Journal of Immunological Methods, 126 (1990) 125-133 Elsevier
125
JIM 05429
Assay of the human leukocyte enzymes myeloperoxidase and eosinophil peroxidase * Paula M. Bozeman 1, Douglas B. Learn 2 and Edwin L. Thomas 2,3 i Division of Cardiopulmonary Medicine, and 2 Department of Biochemistry, St. Jude Children's Research Hospital, Memphis, TN 38101, U.S.A., and ~ Department of Biochemistry, University of Tennessee, Memphis, TN 38163, U.S.A. (Received 9 August 1989, revised received 18 September 1989, accepted 20 September 1989)
Conditions were optimized for measuring the activity of myeloperoxidase (MPO) and the eosinophil peroxidase (EPO) with tetramethylbenzidine (TMB) as the substrate. Detergents caused a small increase in the measured activity of the purified enzymes and were required when isolated neutrophils or eosinophils were assayed. ShaW concentration optima were observed with both ionic and non-ionic detergents, Activity was also influenced by halide ions. Bromide or iodide caused up to a 7-fold increase in EPO activity and a 1.5-fold increase in MPO activity. The effect of bromide is notable because the bromide-containing detergent CETAB is often used to extract the enzymes for assay and purification. Stimulation by bromide or iodide was consistent with peroxidase-catalyzed oxidation of the halides to hypohalous acids (HOBr and HOI), which oxidized TMB. MPO catalyzes the oxidation of chloride to hypochlorous acid (HOC1), which also oxidized TMB, but chloride up to 20 mM had little effect on the assay. Both MPO and EPO catalyze thiocyanate oxidation, but the product (HOSCN) was a poor oxidant for TMB, and thiocyanate inhibited the measured activities. Stimulation by bromide or iodide could be used to facilitate detection of EPO and to distinguish between MPO and EPO. Activities could also be distinguished based on the greater sensitivity of EPO to inhibition by thiocyanate, azide, aminotriazole, and dapsone. Methods reported here may prove useful for measuring leukocyte influx into inflamed tissues, detecting MPO or EPO deficiencies, and measuring enzyme synthesis and secretion. Key words.. Myeloperoxidase; Eosinophil peroxidase; Tetramethylbenzidine; Aminotriazole; Dapsone; CETAB
Introduction Correspondence to." E.L. Thomas, Department of Biochemistry, St. Jude Children's Research Hospital, Memphis, TN
38101, U.S.A. * This work was supported by an American Lung Association Training Fellowship, NIH Grants DE 04235, AI 16795, and CA 21765, and the American Lebanese Syrian Associated
Charities. Abbreviations: CETAB and CETAC, the C1- and Br- salts
of hexadecyl(cetyl)trimethylammonium ion; dapsone, 4,4'-diaminodiphenylsulfone; EPO, eosinophil peroxidase; MPO, myeloperoxidase; PBS, phosphate-buffered saline (0.14 M NaCI and 15 mM potassium buffer, pH 7.2); TMB, 3,3',5,5'-tetra-
methylbenzidine.
The hemoprotein peroxidases are a related group of enzymes that catalyze the peroxide-dependent oxidation of inorganic halide ions and many organic compounds. Myeloperoxidase (MPO), found in the secretory compartment of human neutrophils and monocytes, catalyzes the oxidation of chloride (C1-) by hydrogen peroxide (H202) to yield the highly reactive oxidizing and chlorinating agent hypochlorous acid (HOC1). MPO also catalyzes the oxidation of bromide
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
126 (Br-), iodide (I-), and the pseudohalide ion thiocyanate (SCN-). The eosinophil peroxidase (EPO), found in cytoplasmic granules of human eosinophilic leukocytes, is much less active in C1oxidation but highly active with Br-, I-, and SCN-. The role of MPO and EPO in leukocyte function is to use H202 to oxidize halide ions or SCN- and thus to produce an array of antimicrobial oxidizing and halogenating agents. Nevertheless, the ability to catalyze the oxidation of phenols and aromatic amines is shared by the leukocyte peroxidases with plant peroxidases, for which these organic compounds are the physiologic substrates, Many assays based on measuring the rate of H202-dependent oxidation of phenols and aromatic amines have been developed to measure peroxidase activity. The spectrophotometric assay of tetramethylbenzidine (TMB) oxidation (Andrews and Krinsky, 1982; Suzuki et al., 1983) has a number of advantages. First, TMB is less hazardous than many peroxidase substrates. Second, the assay is highly sensitive, and enzyme concentrations of 1 nM are readily assayed. Third, the TMB assay is linear with time for at least 3 min, whereas many other assays are linear for only seconds. With these other assays, continuous recording of the increasing absorbance followed by extrapolation is required to calculate the initial rate, which is proportional to peroxidase concentration. The TMB assay requires only a single measurement at 3 min. Therefore, the assay is not only convenient but, as shown below, permits a detailed evaluation of assay conditions, The first aim of this study was to optimize conditions for the assay of the purified enzymes and the enzyme content of neutrophils and eosinophils. In particular, the effect of halides and detergents was examined. Biological samples usually have high levels of C1- and lower levels of Br-, I-, and SCN-. Secreted fluids have high levels of SCN-. In addition, Br- is often introduced in the form of the detergent CETAB, which has been used to solubilize leukocytes for peroxidase assays and to extract the enzymes for purification. Both CETAB and SCN- have been reported to interfere in peroxidase assays (Bos et al., 1981; Oberg and Paul, 1985; ManssonRahemtulla et al., 1986).
The second aim was to develop methods that could be used to distinguish between MPO and EPO activity. In recent years, peroxidase assays have been used to monitor the influx of leukocytes into sites of infection and inflammation (Bradley et al., 1982; Krawisz et al., 1984; Goldblum et al., 1985; Grisham et al., 1986). It is usually assumed that the activity is that of MPO and indicates neutrophil influx. However, eosinophils are prominent in skin and mucosal tissues and accumulate at sites of allergic reactions and at the periphery of bacterial inflammatory lesions (Bass, 1975; Kazura, 1980). Assays that do not distinguish between MPO and EPO could overlook important differences in the nature of inflammatory processes.
Materials and methods
Reagents CETAB, CETAC, dapsone, fMLP, lactoperoxidase, TMB, Triton X-100, and aminotriazole were from Sigma Chemical Company (St. Louis, MO, U.S.A.). TMB (0.1 M) was dissolved in N,N-dimethylformamide. Dilutions of H202 (Fisher Chemical Company, Pittsburgh, PA, U.S.A.) were prepared in sterile 0.3 M sucrose with 50 mM acetate buffer, pH 5.4. Dapsone (0.1 M) was dissolved in 0.46 M HC1 and diluted to 1 mM in the sucrose/acetate solution, and the pH was readjusted to 5.4. Catalase crystals (Boehringer Mannheim, Indianapolis, IN, U.S.A.) were washed twice in water and dissolved in PBS.
Leukocyteperoxidases MPO and EPO were purified from human leukemic leukocytes by CETAB extraction of isolated cytoplasmic granules, which were treated with the protease inhibitor phenylmethylsulfonyl fluoride, followed by ion-exchange and gel-exclusion chromatography (Bakkenist et al., 1978; Matheson et al., 1981; Wever et al., 1981; J/Srg et al., 1982). The ratio of absorbances at 280, 415, and 430 nm was 1:0.76:0.85 for MPO and 1:1.08:0.44 for EPO at pH 4.7, indicating high purity and little or no cross-contamination (Bakkenist et al., 1978; Carlson et al., 1985; Thomas and Fishman, 1986). Extinction coefficients of
127 89,000 and 112,000 M -1 • cm -1 were assumed for M P O and EPO at 430 and 415 nm (Ehrenberg and Agner, 1958; Bolscher et al., 1984). All absorbance measurements were at 25°C.
Leukocytes Neutrophils were isolated from peripheral blood of normal volunteers by dextran sedimentation of erythrocytes and Percoll (Pharmacia LKB Biotechnology, Piscataway, NJ, U.S.A.) density gradient centrifugation of leukocytes by a modification of the method of Thomas et al. (1986b) in which the 40, 50, 61, 70, and 80% Percoll solutions were replaced by solutions with densities of 1.060, 1.072, 1.086, 1.109, and 1.113, and only cells from the 1.086/1.109 interface were taken as neutrophils, Eosinophils were isolated by a modification of the method of Roberts and Gallin (1985), in which erythrocytes were removed by dextran sedimentation after incubating blood with fMLP, and Percoil density gradient centrifugation was performed twice to separate eosinophils from residual neutrophils and erythrocytes. Neutrophil and eosinophil preparations were at least 97% pure.
Peroxidase assays Assay mixtures were warmed to 37°C, and 0.05 ml TMB was added. Incubations were started by adding 0.2 ml of H202 to give 0.3 m M H202 in a total volume of 3.5 ml containing 1.4 m M TMB, 0.3 M sucrose, and 50 m M sodium acetate buffer, p H 5.4, with 2 × 105 neutrophils, 1 × 105 eosinophils, or 2 - 3 nM MPO or EPO. Leukocytes were added as 0.5 ml of cell suspensions in PBS. When B r - or I - was added, the number of leukocytes or the peroxidase concentration was lowered to compensate for the increased activity. After 3 min at 37°C, incubations were stopped by adding 0.1 ml of 0.3 m g / m l catalase and 3.4 ml of cold 0.2 M acetic acid. Portions (0.6 ml) were diluted with 0.75 ml of 0.2 M acetic acid to give a final 4.5-fold dilution. The diluted mixtures were clarified by centrifugation and absorbance was measured at 655 nm. The enzyme and cell concentrations were chosen to obtain an absorbance increase of about 1 U / 3 min in the diluted mixtures,
Oxidized forms of halide ions Sodium hypochlorite (Fisher Chemical Company) was diluted 10-fold in 0.1 M K O H to about 50 mM, and the concentration was determined by diluting 50-fold into 10 m M taurine in PBS and measuring the 252 nm absorbance of taurinemonochloramine, assuming an extinction coefficient of 430 M - 1. c m - 1 (Thomas et al., 1986a). The solution in K O H was diluted into cold PBS to obtain a 2 m M mixture of hypochlorous acid and hypochlorite ion ( H O C 1 / O C I - ) . Bromine (10 /~1) was added to 97 ml of cold 0.1 M K O H to obtain a 2 m M hypobromite ( O B r - ) reagent. When the O B r - reagent was added to T M B solutions, equal volumes of 0.1 M acetic acid were added to maintain the p H at 5.4. Iodine (12) crystals were incubated overnight at 25°C under water to obtain a saturated (1 mM) solution (Thomas and Aune, 1977).
Results
Effects of detergents and CIPurified M P O and EPO were soluble in the absence of detergents. However, Fig. 1 shows that low levels of either an ionic or a non-ionic detergent (CETAC or Triton X-100) caused a small increase in the measured activities of both en-
, ~i~.~ 100, ~---
~po
CErAC ~ O
"~ ~ ~_
50 0 ~.. ~/
.
. . ...a~ ~
100, Triton X-100 ePO'~ 50 .
.
.
10
.
.
100
1000
Detergent (~glml)
Fig. 1. Effect of detergents on peroxidase activity. Assay mixturescontained MPO (O) or EPO (11) and the indicated concentrations of CETAC (above) or Triton X-100 (below).
128 zymes. The optimum concentration was 3 0 / t g / m l (0.003% W/V) for both detergents. Higher detergent concentrations of 300 / t g / m l (0.03%) or greater were inhibitory. Because similar results were obtained with two enzymes and two detergents, the effects may be related to the solubility of TMB rather than that of the enzymes. CETAC contains CI-, which is a substrate for MPO and, to a lesser extent, EPO. However, the inhibition observed at high CETAC concentrations did not appear to be due to interference by C1-. First, similar inhibition was obtained with CETAC or Triton X-100. Second, CETAC caused similar inhibition of MPO and EPO despite the differing abilities of the two enzymes to catalyze C1- oxidation. Third, Fig. 2 shows that C1- inhibited MPO but not EPO. Fourth, significant inhibition of MPO required C1- concentrations greater than 20 mM, whereas CETAC inhibited at 1 mM (300 /xg/ml; 0.03%) or higher. Therefore, inhibition by CETAC was an effect of the detergent rather than competition between C1- and TMB as peroxidase substrates, Competition between C1- and TMB also did not account for the inhibition of MPO shown in Fig. 2. Instead, the inhibition appeared to be an effect of ionic strength. For example, 30 mM N a z S O 4 inhibited MPO by 21% and stimulated EPO by 12%, similar to the effects of 30 mM NaC1. The results indicated that CETAC or Triton X-100 at 3 0 / ~ g / m l could be used to obtain optimum levels of activity, and that either detergent
/ 100 ~ ~
=-
"
~ EPo
g o "~_ § 50 ~-
~
,
10
r
i
i
i
20 30 40 50 NaCI(raM) Fig. 2. Effect of CI- added as NaC1. Assaymixtures contained MPO (o) or EPO (11), 30 #g/ml Triton X-100, and NaC1.
700
~.=TAo
.~-..._
5oo ~ 300 ,r/g 100 ~';, -"6 0 . . . . ~ 500 ~_
O
- MPO -- . . . .
300 100 o.
.
0.1
. . . . . . 0.3 1 3 10 CETABor KBr(mM)
30
Fig. 3. Effect of Br- added as CETABor KBr. Assaymixtures containedMPO (O) or EPO (O), and CETAB (above) or KBr and 30 ~tg/ml Triton X-100 (below).
could be used at concentrations up to 3 0 0 / z g / m l without strong inhibition. Moreover, the C1- in the C E T A C did not interfere at these CETAC concentrations. Effects o f C E T A B a n d B r -
Fig. 3 shows that when the Br- salt CETAB rather than the C1- salt C E T A C was added, there was a dramatic increase of up to 7-fold in the measured activity of EPO. The optimum CETAB concentration was 3 mM (1.1 m g / m l ; 0.11% w/v). Similar results were obtained by adding B r - as KBr, indicating that B r - rather than the detergent was responsible for the apparent stimulation. Moreover, results similar to those in Fig. 3 were obtained when CETAB was added in the presence of 30 / t g / m l Triton X-100. Concentrations of CETAB or KBr greater than 3 mM caused inhibition, which was associated with formation of white to blue-green precipitates of TMB and its oxidation products. Fig. 3 also shows that 3 mM KBr caused a smaller (1.5-fold) increase in the measured MPO activity. N o stimulation by 3 mM CETAB was observed, probably as a result of a combined stimulation by B r - and inhibition by detergent. The optimum B r - concentration (3 mM) was quite different from the optimum detergent concentration (0.1 mM), as determined with CETAC.
129
Oxidation of TMB by hypohalous acids
600 5oo
.__
400 ~
EPo/~
,
~
3o0 -~ 2oo oEo 100= "~_ 0 ~ lOOq ~ 0.
. . . .
"~
125
JIM 05429
Assay of the human leukocyte enzymes myeloperoxidase and eosinophil peroxidase * Paula M. Bozeman 1, Douglas B. Learn 2 and Edwin L. Thomas 2,3 i Division of Cardiopulmonary Medicine, and 2 Department of Biochemistry, St. Jude Children's Research Hospital, Memphis, TN 38101, U.S.A., and ~ Department of Biochemistry, University of Tennessee, Memphis, TN 38163, U.S.A. (Received 9 August 1989, revised received 18 September 1989, accepted 20 September 1989)
Conditions were optimized for measuring the activity of myeloperoxidase (MPO) and the eosinophil peroxidase (EPO) with tetramethylbenzidine (TMB) as the substrate. Detergents caused a small increase in the measured activity of the purified enzymes and were required when isolated neutrophils or eosinophils were assayed. ShaW concentration optima were observed with both ionic and non-ionic detergents, Activity was also influenced by halide ions. Bromide or iodide caused up to a 7-fold increase in EPO activity and a 1.5-fold increase in MPO activity. The effect of bromide is notable because the bromide-containing detergent CETAB is often used to extract the enzymes for assay and purification. Stimulation by bromide or iodide was consistent with peroxidase-catalyzed oxidation of the halides to hypohalous acids (HOBr and HOI), which oxidized TMB. MPO catalyzes the oxidation of chloride to hypochlorous acid (HOC1), which also oxidized TMB, but chloride up to 20 mM had little effect on the assay. Both MPO and EPO catalyze thiocyanate oxidation, but the product (HOSCN) was a poor oxidant for TMB, and thiocyanate inhibited the measured activities. Stimulation by bromide or iodide could be used to facilitate detection of EPO and to distinguish between MPO and EPO. Activities could also be distinguished based on the greater sensitivity of EPO to inhibition by thiocyanate, azide, aminotriazole, and dapsone. Methods reported here may prove useful for measuring leukocyte influx into inflamed tissues, detecting MPO or EPO deficiencies, and measuring enzyme synthesis and secretion. Key words.. Myeloperoxidase; Eosinophil peroxidase; Tetramethylbenzidine; Aminotriazole; Dapsone; CETAB
Introduction Correspondence to." E.L. Thomas, Department of Biochemistry, St. Jude Children's Research Hospital, Memphis, TN
38101, U.S.A. * This work was supported by an American Lung Association Training Fellowship, NIH Grants DE 04235, AI 16795, and CA 21765, and the American Lebanese Syrian Associated
Charities. Abbreviations: CETAB and CETAC, the C1- and Br- salts
of hexadecyl(cetyl)trimethylammonium ion; dapsone, 4,4'-diaminodiphenylsulfone; EPO, eosinophil peroxidase; MPO, myeloperoxidase; PBS, phosphate-buffered saline (0.14 M NaCI and 15 mM potassium buffer, pH 7.2); TMB, 3,3',5,5'-tetra-
methylbenzidine.
The hemoprotein peroxidases are a related group of enzymes that catalyze the peroxide-dependent oxidation of inorganic halide ions and many organic compounds. Myeloperoxidase (MPO), found in the secretory compartment of human neutrophils and monocytes, catalyzes the oxidation of chloride (C1-) by hydrogen peroxide (H202) to yield the highly reactive oxidizing and chlorinating agent hypochlorous acid (HOC1). MPO also catalyzes the oxidation of bromide
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
126 (Br-), iodide (I-), and the pseudohalide ion thiocyanate (SCN-). The eosinophil peroxidase (EPO), found in cytoplasmic granules of human eosinophilic leukocytes, is much less active in C1oxidation but highly active with Br-, I-, and SCN-. The role of MPO and EPO in leukocyte function is to use H202 to oxidize halide ions or SCN- and thus to produce an array of antimicrobial oxidizing and halogenating agents. Nevertheless, the ability to catalyze the oxidation of phenols and aromatic amines is shared by the leukocyte peroxidases with plant peroxidases, for which these organic compounds are the physiologic substrates, Many assays based on measuring the rate of H202-dependent oxidation of phenols and aromatic amines have been developed to measure peroxidase activity. The spectrophotometric assay of tetramethylbenzidine (TMB) oxidation (Andrews and Krinsky, 1982; Suzuki et al., 1983) has a number of advantages. First, TMB is less hazardous than many peroxidase substrates. Second, the assay is highly sensitive, and enzyme concentrations of 1 nM are readily assayed. Third, the TMB assay is linear with time for at least 3 min, whereas many other assays are linear for only seconds. With these other assays, continuous recording of the increasing absorbance followed by extrapolation is required to calculate the initial rate, which is proportional to peroxidase concentration. The TMB assay requires only a single measurement at 3 min. Therefore, the assay is not only convenient but, as shown below, permits a detailed evaluation of assay conditions, The first aim of this study was to optimize conditions for the assay of the purified enzymes and the enzyme content of neutrophils and eosinophils. In particular, the effect of halides and detergents was examined. Biological samples usually have high levels of C1- and lower levels of Br-, I-, and SCN-. Secreted fluids have high levels of SCN-. In addition, Br- is often introduced in the form of the detergent CETAB, which has been used to solubilize leukocytes for peroxidase assays and to extract the enzymes for purification. Both CETAB and SCN- have been reported to interfere in peroxidase assays (Bos et al., 1981; Oberg and Paul, 1985; ManssonRahemtulla et al., 1986).
The second aim was to develop methods that could be used to distinguish between MPO and EPO activity. In recent years, peroxidase assays have been used to monitor the influx of leukocytes into sites of infection and inflammation (Bradley et al., 1982; Krawisz et al., 1984; Goldblum et al., 1985; Grisham et al., 1986). It is usually assumed that the activity is that of MPO and indicates neutrophil influx. However, eosinophils are prominent in skin and mucosal tissues and accumulate at sites of allergic reactions and at the periphery of bacterial inflammatory lesions (Bass, 1975; Kazura, 1980). Assays that do not distinguish between MPO and EPO could overlook important differences in the nature of inflammatory processes.
Materials and methods
Reagents CETAB, CETAC, dapsone, fMLP, lactoperoxidase, TMB, Triton X-100, and aminotriazole were from Sigma Chemical Company (St. Louis, MO, U.S.A.). TMB (0.1 M) was dissolved in N,N-dimethylformamide. Dilutions of H202 (Fisher Chemical Company, Pittsburgh, PA, U.S.A.) were prepared in sterile 0.3 M sucrose with 50 mM acetate buffer, pH 5.4. Dapsone (0.1 M) was dissolved in 0.46 M HC1 and diluted to 1 mM in the sucrose/acetate solution, and the pH was readjusted to 5.4. Catalase crystals (Boehringer Mannheim, Indianapolis, IN, U.S.A.) were washed twice in water and dissolved in PBS.
Leukocyteperoxidases MPO and EPO were purified from human leukemic leukocytes by CETAB extraction of isolated cytoplasmic granules, which were treated with the protease inhibitor phenylmethylsulfonyl fluoride, followed by ion-exchange and gel-exclusion chromatography (Bakkenist et al., 1978; Matheson et al., 1981; Wever et al., 1981; J/Srg et al., 1982). The ratio of absorbances at 280, 415, and 430 nm was 1:0.76:0.85 for MPO and 1:1.08:0.44 for EPO at pH 4.7, indicating high purity and little or no cross-contamination (Bakkenist et al., 1978; Carlson et al., 1985; Thomas and Fishman, 1986). Extinction coefficients of
127 89,000 and 112,000 M -1 • cm -1 were assumed for M P O and EPO at 430 and 415 nm (Ehrenberg and Agner, 1958; Bolscher et al., 1984). All absorbance measurements were at 25°C.
Leukocytes Neutrophils were isolated from peripheral blood of normal volunteers by dextran sedimentation of erythrocytes and Percoll (Pharmacia LKB Biotechnology, Piscataway, NJ, U.S.A.) density gradient centrifugation of leukocytes by a modification of the method of Thomas et al. (1986b) in which the 40, 50, 61, 70, and 80% Percoll solutions were replaced by solutions with densities of 1.060, 1.072, 1.086, 1.109, and 1.113, and only cells from the 1.086/1.109 interface were taken as neutrophils, Eosinophils were isolated by a modification of the method of Roberts and Gallin (1985), in which erythrocytes were removed by dextran sedimentation after incubating blood with fMLP, and Percoil density gradient centrifugation was performed twice to separate eosinophils from residual neutrophils and erythrocytes. Neutrophil and eosinophil preparations were at least 97% pure.
Peroxidase assays Assay mixtures were warmed to 37°C, and 0.05 ml TMB was added. Incubations were started by adding 0.2 ml of H202 to give 0.3 m M H202 in a total volume of 3.5 ml containing 1.4 m M TMB, 0.3 M sucrose, and 50 m M sodium acetate buffer, p H 5.4, with 2 × 105 neutrophils, 1 × 105 eosinophils, or 2 - 3 nM MPO or EPO. Leukocytes were added as 0.5 ml of cell suspensions in PBS. When B r - or I - was added, the number of leukocytes or the peroxidase concentration was lowered to compensate for the increased activity. After 3 min at 37°C, incubations were stopped by adding 0.1 ml of 0.3 m g / m l catalase and 3.4 ml of cold 0.2 M acetic acid. Portions (0.6 ml) were diluted with 0.75 ml of 0.2 M acetic acid to give a final 4.5-fold dilution. The diluted mixtures were clarified by centrifugation and absorbance was measured at 655 nm. The enzyme and cell concentrations were chosen to obtain an absorbance increase of about 1 U / 3 min in the diluted mixtures,
Oxidized forms of halide ions Sodium hypochlorite (Fisher Chemical Company) was diluted 10-fold in 0.1 M K O H to about 50 mM, and the concentration was determined by diluting 50-fold into 10 m M taurine in PBS and measuring the 252 nm absorbance of taurinemonochloramine, assuming an extinction coefficient of 430 M - 1. c m - 1 (Thomas et al., 1986a). The solution in K O H was diluted into cold PBS to obtain a 2 m M mixture of hypochlorous acid and hypochlorite ion ( H O C 1 / O C I - ) . Bromine (10 /~1) was added to 97 ml of cold 0.1 M K O H to obtain a 2 m M hypobromite ( O B r - ) reagent. When the O B r - reagent was added to T M B solutions, equal volumes of 0.1 M acetic acid were added to maintain the p H at 5.4. Iodine (12) crystals were incubated overnight at 25°C under water to obtain a saturated (1 mM) solution (Thomas and Aune, 1977).
Results
Effects of detergents and CIPurified M P O and EPO were soluble in the absence of detergents. However, Fig. 1 shows that low levels of either an ionic or a non-ionic detergent (CETAC or Triton X-100) caused a small increase in the measured activities of both en-
, ~i~.~ 100, ~---
~po
CErAC ~ O
"~ ~ ~_
50 0 ~.. ~/
.
. . ...a~ ~
100, Triton X-100 ePO'~ 50 .
.
.
10
.
.
100
1000
Detergent (~glml)
Fig. 1. Effect of detergents on peroxidase activity. Assay mixturescontained MPO (O) or EPO (11) and the indicated concentrations of CETAC (above) or Triton X-100 (below).
128 zymes. The optimum concentration was 3 0 / t g / m l (0.003% W/V) for both detergents. Higher detergent concentrations of 300 / t g / m l (0.03%) or greater were inhibitory. Because similar results were obtained with two enzymes and two detergents, the effects may be related to the solubility of TMB rather than that of the enzymes. CETAC contains CI-, which is a substrate for MPO and, to a lesser extent, EPO. However, the inhibition observed at high CETAC concentrations did not appear to be due to interference by C1-. First, similar inhibition was obtained with CETAC or Triton X-100. Second, CETAC caused similar inhibition of MPO and EPO despite the differing abilities of the two enzymes to catalyze C1- oxidation. Third, Fig. 2 shows that C1- inhibited MPO but not EPO. Fourth, significant inhibition of MPO required C1- concentrations greater than 20 mM, whereas CETAC inhibited at 1 mM (300 /xg/ml; 0.03%) or higher. Therefore, inhibition by CETAC was an effect of the detergent rather than competition between C1- and TMB as peroxidase substrates, Competition between C1- and TMB also did not account for the inhibition of MPO shown in Fig. 2. Instead, the inhibition appeared to be an effect of ionic strength. For example, 30 mM N a z S O 4 inhibited MPO by 21% and stimulated EPO by 12%, similar to the effects of 30 mM NaC1. The results indicated that CETAC or Triton X-100 at 3 0 / ~ g / m l could be used to obtain optimum levels of activity, and that either detergent
/ 100 ~ ~
=-
"
~ EPo
g o "~_ § 50 ~-
~
,
10
r
i
i
i
20 30 40 50 NaCI(raM) Fig. 2. Effect of CI- added as NaC1. Assaymixtures contained MPO (o) or EPO (11), 30 #g/ml Triton X-100, and NaC1.
700
~.=TAo
.~-..._
5oo ~ 300 ,r/g 100 ~';, -"6 0 . . . . ~ 500 ~_
O
- MPO -- . . . .
300 100 o.
.
0.1
. . . . . . 0.3 1 3 10 CETABor KBr(mM)
30
Fig. 3. Effect of Br- added as CETABor KBr. Assaymixtures containedMPO (O) or EPO (O), and CETAB (above) or KBr and 30 ~tg/ml Triton X-100 (below).
could be used at concentrations up to 3 0 0 / z g / m l without strong inhibition. Moreover, the C1- in the C E T A C did not interfere at these CETAC concentrations. Effects o f C E T A B a n d B r -
Fig. 3 shows that when the Br- salt CETAB rather than the C1- salt C E T A C was added, there was a dramatic increase of up to 7-fold in the measured activity of EPO. The optimum CETAB concentration was 3 mM (1.1 m g / m l ; 0.11% w/v). Similar results were obtained by adding B r - as KBr, indicating that B r - rather than the detergent was responsible for the apparent stimulation. Moreover, results similar to those in Fig. 3 were obtained when CETAB was added in the presence of 30 / t g / m l Triton X-100. Concentrations of CETAB or KBr greater than 3 mM caused inhibition, which was associated with formation of white to blue-green precipitates of TMB and its oxidation products. Fig. 3 also shows that 3 mM KBr caused a smaller (1.5-fold) increase in the measured MPO activity. N o stimulation by 3 mM CETAB was observed, probably as a result of a combined stimulation by B r - and inhibition by detergent. The optimum B r - concentration (3 mM) was quite different from the optimum detergent concentration (0.1 mM), as determined with CETAC.
129
Oxidation of TMB by hypohalous acids
600 5oo
.__
400 ~
EPo/~
,
~
3o0 -~ 2oo oEo 100= "~_ 0 ~ lOOq ~ 0.
. . . .
"~
