ANALYTICAL BIOCHEMISTRY 132, 345-352 (1983)

Assay Method for Myeloperoxidase in Human

Polymorphonuclear Leukocytes

KAZUO SUZUKI,' HIROMI OTA, SUMIKO SASAGAWA,

TATSUICHIRO SAKATANI, AND TOSHIO FUJIKURA
Departments of Pathology and Medicine, Radiation Effects Research Foundation.’
5-2 Hijiyama Park, Minadi Ward. Hiroshima 730. Japan

Received January 3, 1983

A simple assay method for measuring myeloperoxidase (MPG) has been developed. MPG is
found in polymorphonuclear leukocytes and is important as a bactericidal agent in the presence
of Hz02 and halide ions. This improved assay method is based on work of Andrews and Krinsky
using tetmmethylbenzidine (TMB) a noncarcinogenic substrate. By assaying MPG under optimal
conditions of TMB at 1.6 mhi, H202 concentration of 0.3 mM, pH 5.4, and incubation temperature
of 37°C. sensitivity of MPO measurements increased eightfold in comparison with the original
TMB method. A method has been established to determine absorbance at 655 nm of the reaction
mixture by incubation for 3 min and then stopping the reaction by the addition of pH 3.0 buffer.
An attempt was also made to raise the sensitivity by using 3,3’-dimethyoxybenzidine (DMB), a
carcinogenic substrate. The improved TMB method was 34 times more sensitive than the DMB
method.
KEY WORDS:myeloperoxiti, tetramethylbenzidine; polymorphonuclear leukocytes: lysosomal
enzyme.

Myeloperoxidase (MP0)3 in polymorpho- as a consequence of phagocytosis. When PMN

nuclear leukocytes (PMN) plays an important are stimulated by various stimulants, MPO is
role in killing bacteria using halide ions as a released with other Iysosomal enzymes from
cofactor (1,2). MPO, which accounts for 5% the cells (4-10).
(by total dry cell weight) of PMN, is an es- In the assay of MPO, the method employing
sential enzyme for normal PMN function. 3.3’-dimethoxybenzidine (DMB) as substrate
MPO is localized in the azurophil granules in has been widely used ( 1 l- 15). Because DMB
PMN (3) and is released in phagolysosomes was a known carcinogen, an alternate sub-
strate, tetramethylbenzidine (TMB) was used
’ To whom all correspondence should be addressed. (16.17).
* The Radiation Effects Research Foundation (formerly Assay conditions such as optimum pH, in-
the Atomic Bomb Casualty Commission) was established cubation temperature and substrate concen-
in April 1975 as a private nonprofit Japanese Foundation,
trations have been improved over the TMB
supported equally by the Government of Japan through
the Ministry of Health and Welfare, and the Government method of Andrews and Krinsky ( 17.18). The
of the United States, through the National Academy of use of released MPO as enzyme source is log-
Sciences under contracts with the Department of Energy. ical. This is because MPO is released into
r Abbreviations used: MPO, myeloperoxidase; PMN, phagosomes (extracellular sites), where bac-
polymorphonuclear leukocytes; DMB, 3,3’dimethoxy-
teria are killed ( 19). MPO is released by stim-
benzidine; Imp-TMB, improved tetramethylbenzidine
method; Org-TMB, original tetramethylbenzidine method; ulation of cytochalasin B and fMet-Leu-Phe.
PBS, phosphate-buffered saline; HBSS, Hank’s balanced both being potent effecters for lysosomal en-
salt solution. zyme release (20). The sensitivity of the MPO
346 SUZUKI ET AL.

assay has increased and in addition large because the enzyme activity was stable under
numbers of samples can be assayed at one the conditions for a month at least.
time.
Measurement of Enzyme Activity
MATERIALS AND METHODS
(a) Improved TMB method (Imp-TMB
Materials
method). The reaction mixture for MPO con-
TMB, DMB, and cytochalasin B were pur- sisted of PMN supematant, 1.6 tIIM TMB, 0.3
chased from Sigma Chemical (St. Louis). N- mM Hz02, 80 mM sodium phosphate buffer
Formyl - methionyl - leucyl - phenylalanine (pH 5.4), 8% N,N-dimethylformamide, and
(Net-Leu-Phe) was obtained from Protein 40% PBS in a total volume of 500 ~1. The
Research Foundation (Osaka, Japan). TMB mixture was incubated for 3 min at 37°C and
was dissolved with N,Ndimethylformamide. then immersed into an ice bath. The reaction
Dextran (M, 200,000, Nakarai Co., Kyoto, was terminated by the addition of 1.75 ml of
Japan) was dissolved in Dulbecco’s phosphate- 200 mM sodium acetate buffer (pH 3.0). MPO
buffered saline (PBS). product was measured in a Nihon Bunko 505
spectrophotometer at a wavelength of 655 nm.
Preparation of PMN The activity was expressed as initial velocity
PMN and erythrocytes were separated from
whole peripheral blood with heparin as an-
ticoagulant (20 units/ml of blood) of healthy
volunteers. A Lymphoprep (Nyegaard Co.,
Oslo, Norway) density gradient was used as
described by Boyum (21). Erythrocytes were
sedimented from PMN with 1.5% (w/v) dex-
tran at room temperature for 10 min. The
supernatant was centrifuged at 400g for 10
min at 2O’C. A 0.75% ammonium chloride
solution containing 20 mM Tris-HCl buffer
(final pH 7.2) and 0.25% autologous plasma
was added to the solution and gently mixed
for 5 to 10 min at 37°C to lyse any remaining
erythrocytes. Plasma was added to maintain
cell viability and decrease aggregation. PMN
were washed twice in PBS and centrifuged at
35Og for 10 min at 20°C. The cells were re-
suspended in Hank’s balanced salt solution
0 2 4 6 6
(HBSS). Using the trypan blue exclusion test, INCUBATION TIME lmml
96% cell viability in the final preparation was FIG. 1. Time course of product formation. The reaction
commonly observed. A final PMN concen- mixture consisted of PMN supernatant, 1.6 mM TMB,
tration of 2 X lo6 cells/ml of 1.0 ml of HBSS 0.3 mM H20r, 80 mM sodium phosphate buffer (pH 5.4),
was prepared. Cytochalasin B (5 &ml) and 8% N,Ndimethylformamide, and 40% PBS in a total vol-
&let-Leu-Phe ( 10e6 M) were added to 1.O ml ume of 500 pl. The mixture was incubated at various
of the cell suspension. After incubation for 15 times. The reaction was terminated by the addition of
1.75 ml of 200 mM sodium acetate buffer (pH 3.0). Bach
min at 37°C with shaking, the suspension was point was obtained from a separate tube. (O), Product
centrifuged at 15OOg for 2 min at 4°C. The formation at 37°C. (A), Product formation at room tem-
supernatant was stored at -20°C until used, perature (26°C).
 MYELOPEROXIDASE ASSAY 347

bated at room temperature. Initial velocity of

the product formation was continuously mea-
sured by increased absorbance at 480 nm in
the spectrophotometer.

RESULTS
Time Course of Product Formation
To determine the rate of product formation,
JI’ a reaction-stopping reagent was added to each
tube after various incubation times. The
amount of reaction product at each time pe-
riod was measured at 655 nm absorbance. In
Fig. 1, the amount of reaction product after

0 5 10 15 20 25 30
FIME UNTIL STOPPER REAGENT ADDITION
(mm

FIG. 2. Time-course of ice bath before addition of stop-

per. The reaction mixture consisted of PMN supematant,
1.6 mM TMB, 0.3 mM H20z, 80 mM sodium phosphate
buffer (pH 5.4), 8% N,Wdimethylformamide, and 40%
PBS in a total volume of 500 pl. After incubation for 3
min at 37”C, each tube was immersed in ice simulta-
neously. Stopper reagent was added to the tubes after
incubation for 0, 5, 10, 15, and 30 min and absorbance
at 655 nm in the reaction mixture was measured.
1
P
of absorbance increase at 655 nm (AA&mini 2

ml of PMN supernatant). 5
2
(b) Original TMB method (Org-TMB *0

method). The assay method was essentially

the same as the method of Andrews and Krin-
sky (17,18). The reaction mixture for MPO
consisted of PMN supernatant, 0.88 mM
TMB, 5 mM H202, 50 mM sodium acetate
buffer (pH 4.5) in a total volume of 2.5 ml.
The mixture was incubated at 26°C. The rate
of oxidized product was measured by increase
of absorbance at 655 nm. 600 700
I
0
WAVELENGTH lnml
(c) DMB method. The reaction mixture
consisted of PMN supematant, 0.4 mM DMB, FIG. 3. Spectra of reaction product. The reaction mixture
0.15 mM Hz02, 80 mM sodium phosphate consisted of PMN supematant, 1.6 mM TMB, 0.3 mM
H202, 80 mM sodium phosphate buffer (pH 5.4). 8% N.N-
buffer (pH 6.2), and 40% HBSS in a total vol- dimethylformamide, and 40% PBS in a total volume. After
ume of 2.5 ml. The reaction was started by termination by addition of stopper reagent, the spectrum
the addition of PMN supematant and incu- was measured at 0 and 30 min and 18 h.
348 SUZUKI ET AL.
F
0.f i-

02 ,-

z
E
2z 0.4 1

5
t 0.3
5
F
sl
o 0.2

“I

0.1
/ \

0 \-
0 10 20 30 40 50 loo
SUPERNATANT AMOUNT ADDED f/d

FIG. 4. Linearity of MPO activity in various amounts of PMN supematant. The reaction was carried
out as described under Materials and Methods except for concentration of PMN supematant. Supematant
was isolated after exposure of PMN suspension (2 X lo6 cells/ml) to cytochalasin B (5 &ml) and tMet-
Leu-Phe ( 10M6M). (O), ImpTMB method. (A), DMB method.

37°C incubation showed a time-dependent twofold higher than that of incubation at room
linear increase up to 3 min. The initial velocity temperature (26’C). From these results, fur-
of product formation at 37°C incubation was ther measurements to determine the initial

0 0.5
Hz02 CONCENTRATION fmM)

FIG. 5. H20r saturation curve in MPO activity. The reaction mixture consisted of PMN supematant,
1.6 mkt TMB, various concentrations of H202, 80 mM sodium phosphate buffer (pH 5.4), 8% N,N-di-
methylformamide, and 40% PBS in a total volume of 500 pi. The reaction was terminated by the addition
of 1.75 ml of 200 mM sodium phosphate buffer (pH 3.0).
 MYELOPEROXIDASE ASSAY 349

1 1

1 2 3 4 5
TMEI CONCENTRATION (mM1

FIG. 6. TMB saturation curve in MPO activity. The reaction mixture consisted of PMN supematant,
various concentrations of TMB, 0.3 mM Hz02, 80 mM sodium phosphate buffer (pH 5.4), 8% N,N-di-
methylformamide, and 40% PBS in a total volume of 500 ~1. The reaction was terminated by the addition
of 1.75 ml of 200 mM sodium acetate buffer (pH 3.0).

velocity of MPO were done by using a 3-min

incubation time and an incubation temper-
ature at 37°C.

Termination of the Reaction

FIG. 7. Optimal pH measured by Imp-TMB and DMB
methods. The reaction was carried out as described under
Materials and Methods except for reaction PH. (O), Imp-
TMB method (80 mM sodium phosphate buffer). (A),
ImpTMB method (80 mM sodium acetate buffer). (O),
DMB method (80 mM sodium phosphate buffer). (A),
DMB method (80 mM sodium acetate buffer).
A study was made to determine whether
the reaction could be terminated by immer-
sion of the solution in an ice bath. After in-
cubation for 3 min at 37”C, the tubes were
immediately immersed in the ice bath. Then
a cold stopper reagent was added to the re-
action mixture after 0, 5, 10, 15, and 30 min,
and the reaction product was measured ab-
sorbance at 655 nm. Figure 2 shows that ab-
sorbance of the reaction product was unaf-
fected up to 10 min after immersion in the
ice bath. Additionally, stopper reagent did not
affect on the absorbance. There was no change
in absorbance when the stopper reagent was
added and the tubes remained for 10, 30,60,
and 120 min at 4°C before measurements.
Also, the product spectrum was unchanged
after the reaction mixture was left for 0 and
30 min, or 18 h (Fig. 3) at 4°C.
350 SUZUKI ET AL.

TABLE 1
RELEASED MPO ACWITY IN SUPERNATANT BY VARIOUS CONCENTRATION
OF PMN MEASURED BY THREE METHODS

MPO Activity

Supematant from PMN ImpTMB Org-TMB DMB

(X 1O6 cells/ml) W655lminlml) W655lminlml) (AA.&min/ml)

1 3.98 0.528 0.117

2 6.48 0.848 0.192
3 11.1 1.28 0.312
4 12.2 1.35 0.368

Note. Various concentrations of PMN (1.0 ml) were exposed to cytochalasin B (5 rcg/ml) and fMet-Leu-Phe
( 10m6M) for 15 min at 37°C. The supematants were obtained by a centrifugation at 15OOgfor 2 min at 4°C. MPO
activity in the supematant was measured by Imp-TMB, Org-TMB, and DMB methods.

Linear Increase of MPO Activity with Saturation Curves for H202 and TMB
Amount of Enzyme Source Figures 5 and 6 show saturation curves for
H202 and TMB, both of which are substrates
Figure 4 shows MPO activity for the
of MPO. The optimal concentration of Hz02
amount of supematant released from 2 X lo6
was 0.3 mM. Higher concentrations inhibited
PMN/ml. The MPO activity increased linearly
the reaction, Another substrate, TMB, re-
up to 40 JLI by the Imp-TMB method and up
crystallized at concentrations above 2.5 mM,
to 100 ~1 by the DMB method.
so that decreased MPO activity was observed
or higher concentration of TMB might inhibit
the enzyme activity. Therefore, 1.6 mM was
TABLE 2
employed as the standard concentration mix-
RELEASED MPO IN SUPERNATANT OF PMN ture.
STIMULATED BY CLTOCHALASIN B
AND fMet-Leu-Phe Optimal pH
The optimal pH for the assay of MPO in
MPO Activity (AA6&min/ml) PMN is not established and has been reported
to vary from pH 4.5 to 7.0 (2,13,15,17,18).
Donor No. Stimulated Unstimulated Accordingly, we determined the optimal pH
for MPO using the Imp-TMB method and
1 12.2 2.14 DMB method. The buffers used were 80 mM
2 10.5 1.84
3 sodium phosphate buffer and 80 mM sodium
6.64 1.43
4 11.1 1.39 acetate buffer. As shown in Fig. 7, the optimal
5 19.1 1.69 pH for MPO was 5.4 by the Imp-TMB method
6 14.3 2.60 and 5.8 by the DMB method. In both meth-
ods, sodium phosphate buffer gave higher val-
Note. PMN suspensions (2 X lo6 cells/ml) of six healthy ues than sodium acetate buffer.
donors were exposed to cytochalasin B (5 &ml) and
fMet-Leu-Phe (10e6 M) for 15 min at 37°C. The super- Comparison of Imp- TMB with Org- TMB
natants were obtained by a centrifugation at 15OOgfor 2 and DMB Methods
min at 4°C. MPO activity was measured by ImpTMB
method. Unstimulated supematant of PMN was obtained Sensitivity between the three methods, the
from PMN exposed to only cytochalasin B. Imp-TMB, the Org-TMB, and the DMB
 MYELOPEROXIDASE
methods, was compared. Supematants were
obtained from different concentrations of
PMN on stimulation by both cytochalasin B
and fMet-Leu-Phe. As shown in Table 1, MPO
activities in the supematants of various cell
concentrations were obtained by the three
methods. The ratios of MPO activity between
the three assay methods at all concentrations
were Imp-TMB:Org-TMB:DMB = 34:4: 1, re-
spectively.
Range of Released MPO from PMN
Table 2 shows the values of MPO activity
in supematant of stimulated or unstimulated
PMN by cytochalasin B and fMet-Leu-Phe.
These values were determined by using 10 ~1
of the supematant obtained from 2 X lo6
PMN/ml in six healthy individuals and ranged
from 1.39 to 19.1 units (AA,,,/min/ml). The
lower limit of measurement was 0.90 units.
This suggests that MPO could be assayed using
only 10 ~1 of supematant of 2 X lo6 PMN/
ml by the Imp-TMB method.
DISCUSSION
The sensitivity of the original method was
improved by more than eightfold when in-
cubated at 37°C with 1.6 mM of TMB, 0.3
mM of H202, and 80 mM of sodium phosphate
buffer, pH 5.4. The reaction was effectively
terminated using sodium acetate buffer, pH
3.0. This termination method made it possible
to rapidly assay a large number of samples.
In the DMB method, which was employed
for comparing both TMB methods, MPO was
assayed by determining the optimal concen-
trations of DMB (0.4 mM) and H202 (0.15
mM). However, the sensitivity of the Imp-
TMB method was 34 times higher than the
DMB method.
The optimal pH was 5.4 for the Imp-TMB
method and 5.8 for the DMB method. The
pH in phagolysosomes has been reported to
be from 6 to 4 (22,23). These optimal pH
levels are effective for the action of MPO in
phagolysosomes and are physiologically sig-
ASSAY 351
nificant. It is assumed that the small difference
in optimal pH between the ImpTMB method
and the DMB method is attributable to the
interaction of substrates and MPO.
Released amounts of MPO from different
concentrations of PMN were measured by
both the ImpTMB and DMB methods. MPO
activity was dependent upon increase in cell
concentration. The increases and the ratios
among the three assay methods at the different
concentrations were almost identical (Table
1). This fact illustrates that MPO assayed by
the three methods is the same enzyme.
MPO is an important lysosomal enzyme
involved in bactericidal activity of PMN ( 1,2).
Assay of this enzyme is essential to understand
the role of lysosomal enzymes in PMN bac-
tericidal activity and in the action of various
natural effects of PMN influencing the acti-
vation of lysosomal enzymes. It is also useful
for clinical examination of MPO dysfunction
because it is possible, using small blood vol-
umes, to rapidly assay large numbers of sam-
ples.
ACKNOWLEDGMENTS
Deep appreciation is expressed to Dr. Anthony V. Pis-
ciotta vice chairman, Radiation Effects Research Foun-
dation, for his kind guidance and assistance throughout
this study.
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