Spectrometric assay for horseradish peroxidase activity based on the linkage of conjugated system formed by oxidative decarboxylation

Article abstract of DOI:10.1016/j.saa.2016.11.012

Horseradish peroxidase (HRP)-catalyzed oxidation of 2,2-bis[3-acethylfilicinic acid-5-yl]acetic acid (BAFA, 4) produces Dehydro-3,3’-diacetyl-5,5’-methylenedifilicinic acid (DDMF, 3). A new photometric hydrogen donor (4) for peroxidase (POD)-catalyzed oxi

Full text of DOI:10.1016/j.saa.2016.11.012

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 174 (2017) 189–194
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Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Spectrometric assay for horseradish peroxidase activity based on the
linkage of conjugated system formed by oxidative decarboxylation
⁎
Masaki Yamaguchi, Shingo Sato
Department of Biochemical Engineering, Graduate School of Science and Engineering, Yamagata University, Jonan 4-3-16, Yonezawa-shi, Yamagata 992-8510, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 April 2016
Received in revised form 25 October 2016
Accepted 12 November 2016
Available online 13 November 2016
Horseradish peroxidase (HRP)-catalyzed oxidation of 2,2-bis[3-acethylfilicinic acid-5-yl]acetic acid
(BAFA, 4) produces Dehydro-3,3’-diacetyl-5,5’-methylenedifilicinic acid (DDMF, 3). A new photometric
hydrogen donor (4) for peroxidase (POD)-catalyzed oxidation was demonstrated to be potentially
useful for spectrocolorimetric and spectrofluorometric determination of HRP. Our developed colorimetric
(absorption at 483 nm) and fluorometric (emission at 529 nm) systems both gave a linear calibration curve for
HRP (y = 0.0025× + 0.0237; R2 = 0.9997, and y = 0.241× + 3.194; R2 = 0.9914, respectively) in the same con-
centration range of 9.1 × 10−8–1.1 × 10−6 nmol/L. The calculated Km and Vmax values of 5.10 × 10−4 M and
5.13 × 10−6 M/min, respectively. The results indicate that the quantification of HRP using BAFA 4 as hydrogen
donor is possible.
Keywords:
Carthamin-precursor model
Oxidative decarboxylation
Horseradish peroxidase
Spectrocolorimetry
© 2016 Elsevier B.V. All rights reserved.
Spectrofluorometry
1. Introduction
used as a visual detection reagent of POD. However, both pigments 1
and 2 are unstable and are present only in small quantities in safflower
The spectrometric determination of peroxidase (POD) using a chro-
mogenic compound is one of the most important analytical methods
and has widespread applications in clinical and environmental chemis-
try. Horseradish peroxidase (HRP) is heme peroxidase [1] and catalyzes
a variety of oxidative transformations of organic and inorganic sub-
strates [2,3]. Peroxidases such as HRP are frequently used as markers
or labels in enzyme-linked assays for biological molecules and other
analytes of interest, e.g., drugs, hormones, steroids, and cancer markers
[4–12]. Detection of these enzymes can be achieved by the use of sub-
strates that produce a detectable product. Chromogenic compounds
such as o-phenylenediamine [13,14] 2,2′-azino-bis(3-ethylbenzothiaz-
oline-6-sulfonic acid) (ABTS) [15], tetramethylbenzidine [16] and
diaminodiphenylmethane [17] produce a colored reaction product,
and fluorogenic substrates produce fluorescent products [4–12]. These
methods offer a safe, convenient, and sensitive means to provide a
quantitative measurement of the amount of enzyme or an enzyme-
labeled analyte in a sample.
petals; moreover, 2 is readily oxidized to 1. Because of their complexity,
the synthesis of both 1 and 2 has not yet been achieved. On the basis of
the experience in synthesis [21,22], we here design more stable, readily
synthesizable, and water-soluble model compounds (DDMF 3 [19] and
BAFA 4 [20]) for the detection of POD, in which the hydroxyl and
glucosyl residues of 1 and 2 are replaced by methyl groups and the
cinnamoyl moieties are replaced by acetyl groups (Scheme 1). Model
compounds 3 and 4 take over the linkage of the conjugated system
due to the sp2-carbon formed by oxidative decarboxylation from natural
dyes. This structure and oxidation reaction mechanism are new and dif-
fer from those of the commercially available reagents used for POD de-
tection. We here describe a spectrometric method for the determination
of HRP activity using BAFA 4 as a hydrogen donor for HRP.
2. Experiment
2.1. Synthesis
The color of the petals of safflower (Carthamus tinctorius L.) gradual-
ly changes from yellow to red. The red pigment is referred to as
carthamin (1) [18] and has been used for a long time as a dye stuff,
rouge, and food colorant. Red pigment 1 is formed by POD-catalyzed ox-
idative decarboxylation of one of the yellow pigments in safflower petal,
which is referred to as carthamin precursor (2) [20,21] (Scheme 1). This
remarkable color change led us to assume that yellow dye 2 could be
2.1.1. Reagents and Apparatus
All solvents used in this synthesis were commercial products.
Reactions were monitored by TLC on 0.25-mm silica gel F254
plates (E. Merck, Japan). UV light_irradiation (254 and 365 nm), a 5%
ethanolic solution of ferric chloride, and a 7% ethanolic solution of
phosphomolybdic acid with heat were used as coloring agents. For the
separation and purification of the reaction products, flash column chro-
matography on silica gel (40–50 μm, Kanto Reagents Co. Ltd., Japan,
silica-gel 60) was performed. Melting points were determined using
⁎
Corresponding author.
E-mail address: shingo-s@yz.yamagata-u.ac.jp (S. Sato).
http://dx.doi.org/10.1016/j.saa.2016.11.012
1386-1425/© 2016 Elsevier B.V. All rights reserved.

190
M. Yamaguchi, S. Sato / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 174 (2017) 189–194
Scheme 1. Biosynthesis carthamin (1) by oxidative decarboxylation of its precursor yellow pigment (2) in safflower petals, and their model compounds (DDMF 3, BAFA 4).
an ASONE micro-melting point apparatus and are uncorrected. IR spec-
tra were recorded on a Horiba FT-720 IR spectrometer using a KBr disk.
NMR spectra were recorded on a JEOL ECX-500 spectrometer using
Me4Si as the internal standard. Mass spectral data were obtained by
fast-atom bombardment (FAB) in a matrix of 3-nitrobenzyl alcohol
(NBA), using a JEOL JMS-AX505HA instrument. High-resolution mass
spectra (HRMS) were obtained under electro spray ionization (ESI) con-
ditions on a JEOL JMS-T100LP.
138.08, 184.08, 197.45, 200.95. FAB-MS (m/z) 403 (M + H)+. HRMS
(ESI+) m/z: [M + Na]+ calcd. for C21H22NaO8 425.12124, found
425.12119. UV–vis λmax (EtOH) 482 nm; log ε 4.46.
2.1.2.2. Synthesis of 2,2-Bis[3-acethylfilicinic acid-5-yl]acetic Acid (BAFA
4). To a stirred solution of 5 (210 mg) in 1 M Na2CO3 aqueous solution
(2 mL), glyoxylic acid (59 mg, 0.6 equiv) was added and the mixture
was stirred at room temperature for 0.5 h. The reaction mixture was
poured into an ice-cold 1 M HCl solution (30 mL) and extracted two
times with EtOAc. After removal of the solvent, the residue was purified
by silica-gel column chromatography (toluene/EtOAc/AcOH = 6:1:0.1)
and recrystallized from benzene to afford 4 (164 mg, 73%) as white
prisms.
2.1.2. Synthesis of 3 and 4
DDMF 3 and BAFA 4 were synthesized as shown in Scheme 2.
2.1.2.1. Synthesis of Dehydro-3,3′-bis(acetyl)-5,5′-methylenedifilicinic Acid
(Dehydro-3,3’-diacetyl-5,5’-methylenedifilicinic acid (DDMF, 3).). To a
stirred suspension solution of 2-acetyl-3,5-dihydroxy-4,4-dimethyl-2,5-
cyclohexadien-1-one (5 [23,24], 100 mg) in triethyl orthoformate
(3 mL), 60% NaH (245 mg, 12 equiv) was added portion wise and the mix-
ture was vigorously stirred at room temperature for 2.5 h. After complete
consumption of 5 as monitored by TLC, the reaction mixture was poured
into an ice-cold 1 M HCl solution (30 mL) and extracted two times with
EtOAc. After removal of the solvent, the residue was purified by silica-
gel column chromatography (toluene/EtOAc/AcOH = 6:1:0.1) and re-
crystallized from benzene to afford 3 (74.7 mg, 78%) as yellow prisms.
2.1.2.1.1. Data of 3. Yellow prisms. Mp = 173.0–174.5 °C (lit [19].
173.0–174.5 °C). IRν (KBr) 3315, 2983, 2945, 2879, 1682, 1651, 1540
2.1.2.2.1. Data of 4. White prisms. Mp = 103–105 °C (lit [21] 102–
105 °C). IRν (KBr) 3480, 2981, 2939, 2872, 1749, 1722, 1643, 1564,
1540 cm−1 1H NMR (pyrine-d5, 500 MHz) δ 1.61 and 1.72 (each 6H,
.
s, CH3 × 4), 2.71 (6H, s, Ac × 2), 6.84 (1H, s, NCH\\). 13C NMR (pyrine-
d5) δ 24.40, 36.25, 28.24, 34.01 (NCH\\), 51.34 (quaternary C), 104.87,
105.41, 175.31, 186.53, 189.52, 197.87, 198.23. FAB-MS (m/z) 449
(M
+ . )
H)+ HRMS (ESI− m/z: [M\\H]− calcd. for C22H23O10
447.12912, found 447.12950. UV–vis λmax (EtOH) 342 nm; log ε 4.25.
2.1.2.3. Acetylfilicinic Acid; 2-Acetyl-4,4-dimethylcyclohexan-1,3,5-trione
(5) [24,25]. Colorless prisms. Mp = 108–109 °C. 1H NMR (DMSO-d6) δ
1.28 (6H, s, CH3 × 2), 2.47 (3H, s, COCH3), 5.43 (1H, s, olefinic H),
18.41 (1H, s, OH). 13C NMR (DMSO-d6) δ 24.6 (CH3), 28.3 (COCH3),
48.7 (C4), 94.9 (C6), 105.2 (C2), 182.5 (C_O), 188.7, 196.7 and 200.5
(C1, 3, 5).
cm−1 1H NMR (pyrine-d5) δ 1.66 (12H, s, \\CH3 × 4), 2.69 (6H, s,
.
\\Ac × 2), 9.14 (1H, s, –CH_). 13C NMR (pyrine-d5) δ 23.00
(CH3 × 4), 28.73 (Ac × 2), 54.77 (quaternary C), 106.79, 112.89,
Scheme 2. Synthesis of 3 and 4.

M. Yamaguchi, S. Sato / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 174 (2017) 189–194
191
2.5
2.2. Analysis
2
2.2.1. Reagents and Apparatus
The reagents used in analysis were commercial products: peroxidase
from horseradish (Wako Pure Chemical Industries, Ltd., Osaka, Japan)
with optimum pH 5.0_8.0 and activity 383 units/mg [5]: 30% H2O2
aqueous solution (Wako Pure Chemical Industries, Ltd.): anthracene
(Sigma-Aldrich, St. Louis, MO, USA).
UV–vis absorption spectra were obtained using a Hitachi U-2010
UV–VIS spectrophotometer. Fluorescence spectra and quantum yield
were measured using a Hitachi F-7000 fluorescence spectrophotometer.
Excitation and emission wavelength band passes were set at 2 nm.
1.5
1
0.5
0
450
500
550
600
650
Wavelength [nm]
2.2.2. Preparation of Sample Solutions for UV–vis and Fluorescence
Spectroscopy (Figs. 1–3, Tables 1 and 2)
Fig. 2. Fluorescence spectra of 3 and 4 in EtOH ( EtOH, 3, 4, both 3.0 × 10−7 M). (For
interpretation of the references to color in this figure legend, the reader is referred to the
web version of this article.)
Solutions of 3 and 4 with a concentration of 3.0 × 10−5 M were pre-
pared in EtOH, MeOH, CHCl3, acetone, and 0.01 M phosphate buffer so-
lution (PBS) (pH 6.5), and were subjected to UV–vis and fluorescence
measurements.
Φ
sample =0.27×(0.136/0.367)×(5997.2/21,396)×(1.32892/
1.36142)=2.7×10−2
2.2.3. Determination of the Fluorescence Quantum Yield of 3 (Table 3) [25]
The fluorescence quantum yields for 3 in EtOH and MeOH were ob-
tained by comparing the area of the emission spectrum for a reference
sample with that for a solution of anthracene excited at the same wave-
length. The fluorescence quantum yields were obtained from the fol-
lowing equation:
The quantum yield of 3 in MeOH was determined to be 2.7 × 10−2
.
2.2.4. Examination of the Detection of HRP Using 4
2.2.4.1. Absorbance at Different Concentrations of H2O2 in the Presence of
HRP. Mixtures of 0.01 M PBS (4.5 mL, pH 6.5) of 4 (1.0 mg) with
0.01 M PBS (0.5 mL, pH 6.5) containing HRP (0.1 mg) were prepared,
20 μL of 0.03, 0.3, 3.0, or 30% H2O2 aqueous solution was added, and
the reactions were mixed. The resulting solutions were sampled at
10 min intervals for 60 min, diluted 10 times with EtOH, and subjected
to UV–vis or fluorescence measurement.
À
Á
À
Â
Ã
Á
Φsample ¼ Φstandard ꢀ Astandard=Asample ꢀ Σ Fsample =Σ½Fstandard
ꢁ
À
Á
2
ꢀ nsample2=nstandard
ð1Þ
where A, F, Σ [F] and n2 denote absorbance, fluorescence intensity, the
peak area of the fluorescence spectra, and refractive index of the used
solvent, respectively. Here, anthracene was as a standard and its quan-
tum yield (Φstandard) in EtOH is 0.27 [25]. Since measurement of both
sample 3 and standard (anthracene) was conducted with both the con-
centration of 3.0 × 10−5 mol/L using EtOH as a solvent, the term of the
refractive index (n2sample/ns2tandard) was removed from the Eq. (1). Each
data determined from the measurement; Astandard:0.136, Asample:0.501,
Σ[Fsample]:2636.4, Σ[Fstandard]:21396 was introduced to Eq. (1).
2.2.4.2. Absorbance at Different Concentrations of PBS (pH 6.5) in the Pres-
ence of HRP. Substrate 4 (1.0 mg) was dissolved in 4.5 mL of H2O and in
0.01, 0.05, 0.1, and 0.5 M PBS (pH 6.5). To each solution, 0.5 mL of a so-
lution of the same concentration containing HRP (0.1 mg) and 20 μL of
0.03% H2O2 was added, and the mixtures were stirred. After stirring
for 1 h, the resulting solution was diluted 10 times with EtOH and sub-
jected to UV–vis spectroscopy measurement. When 0.5 M PBS was used,
the mixture was left to stand overnight to allow the resulting salts to
precipitate, and the supernatant was subjected to analysis.
Φsample ¼ 0:27 ꢀ ð0:136=0:501Þ ꢀ ð2636=21; 396Þ ¼ 9:0 ꢀ 10−3
2.2.4.3. Absorbance at Different pH Values in the Presence of HRP and H2O2.
Substrate 4 (1.0 mg) was dissolved in 4.5 mL of 0.01 M PBS of different
pH (pH 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0). To each solution, 0.5 mL of a
PBS of the same pH containing HRP (0.1 mg) and 20 μL of 0.03% H2O2
were added and the mixtures were stirred. After diluting 10 times
with EtOH, the ABS and fluorescence intensity of the resulting mixtures
The quantum yield of 3 in EtOH was determined to be 9.0 × 10−3
Next, each data measured in MeOH, Asample:0.367, Σ[Fsample]: 5997.2,
nsample:1.3289, nstandard:1.3614 was introduced to Eq. (1).
.
0.5
0.45
0.4
5
4.5
4
0.35
0.3
3.5
3
0.25
0.2
2.5
2
0.15
0.1
1.5
1
0.05
0
0.5
0
250
350
450
550
400
450
500
550
600
650
wavelength [nm]
Wavelength [nm]
Fig. 1. UV–vis spectra of 3 and 4 in EtOH (both 1.6 × 10−5 M, 3, 4). (For interpretation
of the references to color in this figure legend, the reader is referred to the web version of
this article.)
Fig. 3. Excitation and Emission spectra of 3 in EtOH ( excitation,
(For interpretation of the references to color in this figure legend, the reader is referred
to the web version of this article.)
emission).

192
M. Yamaguchi, S. Sato / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 174 (2017) 189–194
Table 1
Table 3
Visible absorption spectra of 3 in different solvents [CHCl3, acetone, EtOH, MeOH, PBS
The fluorescence quantum yield (Φ) of 3 in EtOH and MeOH.
(0.01 M, pH 6.5)].
Solvent
Fluorescence quantum yield (Φ)
Solvent
max (nm)
CHCl3
Acetone
EtOH
MeOH
PBS (0.01 M, pH 6.5)
EtOH
MeOH
9.0 × 10−3
2.7 × 10−2
λ
–*
489
483
470
467
Absorbance (−)
–
0.135
0.740
0.356
0.310
* No absorption in a visible region.
in EtOH and MeOH was 529 nm and the quantum yield (Φ) was deter-
mined to be 9.0 × 10−3 and 2.7 × 10−2, respectively (Table 3). Because
the use of 4 was proposed for the colori- and fluorometric determina-
tion of HRP, HRP quantification methods using this model compound
were evaluated.
were measured at 20 min intervals for 120 min at a wavelength of
482 nm and emission wavelength of 529 nm (excitation wavelength:
492 nm).
In order to determine the optimum conditions for UV–vis and fluo-
rescence measurements, the HRP-catalyzed oxidative decarboxylation
reaction of 4 was examined. In the absence of HRP, the oxidation of 4
(1 mg) with 30% hydrogen peroxide (H2O2) aqueous solution (20 μL)
in 0.1 M PBS (pH 6.5) did not proceed and 4 was stable for at least 8 h.
In the presence of HRP (0.1 mg), the time-course of the ABS at 482 nm
was measured at the presence of 20 μL of the concentrations of 0.03,
0.3, 3, and 30% H2O2 under the same oxidation conditions (Fig. 4).
When the concentration of H2O2 was 0.03%, the highest ABS (1.2) was
observed, which remained constant for 30 min. When the concentration
of H2O2 was over 0.3%, formation of the unknown oxidation products is
possible. When the adding concentration of H2O2 was 0.003%, the ABS
decreased to 1.0 over 1 h. Thus, the optimum H2O2 concentration and
reaction time were determined to be 0.03% and 1 h, respectively. Next,
the optimum concentration and pH of the PBS were examined. The
ABS of 4 at 482 nm was measured for solutions with increasing concen-
trations of PBS from 0 (H2O) to 0.01, 0.05, 0.1, and 0.5 M, in the presence
of 0.03% H2O2 (20 μL) and HRP (0.1 mg) (Table 4). The measurement
was carried out after 10-fold dilution with EtOH. The ABS was higher
at higher concentrations of PBS (pH 6.5). However, because the salt
was precipitated by the addition of EtOH at concentrations N0.05 M,
the optimum concentration of PBS was determined to be 0.01 M.
Thus, the ABS at 482 nm of each reaction mixture in 0.01 M PBS (pH
5.0_8.0) in the presence of HRP (0.1 mg) and 0.03% H2O2 (20 μL) was
measured (Fig. 5), and a maximum ABS of 1.1 was recorded at pH 6.0.
Analysis of the time-course of the ABS at each pH shows that the maxi-
mum ABS at pH 6.0 was maintained for 80 min. Thus, the optimum pH
was determined to be 6.0. In the HRP-catalyzed oxidative coupling reac-
tion of pyrocatechol and aniline, the maximum ABS was from pH 6.5 to
8.5 in the pH region of 4.5_8.5 and the optimum pH chosen was 7.0 be-
cause pyrocatechol is acidic and aniline is basic [1]. Our sample 3 and 4
are acidic and unstable in basicity. Next, the ABS was measured in the
concentration range of 8.98 × 10−2–11.5 U/L HRP with 2-fold dilution,
2.2.4.4. Measurement of the Detection Limit of HRP by Colorimetry and
Fluorometry (Fig. 6). To 4.5 mL of a 0.01 M PBS (pH 6.0) containing 4
(1.0 mg), 0.5 mL of 0.01 M PBS (pH 6.0) containing concentrations of
HRP in the range 8.98 × 10−2–11.5 U/L with 2-fold dilution and 20 μL
of 0.03% aqueous H2O2 were added and the oxidation reaction was
started. Each resulting solution was diluted 10 times with EtOH and
subjected to colorimetric and fluorometric analyses.
2.2.4.5. Determination of Km and Vmax. EtOH solutions of 3 at the concen-
trations of 3.1 × 10−5 to 6.1 × 10−8 M with 2-fold dilution was pre-
pared, their ABS was measured at 482 nm, and their calibration curve
was determined. Next, 0.01 M PBS (pH 6.0) of 4 at concentrations
from 1.1 × 10−3 to 1.7 × 10−5 M with 2-fold dilutions were prepared.
To each, a PBS (1.5 mL) containing HRP at a concentration of 15.7 U/L
and a 0.03% H2O2 aqueous solution (20 μL) were added, and the oxida-
tion reaction was performed for 5 min. The resulting mixtures were di-
luted 10 times with EtOH and the ABS was measured at 1 min intervals.
3. Results and Discussion
3.1. Synthesis [19–24]
The synthesis of 3 and 4 was conducted as shown in Scheme 2. Com-
pounds 3 (yellow) and 4 (white) were afforded via a four_step sequence
in 45 and 43% overall yields, respectively. On the synthesis of 3 by oxi-
dation of 4 using metal oxide such as KMnO4, the reaction proceeded,
however gave a mixture of the complex products including 3. In the ox-
idation using H2O2/peroxidase, water-solubility of 4 was poor under the
synthetic conditions. Dimerization of acetylfilicinic acid 5 using triethyl
orthoformate in the presence of NaH was smoothly proceeded and gave
3 as an only product. Both are recrystallizable from benzene, and the
resulting crystals are stable and can be stored at room temperature for
long periods.
1.6
1.4
1.2
1
3.2. Analysis
The UV–vis spectra of 3 and 4 in EtOH showed a maximum absorp-
tion at 482 nm (log ε 4.46) and 342 nm (log ε 4.25), respectively and no
overlapping signals (Fig. 1). Whereas 4 exhibited no fluorescence, 3
emitted a weak yellowish green fluorescence in EtOH (Figs. 2 and 3).
The UV–vis and fluorescence spectra of 3 were measured in different
solvents [EtOH, MeOH, CHCl3, and PBS, (pH 6.5)], and the maximum ab-
sorbance and fluorescence intensity were observed in EtOH and MeOH,
respectively (Tables 1 and 2). The maximum emission wavelength of 3
0.8
0.6
0.4
0.2
0
Table 2
Fluorescence spectra of 3 in different solvents [CHCl3, acetone, EtOH, MeOH, PBS (0.01 M,
0
20
40
60
80
pH 6.5)].
Time [min]
Solvent
CHCl3 Acetone EtOH MeOH PBS
(0.01 M, pH 6.5)
Fig. 4. Time-dependent absorbance of 4 (1.0 mg) at 480 nm in 5 mL of a 0.1 M PBS (pH 6.5)
in the presence of 20 μL of 0.03–30% H2O2 solutions ( 0.03%, 0.30%, 3.00%, 30%) and
HRP (0.1 mg). (For interpretation of the references to color in this figure legend, the reader
is referred to the web version of this article.)
λ
em (nm)
493
495
530
170.1 834.6
527
529
47.14
Fluorescence intensity (−) 56.94 12.33

M. Yamaguchi, S. Sato / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 174 (2017) 189–194
193
Table 4
Absorption spectra on the oxidation reaction of 4 in H2O and 0.01–0.5 M PBS (pH 6.5) in
the presence of 0.03% H2O2 solution (20 μL) and HRP (0.1 mg).
Solvents
H2O
0.01 M
1.024
0.05 M
1.038
0.1 M
1.086
0.5 M
1.244
Absorbance [−]
0.446
under the optimum conditions. The relationship between the ABS and
the concentration of HRP 1.4 _11.5 U/L was linear (y
=
0.0025× + 0.0237, R2 = 0.9997) and can be used as a calibration
curve. The value of 1.4 U/L represents the cross point and the limit of
this quantification method [1]. The relation between fluorescence inten-
sity and concentration of HRP (excitation: 482 nm; emission: 529 nm)
was measured by fluorometry in a manner similar to that above-
described for the colorimetric assay. Similarly to the colorimetric data,
a linear relationship was observed in the concentration range from 1.4
to 11.5 U/L (y = 0.241× + 3.1942, R2 = 0.9914), which could also be
used as a calibration curve. Although the colorimetric and fluorometric
measurements were performed in the same HRP concentration range
1.4_11.5 U/L (Fig. 6). The limit of this quantification method (detection
limit for HRP), 1.4 U/L (9.1 × 10−14 mol/mL) is analogous to that
using tyramine (2.3 × 10−14 mol/mL), homovanillic acid (1.1 × 10−13
mol/mL), and N,N′-dicyanomethyl o-phenylenediamine (1.0 × 10−13
mol/mL) as fluorescent substrates [4], and pyrocatechol-aniline
(1.42 × 10−13 mol/mL) and phenol-aminoantipyridine (2.31 × 10−13
mol/mL) as colored substrates [1], but higher than that using p-
hydroxyphenylpropionic acid (6.5 × 10−16 mol/mL), tyrosol (1.3 ×
10−15 mol/mL), and amino aluminum phthalocyanine (6.0 × 10−15
mol/mL) [4].
For the determination of the Michaelis-Menten and Lineweaver-
Burk plots, the calibration curve of the ABS at 483 nm vs. HRP concentra-
tion [0.06_31.0 μM in 0.01 M PBS (pH 6.0)] was prepared. The calibration
curve was calculated as y = 0.0485× + 0.0027, and the correlation co-
efficient (R2) was 0.9999. Because of its high linearity, this equation
could be used for the determination of the Michaelis-Menten plot and
of the Lineweaver-Burk plot. The Km and Vmax values were calculated
to be 5.10 × 10−4 M and 5.13 × 10−6 M/min, respectively, using the
equation: y = 99.572× + 0.1951, derived from the Lineweaver-Burk
plot. As compared with the Km (1.25 × 10−2 M) and the Vmax (1.22 ×
10−5 M/min) of the HRP-catalyzed oxidative coupling reaction of pyro-
catechol and aniline [1], the affinity of the substrate for HRP was 24.5-
fold higher and the maximum reaction rate was 2.38-fold slower. This
shows that the catalytic ability of HRP is low for the abstraction of labile
carboxylic and enol proton atoms of 4. However, taking into account the
detection limit for HRP and the affinity of 4 for HRP, the HRP activity for
4 is adequate for practical applications.
Fig. 6. Calibration curve of the absorbance at 483 nm and the fluorescence intensity at
582 nm vs. concentration of HRP (0–11.5 U/L).
4. Conclusion
The HRP-catalyzed oxidative decarboxylation of BAFA 4 proceeded
smoothly to form a yellow DDMF 3 in the presence of 0.03% H2O2
(20 μL) in 0.01 M PBS (pH 6.0, 5 mL) without exposure to light. Nonflu-
orescent BAFA 4 was converted into a fluorescent DDMF 3 with fluores-
cence quantum yield of 0.027 and emission wavelength at 529 nm. The
colori- and fluorometric quantification of HRP using BAFA 4 as hydrogen
donor is demonstrated. Kinetic parameters, Km (1.25 × 10−2 M) and
Vmax (1.22 × 10−5 M/min) are suggested that BAFA 4 is an appropriate
hydrogen donor for the HRP spectrometric assay. The colori- and fluo-
rometric quantification of HRP using 4 as hydrogen donor is demon-
strated. Many substrates for HRP are coupling products of the two
components such as 2,2′-dihydrodiphenyl derivatives, which are one
component is subjected to HRP-catalyzed oxidation with H2O2 and
coupled with another component [4a]. Our developed substrate, BAFA
4 is one component and subjected to the HRP-catalyzed oxidative de-
carboxylation with H2O2 to give colored and fluorogenic DDMF 3.
Thus this reaction is simple and difficult to be influenced. For practical
applications, further studies using vital fluids such as blood are required.
Conflict of Interest
The authors declare no conflict of interest.
Acknowledgement
The authors wish to thank Professor Bunpei Hatano for HRMS mea-
surements and Mr. Koya Maeda and Mr. Kota Sasaki for their technical
assistance.
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