Central Laboratory of the Netherlands Red Cross Blood Transfusion Service and Laboratory for ... Some enzymatic characteristics ofhuman eosinophil peroxidase were compared .... 3.5 to 7.0, KI, H202, and enzyme or cell preparation.
INFECTION AND IMMUNITY, May 1981, p. 427431 0019-9567/81/050427-05$02.00/0
Vol. 32, No. 2
Some Enzymatic Characteristics of Eosinophil Peroxidase from Patients with Eosinophilia and from Healthy Donors ARTHUR J. BOS,' RON WEVER,' MIC N. HAMERS,' AND DIRK ROOSI* Central Laboratory of the Netherlands Red Cross Blood Transfusion Service and Laboratory for Experimental and Clinical Immunology of the University of Amsterdam,' and Laboratory of Biochemistry, B.C.P. Jansen Institute, University of Amsterdam,2 Amsterdam, The Netherlands
Some enzymatic characteristics of human eosinophil peroxidase were compared with those of human myeloperoxidase. Both enzymes catalyzed the oxidation of iodide by hydrogen peroxide. This assay proved to be very sensitive; the activity of 100 eosinophils/ml could be measured. The position of the pH optimum of this reaction waslinearly dependent on the logarithm of the iodide/H202 ratio. At the same substrate ratio, this optimum was about 1 pH unit higher for eosinophil peroxidase than for myeloperoxidase. This difference may be related to the action of myeloperoxidase inside an acidified phagolysosome as opposed to the extracellular action of eosinophil peroxidase on the surface of certain parasites. Under defined conditions (KI, 1.4 mM; H202, 0.18 mM; cetyltrimethylammonium bromide, 0.008% [wt/vol]; pH 6), the activity of eosinophil peroxidase could be measured in a mixed granulocyte suspension independently of myeloperoxidase. Eosinophils from patients with eosinophilia were found to contain as much peroxidase activity as did eosinophils from healthy donors. No enzymatic differences in eosinophil peroxidase were found between the two types of donors. Eosinophils as well as neutrophils from human blood contain large amounts of peroxidase; these peroxidases differ immunologically (19), genetically (13, 18), and spectroscopically (22) from each other. The peroxidase of the neutrophil, myeloperoxidase, is considered to be involved in defense against microorganisms by forming bactericidal products from H202 and halide ions (12). Eosinophils probably play a role in defense against parasites. This can be deduced from the increase in blood eosinophils after helminthic parasite infestation and from the observation that anti-eosinophil serum impairs the defense of preimmunized mice against larvae of Schistosoma mansoni or Trichinella spiralis (11, 14). Moreover, human eosinophils are able to damage schistosomulae in vitro (4, 10). In electron microscope studies, it has been shown that eosinophils adhere to helninth larvae and subsequently discharge their granule content on the surface of the parasite (5, 15). Thus, it is conceivable that eosinophil peroxidase, in analogy with the action of myeloperoxidase, damages
the fact that in these studies eosinophils were not obtained from healthy human donors, but from patients with eosinophilia. The latter cells seem to be activated in comparison with eosinophils from healthy donors (2a). In the present study, we have compared some enzymatic characteristics of myeloperoxidase with those of eosinophil peroxidase from patients with eosinophilia and from healthy donors. With iodide as the electron donor, a very sensitive assay was developed. Under defined conditions, even small amounts of eosinophil peroxidase could be measured independently of myeloperoxidase. In this way, it was found that eosinophils from healthy donors contain as much peroxidase activity as do eosinophils from patients with eosinophilia.
MATERIALS AND METHODS Myeloperoxidase was purified from human blood leukocytes as described elsewhere (2). The concentration was determined spectrophotometrically, using an absorbancy coefficient of 89 mM-'. cm-' at 430 nm (8); purity was determined from the ratio of absorbancy at 430 nm to absorbancy at 280 nm (A430/A,80), which was 0.7 to 0.8. Horseradish peroxidase was obtained from Boehringer Mannheim GmbH, Mannheim, West
parasites by reacting with H202 and a halide. Indeed, eosinophil peroxidase can utilize H202 and iodide to kill schistosomula in vitro (lla). Germany. In previous studies, the enzymatic activity of Fresh blood was obtained from healthy donors and eosinophil peroxidase has been compared with from patients with mild to severe eosinophilia of varthat of myeloperoxidase (9, 16, 21); the interpre- ious etiologies. Granulocytes were isolated as detation of these results is hampered, however, by scribed previously (20). These granulocyte prepara427
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tions contained 1.3 to 89% eosinophils. In each preparation, 1 to 10% lymphocytes were present; these cells do not contain peroxidase. The remaining cells were neutrophils. The cell and enzyme preparations were suspended in 25 mM phosphate buffer (pH 7.0). The cell preparations were freeze-thawed three times, homogenized with a Potter homogenizer, and sonicated. To some portions, CETAB (cetyltrimethylammonium bromide; Fluka AG, Buchs, Switzerland) was added (1.6 to 2% [wt/vol]). Oxidation of iodide was measured by the formation of I3- on a Cary 219 spectrophotometer at 360 nm and room temperature. The reaction medium contained Na2SO4 (0.2 M), phosphate-citrate buffer (50 mM), pH 3.5 to 7.0, KI, H202, and enzyme or cell preparation. Solutions of KI and H202 were freshly made each day. The H202 solutions were prepared by dilution of a 30% stock solution (Brocades-ACF, Maarssen, The Netherlands); the concentration was determined spectrophotometrically by using an absorbance coefficient of 81 mM-'.cm-' at 230 nm (17). In most experiments, the reaction medium contained 1.4 mM KI and 0.18 mM H202. The CETAB concentration (if present) in the reaction medium was between 0.006 and 0.03% (wt/vol); in most experiments, it was 0.008%. The reaction was started by addition of the enzyme or cell preparation in a dilution that gave absorbance changes of about 0.15/min. RESULTS Use of CETAB in the oxidation of iodide. CETAB was added to most cell and enzyme preparations because this detergent is known to
INFECT. IMMUN.
with that of 13. From these observations, we concluded that the enzyme kinetics of the peroxidases could be studied by the oxidation of iodide in the presence of CETAB under the following conditions: I-, 0.48 to 5.6 mM; H202, 0.027 to 0.27 mM; and CETAB, 0.006 to 0.03%
(wt/vol). Oxidation of iodide by myeloperoxidase and eosinophil peroxidase. To study the oxidation of iodide by eosinophil peroxidase, we used a granulocyte preparation, containing 89% eosinophils, 6% neutrophils, and 5% lymphocytes, from a patient with severe eosinophilia of unknown etiology. This eosinophil preparation, as well as isolated myeloperoxidase, oxidized iodide in the presence of H202; at fixed substrate concentrations, sharp pH optima were observed. Addition of CETAB had no effect on the position of the pH optima (not shown). CETAB caused a 2.2-fold increase in the AA360 with isolated myeloperoxidase due to the formation of the I3 -CETAB complex (Table 1). The activity of the eosinophil preparation was increased 5.5fold by the addition of CETAB; this extra increase in activity can probably be ascribed to better solubilization of eosinophil peroxidase. This assay is very sensitive (Table 1); the activity of 100 eosinophils/ml could be measured. Incubation of a CETAB-lysed eosinophil preparation for 15 min at 100°C or addition of sodium azide (final concentration, 1 mM) completely abolished the iodide oxidation. In the oxidation of chloride or bromide by myeloperoxidase, a linear relationship exists between the logarithm of the initial halide/H202 ratio and the optimal pH of the reaction (1, 23).
dissolve myeloperoxidase (2, 7) and eosinophil peroxidase (6, 7) very well. Moreover, the addition of CETAB to low concentrations of myeloperoxidase and eosinophils (
