Determination of myoglobin based on its enzymatic activity by stopped-flow spectrophotometry

Article abstract of DOI:10.1016/j.saa.2004.06.010

A new method has been developed for the determination of myoglobin (Mb) based on its enzymatic activity for the oxidation of o-phenylenediamine (OPDA) with hydrogen peroxide. Stopped-flow spectrophotometry was used to study the kinetic behavior of the oxidation reaction. The catalytic activity of Mb was compared to other three kinds of catalyst. The time dependent absorbance of the reaction product, 2,3-diamimophenazine (DAPN), at a wavelength of 426 nm was recorded. The initial reaction rate obtained at 40 °C was found to be proportional to the concentration of Mb in the range of 1.0 × 10 ^-6 to 4.0 × 10^-9 mol L^-1. The detection limit of Mb was found to be 9.93 × 10^-10 mol L^-1. The relative standard deviations were within 5% for the determination of different concentrations of Mb. Excess of bovine serum albumin (BSA), Ca(II), Mg(II), Cu(II), glucose, caffeine, lactose and uric acid did not interfere.

Full text of DOI:10.1016/j.saa.2004.06.010

Spectrochimica Acta Part A 61 (2005) 1035–1038
Determination of myoglobin based on its enzymatic activity
by stopped-flow spectrophotometry
∗
Qi Zheng , Zhihong Liu, Ruxiu Cai
Department of Chemistry, Wuhan University, Wuhan 430072, PR China
Received 5 March 2004; received in revised form 8 June 2004; accepted 8 June 2004
Abstract
A new method has been developed for the determination of myoglobin (Mb) based on its enzymatic activity for the oxidation of o-
phenylenediamine (OPDA) with hydrogen peroxide. Stopped-flow spectrophotometry was used to study the kinetic behavior of the oxidation
reaction. The catalytic activity of Mb was compared to other three kinds of catalyst. The time dependent absorbance of the reaction product, 2,3-
◦
diamimophenazine (DAPN), at a wavelength of 426 nm was recorded. The initial reaction rate obtained at 40 C was found to be proportional
−
6
−9
−1
−10
−1
.
to the concentration of Mb in the range of 1.0 × 10 to 4.0 × 10 mol L . The detection limit of Mb was found to be 9.93 × 10 mol L
The relative standard deviations were within 5% for the determination of different concentrations of Mb. Excess of bovine serum albumin
BSA), Ca(II), Mg(II), Cu(II), glucose, caffeine, lactose and uric acid did not interfere.
2004 Elsevier B.V. All rights reserved.
(
©
Keywords: Stopped-flow spectrophotometry; Myoglobin; Enzymatic activity
1
. Introduction
an amperometric immunosensor for the rapid detection of
Mb in whole blood [9]. Hashimoto et al. have detected heme
proteins by chemiluminescence [12]. Liang et al. detected
Mb with high-performance size-exclusion chromatography
[13]. To our knowledge, no stopped-flow spectrophotometric
method has been developed to determine Mb thus far. More-
over, the determination of Mb based on its enzymatic activity,
as is described in the present work, is a novel idea.
The search for satisfying substitutes for HRP is one of the
most interesting trends in enzymatic analysis [14–19]. As is
well know, the active center of the naturally occurring per-
oxidase HRP is a Fe(II) contained heme. Therefore, some
heme derivations and synthetic metalloporphyrins have been
applied in simulating HRP in some peroxidatic reactions.
However, because these substitutes lack a three-dimensional
structure, the peroxidatic activity of such mimetic enzymes is
not very satisfactory, and is seriously inhibited by competing
substrates. Fortunately, as we know, the Mb molecule con-
tains a polypeptide subunit and a single heme as the active
center. And what is more attractive, it has the naturally oc-
curring quaternary structure of proteins. Heme we expected
Mb would be a competent mimetic enzyme of HRP.
Myoglobin (Mb) is a small single heme protein present in
high concentration in both cardiac and skeletal muscle, where
it acts as a transportation vehicle for oxygen. Even a small
degree of muscle cell damage will result in a substantial and
rapid increase in the concentration of Mb in plasma. It has
been found that Mb is released into the circulatory system af-
ter cardiac muscle damage due to myocardial infarction. Mb
is the commercially available marker that most effectively
fits the role of an early marker for AMI [1–3]. Since it plays
such an important role in life, Mb has always been of in-
terest from many aspects. The sophisticated oxygen delivery
function, oxygen binding mechanism and the structure of Mb
have been investigated [4–8]. The determination of Mb is also
a quite significant work, and some methods have been pro-
posedforthepurpose[9–11]. Amongthesereportedmethods,
ELISA is the most widely used. However, it requires multi-
ple steps and is time-consuming. O’Regan et al. developed
∗
Corresponding author. Tel.: +86 278 7684184; fax: +86 278 7647617.
E-mail address: zq 1101@sina.com (Q. Zheng).
1
386-1425/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2004.06.010

1
036
Q. Zheng et al. / Spectrochimica Acta Part A 61 (2005) 1035–1038
It was certified in the present work that Mb did show
quite high peroxidatic activity when it was used to cat-
alyze the oxidation of OPDA by H2O2, which generated
pH 5.6 Na2HPO4–citric acid buffer solution to 5 mL. The
oxidation reaction was monitored by the light absorption at
426 nm.
2
,3-diaminophenazine (DAPN), a yellow product with high
absorptivity. The reaction is as follows:
3. Results and discussion
3.1. UV–vis spectra of the system
Since DAPN was the oxidized product of OPDA with Mb
Therefore, a new spectrophotometric method for the deter-
mination of Mb based on its catalytic activity was set up. The
quantitative determination was built on kinetic analysis by
means of stopped-flow, which has been widely recognized as
a powerful technique for kinetic studies [20]. The catalytic
reaction was studied by measuring the time-dependent ab-
sorbance of DAPN, the initial reaction rate was obtained and
found to be proportional to the concentration of Mb within
catalyzing, the absorption of the product was likely to be
utilized to quantitatively determine the concentration of Mb.
The UV–vis spectra of H2O2, Mb, OPDA and DAPN were
recorded and shown in Fig. 1. It can be seen that DAPN had
a strong absorption band that peaked at 426 nm, while none
of the other three reagents absorbed at this wavelength. It in-
dicated that the measurement was free of interference from
other chemicals in this system. For the purpose of kinetic
analysis, the time dependent absorbance of DAPN at 426 nm
was recorded during the reaction process. As is shown in
Fig. 2, within the first 10 min of the reaction, the absorbance
−
6
−9
−1
a concentration range of 1.0 × 10 to 4.0 × 10 mol L
.
The method is simple, sensitive and reliable for the determi-
nation of Mb.
2
. Experimental
2
.1. Apparatus
The SFA-12 HI-TECH scientific kinetic stopped-flow ac-
cessory (HI-TECH, Japan) was fitted to the UVIKON-941
spectrophotometer (Kontron Instrument, Germany). A circu-
lation water-bath was used to control the temperature in the
stopped-flow module and the cell compartment. Amicrocom-
puter was applied to control the operation of the instrument
and to collect and process the data.
2
.2. Reagents
−
1
Fig. 1. UV–vis spectra of H2O2, Mb, OPDA, DAPN in 0.2 mol L
−
7
−1
Na2HPO4–citric acid buffer (pH 5.6). [Mb] = 1.0 × 10 mol L , [OPDA]
All solutions were prepared using deionized water. Unless
−
4
−1
−3
−1
=
1.0 × 10 mol L , [H2O2] = 1.0 × 10 mol L , [DAPN] = 1.0 ×
−
1
indicated otherwise, all dilutions were made in 0.2 mol L
−4
−1
mol L .
10
Na2HPO4–citric acid (pH 5.6). Mb solutions were pre-
pared by dissolving a certain amount of Mb (Sigma
◦
Chemical Co.) in deionized water and stored below 4 C.
o-Phenylenediamine (Shanghai Chemical Agent, Shang-
−
1
hai, China) was sublimed before use; a 0.1 mol L
o-
phenylenediamine stock solution was prepared by dissolv-
ing sublimed o-phenylenediamine in the buffer solution. The
working solution was prepared daily by diluting the stock so-
lution with buffer to the desired volume. Hydrogen peroxide
solutions were prepared by appropriate dilution of the 30%
solution with water (standardized by titration with KMnO4).
2
.3. Procedures
Each color comparison tube was filled with 0.40 mL
Fig. 2. Single-wavelength stopped-flow traces obtained in 10 min. [Mb]
−
2
−1
of 1.00 × 10 mol L
H2O2, 0.50 mL of 1.00 ×
−7
−1
−3
−1
=
1.0 × 10 mol L , [OPDA] = 1.0 × 10 mol L , [H2O2] = 1.0 ×
−2
−1
−3
−1
1
0
mol L , a proper amount of Mb, and then diluted with
10 mol L .

Q. Zheng et al. / Spectrochimica Acta Part A 61 (2005) 1035–1038
1037
Table 1
Catalytic characteristics of Mb and Hb, ␤CD–hemin, hemin
−
3
−1
−1
−6
−1
6
−1
−1
Catalyst
Km (10 mol L
)
Vmax (min
)
[E] (10 mol L
)
Kcat (10 L mol min
)
Hb
Mb
8.75
20.9
47.2
55.9
1.60
0.86
0.643
0.493
0.250
0.20
1.20
1.20
6.40
4.26
0.536
0.411
␤CD–hemin
Hemin
of DAPN continuously increased as oxidization proceeded.
But there had an obvious lag phase at the very beginning,
which, in our opinion, was considered as the typical induced
period of enzymatic reactions. That is to say, Mb in this re-
action system clearly exhibited enzymatic characteristics.
3
.2. Comparison of the catalytic of Mb and Hb,
␤
CD—hemin, hemin
The enzymatic activity of Mb was compared with that
of Hb, ␤CD—hemin and hemin for the catalysis of the re-
action between 1,2-diaminobenzene and hydrogen peroxide.
The Michaelis–Menten constant Km and the maximum rate
Vmax were obtained from an Eadic–Hofatee graph (ν versus
ν/[substrate]), and the transformation constant Kcat was cal-
culated from the equation Vmax = Kcat [E]0, in which [E]0
is the initial concentration of the catalyst. The Km, Vm and
Kcat values for Mb, Hb, ␤CD—hemin and hemin are listed
in Table 1. The order of catalytic activities of the catalysts,
Hb > Mb > ␤CD—hemin > hemin, was obtained using these
parameters. Since the active center of all four catalysts is just
the same iron-porphyrin, the differences in catalytic activity
are mainly determined by molecular conformation. Hb has
the natural quaternary structure, which may explain why the
activity of Hb is higher than that of Mb. However, as a nat-
ural protein with smaller molecular size, Mb would be less
likely than Hb to block the antigen–antibody immunoreaction
because of the spatial effect. As for ␤CD—hemin, it just sim-
ulates the three-dimensional structure of naturally occurring
proteins through the cavum of ␤CD, so its catalytic active is
less than that of Hb and Mb but still better than hemin, its
prosthetic group model.
−
7
−1
Fig. 3. Effect of pH and buffer system. [Mb] = 1.0 × 10 mol L , [OPDA]
−4
−1
−3
−1
=
1.0 × 10 mol L , [H2O2] = 1.0 × 10 mol L
.
Mb. All absorptions for analysis were measured at a constant
◦
temperature of 40 C.
3
.4. Analytical characteristics and application
Under the optimized conditions, the initial reaction rate
was found to be proportional to the Mb concentration in a
wide range, based on which the kinetic method for determin-
ing Mb was built. The results are listed in Table 2.
The interference of other species in the determination of 2
10 mol L Mb was investigated. The results are listed in
Table 3. The values represent the tolerable amount (in molar
ratio) of foreign species relative to Mb, which resulted in a
relative error less than 5%. Excess of BSA, Ca(II), Mg(II),
−7 −1
×
3
.3. Optimization of experimental variables
Different kinds of buffer solutions with varying pH values
were tested for the reaction. The absorbance of DAPN was
plotted versus pH for the different buffers (Fig. 3). The max-
imum absorption occurred in Na2HPO4–citric acid buffer
within a pH range 5.0–5.6. Therefore, a Na2HPO4–citric acid
buffer with pH 5.0 was chosen for subsequent experiments.
Temperature is a very important factor that cannot be ne-
glected in kinetic analysis. The effect of temperature on the
absorption was studied. As is demonstrated in Fig. 4, the ab-
◦
sorption increased before the temperature reached 40 C. The
◦
decrease in the absorption at temperatures higher than 40 C
−7
−1
Fig. 4. Effect of reaction temperature. [Mb] = 1.0 × 10 mol L , [OPDA]
−
3
−1
−3
−1
might have resulted from the loss of the catalytic activity of
= 1.0 × 10 mol L , [H O ] = 1.0 × 10 mol L
.
2
2

1
038
Q. Zheng et al. / Spectrochimica Acta Part A 61 (2005) 1035–1038
Table 2
Calibration curves for the determination of Mb (n = 7)
Liner range of Mb (mol L 1)
−
Liner equation
r2
[OPNA] (mol L
−1
)
[H2O2] (mol L
−1
)
Time (min)
−
3
−4
(×10
)
(×10
)
−
9
−7
−6
7
4
.0 × 10 to 1.0 × 10
r = 0.054 + 4.26 × 10 [Mb]
0.9987
0.9992
1.0
1.0
8
8
5
3.5
−
7
6
1
.0 × 10 to 1.0 × 10
r = 0.069 + 1.28 × 10 [Mb]
Table 3
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(
The authors gratefully acknowledge financial support
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