ANALYTICAL

BIOCHEMISTRY85,

63-70(1978)

A Controlled-Temperature Apparatus for Measurement Hydrogen Peroxide Production C.O.PAT PATTERSON,*J. B. GLOVER,~AND S. E.

STEVENS,

of

JR.$

* Department of Microbiology, Indiana University, Bloomington, Indiana 47401, I’ Department of Zoology, University of Texas, Austin, Texas 78712, and $ Department of Microbiology and Cell Biology, Pennsylvania State University, University Park, Pennsylvania 16802 Received February 14, 1977; accepted October 3, 1977 An apparatus is described which permits continuous assays of hydrogen peroxide production from biological materials, including whole cells. Temperature in the assay mixture is controlled, for work above or below room temperatures. Data obtained by use of the apparatus are presented, as illustration of the effects of temperature upon peroxide output.

Production of hydrogen peroxide by biological systemsis a well-known phenomenon (l-3). In a few instances the physiological significance of this production is understood; in most casesit is not. At least one organelle, the peroxisome, is believed to function primarily to form and detoxify (decompose) HzOz. Study of peroxide metabolism is complicated by several factors, most notably the small quantities of peroxide produced and the rapidity of decomposition following formation. A suitable assay technique must be rapid, reproducible, capable of being run over a wide temperature range, able to detect peroxide production rates in the nanomole per minute range, and neutral to (neither accelerating nor interfering with) reactions producing H202. Characteristics such as rate and time course of peroxide production may be markedly influenced by the temperature at which the reaction proceeds. In particular, assays of peroxide formation by whole cells may give misleading or incomplete results if run only at temperatures other than optimal growth temperature for that cell type. Various techniques have been developed for measurement of H20z production; these include spectrophotometric assays in which formation of a H,02-peroxidase complex or a H%O,-catalase complex is measured, or in which oxidation of cytochrome c is observed. However, these assaysrequire rather expensive instrumentation. Madly and Chance (4) have provided detailed reviews of these assays. Other techniques have also been developed (5,6). The scopoletin method of Perschke and Broda (7) has found wide acceptance due to its 63

0003-2697/78/0891-0063$02.OW0 Copyri#bt 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved

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sensitivity, stability, and relatively simple instrument requirements. Use of this method to analyze photosynthetic production of peroxide has been reported (1). It is of considerable importance to know the precision and accuracy of the scopoletin method in comparison to older methods. Boveris et al. (8) have recently reported their evaluation of the technique; they carried out side-by-side determinations of mitochondrial peroxide production, using the scopoletin assay, the cytochrome c-peroxidase assay, and other methods; they found that the scopoletin technique consistently indicated lower rates of H,O, production than other techniques, apparently due to initial competition by other reductant sources and peroxide-decomposing reactions. They concluded that precautions are necessary to ensure that the scopoletin assay accurately measures peroxide production rates. We now describe our improved assay apparatus which permits rapid, continuous recording and quantitative estimation of HzOz levels in cell suspensions, in addition to regulation of reaction temperature, including temperatures 20°C above or below ambient room temperature. MATERIALS AND METHODS The entire apparatus is illustrated schematically in Fig. 1. Figure 2 describes schematically the internal construction of the assay chamber. The chamber is constructed of aluminum, with bakelite insulation separating components requiring constant room temperature (phototube) from controlled-temperature portions of the apparatus. Our experience with the apparatus has shown no need to insulate the chamber from room temperature, since water flow through the apparatus is great enough to compensate for heat gain or loss from air. A thermometer immersed in the cuvet measures reaction temperature directly. The fluorometric assay (7) makes use of scopoletin (6-methoxy, 7-hydroxycoumarin), a naturally occurring coumarin derivative, which fluoresces strongly in the 460- to 470-nm region when excited with 365 nm, as from a Hg-vapor lamp. Scopoletin is oxidized by H,O, in the presence of peroxidase so that decrease in scopoletin fluorescence is a direct measure of amount of HzOz. The routine assay reaction mixture contained 1 nmol of scopoletin and 80 units of peroxidase in a total volume of 3.3 ml. Fluorescence intensity from scopoletin is pH dependent; it is thus appropriate to buffer the reaction mix to avoid spurious fluorescence drifts. Details of buffering are given in the legend of Fig. 3. We checked and confirmed the previously reported (7) 1:l stoichiometry for the reaction between reduced scopoletin and hydrogen peroxide. That is, precisely 1 mol of HzOz was required for oxidation of 1 mol of scopoletin, so long as scopoletin was present in at least 20% excess. This value was used for all calculations of peroxide production. The blue-green alga Anacystis m&funs (strain TX20 of our laboratory)

CONTROLLED-TEMPERATURE

FOR HzOz

65

FIG. 1. Schematic diagram ofthe complete controlled-temperature assay system. “UV” is a 100-W mercury-vapor lamp (GE HlOO-A4/T) powered by a GE Autotransformer (15G 3070) connected to a Raytheon Voltage stabilizer. The lamp is enclosed in a metal housing (to protect user from stray uv light) and positioned at a distance chosen for desired intensity (about 26 cm) from the near face of the 1.0 x l.O-cm reaction cuvet “C.” A collimating tube of brass (not shown) may be installed between UV and Fs to decrease scattering of 365nm excitation. “Fs” is a filter holder (attached to assay chamber) containing a Baird-Atomic 365nm interference filter plus a Coming No. 586 transmission filter. For some applications (not involving photosynthetic samples) the less expensive Wratten filter No. 18A may be a suitable replacement for the Baird-Atomic 365nm interference filter. “F,” is a ftlter holder (built into assay chamber) containing a CuSO, solution (1 .O M, 1J-cm pathlength in a quartz cell), a Wratten filter No. 47B, two layers of Wratten tilter No. 2B, and a W&ten tilter No. 2A. This filter combination passes light in the 410- to 470-nm range. “PM” is the housing for an Aminco IP21 phototube, connected to an Aminco multirange photomultipiiermicrophotometer, powered from a Raytheon voltage stabilizer. PM is attached to the assay chamber but insulated from it by a 0.5~cm strip of bakelite so that the phototube operates at room temperature. “Fs” is a filter holder (attached to assay chamber) containing appropriate interference and cut-off filters transmitting chosen wavelengths for activation of peroxidegenerating photoreactions, such as photosynthesis. “I” is a high intensity light source such as a Kodak slide projector or a Bausch & Lomb microscope lamp. A water cell may be mounted between F* and I to remove infrared. A sliding shutter between Fz and I allows on-off control of illumination. “LT” is a Lauda thermostat (model K-2) from Messgerate-Werk-Lauda, West Germany. Its reservoir of water at selected temperature circulates through polyethylene tubing to entrance and exit ports in the wall of the assay chamber. Entrance port is smaller than exit port to avoid pressure increases in water cavity of assay chamber. Dotted lines indicate light paths when the chamber is in operation.

was grown in medium C (9) in a continuous culture (turbidostat) apparatus (10) at 3-o”C, aerated with 1% CO2 in air. It has previously been shown that TX20 cells grown under far-red illumination give greatly increased I&O, production as compared to cells grown under ordinary white light (1). Ceils to be used for the assays of peroxide production reported herein were

PATTERSON,

b

GLOVER,

I

AND STEVENS

I

FIG, 2. Exploded view of assay chamber from side (a) and front (b). Labels correspond to those in Fig. 1. Shown in addition are a spring-loaded, locking iris shutter (S) controlling illumination to PM, the base block (B) which is drilled and tapped for mounting the chamber, and entrance and exit ports (W, and W,) for water at selected temperature. In b, asterisk indicates floor of water cavity surrounding the hollow cuvet holder C. In operation this activity is tilled with circulating water at chosen temperature. Also shown are two of the three

CONTROLLED-TEMPERATURE

20.3’

214’

29

3’

67

FOR He02

33 3’

37 4’

41 7’

FIG. 3. Recorder traces of fluorescence decrease (=H20z production) due to TX20 cells. Cells were grown at 41°C under BCJ lamps providing red illumination. Samples were harvested 1 min prior to use and placed directly into assay mixture without centrifug8tion or washing. A fresh sample was used to obtain each record shown 8bove. Two milliliters of 50 mM Na, K phosphate buffer, pH 7.2, were used in each replicate sample. Approximately 5 min incubation of the complete assay mix including cells in darkness allowed complete stirring (by 1% COx in air, bubbled through a Tellon tube) and also provided an initial baseline. Bubbling continued throughout subsequent illumination. H&production was elicited by red illumination from a Kodak 500 projector fitted with a Baird-Atomic filter, maximum transmission at 620 nm, half-band width 11 nm. For each curve, illumin8Gon intensity was 2.9 mW/cmg as measured by a YSI Model 65 Radiometer with detector inserted into the position normally occupied by the cuvet containing the reaction mix. Slight downward and upward dispMcements from the baselines at the beginning and end of each experimental curve result from imperfect shielding of the phototube from 620-nm illumination and provide a precise record of time of light on and light off. Time scale (abscissa) is identical for all replicates; the bar (5-min interval) provides 8 reference scale. Ordinate indicates qtmntity of peroxide produced by 1 ~1 of cells (measured as packed-cell volume 8Bercentrifugatin into a calibrated cagilkuy). For this assay series, multiplic8tion of ordinate vahres by 180 yields (in nanomolesfliter) concentration of peroxide actually observed.

therefore grown under red light (BCJ photographic safe lamps, output > 660 nm). For experiments, exponential phase cells (-0.6 ~1 cells/ml) were harvested and either centrifuged at 33Wg for 10 min, then resuspended in medium Cm (containing only the major salts), or used directly in the assay mixture without modification. Identical results were obtained from either treatment. CeH concentrations were determined by centrifuging the cell suspension for 1 hr at 22OOg in centrifuge tubes with calibrated capiIlary bottoms. tubular projections from C which permit passage of light aad which are sealed to the outer aluminum w8ll of the water cavity by rubber 0 rings. The circuhu bole in the top oftbe cuvet holder permits removal of the cuvet for cleaning; a met8l fhmge guides rephrcement of the cuvet in its former position. In use, this hole is covered, and the cuvet holdertlms da&em& by an 8bnninum c8p in the center in which a 1.hnm bole is drilled to admit a Teflon tube for aeration and stirring. In a, filter holder F2 has been removed for clarity.

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Scopoletin was purchased from Mann Research Laboratories; scopoletin solutions were prepared in 2-liter quantities (2 mg/liter) and stored frozen until use. Peroxidase (type VI, horseradish) was from Sigma Chemical Co. Assay of hydrogen peroxide at extremely low concentrations may be seriously hampered by poorly cleaned glassware. Since this is a fluorometric assay, careful cleaning is of special importance for the reaction cuvet. We routinely use high-purity water which has been distilled, filtered, demineralized, and redistilled in glass for all washing and solution preparation. For cleaning of glassware, we use a mild liquid detergent, diluted 20: 1 in high-purity water, soaking for 5-6 hr, followed by repeated rinses with high-purity water; glassware is then soaked in chromate cleaning solution for 5 min, rinsed repeatedly with high-purity water, and dried in an oven at 40°C. This cleaning regimen has enabled us to avoid serious problems of contamination. All solutions and reaction mixtures contact only glass or Teflon. RESULTS

AND DISCUSSION

Typical data obtained with this assay apparatus are presented. Effects of temperature are evident in experimental results depicted in Fig. 3, displaying peroxide production from whole, undamaged algal cells. Assay temperatures were selected to span the range of growth temperatures suitable for strain TX20 (11). All prior work measuring HzOz production from this alga was carried out at room temperature (24-26°C). The data of Fig. 3 make it obvious that both magnitude and time course of peroxide output are strongly temperature influenced. In particular, Hz02 production is decreased at higher temperatures which are known to permit higher maximum growth rates for this alga. Results of extensive study of H,O, output from TX20 at room temperature were interpreted (1) as due to a leak or imbalance between photochemical generation of reductant and its utilization in reductive carbon fixation. We have not identified the exact site of peroxide generation (immediate electron donor). Other data (1,9) indicate that peroxide is derived in this blue-green alga from the reducing side of Photosystem I of the photosynthetic apparatus. Boveris et al. (12) have shown that in isolated mitochondrial preparations, ubiquinone is a source of superoxide-free radicals which may then be dismuted to Hz02. But since ubiquinone is believed not to be present in blue-green algae, and since no other endogenous quinone participates in electron transfer at the indicated region in these algae, we think it unlikely that this is the source of our observed peroxide. A more likely candidate is ferredoxin; Telfer ef al. (13) have demonstrated that in isolated chloroplasts, Hz02 is the product of autoxidation of ferredoxin. It is significant that at temperatures allowing higher growth rates, peroxide output is decreased, as would be expected if

CONTROLLED-TEMPERATURE

FOR

H202

69

generation and utilization of reducing power proceeded at more nearly equal rates. In short, these data provide confirmation of a hypothesis in a way completely impossible without availability of temperature control and variation. Rapidity of the individual assay runs and ease of temperature control enable quick construction of curves such as those depicted in Fig. 3. Certain limitations inherent in the assay technique should be mentioned. The method relies upon measurement of fluorescence change attending the enzymatic oxidation of scopoletin. Any electron donor capable of directly reducing H,O, , or of serving as substrate for peroxidase, or of nonenzymatic rereduction of oxidized scopoletin, will interfere with accuracy of the assay. For instance the assay cannot be run in the presence of either ascorbate or dithionite. Likewise, assay conditions are restricted to those which do not interfere with peroxidase activity. Fortunately, horseradish peroxidase has broad temperature and pH optima. However, presence of concentrations of cyanide, sulfide, or heavy metals above 0.01 mM would be expected to interfere with the assay. Further, if run with intact cells as in this case, the assay is strictly extracellular, responding only to peroxide reaching the exterior of the cells. Peroxide-consuming reactions within the cell thus interfere with the assay. In catalasecontaining cells or preparations (as here), measured values of H202 must be regarded as minima. For the experiments reported here, only the presence of catalase and endogenous reductants within the cells interfered with the accuracy of the assay. Control experiments (not shown) indicated that fluorescence change was less than ? 1% during the duration of the assay if cells were omitted, or if peroxidase was omitted, from the reaction mix, or if the complete reaction mix remained in darkness (photosynthetically active illumination never turned on). Likewise, if oxygen was removed from the reaction mix by vigorous bubbling with Nz + CO, before and during illumination, no change in scopoletin fluorescence could be observed. We have found this apparatus and technique very useful for our work with algal cells. In addition, the apparatus is so designed that it can be used for other photochemical or enzymatic reactions producing H,O,. We have been most interested in measuring HIOz concentrations in the nanomolar to micromolar range. Sensitivity is adjustable, both higher and lower than used for this work. The low solubility of scopoletin in water (2 mg/liter) determines the upper end of the sensitivity range (least sensitive condition), while the characteristics of the detecting phototube and filter combinations (response to 460-470 vs response to 365 nm) establish the lower end of the range (most sensitive condition). Within these limits, sensitivity can be readily adjusted by scopoletin concentration. Our experience with the apparatus indicates that picomole amounts of HzOz are readily measured. Thus the technique allows kinetic measurements by a

70

PATTERSON,

nondestructive method, production rates.

GLOVER,

over

AND STEVENS

a wide range of concentrations

and

ACKNOWLEDGMENTS We thank Drs. J. Myers and J. L. Fox for use of their laboratory facilities. This work was supported in part by a grant, PHS SO5 RR 7031, from the Indiana University Biomedical Sciences Support Program to COPP and by Public Health Service postdoctoral fellowship GM-521 17 to SES. Additional support was provided by GM-113OOof the National Institutes of Health.

REFERENCES 1. Patterson, C. 0. P., and Myers, J. (1973) Plant Physiol. 51, 104-109. 2. Koenigs, J. W. (1974)Arch. Mikrobiol. 99, 129-145. 3. Rotilio, G., Calabrese, L., Finazzi Agro, A., Argento-Cent, M. P., Autuori, F., and Mondori, B. (1973) Biochem. Biophys. Acta 321, 98-102. 4. Maehly, A. C., and Chance, B. (1954) in Methods of Biochemical Analysis, (Glick, D., ed.), Vol. I, pp. 357-424, Interscience Publishers, New York. Also see Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. II, pp. 764-775 and 801-813, Academic Press, New York. 5. Weetall, H. H., and Weliky, N. (1966)Anaf. Eiochem. 14, 160-162. 6. Mottola, H. A., Simpson, B. E., and Gorin, G. (1970) Anal. Chem. 42, 410-411. 7. Perschke, H., and Broda, E. (1961) Nature (London) 190, 257-258. 8. Boveris, A., Martino, E., and Stoppani, A. 0. M. (1977) Anal. Biochem. 60, 145- 158. 9. Stevens, S. E., Jr., Patterson, C. 0. P., and Myers, J. (1973) J. Phycol. 9, 427-430. 10. Myers, J., and Clark, L. B. (1944) J. Gen. Physiol. 28, 103-112. 11. Kratz, W. A., and Myers, J. (1955) Amer. J. Bat. 42, 282-287. 12. Boveris, A., Cadenas, E., and Stoppani, A. 0. M. (1976) Biochem. J. 156, 435-444. 13. Telfer, A., Cammack, R., and Evans, M. C. W. (1970) FEB.9 Left. 10, 21-24.

A controlled-temperature apparatus for measurement of hydrogen peroxide production.

ANALYTICAL BIOCHEMISTRY85, 63-70(1978) A Controlled-Temperature Apparatus for Measurement Hydrogen Peroxide Production C.O.PAT PATTERSON,*J. B. GLO...
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