Assay method for myeloperoxidase in human polymorphonuclear leukocytes

ANALYTICAL

132, 345-352

BIOCHEMISTRY

(1983)

Assay Method for Myeloperoxidase in Human Polymorphonuclear Leukocytes KAZUO SUZUKI,' HIROMI OTA, SUMIKO SASAGAWA, TATSUICHIRO Departments

of Pathology 5-2 Hijiyama

SAKATANI, AND TOSHIO FUJIKURA and Medicine, Park, Minadi

Radiation Effects Research Foundation.’ 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 polymorphoas 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 esIn 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 substrate, tetramethylbenzidine (TMB) was used (16.17). ’ To whom all correspondence should be addressed. * The Radiation Effects Research Foundation (formerly Assay conditions such as optimum pH, inthe Atomic Bomb Casualty Commission) was established cubation temperature and substrate concenin April 1975 as a private nonprofit Japanese Foundation, trations have been improved over the TMB supported equally by the Government of Japan through method of Andrews and Krinsky ( 17.18). The the Ministry of Health and Welfare, and the Government use of released MPO as enzyme source is logof the United States, through the National Academy of 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 bacpolymorphonuclear leukocytes; DMB, 3,3’dimethoxyteria are killed ( 19). MPO is released by stimbenzidine; Imp-TMB, improved tetramethylbenzidine ulation of cytochalasin B and fMet-Leu-Phe. method; Org-TMB, original tetramethylbenzidine method; both being potent effecters for lysosomal enPBS, phosphate-buffered saline; HBSS, Hank’s balanced salt solution. zyme release (20). The sensitivity of the MPO

346

SUZUKI

assay has increased and in addition large numbers of samples can be assayed at one time. MATERIALS

AND METHODS

Materials TMB, DMB, and cytochalasin B were purchased from Sigma Chemical (St. Louis). NFormyl - methionyl - leucyl - phenylalanine (Net-Leu-Phe) was obtained from Protein Research Foundation (Osaka, Japan). TMB was dissolved with N,Ndimethylformamide. Dextran (M, 200,000, Nakarai Co., Kyoto, Japan) was dissolved in Dulbecco’s phosphatebuffered saline (PBS). Preparation

of PMN

PMN and erythrocytes were separated from whole peripheral blood with heparin as anticoagulant (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) dextran 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 resuspended in Hank’s balanced salt solution (HBSS). Using the trypan blue exclusion test, 96% cell viability in the final preparation was commonly observed. A final PMN concentration of 2 X lo6 cells/ml of 1.0 ml of HBSS was prepared. Cytochalasin B (5 &ml) and &let-Leu-Phe ( 10e6 M) were added to 1.O ml of the cell suspension. After incubation for 15 min at 37°C with shaking, the suspension was centrifuged at 15OOg for 2 min at 4°C. The supernatant was stored at -20°C until used,

ET AL.

because the enzyme activity was stable under the conditions for a month at least. Measurement

of Enzyme Activity

(a) Improved TMB method (Imp-TMB method). The reaction mixture for MPO consisted of PMN supematant, 1.6 tIIM TMB, 0.3 mM Hz02, 80 mM sodium phosphate buffer (pH 5.4), 8% N,N-dimethylformamide, and 40% PBS in a total volume of 500 ~1. The mixture was incubated for 3 min at 37°C and then immersed into an ice bath. The reaction was terminated by the addition of 1.75 ml of 200 mM sodium acetate buffer (pH 3.0). MPO product was measured in a Nihon Bunko 505 spectrophotometer at a wavelength of 655 nm. The activity was expressed as initial velocity

0

2

4 INCUBATION

6 TIME

6 lmml

FIG. 1. Time course of product formation. The reaction mixture consisted of PMN supernatant, 1.6 mM TMB, 0.3 mM H20r, 80 mM sodium phosphate buffer (pH 5.4), 8% N,Ndimethylformamide, and 40% PBS in a total volume of 500 pl. The mixture was incubated at various times. The reaction was terminated by the addition of 1.75 ml of 200 mM sodium acetate buffer (pH 3.0). Bach point was obtained from a separate tube. (O), Product formation at 37°C. (A), Product formation at room temperature (26°C).

MYELOPEROXIDASE

347

ASSAY

bated at room temperature. Initial velocity of the product formation was continuously measured by increased absorbance at 480 nm in the spectrophotometer. RESULTS

Time Course of Product Formation JI’

0

5 10 15 20 FIME UNTIL STOPPER REAGENT (mm

25 ADDITION

To determine the rate of product formation, a reaction-stopping reagent was added to each tube after various incubation times. The amount of reaction product at each time period was measured at 655 nm absorbance. In Fig. 1, the amount of reaction product after

30

FIG. 2. Time-course of ice bath before addition of stopper. 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 simultaneously. 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 ml of PMN supernatant). (b) Original TMB method (Org-TMB method). The assay method was essentially the same as the method of Andrews and Krinsky (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. (c) DMB method. The reaction mixture consisted of PMN supematant, 0.4 mM DMB, 0.15 mM Hz02, 80 mM sodium phosphate buffer (pH 6.2), and 40% HBSS in a total volume of 2.5 ml. The reaction was started by the addition of PMN supematant and incu-

2 5 2 *0

600 WAVELENGTH

700 lnml

I 0

FIG. 3. Spectra of reaction product. The reaction mixture consisted of PMN supematant, 1.6 mM TMB, 0.3 mM H202, 80 mM sodium phosphate buffer (pH 5.4). 8% N.Ndimethylformamide, and 40% PBS in a total volume. After termination by addition of stopper reagent, the spectrum 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 SUPERNATANT

40 50 AMOUNT ADDED

\

loo 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 tMetLeu-Phe ( 10M6M). (O), ImpTMB method. (A), DMB method.

37°C incubation showed a time-dependent linear increase up to 3 min. The initial velocity of product formation at 37°C incubation was

0

twofold higher than that of incubation at room temperature (26’C). From these results, further measurements to determine the initial

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-dimethylformamide, 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

349

ASSAY

1

1

2

1

3

TMEI CONCENTRATION

4

5

(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-dimethylformamide, 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 time and an incubation temperature at 37°C.

incubation

Termination

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), ImpTMB 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).

of the Reaction

A study was made to determine whether the reaction could be terminated by immersion of the solution in an ice bath. After incubation for 3 min at 37”C, the tubes were immediately immersed in the ice bath. Then a cold stopper reagent was added to the reaction mixture after 0, 5, 10, 15, and 30 min, and the reaction product was measured absorbance at 655 nm. Figure 2 shows that absorbance of the reaction product was unaffected 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 TABLE RELEASED

ET AL. 1

MPO ACWITY IN SUPERNATANT BY VARIOUS CONCENTRATION OF PMN MEASURED BY THREE METHODS MPO Activity

Supematant from PMN (X 1O6 cells/ml) 1 2 3 4

ImpTMB W655lminlml)

Org-TMB W655lminlml)

3.98 6.48 11.1 12.2

0.528 0.848 1.28 1.35

DMB (AA.&min/ml) 0.117 0.192 0.312 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 Amount of Enzyme Source Figure 4 shows MPO activity amount of supematant released from PMN/ml. The MPO activity increased up to 40 JLI by the Imp-TMB method to 100 ~1 by the DMB method.

for the 2 X lo6 linearly and up

TABLE 2 RELEASED MPO IN SUPERNATANT OF PMN STIMULATED BY CLTOCHALASIN B AND fMet-Leu-Phe

MPO Activity (AA6&min/ml) Donor No.

Stimulated

1 2 3 4 5 6

12.2 10.5 6.64 11.1 19.1 14.3

Unstimulated 2.14 1.84 1.43 1.39 1.69 2.60

Note. PMN suspensions (2 X lo6 cells/ml) of six healthy donors were exposed to cytochalasin B (5 &ml) and fMet-Leu-Phe (10e6 M) for 15 min at 37°C. The supernatants were obtained by a centrifugation at 15OOgfor 2 min at 4°C. MPO activity was measured by ImpTMB method. Unstimulated supematant of PMN was obtained from PMN exposed to only cytochalasin B.

Saturation

Curves for H202 and TMB

Figures 5 and 6 show saturation curves for H202 and TMB, both of which are substrates of MPO. The optimal concentration of Hz02 was 0.3 mM. Higher concentrations inhibited the reaction, Another substrate, TMB, recrystallized at concentrations above 2.5 mM, so that decreased MPO activity was observed or higher concentration of TMB might inhibit the enzyme activity. Therefore, 1.6 mM was employed as the standard concentration mixture. Optimal pH The optimal pH for the assay of MPO in PMN is not established and has been reported to vary from pH 4.5 to 7.0 (2,13,15,17,18). Accordingly, we determined the optimal pH for MPO using the Imp-TMB method and DMB method. The buffers used were 80 mM sodium phosphate buffer and 80 mM sodium acetate buffer. As shown in Fig. 7, the optimal pH for MPO was 5.4 by the Imp-TMB method and 5.8 by the DMB method. In both methods, sodium phosphate buffer gave higher values than sodium acetate buffer. Comparison and DMB Sensitivity Imp-TMB,

of Imp- TMB with Org- TMB

Methods between the three methods, the 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, respectively. 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.

351

ASSAY

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 bactericidal activity and in the action of various natural effects of PMN influencing the activation of lysosomal enzymes. It is also useful for clinical examination of MPO dysfunction because it is possible, using small blood volumes, to rapidly assay large numbers of samples.

DISCUSSION

The sensitivity of the original method was improved by more than eightfold when incubated 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 concentrations of DMB (0.4 mM) and H202 (0.15 mM). However, the sensitivity of the ImpTMB 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-

ACKNOWLEDGMENTS Deep appreciation is expressed to Dr. Anthony V. Pisciotta vice chairman, Radiation Effects Research Foundation, for his kind guidance and assistance throughout this study. REFERENCES 1. Klebanoff, S. J. (1968) J. Bucteriol. 95, 2131-2 138. 2. Klebanoff, S. J. (1975) Semin. Hematol. 12, 117-142. 3. Bretz. U., and Baggliolini. M. (1974) J. Cell. Biol. 63,251-269. 4. Weissmann, G., Zurier, R. B., and Hoffstein, S. (1973) Agents Actions 3, 370-379. 5. Becker, E. L., and Henson, P. M. (1973) Adv. Immunol. 17, 93-193. 6. Goldstein, I. M. (1976) Prog. Allergy 20, 301-340. 7. Rosen, H., and Klebanoff, S. J. (I 977) J. Biol. Chem. 252,4803-48 10. 8. Baehner, R. L., Karnovsky, M. J., and Kamovsky. M. L. (1969) J. Clin. Invest. 48, 187-192. 9. Weissmann, G., Zurier, R. B., Spieler, P. J., and Goldstein, I. M. ( 197 I) J. Exp. Med. 134, 149s165s. 10. Henson, P. M. (1971) J. Immunol. 107. 1535-1546.

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