Method for Determination of Hydrogen Peroxide
Transcription
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 1. Savage, D. J., The determination of hydrogen peroxide in radiation experiments on aqueous solutions, Analyst 76, 224-226 (1951). 2. Teller, J. D., Direct, quantitative, colorimetric determination of serum or plasma glucose. Abstr. 130th Meeting Am. Chem. Soc., 69C (1956). 3. Huggett, A. St. G., and Nixon, D. A., Use of glucose oxidase, peroxidase, and o-dianisidine in determination of blood and urinary glucose. Lancet ii, 368-370 (1957). 4. Kaplan, N. 0., Enzymatic determination of free sUgars. In Methods in Enzymology 3, S. P. Colowick and N. 0. Kaplan, Eds., Academic Press, New York, NY 1957, pp 107-110. 5. Saifer, A., and Gerstenfeld, S., Laboratory Methods. The photometric microdetermination of blood glucose with glucose oxidase. J. Lab. Clin. Med. 51, 448-460 (1958). 6. Washko, M. E.,and Rice,E. W., Determinationofglucoseby an improved enzymatic procedure. Clin. Chem. 7, 542-545 (1961). 7. Bergmeyer, H. U., Gawehn, K., and Crassl, M., Glucose Oxydase. In Methoden der Enzymatischen Analyse, H. U. Bergmeyer, Ed., VerlagChemie, Weinheim, F.R. G.,1970,p 416. 8. Kleppe, K., The effect of hydrogen peroxide on glucose oxidase from Aspergillus niger. Biochemistry 5, 139-143 (1966). 9. Johnston, R. B., and Baehner, R. L., Chronic granulomatous disease: Correlation between pathogenesis and clinical findings. Pediatrics 48, 730-739 (1971). 10. Salmon, S. E., Cline, M. J., Schultz, J., and Lehrer, R. I., Myeloperoxidase deficiency. Immunologic study of a genetic leukocyte defect. N. EngI. J. Med. 282, 250-253 (1970). 11. Wolff, S. M., Dale, D. C., Clark, R. A., et aL, The Chbdiak-Higashi syndrome: Studies of host defenses. Ann. Intern. Med. 76, 293-306 (1972). 12. Gibson, Q. H., Swoboda, B. E. P., and Massey, V., Kinetics and mechanism of action of glucose oxidase. J. Biol. Chem. 239,3927-3934 (1964). 13. Witte, D. L., Brown, L. F., and Feld, R. D., Effects of bilirubin on detection of hydrogen peroxide by use of peroxidase. Clin. Chem. 24, 1778-1782 (1978).