D. Tarasek, et al.
BioorganicChemistry97(2020)103692
OH
OH
2.2. Spectrophotometric analysis of the oxidation of 4-aminoantipyrine/
phenolic substrates by HRP in the presence of p-diphenols
O
HRP was dissolved in 50 mM sodium phosphate buffer, pH 7.0 at
1 mg/ml initial concentration, then diluted to either 0.04 mg/ml or
0.2 mg/ml in the same buffer. Phenol and DHBS were dissolved in
50 mM sodium phosphate buffer, pH 7.0, at 20 mM initial concentra-
tion. HTIB was dissolved in DMSO at 20 mM initial concentration. 4-
Aminoantipyrine was dissolved in DMSO at 10 mM initial concentra-
tion. Hydrogen peroxide was diluted from 30% stock solution with
deionized water and its concentration was determined with the per-
manganate method. p-Diphenols were dissolved in DMSO at 20 mM
initial concentration and then diluted to 16, 12, 8, and 4 mM in DMSO.
The final DMSO concentration was therefore 1% (v/v) in reactions with
phenol and DHBS and 1.5% (v/v) in reactions with HTIB. For calcium
dobesilate the concentrations given correspond to the concentrations of
2,5-dihydroxybenzenesulfonate present in this salt, which was used to
allow comparison of results with other p-diphenols used in this study.
Reactions were carried out at room temperature in 3 ml of 50 mM
sodium phosphate buffer, pH 7.0 containing 50 μM 4-aminoantipyrine,
100 μM phenolic substrates, 50–110 μM H2O2, and either 0.4 μg/ml
(0.08 U/ml for phenol) or 2 μg/ml (0.4 U/ml for DHBS and HTIB) of
HRP. The tested p-diphenols were applied at 20, 40, 60, and 80 μM. The
reactions were initiated by addition of the enzyme and absorbance was
measured at either the maximum of the antipyrilquinoneimine chro-
mophore (505 nm for phenol, 513 nm for DHBS, and 516 nm for HTIB)
or the maximum of the p-quinones (247 nm) for 10–30 min.
Additionally the same reactions were performed and spectra from 230
to 680 nm were recorded immediately after mixing the reagents and
then in 1 min intervals for 10–30 min. Spectra of the mixtures before
addition of the enzyme were also recorded.
O
OH
HO
HO
HO
1
2
OH
HO
O
S
O
Ca++
-O
S
O-
O
O
HO
OH
3
OH
O
S
+
O-
NH2
O
HO
4
Fig. 1. Structures of compounds tested in this study: 1 – homogentisic acid
((2,5-dihydroxyphenyl)acetic acid), 2 – gentisic acid (2,5-dihydroxybenzoic
acid), 3 – calcium dobesilate (calcium 2,5-dihydroxybenzenesulfonate), and 4 –
etamsylate (N,N-diethylammonium 2,5-dihydroxybenzenesulfonate).
2.3. Measurements of hydrogen peroxide concentration
the chromogenic substrates used [29]. However, the exact mechanisms
have not yet been established.
Hydrogen peroxide consumption was measured under the same
conditions as the reactions monitored spectrophotometrically with the
modified xylenol orange method including sorbitol [34]. Aliquots of the
reaction mixtures were removed at 1 min intervals, mixed with the
xylenol orange reagent, incubated in the dark for 30 min and the ab-
sorbance was measured at 560 nm. Under the reaction conditions
(25 mM sulfuric acid) the p-diphenols used in this study did not inter-
fere with this assay.
We have recently shown that catechols, such as carbidopa, L-dopa,
and dopamine, interfere with reactions catalyzed by bovine myeloper-
oxidase (MPO), bovine lactoperoxidase (LPO) and horseradish perox-
idase (HRP) by reducing the oxidation products or intermediates of
standard substrates, such as o-dianisidine, ABTS, and 4-aminoanti-
pyrine/phenol. We have also postulated that the same mechanism may
be involved in interference caused by p-diphenols – homogentisic acid,
gentisic acid, calcium dobesilate, and etamsylate [33] (Fig. 1). We have
reaction. Here we show that the mechanisms of interference and its
magnitude vary and strongly depend on the redox properties of these
compounds resulting from the electron-donating or electron-with-
drawing character of their substituents.
2.4. Analysis of the oxidation of p-diphenols
Reactions were carried out at room temperature in 3 ml of 50 mM
sodium phosphate buffer, pH 7.0 containing 1% (v/v) DMSO, for 50 or
100 μM homogentisic acid, gentisic acid, calcium dobesilate or etam-
sylate with 50 μM H2O2 in the absence or presence of HRP at either
2. Materials and methods
0.4 μg/ml (0.08 U/ml) or 2 μg/ml (0.4 U/ml) concentration.
Additionally, to accelerate the oxidation of gentisic acid, calcium do-
besilate and etamsylate reactions were carried out with 100 μM H2O2 in
the presence of 20 μM phenol and 2 μg/ml (0.4 U/ml) of HRP.
Reactions were initiated by the addition of the enzyme. UV/Vis spectra
from 230 to 680 nm were recorded immediately after mixing the re-
agents and then at 1 min intervals for 10–30 min. Absorbance at
247 nm was measured in separate experiments. Hydrogen peroxide
consumption was measured as described above. All experiments were
performed at least in triplicates.
2.1. Instruments and reagents
Spectrophotometric analysis was performed in
a Jasco V-650
UV–Vis spectrophotometer. NMR spectra were recorded in a Bruker
Avance 400 NMR spectrometer. HRP (type II, 200 U/mg) and 4-ami-
noantipyrine were purchased from Merck. Homogentisic acid ((2,5-di-
hydroxyphenyl)acetic acid), gentisic acid (2,5-dihydroxybenzoic acid),
calcium dobesilate (calcium 2,5-dihydroxybenzenesulfonate), and
etamsylate (N,N-diethylammonium 2,5-dihydroxybenzenesulfonate)
were obtained from TCI (Tokyo Chemical Industries). Sodium 3,5-di-
chloro-2-hydroxybenzenesulfonate (DHBS) and 3-hydroxy-2,4,6-triio-
dobenzoic acid (HTIB) were obtained from Fluorochem. Hydrogen
peroxide, phenol, xylenol orange, sorbitol, buffer components and sol-
vents were purchased from Avantor Performance Materials (Gliwice,
Poland).
2.5. Preparation of p-benzoquinone acetic acid (cyclohexa-2,5-diene-1,4-
dioneacetic acid, BQA)
Homogentisic acid (33.6 mg, 0.2 mmol, 20 mM) was incubated with
H2O2 (24 mM, 1.2 eq.) and 40 μg/ml (8 U/ml) of HRP in 10 ml of
100 mM sodium phosphate buffer, pH 7.0 for 10 min. The reaction
2