Acad Sci USA 100, 1067–1071.
3 Lohman K & Meyerhof O (1934) U
¨
ber die enzyma-
tische umwandlung von phosphoglyzerinsa
¨
ure in brenz-
traubensa
¨
ure und phosphorsa
¨
ure (Enzymatic
transformation of phosphoglyceric acid into pyruvic and
phosphoric acid). Biochem Z 273, 60–72.
4 Richard JP (1984) Acid-base catalysis of the elimination
and isomerization reactions of triose phosphates. JAm
Chem Soc 106, 4926–4936.

J Biol Chem 279, 22209–22217.
14 Martins AM, Cordeiro CA & Ponces Freire AM (2001)
In situ analysis of methylglyoxal metabolism in Saccharo-
myces cerevisiae. FEBS Lett 499, 41–44.
15 Mclellan AC, Phillips SA & Thornalley PJ (1992) The
assay of methylglyoxal in biological-systems by derivati-
zation with 1,2-diamino-4,5-dimethoxybenzene. Anal
Biochem 206, 17–23.
16 Bakker BM, Michels PAM, Opperdoes FR &
Westerhoff HV (1997) Glycolysis in bloodstream form
Trypanosoma brucei can be understood in terms of the
kinetics of the glycolytic enzymes. J Biol Chem 272,
3207–3215.
17 Martins AM, Cordeiro C & Freire AP (1999) Glyoxa-
lase II in Saccharomyces cerevisiae: in situ kinetics using
the 5,5¢-dithiobis (2-nitrobenzoic acid) assay. Arch Bio-
chem Biophys 366, 15–20.
18 Vickers TJ, Greig N & Fairlamb AH (2004) A trypa-
nothione-dependent glyoxalase I with a prokaryotic
ancestry in Leishmania major. Proc Natl Acad Sci USA
101, 13186–13191.
19 Iozef R, Rahlfs S, Chang T, Schirmer H & Becker K
(2003) Glyoxalase I of the malarial parasite Plasmodium
falciparum: evidence for subunit fusion. FEBS Lett 554,
284–288.
20 Martins AM, Mendes P, Cordeiro C & Freire AP
(2001) In situ kinetic analysis of glyoxalase I and
glyoxalase II in Saccharomyces cerevisiae. Eur J Bio-
chem 268, 3930–3936.
M. Sousa Silva et al. The glyoxalase pathway in Leishmania infantum

tion of trypanothione from glutathione and spermidine
in Trypanosoma cruzi. J Biol Chem 277, 35853–35861.
29 Oza SL, Ariyanayagam MR, Aitcheson N & Fairlamb
AH (2003) Properties of trypanothione synthetase from
Trypanosoma brucei. Mol Biochem Parasitol 131, 25–33.
30 Mclellan AC & Thornalley PJ (1992) Synthesis and
chromatography of 1,2-diamino-4,5-dimethoxybenzene,
6,7-dimethoxy-2-methylquinoxaline and 6,7-dimethoxy-
2,3-dimethylquinoxaline for use in a liquid-chromato-
graphic fluorometric assay of methylglyoxal. Anal Chim
Acta 263, 137–142.
31 Vander Jagt DL, Han LP & Lehman CH (1972) Kinetic
evaluation of substrate specificity in the glyoxalase-I-
catalyzed disproportionation of ketoaldehydes. Biochem-
istry 11, 3735–3740.
32 Castro H, Sousa C, Novais M, Santos M, Budde H,
Cordeiro-da-Silva A, Flohe
´
L & Tomas A (2004) Two
linked genes of Leishmania infantum encode tryparedox-
ins localised to cytosol and mitochondrion. Mol Bio-
chem Parasit 136, 137–147.
33 Bradford MM (1976) Rapid and sensitive method for
quantitation of microgram quantities of protein utilizing
principle of protein-dye binding. Anal Biochem 72, 248–
254.
34 Tang D, Shafer MM, Vang K, Karner DA & Arm-
strong DE (2003) Determination of dissolved thiols
using solid-phase extraction and liquid chromatographic
determination of fluorescently derivatized thiolic com-