Spectrophotometric determination of H2O2-generating oxidases using oxyhemoglobin as oxygen donor and indicator

Int. J. Bmhem., Vol. 11. pp. 121 lo 126 0 Pergamon Press Lfd 1980. Printed in Great

Britain

SPECTROPHOTOMETRIC DETERMINATION OF H,O,-GENERATING OXIDASES USING OXYHEMOGLOBIN AS OXYGEN DONOR AND INDICATOR OCTAVIANBAR&* and MANASEDAN~REANU’ ‘Maryland Psychiatric Research Center, Baltimore, MD21228, U.S.A. and ‘Department of Biophysics, Medical and Pharmaceutical Institute, R-3400 Cluj-Napoca, Romania (Received 28 June 1979)

Abstract-l. Spectrophotometric determination of oxygen uptake using oxyhemoglobin as oxygen donor and indicator was used for assay of H,O,-generating oxidases like monoamine oxidase and glucose oxidase. 2. In order to decompose H,O, formed during the oxygen uptake, catalase and methanol (or ethanol) was added to the respiratory system. At pH values higher than 7.5 the oxydation of deoxygenated hemoglobin to methemoglobin was less than 3%. 3. Oxidases with low K, for oxygen can be assayed using the spectrophotometric method if suitable correction factors are introduced into the calculation of oxygen uptake. The correction factor represents the ratio of the rate of formation (or dissappearance) of one of the reactants and the rate of oxyhemoglobin deoxygenation, measured under identical experimental conditions.

INTRODUCTION The spectrophotometric method for assay of oxygen uptake, based on the utilization of oxyhemoglobin, to serve both as a source of oxygen and as an indicator of respiration, is characterized by a wide range of sensitivity as well as by relative simplicity (BLrzu & Borza, 1967; Barzu et al., 1968; Bhrzu & Satre, 1970; Bhrzu & Cioara, 1971; BLrzu et al., 1972). By decreasing the reaction volume down to 1 nl it was possible to measure the oxygen uptake of a single cell (Hultborn, 1972; Herlitz & Hultborn, 1973; Hultborn & Hyden, 1974). A particular situation, not discussed in previous work, is created by enzymatic systems generating H202 during oxygen uptake. It is well known that Hb is sensitive, while Hb02 not, to the oxidative action of H202 (White et al., 1959). As a consequence, a subsequent conversion of Hb into MetHb is expected to take place during the deoxygenation of HbOz by these oxygen consuming systems. In this work we examined the experimental conditions under which the oxidation of benzylamine by monoamine oxidase (monoamine:O, oxidoreductase (deaminating), EC 1.4.3.4) interferes with the pigment molecule. To avoid the risk of Hb conversion into MetHb, catalase was added together with ethanol or methanol to the reaction mixture: * Permanent address: Department of Biochemistry, Medical and Pharmaceutical Institute, R-3400 ClujNapoca, Romania. t Abbreviations; HbO,-oxyhemoglobin; Hb-deoxy genated hemoglobin; Hb,-total hemoglobin, i.e. HbO, + Hb; MetHb-methemoglobin; y-the fractional saturation of the pigment with oxygen, HbO,/Hb,.

(A)

C,Hs--CH2NH,

C,H5--CH0 (B)

+ 0,

+ H,O =

oxidase

+ HzO, + NH,

H202 + RCH20H - cata’ase 2 H,O + RCHO.

In this way the affinity of catalase for H20, is substantially increased (Nicholls & Schonbaum, 1963) and the stoichiometry of the oxygen consuming system improved, i.e. one mol of oxidized substrate corresponds to 1 mol of oxygen taken up, while when catalase is added alone, 1 mol of oxidized substrate corresponds to !/2 mol of oxygen. MATERIALSAND

METHODS

Purified bovine kidney mitochondrial monoamine oxidase (Erwin & Hellerman, 1967) was kindly provided by Drs L. Hellerman and D. R. Patek (John Hopkins University, Baltimore, Maryland). Beef liver catalase and yeast glucose oxidase were supplied by Worthington and Boehringer, respectively. Human hemoglobin was prepared by the ammonium sulfate method previously described (BLrzu et al., 1968) without any attempt to remove the organic phosphates. Kinetic measurements were made at room temperature (23°C) with Gilford 2400 spectrophotometer equipped with a recorder (full scale deflection 0.1 absorbance units), by alternative recordings at 420.6 nm (isosbestic point for Hb and HbO,) and 430nm, in a final vol of 0.3-3 ml and optical paths between 0.2 and 1.0 cm. Some experiments were made with an Aminco-Chance dual wavelength spectrophotomeer for the same wavelength pairs. The reaction mixture containing buffer, HbO, and enzyme (or substrate) was bubbled with a fine nitrogen current in order to reduce y to the desired value (usually to 0.75) and the sample was covered with paraffin oil. After 2-3 min necessary for optical density equilibration, the reaction was started by addition of the substrate (or 121

122

OCTAVIAN BAazu and MANASE D~N~REANU Table

1. Effect of pH, catalase and methanol on the rate of Hb02 deoxygenation and MetHb formation, during the oxidation of benzylamine by bovine kidney monoamine oxidase

MetHb formed PH

HbOl deoxygenated - MeOH + MeOH

6.5 7.0 1.6 8.1

17.6 31.3 47.0 49.1

34.7 63.5 89.8 98.0

MetHb formed - MeOH + MeOH 4.5 4.4 4.8 4.7

4.2 4.1 2.3 2.2

HbOz deoxygenated

x loo

- MeOH

+ MeOH

26.1 14.0 10.2 9.6

12.1 6.5 2.6 2.2

The reaction medium (final vol, 1.5 ml) contained in a 0.5 cm path cuvette at 23°C: 100 mM phosphate buffer at different pH values, 0.05 mM Hb02, 4-15 pg enzyme, 100 mM methanol (MeOH) and 300U catalase. After deoxygenation of HbOz by about 30x, the reaction was started with benzylamine (0.2 mM final concentration). The rate of HbOt deoxygenation and MetHb formation are expressed as nmol/min/mg of protein.

enzyme) with a Hamilton type microsyringe. Other experimental details are given in the legends to the tables and figures. The following millimolar extinction coefficients (ems,),on heme basis, at 420.6 and 430 nm were used in our calculations: 113 and 133 for Hb; 113 and 52 for HbO,; 49 and 18 for MetHb. Taking into account the fact that HbOl is practically insensitive to H,O, oxidation (White er al., 1959) and considering that the products of reaction are linearly related to the time in the intervals we proceeded the experiments, we can write the rate of MetHb formation (a,): t’r(pmols/min) =

AA420.6 d.A$y,yHb

x Vol

The rate of Hb02 deoxygenation (u2) corrected for MetHb formation ( - AHbO,/At = AHb/At + AMetHb/At) is : u,(pmols/min) =

AAaJO +

mzMe’Hb h;&y’nb

Vol is the reaction volume (ml), d the light path length (cm) and AA,,,.6 and AA,,, the absorption changes per min at 420.6 and 430nm. Isolation of rat liver mitochondria, determination of proteins and cytochrome oxidase activity were previously described (Blrzu et al., 1968, 1971, 1972). Oxygraphic measurements were made with a Clark type oxygen electrode at volumes between 0.5 and 2 ml.

values of u2 and minimal values of v, being approx 100 mM (0.3%). At this concentration methanol does not affect the oxidase activity. Similar results were obtained with ethanol using glucose oxidase or monoamine oxidase as an enzyme source. Comparative determination of monoamine oxidase, glucose oxidase and cytochrome oxidase by diferent procedures (AO,, AHbOz and Abenzylamine)

Having in view that the velocity of HbO, deoxygenation depends upon the concentration of the pigment, its affinity for oxygen as well as upon K”, (Bbrzu & Borza, 1967; Bbrzu et al., 1972) we followed v2 at different concentrations of Hb02 in the presence of the three enzymes. In all cases the plot of l/v, against I/Hb, gives a straight line, which can serve for calculation of Ktb (Fig. 2). The experimental values are close for the three enzymes: 0.034mM Hb for monoamine oxidase; 0.038mM Hb for glucose oxidase and 0.036 mM Hb for cytochrome oxidase from rat liver mitochondria. On the basis of this data we have selected the most convenient experimental con-

RESULTS

The effect of catalase plus methanol on Hb oxidation by H,Oz Table 1 shows the dependence of v, and us as a function of pH during the oxidation of benzylamine by beef kidney monoamine oxidase. As expected the

rate of HbO, deoxygenation is twofold higher in the presence of methanol and catalase than that with catalase alone. At pH values lower than 7 the rate of MetHb formation is high even in the presence of methanol. At pH 7.6 the oxidation of Hb is quite low to be neglected in routine measurements. Both precision and reproducibility of the measurements are obviously improved when one takes into calculations the corresponding corrections made at the two wavelengths mentioned above or obtained with a dual wavelengths spectrophotometer. Figure 1 shows the optimal concentration of methanol for maximal

Methanol

concentration

(mM

1

Fig. 1. Effect of methanol concentration on the rate of Hb02 deoxygenation and MetHb formation. The reaction medium (final vol of 3 ml) contained in a 1 cm path cuvette; 1COmM phosphate buffer (pH 7.6), 0.02 mM HbOz, 1.67 mM glucose, 300 U catalase and different concentrations of methanol. The reaction was started with 10 pg glucose oxidase.

Assay of oxygen consumption

-20

0

20

123

&3

1I Hb02

60

1mM-’ 1

Fig. 2. Effect of HbO, concentration on the rate of pigment deoxygenation by glucose and glucose oxidase. The reaction medium (final vol of 1.5 ml) contained in a 0.5 cm path cuvette: 100 mM phosphate buffer (pH 7.6), 100 mM methanol, 3 mM glucose, 300 U catalase, and different HbOl concentrations (0.012-0.080 mM). The reaction was strated with 7.2 pg glucose oxidase. ditions with respect to sensitivity and accuracy; thus, HbOl concentrations proves to be optimal when it lies between 0.06 and 0.15 mM, which requires cuvettes having a d value of OS-O.2 cm. When respiratory systems with high affinity for oxygen are used (Ki < 10m6 M), the velocity of HbOz deoxygenation (extrapolated to infinite pigment concentration) corresponds to the oxygen uptake, expressed as pmols gas. This situation was checked for the mitochondrial respiratory system (Blrzu et al., 1972; Hultborn, 1972). Since both glucose oxidase and monoamine oxidase have a low affinity for oxygen (Gibson et al., 1964; Tipton et al., 1972) we followed under identical experimental conditions the rate of both Hb02 deoxygenation and of benzaldehyde formation by measuring the absorption increase at 250nm (emM= 12). At this wavelength

HbO,, Hb and MetHb do not exhibit considerable differences with respect to their E,,,~, to interfere in the reaction of benzylamine oxidation. As shown in Table 2 the activity of monoamine oxidase as deduced from the rate of Hb02 deoxygenation (extrapolated to infinite pigment concentration) is lower by 19% than the value obtained on the basis of benzaldehyde formation. In the same table is presented the good concordance between the HbOz method and oxygraphy in estimating mitochondrial cytochrome oxidase activity. Kinetic properties of monoamine oxidase and glucose oxidase assayed by d@rent procedures (A02 and Abenzylamine) It is well documented that both monoamine oxidase and glucose oxidase are enzymes of a ping-pong

Table 2. Comparison of monoamine oxidase and cytochrome oxidase activity measurements as determined by Abenzylamine, A02 and AHbO* Monoamine oxidase’

Cytochrome oxidaset

AHbOZ Experiment

ABA

AHb02

1 2 3 4 5

0.130 0.102 0.050 0.164 0.122

0.106 0.080 0.042 0.130 0.101

ABA

AHbOl

x 100

82 78 84 19 83

A@

AHb02

0.66 0.80 1.03

0.64 0.80 1.01

AO,

x 100

91 100 98

* The reaction mixture contained at 1.5 ml final vol; 0.5 cm path cuvette and 23°C: 100 mM phosphate buffer (pH 7.6), 1OOmM methanol, 300U catalase, 0.019-0.06mM HbOz and O.OOl~).lOmM benzylamine (BA). After deoxygenation of HbOz by about 30x, the reaction was started with 25 pg purified monoamine oxidase. Parallel samples were recorded at 250 nm (ABA) and 430420.6 nm (AHbOz). The results are expressed as pmols BA oxidized/min/mg of protein, and as pmols Hb02 deoxygenated/min/mg of protein (extrapolated for infinite pigment concentration, considering Kib as 0.034 mM), respectively. t The reaction medium contained at 1.5 ml final vol; 0.5 cm path cuvette and 23°C: 75 mM phosphate buffer (pH 7.2) 3 mM Na ascorbate, 0.02 mM cytochrome c, 0.045 mM HbOZ and 0.018-0.03 mg rat liver mitochondrial protein previously solubilized with Lubrol WX (0.5 mg/mg of protein). The results are expressed as pmols Hb02 deoxygenated/min/mg of protein, In separate experiments the oxygen uptake (~mols/min/mg of protein) was measured oxygraphycally.

OCTAVIANBARZU and MANASEDAN~REANU

124

0.5

-o.i

0

0.02

O.OL

0.06

i I

- 0.25

0

0.25

0.75

0.50 1/Glucose

1.00

1.25

(mM_‘)

Fig. 3. Dependence of glucose oxidase activity on the concentration of glucose at different oxygen concentrations. The experiments were made at pH 7.6 and 1.5 ml final vol in a medium containing 100 mM phosphate buffer, 300 U catalase and different glucose concentrations. When glucose oxidase activity was measured oxygraphycally (0.24 mM O,), 18 pg enzyme were used and methanol omitted. In the spectrophotometric assay 0.048 mM HbO,, 100 mM methanol and 7.2 pg enzyme were used. The results are expressed as pg atoms oxygen/min/mg of protein, and as pmols HbO,/min/mg of protein (extrapolated for infinite pigment concentration), respectively.

kinetics (Gibson et al., 1964; Weibel & Bright, 1971; Oi & Yasunobu, 1973). As a consequence, the dependence of l/u upon l/benzylamine or l/glucose, at different oxygen concentrations, is described by a family of parallel straight lines. Decrease of substrate concentration leads to a diminuation of K”, and, conversely, a diminuation of oxygen concentration will decrease the K, value for the substrate. Indeed, by measuring oxygraphically the activity of glucose oxidase at 0.24mM 0, (air saturated water), we found for glucose a K, value of 17 mM, in agreement with values previously reported (Tipton, 1972; Keilin & Hartree, 1948). Under the conditions of the spectrophotometric measurements, at a 70% saturation of the pigment with oxygen (corresponding to an oxygen concentration of about 0.04 mM), K, for glucose was only 0.8 mM. The same situation was also proved for monoamine oxidase, whose activity was measured at 250 nm (benzylamine oxidation) under the condition of saturation with pure oxygen (1.38 mM), saturation

with air (0.24mM oxygen) or at 430nm (HbOz deoxygenation) at two different values of the pigment saturation with oxygen (Fig. 3 and Table 3). The parallelism of the straight line family, representing the plot of l/u against lfienzylamine is also given by the constant ratio VJK~mY1amine, irrespective of the 0, concentration at which the measurements were made. DlSCL’SSION

The measurement of oxygen uptake, most currently using manometric and oxygraphic procedures, is still an important tool for investigating the oxidative processes in a wide variety of biological preparations such as tissue fragments, isolated cells, isolated organelles and purified enzymes. The utilization of the spectrophotometric method, employed for studying the mitochondrial (Bbrzu et al., 1972; Benga et al., 1972; HodPrnSu et al., 1973; Nessi et al., 1977), cellular (Hultborn, 1972; Hultborn & HydCn, 1974) or tis-

Table 3. Kinetic parameters of purified kidney monoamine oxidase at different oxygen concentrations O2 concentration (mM) 1.38 0.24 0.04* 0.016*

Kbenqlaminp “imM)

V, (pmols/ min/g protein)

0.222 0.147 0.025 0.015

2.08 1.37 0.23t 0.13t

V,lG 9.4 9.3 9.2 8.7

* Approximated from the dissociation curve of hemoglobin. measurements being made at 75% saturation and 25% saturation, respectively. t Extrapolated for infinite HbOl concentration.

Assay of oxygen consumption

sular (Raddacz & Kucera, 1977) respiration may be extended to other systems, including those generating H,02, such as the enzymes described in this work. Protection of Hb against the oxidative action of H202 is achieved in the presence of catalase and appropriate hydrogen donors like ethanol or methanol. When the affinity for oxygen of the investigated oxidase is low (K”, > 10m6 M) the necessity arises to determine a correction factor in order to convert the rate of HbO, deoxygenation into moles or atoms of oxygen. This factor represents the ratio of the rate of formation (or disappearance) of one of the reactants to the rate of HbO, deoxygenation, measured under identical experimental conditions. When the affinity of oxidases for substrate is dependent upon O2 concentration, the kinetic parameters determined under the conditions of saturation with air or pure oxygen should be reconsidered for the conditions of spectrophotometric measurement of oxygen uptake; this is so because the last procedure operates at relatively low oxygen tensions, which are in fact the physiological ones for mammalian systems (Barzu, 1978). A comparison of the kinetic parameters obtained by measuring oxygen consumption (including the spectrophotometric procedure) might offer new information regarding the mechanism of reaction or the physiological significance of different oxidases (Gibson et al., 1964; Oi & Yasunobu, 1973; Jain et al., 1973; Hellerman et al., 1972). The high sensitivity of the spectrophotometric method, along with recent technical improvements like the rapid mixing of the reactants, maintenance of the optical homogeneity even of such preparations as cellular suspensions (Bbrzu, 1978) allow the procedure to be extended to a variety of studies. Among them, one may take into consideration oxidases with a low specific activity, whose assay involves laborious calorimetric, fluorimetric or radiometric procedures such as liver phenylal~inhydroxyla~, brain or adrenal tyrosin-hydroxylase, microsomal hydroxylases involved in drug metabolism. CONCLUSION

The spectrophotometric method for assay of oxygen consumption based on the utilization of HbO, as oxygen donor and indicator, may be extended to any respiratory system including those generating H,O,, such as monoamine oxidase or glucose oxidase. Protection of Hb against the oxidative action of H,O, is achieved in the presence of catalase and appropriate hydrogen donors like ethanol or methanol. ~c~~ow~e~~e~e~f-one of us (O.B.) is grateful to Dr R. W. VonKorff for kind hospitality at the Department of Biochemistry, Maryland Psychiatric Research Center, as well as for many suggestions and stimulating discussions. REFERENCES B~RZU 0. & BORZAV. (1967) Spectrophotometric method for assay of mitochondria1 oxygen uptake using oxyhemoglobin as indicator and oxygen donor. Anafyt. Bio.. them. 21. 344-357. BI~RZUO.,’ MURESANL. 8~ TARMUREC. (1968) Spectrophotometric method for assay of mitochondrial oxygen uptake. II. Simultaneous determination of mitochondrial swelling, respiration and phosphate esterification. Anulyr. Biochem.

24, 249-258.

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FREIJ. (1977) Leucocyte energy metabolism. VII. Respiratory chain enzymes oxygen consumption and oxidative phosphorylation of mitochondria isolated from leucocytes. Enzyme 22, 183-195. NICHOLLSP. & SCHONBAUM G. R. (1963) In The Enzymes, 2nd edn, (Edited by BOYERP. D., LARDYH. A. & MYRBXCK K.) Vol. 8, pp. 1477225. Academic Press, New York. Or S. & YASUNOBU K. T. f1973) Mechanistic aspects of the oxidation of amines by monoamine oxidase. Biochem. biophys. Res. Commun. 53, 631-637. RADDACZE. & KUCERAP. (1977) Continuous microspectrophotometric measurement of oxygen consumption of in vitro cultured chick embryo. Experientia 33, 9. TIPTON K. F. (1972) In Advances in Biochemical Psychopharmacology, Vol. 5, pp. 11-24. Raven Press, New York.

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