Method for Determination of Hydrogen Peroxide

CLIN. CHEM. 26/5, 658-660 (1980)
Method for Determination of Hydrogen Peroxide, with Its Application
Illustrated by Glucose Assay
Ernst Graf and John T. Penniston1
We describe a simple colorimetric method for determining
micromolar quantities of hydrogen peroxide, based on the
oxidation of iodide in the presence of ammonium molybdate and photometry of the resulting blue starch-iodine
complex. Color development is linearlydependent on
analyte concentration, but only slightly time dependent,
and the color of the complex formed is stable for several
hours. In the range of wavelengths that may be used (570
to 630 nm), lack of interferencefrom other biological
compounds makes thismethod seem suitable for routine
analyses.As one illustrative
application
of the method we
quantitated glucose by measuring hydrogen peroxide
produced from it by glucose oxidase catalysis. This method
of quantitating glucose is more than five times as sensitive
as the commonly used dianisidinemethod. With the appropriate hydrogen peroxide-producing oxidases, this
method may be used to directly measure amino acids,
purines, uric acid, xanthine, and hypoxanthine.
in 0.5 molfL H2S04 [247.2mg
of (NH4)6Mo7024.4H2O
and 5.6
of concentrated
H2S04, diluted to 200 mL with waterj;
starch solution (prepared daily) consisting of 5.0 g of soluble
starch (Lintner) made into a slurry with 10.0 mL of water and
then added, with stirring, to 90 mL of boiling water; hydrogen
peroxide stock solution consisting of 1X mL of 300 g/L hymL
drogen peroxide diluted to 1 L with water (the exact concentration,
determined
iodometrically,
was 8.347 mmolfL; the
thiosulfate solution had been standardized
against potassium
iodate);
2.21 mmolfL
glucose stock solution in 0.1 mol/L
aqueous sodium acetate, pH 5.0; 10 g/L glucose oxidase stock
solution in 0.1 mol/L aqueous sodium acetate,
pH 5.0. Glucose
oxidase (EC 1.1.3.4) type II, from AspergiUus
niger, was
purchased from Sigma Chemical Co., St. Louis, MO 63178; it
had a specific
activity
of 26 kU/g.
Procedure
Standard
hydrogen
peroxide
solutions
were prepared
by
diluting the above stock solution with various amounts
AdditIonal Keyphrases: starch-iodine complex . spectrophotometryother potential applications (diseases and analytes)
determination
of hydrogen peroxide by use
was first reported
by Savage (1). We describe two modifications
of this method
and a more complete
Quantitative
of iodide and starch
of the optimum wavelength, time dependence,
and the significance
of the order in which reagents are
added.
We also describe the application
of the starch-iodine
method to the analysis for glucose, using glucose oxidase to
exemplify how various
substrates that can be acted on by
specific oxidases
to produce hydrogen peroxide may be assayed. Most commonly,
the hydrogen peroxide so generated
is analyzed by the o-dianisidine method (2-7), in which peroxidase catalyzes the oxidation of o-dianisidine (or of some
other suitable chromogenic oxygen acceptor) to form a colored
product with an absorption maximum
at 420 nm. Our method
presents several advantages over the dianisidine method, as
we will discuss.
investigation
Materials and Methods
Materials
All chemicals were reagent grade.
We used the following solutions:
1.0 mol/L KI (prepared
daily); 50 mmolfL HC1; 1.0 mmol/L ammonium
molybdate
Section
of Biochemistry,
ClinicfFoundation,
Department
of
Cell
MN 55901.
‘To whom correspondence
should be addressed.
Received Dec. 7, 1979; accepted Jan. 25, 1980.
Rochester,
658 CLINICALCHEMISTRY,Vol. 26, No. 5, 1980
Biology,
Mayo
of
water. These solutions were used to produce hydrogen peroxide standard curves by the following two methods:
Method A: To 10 zL of standard
hydrogen peroxide solution add, in order, 2.0 mL of HC1, 0.2 mL of KI, 0.2 mL of
ammonium molybdate in H2S04, and 0.2 mL of starch solution, in the concentrations
specified
after adding the KI, measure
1.0-cm cuvets at 570 nm.
Method
B: The
above.
Twenty
the absorbance
minutes
vs water
in
standard
curve was prepared
as in method
waiting period of 20 mm between
of ammonium
molybdate
and the addition
of
A, but with an additional
the addition
starch.
The absorption
maximum of the iodine-starch
determined by scanning a solution as prepared
from 850 to 400 nm with a spectrophotometer.
The color development
four different
termined
and compliance
concentrations
of hydrogen
over a 4-h period by measuring
complex was
by method
A
with Beer’s law of
peroxide
were de-
the absorbance
(vs
water) at 570 nm of standards
prepared
by method A.
Standard
glucose solutions
and standard
glucose oxidase
solutions were prepared
by diluting the stock solutions
with
sodium acetate solution
(0.1 mol/L, pH 5.0). The optimum
glucose oxidase concentration
was determined
by adding 40
iL of glucose (2.2 mmol/L)
to 20 L of glucose oxidase standards and incubating
the mixtures
of 37 #{176}C
for 60 mm. The
hydrogen peroxide generated
was then analyzed as described
under method A. A glucose standard
curve was established
from data obtained
by adding 40 tL of the above glucose
standards
to 20 zL of glucose oxidase (250 mg/L), incubating
the mixtures at 37 #{176}C
for 60 mm, and measuring
the hydrogen
peroxide
produced.
In all glucose determinations
the absorbance
was measured
vs water at 570 nm, in 1.0-cm cu-
vets.
E
C
0
I’,
0
10
a,
U
C
0
0.5
0
4
0
20
1.0
pg
Fig. 1. Standard curves for H202 prepared b method A (0) and
by method B (#{149})
2.4
1.2
0
jig H202/2.6l ml
H202/2.61
ml
Fig. 3. Compliance with Beer’s law after incubation for 1.5 mm
(X), 3 mm (A), 60 mm (S), or 240 mm (0)
Method A was used
Results
A standard
curve for hydrogen peroxide determined
by
method A is shown in Figure 1. The non-zero intercepts
in
Figure 1 are ascribable to the slight turbidity of the starch
solutions (the absorbance was measured vs water). The absorbance was linearly proportional
to hydrogen peroxide up
to concentrations
exceeding 0.1 mg/L. From the slope of the
curve at high concentrations,
we calculated a molar absorptivity of E = 39.45 mmolLcm1.L
at 570 nm. Most of the color
develops within the first 8 mm; the absorbance
then increases
slowly over the next 4 h (Figure 2). The rate of color development
is concentration
independent,
and the linear relationship
between
color and concentration
is maintained
at
various incubation times (Figure 3). Thus, the time-dependent
increase in absorbance
will not be a problem in routine analyses as long as the interval between addition of K! and pho-
which removes both product and enzyme (8). The rea#{235}tion
by
which the enzyme is destroyed
is rather fast because
the
maximum
amount of hydrogen peroxide is observed at the
same enzyme concentration,
even when K! and ammonium
molybdate are present during the glucose oxidase reaction.
The optimum glucose oxidase concentration was used to set
up the standard curve for glucose assay (Figure 5). The curve
is linear down to 0.6mg of glucose per liter. From the slope we
E
C
0
tometric measurement of standards and unknown samples
does not vary by more than ito 2 mm. Sensitivity
may be increased when hydrogen peroxide is determined by method B
(Figure i); the absorptivity
calculated from this slope was
0
a,
a
C
0
.0
0
58.38 mmol’.cm”L.
In
.0
The optimum amount of glucose oxidase for the determination of 15.88 xg of glucose in 2.66 mL of assay medium was
5.0 zg (Figure 4). At higher enzyme concentrations the amount
of assayable hydrogen peroxide decreases
to less than 10% of
the maximum value, probably because of the oxidation of
methionine residues of glucose oxidase by hydrogen peroxide,
4
jig Glucose Oxidase added
Fig. 4. Effects of glucose oxidase concentration on colordevelopment of 15.9 g of glucose in a total volume of 2.66 mL
H,O, assayed by method A
E
I.
1.0
C
0
E
C
0
0
II’)
a,
a
C
0
0
a,
U
.0
0
C
0
.0
In
.0
4
0
U)
.0
60
120
‘
Time
‘
ISO
‘
‘
240
(mm)
Fig. 2. Rate of color developmentatfourdifferent
H202 concentrations: 0.57 tg (X), 1.14g (A), 1.70 zg (#{149}),
and 2.27 ig
(0) in a total assay medium of 2.61 mL
Method A was used
4
0
0
5
pg Glucose/2.66
ml
Fig. 5. Standardcurveforglucoseinthepresenceof 5 g
glucose oxidase, assayed by method A
CLINICAL CHEMISTRY,Vol. 26, No. 5, 1980
of
859
calculated
corresponds
of 29.14 mmol.cm.L,
to 73.9% oxidation of the glucose.
an absorptivity
which
Discussion
Our method for determining
concenand easily
adaptable to routine analysis for hydrogen peroxide. A simple
assay for hydrogen peroxide and peroxidase may be useful in
the study of certain dermatological disorders such as chronic
granulomatous disease, myeloperoxidase
deficiency, and
Ch#{233}diak-Higashi
syndrome. These diseases may be caused
by an inability to generate hydrogen peroxide (9), a lack of
eosinophil peroxidase (10), and a lack of myeloperoxidase (11),
respectively.
In our application of this method, determinations of glucose
concentrations with glucose oxidase produced hydrogen
peroxide in a yield of 73.9% of theoretical. This oxidation yield
is large enough for good reproducibility,
and the known inhibitory effects of hydrogen peroxide and of gluconolactone
on glucose oxidase (8, 12) thus do not in any way hinder the
usefulness
of this assay. This method of quantitating glucose
is more than fivefold as sensitive as the commonly used odianisidine method, in which peroxidase (EC 1.11.1.7) catalyzes the oxidation of o-dianisidine to form a colored product
having an absorption maximum
at 420 nm. The peroxidase
reaction is inhibited by bilirubin (13), uric acid, ascorbic acid,
catechols, glutathione, and other hydrogen donors. The absence of peroxidase in our method obviates these problems
of interference.
Our assay for hydrogen peroxide could be particularly useful
for measuring certain metabolites by coupling it to specific
enzymic reactions that yield hydrogen peroxide. To compare
the respective sensitivities in the quantitation of some of these
metabolites, we calculated what concentrations of each would
be required to give an absorbance of 0.5 A at 570 nm in 1.0-cm
cuvets, based on the absorptivity of 58.38 mmol1.cm’.L.
Assuming that 1,5 mL of a sample was analyzed in a total assay
medium of 2.6 mL by method B, and assuming a 75% yield of
hydrogen peroxide, the following concentrations, in milligrams
of metabolite per liter of original sample, are needed to obtain
an absorbance of 0.5: 3.56mg of glucose, 3.13mg of uric acid,
3.01 mg of xanthine, 1.35 mg of hypoxanthine, and 2.71 mg of
amino acids (mean molecular mass of amino acids = 136.75
daltons). The increased sensitivity and favorable wavelength
trations
880
is sensitive,
hydrogen
fast, inexpensive,
CLINICAL CHEMISTRY,
peroxide
reproducible,
Vol. 26, No. 5, 1980
of colorimetric analysis of this coupled enzyme and hydrogen
peroxide assay may make possible an improved quantitative
analysis for xanthine, hypoxanthine, and uric acid. Assays
currently used in the clinical diagnosis of pathological states
such as hyperxanthinuria
are cumbersome and not very reliable. At this time, the development of such an assay is being
investigated at the Mayo Clinic.
This work was supported
by the Mayo
Foundation.
References
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experiments on aqueous solutions, Analyst
76, 224-226 (1951).
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colorimetric determination
of
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(1956).
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peroxide by use of peroxidase. Clin. Chem. 24,
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