Improved assay for catalase based upon steady-state substrate concentration

ANALYTIC4L

45,

BIOCHEMISTRY

Improved

Assay

469-479

for

(1972)

Catalase

Substrate JOSEPH Bmic

Science

L. HAINING

Research

Based

upon

Steady-State

Concentration AND

JIM

S. LEGAK

Laboratory, T’eterans AdnaiG.strafiou Jackson, Mississippi 39216

Center,

Received May 27, 1971 The routine assay of catalase (hydrogen-peroxide: hydrogen-peroxide oxidoreductase, EC 1.11.1.6), especially in crude tissue preparations, is hampered by a number of factors, the most problematic being the necessity of expressing enzyme activity in terms of a first-order rate constant (or half-time) and employing relatively short reaction times at low substrate concent.rations. These problems result from the unusual kinetics of the catalase-H,Oz reaction and the formation of inactive enzymesubstrate complexes (1,2). The latter complication is a function of both substrate concentration and temperature (3). This paper describes a modification of the polarographic assay of catalase originally developed by Bonnichsen, Chance, and Theorell (4) that largely circumvents these as well as other problems presented by extant assay methods for this enzyme. Hydrogen peroxide can be measured without interference by oxygen, by means of a platinum electrode polarized as an anode at 0.8-l V (5). The assay described here is based upon this principle together with automatic titration of a buffered H,O,-catalase solution with the substrate. That, is, the substrate concentration is held constant by continuous addition of a more concentrated solution of H,O, without significant change in the volume of the reaction mixture. The volume of titrant required to maintain a steady-state concentration of H,O, is recorded as a function of time, thereby permit,ting calculation of the rate of reaction in terms of substrate utilization. The assay can be carried out at relatively low temperatures and substrate concentrations. MATERL4LS

AND METHODS

The assay reaction mixture was composed of 73.5 ml of phate buffer, pH 7.2, with or without Triton X-100 (0.05% of various dilutions of “3%” H20, in distilled water, and of catalase-containing solution in distilled water, in a 100 469 @ 1972 by -4cademic Press, inc.

0.02 M phosv/v), 1.5 ml up to 0.1 ml ml beaker.

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Two sources of catalase were employed: purified beef liver catalase (Worthington Biochemical Corp., code CTR) and homogenates of several rat tissues. The tissues were quickly removed from 3 month old male Fischer 344 rats guillotined after stunning, chilled thoroughly, trimmed of fat, and disrupted in ice-cold distilled water in a prechilled Omni-Mixer (Sorvall) blender held in an ice bath for 2 min. Various ratios of tissue-to-water were utilized as indicated. One or the other of two “oxygen analyzers,” the Beckman model 777 (gold/silver electrodes) or the Yellow Spring model 53 (platinum/silver electrodes) served to monitor the substrate concentration. In both cases the polarity of the measuring electrode, normally 0.8 V negative, was reversed by interchanging the electrode connections to the electronic unit. The recorder output of the “oxygen analyzer” was connected to a Radiometer type TTTlc titrator (The London Co.), which was connected with a Radiometer type ABU lb Auto-burette (2.5 ml capacity). The flexible cable connection of the Auto-burette’s digital volume counter was fitted instead with a 1.7 cm diameter pulley wheel. The pen drive gears of a Sargent model SRL recorder were removed and the shaft of the “cable drum” fitted with a 20 cm diameter pulley wheel. Braided silk suture material was used as a belt to connect the two pulley wheels. For each assay, the buffer-substrate was maint,ained at the desired temperature in a Lauda K-B/R (Brinkmann) refrigerated water bath. The naked “oxygen” electrode unit (devoid of membrane, electrolyte gel, and cap) was suspended securely in the buffer-substrate opposite the tip of the Auto-burette delivery tubing (ca. 1 mm i.d. polyethylene). The titrant (nominally 3% H,O,) was brought to room temperature prior to filling the Auto-burette with it so as to minimize bubble formation in the burette. The speed of a motor-driven constant-torque (DC) glass stirrer suspended in the buffer-substrate was increased to the point that produced a maximum response of the “oxygen analyzer” (a requirement for use of the electrodes in a liquid medium). The recorder chart motor (1 in./min) was started and the “proportional band” and endpoint (mV value of the buffer-substrate) settings of the titrator adjusted so as to maintain the initial meter reading by automatic addition of the titrant to the buffer-substrate. After several minutes of recording the spontaneous (baseline) electrolytic decomposition of H,O,, the enzyme solution was added and the automatic titration continued until 0.5 ml, at most, of titrant had been added. With the pulley wheels employed, this volume of titrant corresponds to the full chart width (25 cm with 10 chart divisions/cm) of the recorder paper and represents 0.7% (or less) of the initial volume of the assay reaction mixture. The slope of the baseline graph, if any, was subtracted from that of the catalase-produced recording to yield the enzyme-catalyzed reaction rate in terms of milli-

IMPROVED

CATALASE

471

ASSAY

liters titrant per unit time. An absorptivity of 0.067 cm-l mM-l at 230 nm (3) was employed to determine the actual HzO, concentration of the titrant (and substrate) and hence calculation of the rate of addition (utilization) of H,O, in terms of micromoles decomposed per minute. Enzyme concentration is expressed in terms of kilounits (kU), where one unit of enzyme is that amount which catalyzes the transformation of 2 pmoles of H,O, per minute (6) .I RESULTS

Figure 1 shows that the Lioxygen analyzers” responded in a linear manner to the H,Oz concentration. Gassing the buffer with pure oxygen produced no response by the reversed-polarized “analyzers.” A tracing of a typical recording of automatic titration of an assay mixture with H,O, at 25°C is presented in Fig. 2. The initial slope represents the rate of consumption of substrate by the electrode; thereafter, that occurring after the addition of catalase. The “spontaneous” decomposition of H,O, was usually negligible when the Yellow Spring electrode was employed owing, no doubt, to its relatively smaller surface area compared to the Beckman electrode. This was especially true at low temperature and low substrate concentration, even for the larger surface area Beckman electrode. The reproducibility of the technique was examined with both the puri-

0

0.4

0.8 H202

1.2

1.6

2.0

2.4

i mM )

FIG. 1. Relationship between Hz02 concentration and response of reversed-polarized “oxygen analyzers” at 25°C. Closed circles represent Yellow Spring Instrument’s system at highest (broken line) and lowest (solid line) sensitivity settings. Open circles represent Beckman Instrument’s system operating in minimum sensitivity range.

‘It should be carefully noted that this definition of a unit of catalase activity is in accsord wit,h the recommendations of the International Union of Biochemistry for bimolecular reactions (6) whereas the Worthington Biochemical Corp. “unit” as well as that of Ganschow and Schimke (7) is based upon 1 pmole of H,O, decomposrd per minute.

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FIG. 2. Free-hand tracing of actual recording of automatic titration assay of catalase. The reaction mixture consisted of 2.4 mM HzO, in phosphate buffer (see text) and 1.0 mg rat liver. The titrant was 0.95 M HgOz, which was auto,matically added at the rate of 39.9 /Lmoles/min due to the presence of this homogenate sample. Beckman electrode system.

fied beef liver catalase and a rat liver homogenate, at 25°C. Six consecutive assays of the former preparation yielded a range of 3.1 to 3.4 kU/mg with a mean and standard deviation of 3.2 + 0.09 kU/mg. Eight successive assays of the homogenate varied between 15.3 and 17.4 kU/gm with the mean and standard deviation being 16.4 +- 0.7 kU/gm liver. The activity of a recent.ly obtained beef liver catalase preparation was assayed several times employing the spectrophotometric method described by its producer (Worthington), viz., 20 mM H,Oz in 0.016 M phosphate buffer, pH 7.0, at 25”. The first assay yielded an activity of 28 kU/mg, corresponding well to the labeled value of 54,800 pmoles H,Oe/min/mg, but the activity of the 1:50,000 dilution of the preparation consistently declined to a value of 47,000 pmolesJmin/mg during 30 min standing in an ice bath. When the same preparation was diluted only 5-fold and assayed by the present method (2.4 mM H,Oz), an activity of 3.1 kU/mg was obtained initially followed by values of 2.9, 3.0,and 3.2 kU/mg at approximately 20 min intervals. Figure 3 shows the proportionality between catalase concentration and rate of reaction observed with both the commercially obtained beef liver enzyme and a rat liver homogenate. The choice of concentrations of the two enzyme preparations illustrates the wide range of stoichiometry attainable. The reaction times in t’his experiment varied from 3.5 to 9 min, depending upon the enzyme concentration. Figure 4 illustrates the expected proportionality between reaction rate and H20Z concentration observed at 5°C. This temperature was selected

0

mg Liver 1

2

FIG. 3. Proportionality between reaction rate and enzyme concentration. The assays were performed with 2.4 mM 802 in buffer at 25°C using the Beckman Instrument system and 0.95M H,Oz as titrant. Abscissas indicate amount of rat liver (closed circles) or purified beef liver catalase (open circles) in the reaction mixtures.

for this experiment in order to minimize formation of inactive enzymesubstrate complexes at the higher substrate concentrations. The maximum reaction rate observed at this particular enzyme concentration approaches the limiting rate of the automatic titrator when “3%” H,O, is used as the titrant. The lower limit of substrate concentration at 5” could be proportionately extended downward to 0.2 mM by using the

FIG. 4. Relationship between substrate concentration and reaction velocity. The assays were carried out at 58°C with 4.5 pg purified beef liver catalase using the Yellow Spring system and 0.95M Hz01 as titrant.

474

HAININC

a

0

20 Meter

AND

LEGAN

40 Reading

60 “Endpoint”

80

Fro. 5. Automatic titration rate in relation to end-point setting of reversedpolarized “oxygen electrode.” Each reaction mixture contained the same enzyme and substrate concentration. The broken line is the mean of the individual assays using the Yellow Spring system at 25°C.

lower range scales of the Beckman analyzer and full clockwise setting of the calibration knob, or platinizing the Yellow Spring platinum electrode. However, all the data contained in this report were obtained by use of the regular nonplatinized platinum electrode or the Beckman analyzer set on the “100%” or “1000 mm” scales. It was also deemed desirable to ascertain that at constant enzyme and substrate concentration the observed reaction rate was the same regardless of the initial reading (end-point) of the “oxygen” analyzers. To do so the calibration or sensitivity knobs of the analyzers were adjusted to produce a midscale reading with a certain substrate concentration. A given amount of enzyme was then added to the buffersubstrate and the titration recorded. This was repeated a number of times with the only variation being the arbitrary initial meter and endpoint settings. It can be seen from Fig. 5 that it does not matter what the initial meter reading is as long as the titrator is adjusted to maintain that potential. Table 1 contains the results of assaying by this method homogenates of various rat tissues and hemolyzed rat blood. The incorporation of Triton X-100 either into the reaction mixt,ure or during homogenization did not result in any higher activity than in its absence. TABLE 1 Catalase Activity of Several Rat Tissue Homogenates Determined Automatic Titration (2.4 mM HzOz, 25°C) Tissue

NO

Tissue: water

Liver Kidney Heart Blood

5 5 5 5

1:50 1:20

kU/gm

1:lO 1:lO

0 Number of animals from which mean activity was determined. b kU/ml blood f S.E.M. (standard error of the mean).

14.55 3.13 0.35 2.04

by

+ S.E.M. f k f f

0.63 0.32 0.05 0.04b

IMPROVED

CATALASE

ASSAY

475

DISCUSSION The peculiar kinetics of the catalase-H202 reaction together with the formation of inactive enzyme-substrate complexes as a function of time, temperature, and substrate concentration has heretofore made it necessary to employ assay conditions involving very short reaction times and/or yielding enzyme activity measurements in terms of a first-order rate constant. While the “initial-rate” requirement is no longer difficult, to attain, thanks to the prevalence of spectrophotometers with recording capabilities, the two most popular assays for cat.alase still have disadvantages unrelated to the form of expression of the data. Thus, the titrimetric assay utilizing KMn04 not only is cumbersome by modern standards but also does not permit continuous measurement of residual H,O, throughout the assay period and is subject to serious interference by organic matter in t,issue extracts (3). The spectrophotometric measurement of H,O, decomposition may also be complicated by high blanks when applied to turbid tissue preparations (2,3). In addition, bubble formation may be a serious problem in the spectrophotometric assay and the use of relatively high substrate concentrations is precluded by this factor (59). The question of catalase inactivation via adsorption to glass surfaces, especially with dilute preparations of the enzyme (3), as well as t,he desirable use of low temperature may further restrict the utility of the spectrophotometric assay. The assay method described in the present study actually or potentially offers several improvements over the extant assays of catalase: A unique advant,age is that the method yields enzyme activity measurements directly in terms of the rate of substrate transformation. The apparent rate constant of reaction, yielded by the other assay methods, has led to a number of arbitrary unit definitions (3,9,10) and a knowledge of the specific rate constant for the particular catalase (obtainable only with an absolutely pure preparation of the enzyme) is required for translating t.he observed rate constant into more conventional units of enzyme activity 13,5,10,11).2 A singular disadvantage of utilizing the apparent rate constant itself as a measure of catalase activity is that the catalase content. of samples from different sources (species and tissue) may not be directly comparable on this basis. The spectrophotomet.ric assays for catalase appear to be limited to ‘In several cases, at least, the “initial rate” of the catalase-HzO, reaction has been equated to the absolute reaction rate (2.7,12). However. these methods require reaction times of the order of approximately 1 min or less and relatively high initial H,O, concentrations together with careful manipulation of t,he enzyme concentration in order to ensure linearity during the “initial” reaction period.

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considerably higher substrate concentrations than required by the present method. Thus, while Beers and Sizer (8, p. 135) describe their reaction mixture as being composed of a 1:3 dilution of 5 X 1O-3 M H,O,, i.e., ca. 1.7 mM, their Fig. 1 states the initial substrate concentration as being approximately 15 mM. The latter value is consistent with the initial absorbance of the reaction mixtures shown in their figure.3 Other spectrophotometric assays for catalase specify substrate concentrations ranging from 10 to 20 mM (7,9,10,12). Chance (5,ll) recommends a concentration of 2 mM H,O, in order to reduce bubble formation and enzyme inactivation. However, with a 1 cm cuvette this concentration of substrate produces an initial absorbance of only about 0.1 and requires use of a very stable spectrophotometer. It. was implied that this technique is not suitable for crude cell extracts (11). Rorth and Jensen (1) and Goldstein (2) have described assays for catalase based upon polarographic measurement of oxygen production. The first of these yields activity measurements in terms of first-order rate constants while t.he second requires a relatively short reaction time (30 set) and high (33 mM) H,O, concentration so as to produce a constant rate of oxygen formation during this period (see footnote 2). Although one of the methods (1) did not specify conditions of temperature, the other (2) indicated that. the temperature range was restricted to 25” to 40°C due to limitations of the working range of the electrode. Another advantage of the present method is that a wide range of reaction temperatures is easily obtainable without the problems created by cuvette-window fogging, bubble formation, or temperature compensation limitations of the transducer. Since the present method is based upon maintaining a constant redox potential by the measured addition of the analyte, differences in the absolute output to the titrator at different temperatures do not have to be corrected for. Indeed, the decreased spontaneous decomposition (electrode consumption) of H,O, at lower temperatures favors the otherwise desired low-temperature conditions. Bubble formation, which is pronounced, did not pose any problems in the present study as long as care was taken to ensure that. the elect.rode surfaces were well below the surface of t’he assay reaction mixture. The presence of Triton X-100 in the mixture did increase the risk of “noise” due to bubbles, by stabilizing the froth. It should also be pointed out that use of a magnetic stirring bar instead of a motor-driven propellertype stirrer accentuated bubble formation. ’ On p. 135 of Beers and Sizer (8) it is stated that a 5 X 10.’ M solution of HSO, is prepared by diluting 0.15 ml of Superoxol with 2.5 ml of buffer. Since Superoxol (Merck) is a 30% solution of HZO,, the buffered substrate was actually approximately 5 X IO-* M in H,Oz.

IMPROVED

CATALASE

477

ASSAY

Although no effort was made in the present study to take into consideration the phenomenon of catalase inactivation via adsorption to glass (3), it is obvious that this factor can be mitigated in this case by using suit,able inert or coated-glass reaction vessels and stirrers. The fact that the titration ‘Lcurves” (Fig. 2) remained linear throughout the course of the assay period indicates that enzyme inactivation was not a serious problem under the conditions employed in these experiments. In addition, the relat’ively large volume of the buffer-substrate solution permits the desired (3) minimum dilution of catalase preparations until just prior to assay. The largest volume of enzyme solution (0.1 ml) ut.ilized in this study results in a 750-fold dilut.ion only at the inst,ant of commencing the assay. We have made it a practice in this study to dilute the commercial catalase suspension or homogenize the tissue in a minimum volume so that l&50 ~1 gave the desired final dilution when added to the buffer-substrate. Experiments not included in t,his report indicated that 1: 1000 and 1: 100 dilutions of the purified catalase lost significant act’ivity while standing in an ice bath for 15-30 min but a 1: 10 dilution remained stable for several hours. The catalase activity of the tissue homogenates appeared to be relatively more stable. It was found that the sensitivity of the electrode(s) diminishes as a result of prolonged or repeated exposure to the enzyme preparations, especially in the case of tissue homogenates. It was ascertained t.hat this was not due to exposure of the electrodes to H,O, or instability of the substrate stock solution. The platinum anode appeared t,o be more susceptible to inactivation than the gold one. Although the rate of “poisoning” of the electrodes was not sufficient. to significantly alter the response of the titrator during a given assay, it was found necessary to occasionally reactivate the electrodes by cleansing with scouring powder as must. he done less frequently when they are employed as oxygen analyzers. FolIowing such rejuvenation the electrodes must be stabilized by means of short-term incubation in the buffer-substrate, in the polarized state.4 The stability of the system can most easily be monitored by connecting the recorder output of the “oxygen” analyzer to a 50 mV recorder. Such a secondary recorder operating in parallel with the titrator during assays also assures one of the constancy of maintenance of the end-point, and, after taking the Gtrator out of the circuit, permits recording of the subsequent decay in substrate concentration for kinetir studies, if desired. That is. both the absolute ’ If 1.11~ rejuvenated rlrctrod(~ lo wad on scale, the electrode homogenate in order to decrease

is so srnsiti\-r that. the meter cannot ran be “doped” by brief esposure its sensitivity sufficiently.

br to

adjustrtl a &sue

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reaction velocity and the rate constant for catalatic H,O, decomposition can be determined on the same reaction mixture. The decrease in sensitivity of the electrodes is not necessarily undesirable. Besides enzyme concentration, the maximum substrate concentration utilizable in the present method is dependent upon two factors: the temperature of the reaction mixture and the sensitivity of the electrode system. The latter is a function not only of temperature but also of electrode geometry, amplifier options, and degree of “poisoning” of the electrodes. A combination of low temperature and otherwise decreased electrode sensitivity permits extension of the upper limit of H,Oz concentration utilizable in this assay for kinetic studies, for example. While the rate of reaction at 5°C is about 80% of that at 25”, the initial reading (end-point) for a given substrate concentration is reduced considerably more and thus allows use of H,Oz concentrations that would be ‘Loff-scale” at 25”. On the other hand, the elect.rode systems employed in this study become saturated at 25” at H,Oz concentrations below that usable in some of the spectrophotometric assays (7,8,12) but it is anticipated that these or other polarographic systems can be modified or developed to permit the applicat,ion of the principles described here to substrate concentrations equal to or greater than the maximum now attainable for kinetic studies. SUMMARY

A modification of the polarographic assay for catalase is described that is based upon automatic titration of a buffered HzOz-catalase reaction mixture with a more concentrated H,O, solution such that there is no significant change in the volume of the reaction mixture. The recorded rate of addition of the titrant required to maintain a steady-state substrate concentration yields enzyme activity measurements in terms of actual reaction rate instead of the less satisfactory rate constant for H,O, decomposition, as is the case for most extant assays for catalase. An additional advantage of the new method is that the reaction can be easily carried out at considerably lower temperature and substrate concentrations than can be employed practicably in other types of assays for this enzyme. Both of these features are desirable to minimize the formation of inactive catalase-H,Oz complexes. The improved assay works satisfactorily for measuring catalase activity in rat tissue homogenates. ACKNOWLEDGMENTS We thank Dr. Britton to us that commercially

Chance available

of the University electrode circuits

of Pennsylvania would probably

for suggesting give adequately

IMI’ROXTI)

CATALAdE

479

ASSAY

accurate results in comparison to his rat her romplicated trode circuit for the polarographic assay of rntalase.

custom-made

microelec-

REFERENCES AND JENSEN, P. K., Bzbchim. Biophys. Acta 139, 171 (1967). D. B., Anal. Biochem. 24, 431 (1968). MAEHLY, A. C., in “Methods of Biochemical Analysis” (D. Glick, ed.), Vol. I, p. 358. Interscience, New York. 1954. BONNICHSEN, R. K., CHANCE, B., AND THEORELL, H., Acta Chem. Stand. 1,

1. RORTH, 2. GOLDSTEIN, 3. 4.

M.,

685 (1947). 5. CHANCE, B.,

6. 7.

8.

9. 10. 11.

12.

in “Methods of Biochemical Analysis” (D. Glick, ed.), Vol. I, p. 408. Interscience, New York, 1954. “Enzyme Nomenclature: Recommendations 1964 of the International Union of Biochemistry,” p. 7. American Elsevier, New York, 1965. GANSCHOW, R., AND SCHIMKE, R. T., in “Regulatory Mechanisms for Protein Synthesis in Mammalian Cells” (A. San Pietro, M. R. Lamborg, and F. T. Kenney, eds.), p. 377. Academic Press, New York, 1968. BEERS, R. F., JR., AND SIZER, I. W., J. Biol. Chem. 195, 133 (1952). Lijc~, H., in “Methods of Enzymatic Analysis” (H.-U. Bergmeyer, ed.), p. 885. Academic Press, New York, 1965. PRICE, V. E., STERLING, W. R., TARANTOLA, V. A., HARTLEY, R. W., JR., AND RECHCIGL, M., JR., J. Biol. Chem. 237, 3468 (1962). CHANCE, B., AND MAEHLY, A. C., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. II, p. 764. Academic Press, New York, 1955. “1968 Data Sheets for the Worthington Manual.” Worthington Biochemical Corp., Freehold, N. J., 1968.