A spectrophotometric method for determination of catalase activity in small tissue samples

ANALYTICAL

BIOCHEMISTRY

174,33 l-336 (1988)

A Spectrophotometric

Method for Determination Small Tissue Samples

LARS H. JOHANSSON Department

of Medical

Ce// Biology.

AND L. A. H&AN

University

of Uppsala,

of Catalase Activity in

BORG'

Box 5 71, S-75 I 23 Uppsala,

Sweden

Received February 16, 1988 A simple and rapid method for determination of catalase activity in small tissue samples is described. Using a new approach, we have exploited the peroxidatic function of catalase for the determination of enzyme activity. The method was based on the reaction of the enzyme with methanol in the presence of an optimal concentration of hydrogen peroxide. The formaldehyde produced was measured spectrophotometrically with 4-amino-3-hydrazino-5-mercapto1,2,4triazole (Purpald) as a chromogen. With this method, a detection limit of 12.5 ng of purified catalase from bovine liver was possible, and it was successfully applied to microgram amounts of mouse liver and pancreatic islet homogenates. The catalase activity in liver was about 50 times higher than that in pancreatic islets. 0 1988 Academic Press. Inc. KEY WORDS: catalase; spectrophotometry; Purpald; liver; pancreatic islets.

strates for catalase, other enzymes with peroxidatic activity do not utilize these substrates ( 14). Current methods for quantitative determination of catalase activity are based on the catalatic function of the enzyme. Such methods involve measurements of either hydrogen peroxide consumption by titrimetry or spectrophotometry or of oxygen production by manometric techniques or by oxygen-sensitive electrodes. The latter methods are particularly sensitive but require combersome procedures or sophisticated equipment. In a new approach, we have evaluated the possibility of utilizing the peroxidatic function of catalase for determination of enzyme activity in small tissue samples. We have used methanol as the hydrogen donor and measured the production of formaldehyde spectrophotometritally with 4-amino-3-hydrazino-5-mercapto1,2,4-triazole (Purpald) as a chromogen. Previous observations indicate that this substance specifically forms a bicyclic heterocycle with aldehydes, which at oxidation changes from colorless to a magenta or purple color ( 15).

Catalase (EC 1.11.1.6) is able to decompose hydrogen peroxide by two types of reactions. Both reactions include a first step of formation of an intermediate (compound I) consisting of the enzyme and hydrogen peroxide (1). The catalatic activity, which is a unique property of catalase, catalyzes a reaction with a second molecule of hydrogen peroxide, resulting in the production of oxygen and water. The peroxidatic activity of the enzyme, first described by Keilin and Hartree (2,3), catalyzes reactions of compound I with hydrogen donors other than hydrogen peroxide. Details of enzyme structure, reaction mechanisms, kinetic characteristics, and the physiological role of catalase have been the subject of numerous reviews (4-12). Obviously, the peroxidatic activity of catalase is most evident at relatively low concentrations of hydrogen peroxide (3,13), and a number of different hydrogen donors may serve as substrates. Lower alcohols, such as methanol and ethanol, are particularly reactive (1,3). While the aliphatic alcohols serve as specific sub’ To whom correspondence should be addressed. 331

0003-2697188 $3.00 Copyright 0 1988 by Academic Press, Inc. All tights of reproduction in any form reserved.

332

JOHANSSON

AND BORG

formaldehyde and Purpald was oxidized by adding 50 ~165.2 mM potassium periodate in Chemicals. Purified catalase (EC 1. I 1.16) 470 mM potassium hydroxide to each tube. from bovine liver, 2,2’-azinobis(3-ethylbenzAny particulate material in the tubes was sedthiazoline-6-sulfonic acid) (ABTS), diamimented by centrifugation at 9500g for 10 monium salt, and 4,4’-dicarboxy-2,2’-bimin, and using glass standard microcuvettes quinoline, disodium salt, were purchased of 2-mm width and 1O-mm light path, the abfrom Sigma Chemical Company, St. Louis, sorbance of the clear liquid was measured at Missouri; 4-amino-3-hydrazino-5-mercapto550 nm in a Zeiss PMQ II spectrophotometer 1,2,4-triazole (Purpald) from Aldrich-Chemie, (Carl Zeiss, Oberkochen, FRG). Steinheim, FRG; collagenase (EC 3.4.24.3) Preparation of tissue samples.Tissue samfrom Boehringer-Mannheim, Mannheim, ples were obtained from adult male NMRI FRG; and bovine serum albumin, fraction V, mice (ALAB, Sollentuna, Sweden). The mice from Miles Laboratories, Slough, UK. All were killed by cervical dislocation. After other chemicals, of analytical grade, were decapitation and bleeding the livers were from E. Merck, Darmstadt, FRG. quickly excised and minced in ice-cold 25 Procedurefor determination of catalase ac- mM KI-12P04-NaOH buffer, pH 7.0, and aptivity. Purified catalase or homogenized tis- proximately 20% (w/v) homogenates were sues were incubated with methanol and hy- prepared. The homogenates were then didrogen peroxide in 250 mM KH2P04-NaOH luted with buffer as appropriate. Pancreatic buffer, pH 7.0. By the standard procedure, 50 islets were isolated by a collagenase (EC ~1 buffer, 50 ~1 100% (w/v) methanol, giving 3.4.24.3) digestion technique ( 16). Batches of a final concentration of 5.9 M, and 10 ~1 400-2500 islets were homogenized on ice in 0.27% (w/v) hydrogen peroxide, giving a final 500 ~1 25 mM KH2P04-NaOH buffer, pH concentration of 4.2 IIIM, were mixed in 7.0. The islet protein content was 36.2 + 0.10 small polystyrene test tubes. The enzymatic pg/ 100 islets (mean + SE; four observations). reaction was initiated by addition of 100 ~1 of Homogenization was performed in agroundglass tissue homogenizer (Kontes Glass Co., a catalase-containing sample. For evaluation of methodological details, the sample con- Vineland, NJ) of 8-ml capacity for the livers sisted of 0.125-2.0 pg/ml purified catalase and of 2-ml capacity for the islets. To liberate from bovine liver, with an activity of 2500 catalase from subcellular particles all homounits/mg, dissolved in 25 InM KH2P04genates were treated in an ultrasonic ice bath NaOH buffer, pH 7.0. Tissue samples were (35 kHz; 95 W) for 15 s. Extreme care was prepared as described below. Standard sam- taken to avoid inactivation of catalase by ples consisted of 26.6- 133 pM formaldehyde overheating the homogenates. solutions in 25 mM KI-12P04-NaOH buffer, Determination of hemoglobin. Since erythpH 7.0. Pure buffer was used as blanks. The rocytes contain catalase, the presence of residual blood in the tissue preparations was reaction mixture was incubated with continuous shaking for exactly 20 min at room tem- taken into account. The content of hemogloperature (20°C). The enzymatic reaction was bin in the tissue homogenates was deterterminated by addition of 50 ~1 7.8 M potas- mined by the method described by Marklund sium hydroxide solution to each tube. Imme(17) and the catalase activity originating from erythrocytes was calculated from meadiately thereafter the tubes were supplied with 100 ~1 each of 34.2 mrvr Purpald in 480 surements performed on mouse blood. The liver homogenates contained 1250 + 142 ng/ mM hydrochloric acid, and a second incubation with continuous shaking was performed ~1 (mean * SE; three observations) hemoglofor 10 min at 20°C. To obtain a colored com- bin, which corresponded to 0.013 + 0.002% pound, the product of the reaction between (mean f SE; three observations) of their total MATERIALS

AND METHODS

SPECTROPHOTOMETRIC

DETERMINATION

FIG. I. Absorbance as a function of the time for reaction between formaldehyde and Purpald. Oxidizing reagent was added at different time points. Samples consisted of I &ml purified catalase from bovine liver and were incubated with 5.9 M methanol and 4.2 mM hydrogen peroxide. Means f SE of 20 observations.

catalase activity. The hemoglobin content of the pancreatic islet homogenates was, however, below the detection limit of the hemoglobin assay, which was 10 ng/hl. For the liver samples, the enzyme activity of the erythrocytes was subtracted from the total catalase activity. Determination ofprotein. The protein content of the tissue preparations was determined by the method of Smith et al. ( 18) using 4,4’-dicarboxy-2,2’-biquinoline, or 2,2’bicinchoninic acid, to enhance the sensitivity of the biuret reaction. Protein standards consisted of bovine serum albumin.

OF CATALASE

ACTIVITY

333

gated system, which probably causes the distinct light absorption in the visible region ( 15). Potassium periodate was used as an oxidizing agent, and full absorbance was developed almost instantaneously. The colored product was stable for at least 3 h. By varying the length of the second incubation step, when the reaction between formaldehyde and Purpald took place, we found that the reaction was completed within 10 min (Fig. l), and this duration of the incubation was used in all subsequent experiments. By employing standard samples containing 2.66- 13.3 nmol formaldehyde a linear relationship between the amount of aldehyde and absorbance was found (Fig. 2). In further experiments, standard curves, similar to that shown in Fig. 2, were used to determine the amount of formaldehyde formed by catalase. The catalatic and peroxidatic activities of catalase are not independent of each other. Oshino et al. (13) have shown that the relationship between the two activities of catalase is a function of the ratio of hydrogen peroxide and other hydrogen donor concentration. From their experiments, it also appeared that neither activity was saturable. Indeed, when incubating purified catalase from bovine liver with various concentrations of methanol at a fixed hydrogen peroxide concentration of 4.2

RESULTS AND DISCUSSION

The present approach to determine catalase activity utilized the peroxidatic function of the enzyme. Methanol was used as hydrogen donor, and Purpald was applied as a reagent for formaldehyde produced by the enzymatic activity. Purpald reacts very specifically with aldehydes in alkaline environment (15). Both the hydrazine and the amino group of Purpald bind to the aldehyde and an aminal is formed. Subsequent oxidation of the aminal leads to the formation ofa bicyclic heterocycle containing an extensive conju-

FIG. 2. Absorbance as a function of the formaldehyde content in standard samples. Samples were incubated with 5.9 M methanol and 4.2 mM hydrogen peroxide. Means + SE of 20 observations.

334

JOHANSSON

FIG. 3. Relationship between formaldehyde production and concentration of hydrogen peroxide. Samples consisted of I &ml purified catalase from bovine liver and were incubated with 5.9 M methanol and hydrogen peroxide as indicated. Means k SE of 20 observations.

mM, we found a linear relationship between methanol concentration and formaldehyde production up to 6 M methanol. At even higher concentrations of methanol there was, however, a decreased peroxidatic activity of the enzyme, and this activity was almost completely inhibited at 7.5 M methanol. This inhibition of the enzymatic activity probably resulted from unspecific changes of the enzyme in the concentrated methanolic solution. Variations in hydrogen peroxide concentration at a fixed concentration of methanol of 5.9 M had significant effects on the peroxidatic oxidation of the alcohol (Fig. 3). At concentrations of hydrogen peroxide up to 2.3 mM there was a very rapid increase in the peroxidatic activity. Between 2.3 and 4.7 mM hydrogen peroxide there was little change in this activity, whereas at higher concentrations of hydrogen peroxide it decreased gradually. The rapid enhancement of the peroxidatic activity at increasing hydrogen peroxide concentrations up to 2.3 mM could be explained by the production of increasing steady-state concentrations of compound I. The enzymatic activity would then reflect the binding of hydrogen peroxide to the enzyme. It is known that maximal binding is reached when 30-40% of catalase hematin is occupied by hydrogen peroxide (19-21). Under the present conditions, this apparently occurred

AND BORG

at a concentration of about 2 mM hydrogen peroxide. The decrease in peroxidatic activity, when the hydrogen peroxide concentration was increased above 4.7 mM, would depend on an increasing proportion of catalatic activity (13). Optimal conditions for utilizing the peroxidatic function of catalase for determination of enzyme activity were thus found at concentrations of methanol and hydrogen peroxide of 5.9 M and 4.2 mM, respectively. At these concentrations of methanol and hydrogen peroxide there was an essentially constant velocity of formaldehyde production for at least 20 min, when purified catalase from bovine liver was incubated at room temperature (20°C) (Fig. 4). The enzyme activity gradually decreased, however, when the incubation was performed at 37°C. Indeed, incubations of purified catalase performed before the determination of enzyme activity showed that in buffer the enzyme rapidly lost activity at both 20 and 37°C (Table 1). However, at 0°C the activity remained constant for at least 60 min. When 4.2 mM hydrogen peroxide was added to the incubation buffer, the decrease in enzyme activity was also more pronounced at 0°C (Table 1). These findings emphasize the extreme importance of maintaining catalase-containing samples at low temperature. In particular, heating during ul-

; 51

/

FIG. 4. Relationship between formaldehyde production and incubation time at two different temperatures. Samples consisted of 1 &ml purified catalase from bovine liver and were incubated with 5.9 M methanol and 4.2 mM hydrogen peroxide. Means f SE of 20 observations.

SPECTROPHOTOMETRIC

DETERMINATION

OF CATALASE

335

ACTIVITY

TABLE 1 STABILITYOFCATALASEINSOLUTION'

Catalase activityb Without hydrogen peroxide Incubation time (mm) 0 10 30 60

0°C 100 + 98 f 100 + 97 *

2.3 2.4 1.7 1.2

With hydrogen peroxide 2o’c

20°C

37°C

0-Z

100 + 1.7 43 i- 1.8 2 z+z0.2 ND

100 f 1.0 17a0.8 ND’ ND

100 z!z2.3 94 rtr 1.5 73 ?I 0.9 52 +- 0.5

100 * 1.7 19rt 1.0 ND ND

37°C lOOk 0.5 13kO.5 ND ND

’ Solutions of I pg/ml purified catalase from bovine liver in 25 IrIM KH2P04-NaOH buffer, pH 7.0, were incubated in the absence and presence of 4.2 mM hydrogen peroxide at three different temperatures, as indicated. The catalase activity was determined after the incubations. b Activity is expressed as percentage of the activity at time zero of incubation. Means ~frSE of 10 observations. ’ No detectable activity.

trasonic disruption of tissue samples should be avoided. Our findings also demonstrate an inhibitory effect of hydrogen peroxide on catalase activity, which can occur in tissue homogenates even at 0°C. This effect can be ascribed to conversion of the primary catalasehydrogen peroxide intermediate into an inactive form (compound II), which is relatively stable (22). In contrast to hydrogen peroxide, aliphatic alcohols are electron donors that do not promote formation of compound II (23). Moreover, compound II is decomposed by such alcohols and catalase activity restored (22). Methanol is therefore effective both in prevention of the formation of compound II

0

IO

100

150 Cslalow

200 In*>

0

1

and in the decomposition of compound II already formed (24). High concentrations of methanol should thus minimize problems with the presence of compound II during determination of catalase activity. As shown in Fig. 4, we virtually avoided such problems, when the enzyme reaction was carried out at 20°C. The gradual decrease in enzyme activity during incubation in the presence of methanol at 37°C probably resulted from decomposition of the enzyme in solution. The dose-response relationship for purified catalase from bovine liver was strictly linear from 12.5 to at least 200 ng of enzyme sample (Fig. 5A). Homogenates of mouse

2

3 !“ro,*in

4 ,po

0

50

‘DO

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2m $0,

FIG. 5. Relationship between formaldehyde production and amounts of purified catalase from bovine liver (A; means f SE of 20 observations), mouse liver homogenate (B; 9 observations), and mouse pancreatic islet homogenate (C, 12 observations). Samples were incubated with 5.9 M methanol and 4.2 mM hydrogen peroxide.

336

JOHANSSON

liver (Fig. 5B) and pancreatic islets (Fig. 5C) also displayed a linear relationship between the amount of tissue and catalase activity over a wide range. The activity was 2.42 +- 0.060 pkat/pg protein (mean -+ SE; three observations) in liver and 0.049 +- 0.002 pkat/pg protein (mean + SE; four observations) in pancreatic islets. Hence, mouse liver contained about 50 times higher catalase activity than mouse pancreatic islets. This finding confirms other observations (25). In separate experiments, we observed that methanol under the present conditions was not oxidized to formaldehyde, when mouse liver homogenates were incubated in the absence of hydrogen peroxide. This observation is in good agreement with findings of no (26) or very low (27) liver alcohol dehydrogenase (EC 1.1.1.1) activity with methanol as substrate. Furthermore, when 133 PM formaldehyde was added to samples of mouse liver homogenates in the presence of either hydrogen peroxide or methanol, the formaldehyde concentration remained essentially constant during prolonged incubations. Thus, it is unlikely that other enzyme activities in tissue homogenates affect the results obtained by the present method. In conclusion, we have found that the peroxidatic function of catalase is very well suited to the determination of the enzyme activity in small tissue samples. Moreover, the determination can be performed by simple methodology and does not require specialized equipment. ACKNOWLEDGMENTS This work was supported by grants from the Swedish Diabetes Association, the Family Emfors Fund, the Swedish Hoechst Diabetes Fund, and the Swedish Medical Research Council (12X-109, 12X-6538).

REFERENCES 1. Chance, B. (1947) Acfa Chem. &and. 1,236-267. 2. Keilin, D., and Hartree, E. F. (1936) Proc. R. Sot. London Sect. B 119,14 I- 159.

AND BORG 3. Keilin, D., and Hartree, E. F. (1945) Biochem. J. 39, 293-301. 4. Nicholls, P., and Schonbaum, G. R. (1963) in The Enzymes (Boyer, P. D., Lardy, H., and Myrbkk, K., Eds.), 2nd ed., Vol. 8, Part B, pp. 147-225, Academic Press, New York/London. 5. Brill, A. S. (1966) in Comprehensive Biochemistry (Florkin, M., and Stotz. E. H., Eds.), Vol. 14, pp. 447-479, Elsevier, Amsterdam/London/New York. 6. Jones, P., and Suggett, A. (1968) Biochem. J. 110, 621-629. 7. Deisseroth, A., and Dounce, A. L. (1970) Physiol. Rev. SO,3 19-375. 8. Schonbaum. G. R., and Chance, B. (1976) in The Enzymes (Boyer. P. D., Ed.), 3rd ed.. Vol. 13, Part C, pp. 363-408, Academic Press, New York/London. 9. Jones, P., and Dunford. H. B. (1977) J. Theor. Biol. 69,451-470. 10. Chance, B.. Sies, H., and Boveris, A. (1979) Physiol. Rev. 59,527-605. 11. Dounce, A. L. (1983) J. Theor. Biol. 105,553-567. 12. Fita, I., and Rossmann, M. G. (1985) J. Mol. Biol. 185,21-37.

13. Oshino, N., Oshino, R., and Chance, B. (1973) Biothem. J. 131,555-563. 14. Mason, H. S. (1957) Adv. Enzymol. 19,79-233. 15. Dickinson, R. G., and Jacobsen, N. W. (1970) Chem. Commun., 17 19- 1720. 16. Schnell, A. H., Swenne, I., and Borg, L. A. H. (1988) Cell Tissue Res. 252,9- 15. 17. Marklund, S. (1979) Clin. Chim. Acta 92,229-234. 18. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K.. Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985) Anal. Biochem. 150,76-85. 19. Kremer, M. L. (1970) Biochim. Biophys. Acfa 198, 199-209. 20. Chance, B., and Oshino, N. (197 1) Biochem. J. 122, 225-233. 21. Sies, H.. Biicher, T., Oshino, N., and Chance, B. (1973)Arch. Biochem. Biophys. 154, 106-I 16. 73 Chance, B. (1950) Biochem. J. 46,387-402. 23. Keilin, D., and Nicholls, P. (1958) Biochim. Biophys. Acta 29,302-307. 24. Adams, D. H.. and Burgess, E. A. (1959) Enzymologia 20,34 I-354. 25. Grankvist, K., Marklund, S. L., and Tiiljedal, I.-B. (1981) Biochem. J. 199,393-398. 26. Theorell, H., and Bonnichsen, R. (195 1) Acta Chem. Stand. 5,1105-l 126. 27. Kini, M. M., and Cooper, J. R. (1961) Biochem. Pharmacol. 8,207-2 15. AL.