Rapid and direct spectrophotometric method for kinetics studies and routine assay of peroxidase based on aniline diazo substrates

Article abstract of DOI:10.3109/14756366.2015.1103234

Peroxidases are ubiquitous enzymes that play an important role in living organisms. Current spectrophotometrically based peroxidase assay methods are based on the production of chromophoric substances at the end of the enzymatic reaction. The ambiguity regarding the formation and identity of the final chromophoric product and its possible reactions with other molecules have raised concerns about the accuracy of these methods. This can be of serious concern in inhibition studies. A novel spectrophotometric assay for peroxidase, based on direct measurement of a soluble aniline diazo substrate, is introduced. In addition to the routine assays, this method can be used in comprehensive kinetics studies. 4-[(4-Sulfophenyl)azo]aniline (λ _max = 390 nm, ? = 32 880 M^?1 cm^?1 at pH 4.5 to 9) was introduced for routine assay of peroxidase. This compound is commercially available and is indexed as a food dye. Using this method, a detection limit of 0.05 nmol mL^?1 was achieved for peroxidase.

Full text of DOI:10.3109/14756366.2015.1103234

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Rapid and direct spectrophotometric method for
kinetics studies and routine assay of peroxidase
based on aniline diazo substrates
Fatemeh Mirazizi, Azita Bahrami, Kamahldin Haghbeen, Hossein Shahbani
Zahiri, Mehdi Bakavoli & Raymond L. Legge
To cite this article: Fatemeh Mirazizi, Azita Bahrami, Kamahldin Haghbeen, Hossein Shahbani
Zahiri, Mehdi Bakavoli & Raymond L. Legge (2015): Rapid and direct spectrophotometric
method for kinetics studies and routine assay of peroxidase based on aniline diazo substrates,
Journal of Enzyme Inhibition and Medicinal Chemistry, DOI: 10.3109/14756366.2015.1103234
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Published online: 02 Nov 2015.
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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–8
2015 Taylor & Francis. DOI: 10.3109/14756366.2015.1103234
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RESEARCH ARTICLE
Rapid and direct spectrophotometric method for kinetics studies and
routine assay of peroxidase based on aniline diazo substrates
Fatemeh Mirazizi1, Azita Bahrami2, Kamahldin Haghbeen3, Hossein Shahbani Zahiri4, Mehdi Bakavoli5, and
Raymond L. Legge6
1Department of Chemistry, Islamic Azad University of Mashhad, Mashhad, The Islamic Republic of Iran, 2National Institute for Genetic Engineering
3
and Biotechnology, Tehran, The Islamic Republic of Iran, Biochemistry and Biophysics, National Institute for Genetic Engineering and
Biotechnology, Tehran, The Islamic Republic of Iran, 4National Institute for Genetic Engineering and Biotechnology, Tehran, The Islamic Republic of
5
6
Iran, Department of Chemistry, Ferdowsi University of Mashhad, Mashhad, The Islamic Republic of Iran, and Department of Chemical
Engineering, University of Waterloo, Waterloo, Ontario, Canada
Abstract
Keywords
Peroxidases are ubiquitous enzymes that play an important role in living organisms. Current
spectrophotometrically based peroxidase assay methods are based on the production of
chromophoric substances at the end of the enzymatic reaction. The ambiguity regarding the
formation and identity of the final chromophoric product and its possible reactions with other
molecules have raised concerns about the accuracy of these methods. This can be of serious
concern in inhibition studies. A novel spectrophotometric assay for peroxidase, based on direct
measurement of a soluble aniline diazo substrate, is introduced. In addition to the routine
assays, this method can be used in comprehensive kinetics studies. 4-[(4-
Sulfophenyl)azo]aniline (lmax ¼ 390 nm, e ¼ 32 880 Mꢀ1 cmꢀ1 at pH 4.5 to 9) was introduced
for routine assay of peroxidase. This compound is commercially available and is indexed as a
food dye. Using this method, a detection limit of 0.05 nmol mLꢀ1 was achieved for peroxidase.
Diazo of aniline, direct assay, peroxidase,
spectrophotometry
History
Received 11 June 2015
Revised 8 September 2015
Accepted 17 September 2015
Published online 30 October 2015
Introduction
for enhancing the physicochemical features of the known enzymes
through molecular approaches6,7, genetic engineering methods8,
and techniques such as directed evolution9. The success of these
approaches is heavily dependent on rapid and reliable assay
methods.
Peroxidases have found a special place in biotechnology, making
them one of the most essential enzymatic groups. Using a
hydroperoxide such as H2O2, peroxidases catalyze the oxidation
of a wide variety of electron donor substrates; including phenols,
aromatic amines, thioanisoles, and iodide. The reaction is a three-
step cyclic process. Initially, the enzyme is oxidized by H2O2
then reduces back to the native form in two sequential steps,
involving the formation of two enzyme intermediates,
Compounds I and II1.
Several assay methods exist for peroxidases but most of them
are based on spectrophotometric approaches because of their low-
cost and high precision. In these methods, the catalyzed reaction
with the ZH is continuously monitored. Different features of some
of the better known spectrophotometric assays are summarized in
Table 1. As will be seen none of the substrates employed in these
methods are themselves chromophores. Consequently, all these
methods rely on the formation of chromophoric compounds,
which result from the radicals produced at the end of the
enzymatic reaction. The 2,20-azino-bis(3-ethylbenzothiazoline-6-
sulphonic acid) (ABTS) radical cation is chromophoric but not
stable. The fate of the 3,30,5,50-tetramethylbenzidine (TMB)
radical is not clearly resolved; however, there is evidence that a
blue color imine is formed directly from the corresponding
radical27,28. The chromophoric products of the other methods are
the result of intermolecular reactions between either the radicals
themselves, or the radicals and a helper compound such as
4-aminoantipyrine (4AA).
HRP þ H2O2 ! Compound I þ H2O
Compound I þ ZH ! Compound II þ Zꢁ
Compound II þ ZH ! HRP þ Zꢁ þ H2O
ð1Þ
ð2Þ
ð3Þ
Because of their unique physicochemical properties and their
ability to oxidize different organic hydrogen donors (ZH),
peroxidases have been employed in many industrial processes2,3
There is ongoing research with respect to discovering novel
enzymes via scanning and high-throughput screening methods4,5
Most of the current research in enzyme biotechnology are looking
.
.
The radical species produced can result in a complex profile of
reaction products. This includes dimers, trimers, and higher
oligomers that may act as reducing substrates in subsequent
reactions. This complexity increases ambiguity regarding the
Address for correspondence: Kamahldin Haghbeen, National Institute for
Genetic Engineering and Biotechnology, Biochemistry and Biophysics,
Tehran, Islamic Republic of Iran. E-mail: Kamahl@nigeb.ac.ir

2
F. Mirazizi et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
Table 1. Features of some selected peroxidase assay methods from the literature.
C
K.a.T. (Ref.)
N.K.
Solubility
P or S
Helper
lmax (nm)
e Mꢀ1 cmꢀ1
C.Enz.
Ref.
10
12
14
16
18
20
22
24
26
ABTS
DMA
Guaiacol
Phenol
ortho-Dianisidine
Pyrocatechol
Pyrogallol
TMB
–
–
–
–
–
–
–
–
–
High
Low
Low
Low
Low
Good
High
Low
Good
P
P
P
P
P
P
P
P
P
–
–
420
540
470
516
550
510
430
653
417
36 000
7180
26 600
7870
11 300
5000
2470
39 000
16 700
Laccase
ꢀ (11
)
–
Laccase
Tyrosinase
–
Tyrosinase
Laccase
+ (13
+ (15
+ (17
+ (19
+ (21
)
)
)
)
)
–
4AA
–
Aniline
–
–
–
ꢀ (23
)
)
–
–
OPD
+ (25
C, chromophoric; K.a.T., known as toxic; N.K., not known; P, product measurement; S, substrate consumption; C.Enz., competitive phenolase for
peroxidase; Ref., reference.
N2 Cl-
+
NH2
Figure 1. Synthetic routes for preparation of
the aniline diazo dyes. See Materials and
methods section for details.
method
"
1) HCl + NaNO2
2) Na2CO3 + HCl + NaNO2
a) Y= OCH3
b) Y= CH3
c) Y= H
-5-0 °C
d) Y= SO3H
e) Y= SO2NH2
Y
Y
1
NH2
Ha
Hb
NH2
NHCH2SO3Na
N
N
1) 1, -5-0°C
HCHO + NaHSO3
60-70°C
2) NaOH, H2O, Δ
Hc
Hd
2
Y
3
identity of the final chromophoric products29,30. Solubility and (AnalytikJena, Germany), equipped with a temperature control-
toxicity of the organic substrates are also matters of concern. ling accessory. Infrared spectra were recorded using a Brucker
Regardless of these disadvantages, any of these assays can be (Tensor 27) FTIR spectrometer. Extinction coefficients of the
used as routine assays for peroxidase but not for comprehensive dyes were determined at their lmax(s) in phosphate buffer
kinetic purposes. In advanced kinetic studies, it is desirable to see solutions (PBS) at pH 7 and 20 ꢁC. The HRP enzymatic reactions
how a small modification in the structure of the substrate or were carried out in PBS (0.01 M, pH 7) at 20 ꢁC.
electron density on the binding terminal affects rate of the
reaction. In this paper, a direct spectrophotometric method, based Synthesis of diazo dyes of aniline
on the consumption of a water-soluble and safe chromophoric azo
A modified synthetic procedure was developed for the facile
substrate is introduced for use in a routine assay of peroxidase.
preparation of diazo derivatives of aniline (3) (Figure 1). This
Using the general structure of the azo substrates, structure 3 in
method consisted of four simple and high yielding steps: making
(Figure 1), these substrates can also be used for advanced kinetic
the diazonium salts with the general structure of 1, protecting the
studies of peroxidases.
aniline amino group (2) and coupling reactions 1 with 2 followed
by removing the protecting group.
Materials and methods
Chemical, enzyme, and instruments
Preparation of diazonium salts
Horseradish peroxidase (HRP, EC 1.11.1.7; 100 U mgꢀ1), and all Diazonium salts were synthesized by two different methods. 1a, 1b, 1c,
the prerequisite chemicals for synthesis, were obtained from 1e were synthesized through method 1 described in Figure 1 as
Merck (Darmstadt, Germany). The azo derivatives of aniline were previously described with no further modifications31. 1d was
synthesized as described below. The chemical structures were synthesizedaccordingtotheproceduredescribedbyJayakannanetal.32
characterized based on various spectroscopic methods. NMR
analysis was performed on a 500 MHZ Brucker spectrometer
Synthesis of sodium anilinomethanesulfonate (2)
(Ultrashield) spectrometer. Proton and carbon chemical shifts are
relative to tetramethylsilane. Spectrophotometric measurements The procedure described by Zheng et al. was modified and used
were conducted on
a
Specord 210 spectrophotometer for the synthesis of sodium anilinomethanesulfonate33. Sodium

DOI: 10.3109/14756366.2015.1103234
Spectrophotometric method and routine assay of peroxidase
3
bisulfite (0.1 mol) was added to a solution of formaldehyde organic co-solvents as described in Table 2. The dye stock
(0.1 mol) and distilled water (40 mL) with stirring. The mixture solutions were prepared by dissolving the desired dye (1 mg) in
was heated to 65 ꢁC for 1 h. To this mixture, aniline (0.08 mol) the appropriate co-solvent (4 mL). Desired dye concentrations
was added drop wise over 15 min and stirred for 2 h at 65 ꢁC. The were obtained by diluting the dye solutions in PBS (0.01 M,
reaction mixture was cooled to about 0 ꢁC which resulted in a pH 7). To determine the impact of the co-solvent on the
bright gray precipitate that was filtered and recrystallized with peroxidase activity, the enzymatic reaction was conducted in the
water.
media containing various amounts of co-solvent. Results showed
the rate of the peroxidase reaction remained unchanged, even in
medium containing up to 10% co-solvent. The amount of co-
solvent did not exceed 7% of the reaction mixture in these
experiments. The stability of the prepared dyes solutions was
examined by monitoring the UV-visible spectrum and measuring
the change of the absorption at the lmax of each dye for 60 min at
20 ꢁC. In all cases, no change in the absorption spectra of the dye
solutions, due to precipitation or auto-oxidation, was observed.
The stock solution of hydrogen peroxide (0.1 M) was prepared by
diluting H2O2 (0.5 mL, 30 wt%) to 50 mL in PBS (0.01 M, pH 7).
Synthesis of diazo derivatives of aniline (3)
2 (0.01 mol) was dissolved in double-distilled water (20 mL)
containing sodium acetate (10 g) and cooled to 0 ꢁC. The desired 1
was then added slowly to this solution at 0 ꢁC. The resulting
colorful mixture was agitated overnight at room temperature. The
precipitate was filtered and washed with water. The crude product
was dissolved in an aqueous solution of NaOH (1%, 50 mL) and
the mixture refluxed until a change in color was evident (1–4 h).
The solution was cooled to room temperature and the precipitate
collected and recrystallized from the appropriate solvent.
Procedure for peroxidase assay and kinetic studies
3a: 4-[(4-methoxyphenyl)azo]aniline (CasNO 2592-28-1)
was recrystallized from ethyl acetate as brownish–yellow crystals. The direct assay of HRP was conducted by following the
Yield: 44%, mp: 140–142 ꢁC. IR in KBr tablet contained: [NH2 oxidation of the diazo derivatives of aniline with the general
(br. 3384 cmꢀ1), CHAr (3042 cmꢀ1), CH3 (2834 cmꢀ1), -C ¼ CAr structure 3 illustrated in Figure 1 in PBS (0.01 M, pH 7) at 20 ꢁC.
(1602 and 1510 cmꢀ1), -N ¼ N- (1345 cmꢀ1), -C-N- All enzymatic reactions were conducted in conventional quartz
(1247 cmꢀ1)]. 1HNMR in DMSO-d6; d[-OCH3 (3.81, 3H, s), UV-visible cuvettes. Total volume of the reaction mixture was
Ha(6.61, 2H, d, J ¼ 8.75 Hz), Hb(7.57, 2H, d, J ¼ 9 Hz), HC(7.65, 3 mL containing at most 7% co-solvent if needed. Experiments
2H, d, J ¼ 9.25 Hz,), Hd(6.81, 2H, d, J ¼ 8.25 Hz), NH2(4.1, s, were carried out in the presence of a constant concentration of
2H)]. 3b: 4-[(4-methylphenyl)azo]aniline (CasNO 722-25-8) HRP (100 mL) and H2O2 (20 mL) with seven different diazo
was recrystallized from a mixture of ethanol and water (2.5:1) as substrate concentrations in a range of 7.5–100 mM. The desired
yellow crystals. Yield: 63%, mp: 142–145 ꢁC. IR in KBr tablet volume of the dye solution was added into the quartz cuvette and
contained: [NH2 (str. 3381, 3476 cmꢀ1), CHAr (3206 cmꢀ1), CH3 diluted to 2880 mL with PBS (0.01 M, pH 7). To this mixture,
(2918 cmꢀ1), -C ¼ CAr (1620 and 1596 cmꢀ1), -N ¼ N- H2O2 (20 mL) was added prior to the addition of the HRP solution
(1417 cmꢀ1), -C-N- (1233 cmꢀ1)]: 1HNMR in CDCl3; d[-CH3 (100 mL). The reference cell contained PBS (0.01 M, pH 7). The
(2.40, 3H, s), Ha (6.73, 2H, d, J ¼ 8.7 Hz,), Hb(7.73, 2H, d, experiments were repeated five times under identical conditions.
J ¼ 8.4 Hz), HC(7.81, 2H, d, J ¼ 8.7 Hz), Hd(7.24, 2H, d,
J ¼ 8.7 Hz), -NH2(4.82, d, 2H)]. 3c: 4-aminoazobenzene ously monitoring the oxidation of the dye at the corresponding
(CasNO 60-09-3) was recrystallized from a mixture of ethanol
max at 20 ꢁC for at least 4 min. The rate of the reaction at constant
The rate curves for each reaction were obtained by continu-
l
and water (2.5:1) as yellow crystals. Yield: 67%, mp: 120–123 ꢁC. temperature was calculated under steady state conditions
IR in KBr tablet contained: [NH2 (str. 3382, 3477 cmꢀ1), CHAr (Figure 2A). Michaelis–Menten constants for the substrates
(3206 cmꢀ1), -C ¼ CAr (1595 cmꢀ1), -N ¼ N- (1415 cmꢀ1), -C-N- were calculated from the corresponding Lineweaver–Burk plots.
(1234 cmꢀ1)]; 1HNMR in DMSO-d6; d[Ha(6.66, dd, 2H, All data presented are an average of n ¼ 5 experiments.
J ¼ 8.76 Hz, J ¼ 2 Hz), Hb(7.65, 2H, dd, J ¼ 8.75 Hz, J ¼ 2 Hz),
HC(7.73, 2H, dd, J ¼ 7.4 Hz, J ¼ 1.2 Hz), Hd(7.49, 2H, t, J ¼ 8 Hz),
Results and discussion
HY(7.39, 1H, t, J ¼ 7.4 Hz), -NH2(6.1, s)]. 3d: 4-[(4-
sulfophenyl)azo]aniline (CasNO 104-23-4) was recrystallized Rationale for selecting diazo derivatives of aniline as
from water as yellow crystals. Yield: 70%, mp: 290 ꢁC. IR in KBr peroxidase substrates
tablet contained: [NH2 (br. 3445 cmꢀ1), CHAr (2923 cmꢀ1),
Experiments confirmed that hydrogen peroxide alone was not able
-C ¼ CAr
(1641 cmꢀ1),
-N ¼ N-
(1429 cmꢀ1),
-C-N-
to oxidize the diazo dyes in aqueous solution at pH(s) close to 7.
Adding HRP into the mixture of H2O2 and the diazo compound
initiated a rapid oxidation of the dye. Other oxidative enzymes,
such as mushroom tyrosinase only oxidized diazo derivatives of
phenol and catechol34. Neurospora crassa laccase35 was effective
on diazo derivatives of catechol and guaiacol. Using conditions
previously introduced34,35, N. crassa laccase and mushroom
tyrosinase had little effect on the diazo derivatives of aniline (data
not shown here). In contrast, it was only HRP which could oxidize
diazo derivatives of aniline with the general structure of 3
(Figure 1) in the presence of H2O2. Noteworthy is that phenolases
usually interfere with peroxidase assays when a natural extract is
being assayed. Therefore, the accuracy of peroxidase assays which
are based on using substrates such as ABTS, guaiacol, phenol, and
(1122 cmꢀ1), -SO2 (1270 cmꢀ1)]. 1HNMR in in DMSO-d6:
d[Ha(7.03, 2H, d, J ¼ 8.6 Hz), Hb, Hc,(7.75, 4H, s),
Hd(7.80, 2H, d, J ¼8.6 Hz), -NH2(4.97, s)]. 3e: 4-[(4-
sulfonamidophenyl)azo]aniline (CasNO 2435-64-1) was recrys-
tallized from a mixture of ethanol and water (1:1) as yellow
crystals. Yield: 68%, mp: 266–268 ꢁC. IR in KBr tablet contained:
[NH2 (br. 3345 cmꢀ1), CHAr (3201 cmꢀ1) -C ¼ CAr (1601),
-N ¼ N- (1423 cmꢀ1), -C-N- (1137 cmꢀ1) -SO2 (1299 cmꢀ1)].
1HNMR in DMSO-d6: d[Ha(6.68, 2H, d, J ¼ 9 Hz), Hb(7.70, 2H,
d, J ¼ 8.75 Hz), HC(7.85, 2H, d, J ¼ 8.5 Hz), Hd(7.94, 2H, d,
J ¼ 8.75 Hz), -NH2(6.3, s), SO2NH2 (7.4, 2H, s)].
Preparation of enzyme and dye solutions
Freshly prepared enzyme solutions were used in this work. The pyrocatechol are affected as these compounds participate in
enzyme stock solution was prepared by dissolving HRP (1.5 mg) enzymatic reactions with tyrosinase and laccase producing very
in PBS (3 mL, 0.01 M, pH 7) and kept on ice. Most of the aniline active intermediates34,35
.
HRP reactions with diazo derivatives of phenol, guaiacol, and
diazo dyes are not readily soluble in water; therefore, the stock
solutions of these compounds were prepared in the appropriate catechol are too rapid to be measured easily using simple

4
F. Mirazizi et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
Table 2. Extinction coefficients, kinetic rates, and Michaelis–Menten parameters for the aniline diazo substrates. See experimental section for details.
dC/min
(mMꢂminꢀ1
Vmax
(mMꢂminꢀ1
49
Specificity
constant ratioz
Substrate
lmax (nm)
e (Mꢀ1ꢂcmꢀ1)y
)
Km (mM)
)
Solvent
IIIa
IIIb
IIIc
IIId
IIIe
384
381
379
390
400
13 030
19 560
29 810
32 880
18 020
37.6
24.5
15.9
3.65
3.33
0.18
0.11
0.07
0.24
0.46
3.37
1.39
2.1
1
DMSO
2-propanol
2-propanol
H2O
12.2
12.3
19.4
39.4
1
2-propanol
ySolution was PBS (0.01 M, pH 7) containing the desired co-solvent (10% at most). Measurements were performed in the presence of the same amount
of enzyme at 20 ꢁC. Therefore, specificity constant was obtained from dividing Vmax to Km.
zTo obtain specificity constant ratio, the lowest specificity constant was considered 1.
(A)
(B)
1.2
0.9
0.6
0.3
0
Figure 2. (A) Velocity curves of the aniline
diazo dyes during the reaction with HRP in
the presence of a constant amount of enzyme,
substrate (55 mM) in PBS (0.01 M, pH 7) at
20 ꢁC. S.S ¼ for steady state. (B) Overlaid
spectra of 3d at pH 4.5, 7, and 9 at 20 ꢁC. (C)
Detection limit for HRP in the presence of 3d
(20 mM) in PBS (0.01 M, pH 7) at 20 ꢁC.
1.7
1.4
1.1
0.8
0.5
S.S
3d
3e
3c
3b
3a
50
150
250
350
220
320
420
520
620
Time (s)
Wavelength (nm)
(C)
0.18
0.12
0.06
0
y = 0.3299x + 0.0021
R2 = 0.9956
0.00 0.10 0.20 0.30 0.40 0.50 0.60
[HRP] nmol.mL-1
spectrophotometers. In contrast, HRP reactions with compounds 3 aromatic ring, the amino group of aniline is protected by a
are kinetically slower so progress of the reaction can be easily sulfomethyl group, compound 2. Finally, the coupling reaction
monitored spectrophotometerically (Figure 2A). Reasons for the between 1 and 2 results in formation of high yields of the diazo
difference in the rates of the HRP reactions with diazo derivatives dyes of aniline 3.
of aniline and phenols have been extensively discussed36. It is
understood that the rate of the oxidation of compounds 3 by HRP
Development of the peroxidase assay
is influenced by the nature of the substituent Y (Table 2).
Derivatives of 3 carrying electron donating substituents (EdS) Figure 3(A–E) provide the overlaid spectra collected during the
have faster reactions with HRP and produce steeper velocity oxidation of compounds 3 by HRP. The UV-visible spectrum of
curves, while those carrying electron withdrawing substituent each dye shows only one peak in the broad region between 300
(EwS) show longer steady state as a result of slower reactions and 550 nm with a lmax around 380–400 nm. The optical density
(Figure 2A).
of each dye decreases at its lmax while oxidized by HRP. This
Although some derivatives of 3 are commercially available, allows the progress of the reaction to be monitored directly by
the synthetic route to these compounds is not complicated. following the depletion of the substrate. Figure 2(A) shows that
Therefore, in more advanced and comprehensive kinetic studies the velocity curve of the enzymatic reaction reaches a plateau.
where a specific derivative of 3 is required, it is possible to This is because of the advent of another absorbing species
synthesize the desired substrate by following the modified (product molecules) in the reaction mixture. Formation of the
procedure described in the experimental section. Generally, the isosbestic point in the region of 350–366 nm confirms the
synthesis of azo dyes involves a process known as diazo coupling. interference of the product(s) absorption with that of the substrate
In this process, a diazonium salt, such as 1, is coupled with the (Figure 2A). But this phenomenon does not affect the accuracy of
aromatic ring of a phenol or aniline31. In the case of aniline, the this method. Upon enzymatic oxidation of an aniline diazo dye,
diazonium salt can react with both the aromatic ring and the double bond of the diazo functional group (-N ¼ N-) is cleaved
the amino group37. To direct the coupling reaction toward the and the amino group is oxidized to nitro38. The degradation

DOI: 10.3109/14756366.2015.1103234
Spectrophotometric method and routine assay of peroxidase
5
381
(A)
(B)
352
1
1
0.75
0.5
384
350
0.75
0.5
0.25
0.25
0
0
250
350
450
550
650
250
350
450
550
650
wavelength (nm)
379
wavelength (nm)
390
361
363
(C)
(D)
1
1
0.75
0.5
0.75
0.5
0.25
0.25
0
0
250
350
450
550
650
250
350
450
550
650
wavelength (nm)
wavelength (nm)
(E)
400
1
366
0.75
0.5
0.25
0
250
350
450
550
650
wavelength (nm)
Figure 3. Overlaid spectra recorded during the HRP reaction with (A) 3a, (B) 3b, (C) 3c, (D) 3d, (E) 3e in PBS (0.01 M, pH 7) at 20 ꢁC.
products, nitro derivatives of benzene and phenol, have lmax(s) that it is insensitive to pH changes over a broad range of 4.5 to 9
between 350 and 360 nm39. This means that there is about 20– (Figure 2B). This allows 3d to be used for the assay of acidic,
30 nm difference between the lmax(s) of the diazo substrate and its neutral, and basic peroxidases with the same extinction coefficient
corresponding degradation products. This allows direct monitor- value. With 3d as substrate, a detection limit of 50 pmolꢂmLꢀ1
ing of the reaction’s progress and more precise estimation of was achieved for HRP (Figure 2C).
substrate consumption, especially during the early stages of the
reaction.
Some of the peroxidase methods featured in Table 1 are
criticized because of carcinogenicity or mutagenicity of the
Sensitivity of a spectrophotometric method depends on the organic substrates employed. 3d is soluble in water and is listed
sharpness of the adsorption peak and the extinction coefficient (e) under the name of food yellow 6 in the substance services of US
of the chromophore at that peak34. Table 2 provides the extinction Environmental Protection Agency and is approved as a coloring
coefficients of compounds 3 in PBS at pH 7 and 20 ꢁC. agent for food, drugs, and cosmetics40. Considering these features
Compound 3d has the highest extinction coefficient value. and the reproducible linear spectrophotometric responses, 3d is
During the HRP-catalyzed reaction, one isosbestic point forms proposed as the preferred substrate for reliable and routine assay
at 27 nm from the lmax of 3d at 363 nm (Figure 3D). The velocity of peroxidases.
curve of 3d (Figure 2A) shows that steady state was not affected
by the advent of the products in the reaction mixture. As a result,
Comparison of the methods
3d exhibited a long steady state of more than 100 s under the
experimental conditions. A longer steady state infers a slower In addition to the fluorometric assay-kit using AmplexÕ Red41,
reaction rate that is attributed to the presence of the EwS (-SO3H) there are two spectrophotometric methods which are commonly
in the chemical structure. This sulfo substituent also increases the used for assaying peroxidases: TMB and the combination of
solubility of 3d in water (Table 2) obviating the need for a co- phenol and 4AA. The use of phenol in the latter method makes it
solvent. The spectrophotometric behavior of 3d would indicate unpleasant. Phenol is toxic and has low solubility in water15. In

6
F. Mirazizi et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
addition, phenol is oxidized by both tyrosinase and laccase34,35
and its reaction with 4AA is not specific which means that 4AA
can produce the quinoid product by reacting with other phenolic
2
1.5
1
substances that can be present in natural extracts16,42
.
The method based on TMB has several advantages as
compared to phenol and 4AA. The TMB method relies on a
single organic substrate24 so there is no need to use a helper
molecule such as 4AA to produce a chromophoric product. The
extinction coefficient of the enzymatic oxidation product of TMB
is greater than that of the quinoid product of phenol oxidation in
the presence of 4AA24. TMB was identified as a suitable substrate
as a result of several studies which were carried out on the
enzymatic oxidation of aniline derivatives. Most of the anilines
that have been studied, such as benzidine and its derivatives, are
mutagenic and carcinogenic11,43. TMB has been found to be non-
mutagenic27 although it may be carcinogenic. Nevertheless, there
are some limitations to the TMB method. TMB has a very low
solubility in water so some modifications are required to increase
its solubility. For example, TMB can form a complex with dextrin
that is water soluble and responds to peroxidase44.
0.5
0
0
0.05
0.1
0.15
1/[3 ] M-1
μ
d
Figure 4. Lineweaver–Burk plot of the kinetic data for the HRP reactions
with various concentrations (10–100 mM) of 3d (R2 ¼ 95.3,
SEMax
¼
0.006) in PBS (0.01 M, pH 7) at 20 ꢁC. SEMax reports the
maximum standard deviation observed for the corresponding data.
molecule by H2O2 in the coordination sphere of the heme iron in
the active site (Figure 5). Due to formation of a bond between
Fe3+ and one of the peroxide oxygens, the O–O bond is
heterolytically cleaved. The hydroxide ion forms a water molecule
and leaves the active site. The bound oxygen participates in a fast
intramolecular electron transfer and acquires two electrons, one
withdrawn from the iron and the other from the porphyrin ring48.
The resulting intermediate, Compound I, is a strong electrophile.
An EdS facilitates the instant electron transfer from the substrate
to the HRP. The specificity constants (kcat/Km) also support faster
reactions of substrates carrying EdS. For instance, in a mixture of
3a and 3d, the molecules of 3a react more than three times faster
than the 3d molecules with HRP. This feature does not guarantee a
lower Km value for the diazo substrate carrying an EdS.
Despite the fact that the substrates with EdS have lower Km
values, compared to those carrying EwS, the data in Table 2
indicate that 3c, which has neither EdS nor EwS, has the lowest
Km value. A correlation between the half potential of the reducing
substrate (ZH) and the rate of the reaction with the activated HRP
has been observed43,49. However, no clear correlation between the
binding constant of the reducing substrate with compound I, and
the rate of the reaction, has been found50. Considering the results
reported here, two assumptions can be proposed to explain the
low Km value for 3C.
First, studies show that there is possibility for formation of an
extra intermediate (Compound III illustrated in Figure 5) at higher
concentrations of hydrogen peroxide or upon direct reaction of
ZH with the enzyme51. Compound III is capable of initiating
some radical reactions52. This phenomenon increases the com-
plexity of HRP reactions and might have affected the kinetic
constants of the substrates examined. But, all enzymatic reactions
were carried out in the presence of a constant amount of H2O2
(0.7 mM) throughout this study. The high reproducibility of the
results and the linear responses over the selected range of
substrate concentrations (Figure 4A and B) do not support this
assumption.
Second, structural examinations show that the binding of the
reducing substrate to the activated HRP involves specific
interactions with the residues in the active site pocket of the
enzyme50. This means that the structural details of ZH have a
strong impact on the observed binding constant. For instance,
when the reducing substrate is ferulic acid, after binding the
aromatic residue of ferulic acid is oriented toward the entrance of
the active site in such a way that the oxygen of the methoxy
substituent is involved in hydrogen binding with some residues in
the active site pocket53. Compound 3 has a planar structure
similar to that of ferulic acid. The methoxy group of ferulic acid is
The enzymatic action of HRP on TMB produces an unstable
)
chromophoric product (lmax ¼ 653 nm, e ¼ 39 000 Mꢀ1 cmꢀ1 24,44
as well as for dextrin complex. The reaction must be acidified to
produce a stable product. This makes the method a two-step
method but results in formation of a stable chromophoric product
)
(lmax ¼ 450 nm, e ¼ 59 000 Mꢀ1 cmꢀ1 44,45. Recent studies ser-
iously question accuracy of the product based assay methods for
peroxidase. As mentioned, the first product of the enzymatic
oxidation of an organic substrate (ZH) such as TMB is its radical
(Zꢁ). It has been shown that molecules with radical scavenging
ability such as phenolic acids affect the results of the assay by
reducing Zꢁ species in the assay mixture30,46. The method
described in this paper is based on the oxidation of a stable
substrate. The substrate identified here that is suitable for routine
peroxidase assays, 3d, is soluble in water, chromophoric with a
large extinction coefficient. Fresh solutions can be easily prepared
and the solution is stable even in the presence of hydrogen
peroxide. The oxidation of 3d with peroxidase can be monitored
directly and precisely by following its optical density at 390 nm
over a wide pH range. Given that, this compound is commercially
available under the food dyes category40 implies higher safety
than others in Table 1.
Although all the methods described in Table 1 can be used for
routine peroxidase assays, by using the facile synthetic method
introduced here it is possible to synthesize a suite of substrates
carrying different EdS and EwS at different positions on the
aromatic rings of structure 3. This approach can be very
informative from a kinetics perspective.
Kinetic parameters of aniline diazo substrates
An important advantage of this direct method is that it allows the
accurate study of peroxidase kinetics in a comprehensive manner.
The physicochemical properties of novel peroxidases are very
important2,47. Accurate kinetic studies shed light on the reaction
mechanisms through which peroxidases respond to different
substrates. These studies are usually carried out in a comparative
style using a well-studied peroxidase such as HRP14. Such studies
usually result in important kinetic constants that can be mean-
ingful from a mechanistic perspective. The kinetic parameters of
each diazo substrate (Table 2) were calculated from the corres-
ponding Lineweaver–Burk plots of the kinetic studies shown in
Figure 4.
The instantaneous rate values (dC/min) corroborate that the
dyes carrying EdS exhibit a faster reaction rate with HRP. The
peroxidase reaction starts with the displacement of a water

DOI: 10.3109/14756366.2015.1103234
Spectrophotometric method and routine assay of peroxidase
7
O
Figure 5. Proposed mechanisms for the
catalytic activity of heme peroxidase in the
Fe4+
presence of hydrogen peroxide49
.
Fe2+
H+
H2O
Ferrous state
Compound I
Z
ZH
ZH
O2
H2O2
Fe3+
Z
Ground state
HOO
H+
-
Z, H2O
O
O
O
ZH
Fe4+
Fe3+
Compound II
Compound III
H2O2
H2O
5. Reymond JL, Fluxa VS, Maillard N. Enzyme assays. Chem
Commun 2009;7:34–46.
6. Taylor SV, Kast P, Hilvert D. Investigating and engineering enzymes
by genetic selection. Angew Chem Int Ed Engl 2001;40:3310–35.
at the ortho position of the phenolic ring, while the Y substituent
is at the para position of the second aromatic ring of 3. This may
cause unfavorable interaction(s) between the Y substituent of the
diazo substrates carrying either EdS or EwS, regardless of its
nature, and the HRP active site pocket.
´
7. Fortea MI, Pellicer JA, Serrano-Martınez A, et al. Red cabbage
(Brassica oleracea) as a new source of high-thermostable peroxidase.
J Agric Food Chem 2012;60:10641–8.
8. Bloom JD, Meyer MM, Meinhold P, et al. Evolving strategies for
enzyme engineering. Curr Opin Struct Biol 2005;15:447–52.
9. Leemhuis H, Kelly RM, Dijkhuizen L. Directed evolution of
enzymes: library screening strategies. IUBMB Life 2009;61:222–8.
10. Childs RE, Bardsley WG. The steady-state kinetics of peroxidase
with 2,20-azino-di-(3-ethyl-benzthiazoline-6-sulphonic acid) as
chromogen. Biochem J 1975;145:93–103.
11. Bomhard EM, Herbold BA. Genotoxic activities of aniline and its
metabolites and their relationship to the carcinogenicity of aniline in
the spleen of rats. Crit Rev Toxicol 2005;35:783–835.
Conclusion
A new method was developed for assaying peroxidase activity
using diazo derivatives of aniline with the general structure of 3 as
the substrate. Using this method, peroxidase activity can be
measured directly through oxidation of the chromophoric sub-
strate. Compounds 3 can be prepared in high yield by a simple
procedure described in this paper. These compounds have rather
sharp absorbance peaks in the visible region and also have
large extinction coefficients. Reaction of HRP with 3d, which 12. Shivakumar A, Rangappa D, Krishna H, Nagaraja P. Development
and kinetic validation of an assay for the quantitative determination
of peroxidase: application in the detection of activity in crude plant
is a water-soluble food dye with an extinction coefficient of
32 880 Mꢀ1ꢂcmꢀ1 at 390 nm at a pH of 4.5 to 9, was introduced
tissues. Enzyme Microb Technol 2010;47:243–8.
´
for routine peroxidase assays. However, any of the compounds 3
13. Martıne Enriquez ME, Del Villar A, Chauvet D, et al. Acute toxicity
of guaiacol administered subcutaneously in the mouse. Proc West
Pharmacol Soc 2009;52:92–3.
can be used in comprehensive peroxidase kinetic studies. Kinetics
parameters introduced in this work can be used for comparative
kinetic studies of novel peroxidases.
14. Farhadi S, Haghbeen K, Marefatjo MJ, et al. Anionic peroxidase
production by Arnebia euchroma callus. Biotechnol Appl Biochem
2011;58:456–63.
15. Michaowicz J, Duda W. Phenols – sources and toxicity. Pol J
Environ Stud 2007;16:347–62.
16. Metelitza DI, Litvinchuk AV, Savenkova MI. Peroxidasecatalyzed
co-oxidation of halogen-substituted phenols and 4-aminoantipyrine.
J Mol Catalysis 1991;67:401–11.
17. Martelli A, Robbiano L, Carrozzino R, et al. DNA damage induced
by 3,30-dimethoxybenzidine in liver and urinary bladder cells of rats
and humans. Toxicol Sci 2000;53:71–6.
Acknowledgements
This work was carried out in the Chemistry and Biochemistry laboratories
of the National Institute of Genetic Engineering and Biotechnology. The
collaboration with Dr Richard W. Smith and Mrs. Janet Venne in the Mass
Spectroscopy and WATSPEC Nuclear Magnetic Resonance Spectroscopy
units at the University of Waterloo is greatly appreciated.
Declaration of interest
18. Fornera S, Yazawa K, Walde P. Spectrophotometric quantification of
lactose in solution with a peroxidase-based enzymatic cascade
reaction system. Anal Bioanal Chem 2011;401:2307–10.
19. Chichirau A, Flueraru M, Chepelev LL, et al. Mechanism of
cytotoxicity of catechols and a naphthalenediol in PC12-AC cells:
the connection between extracellular autoxidation and molecular
electronic structure. Free Radic Biol Med 2005;38:344–55.
The authors report no declarations of interest.
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