An improved spectrophotometric assay for superoxide dismutase based on epinephrine autoxidation

ANALYTICAL BIOCHEMISTRY 90, 81-89 (1978)

An Improved Spectrophotometric Assay for Superoxide Dismutase Based on Epinephrine Autoxidationl

MING SUN AND SEYMOUR ZIGMAN

Department of Ophthalmology und Biochemistry, University of Rochester School

of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, Nercj York 14642

Received December 19. 1977

Superoxide dismutase activity was assayed in terms of its ability to inhibit the radical-mediated chain-propagating autoxidation of epinephrine. The enzyme assay based on adrenochrome absorption at 480 nm has been improved by measuring the absorption change at 320 nm. This alternative procedure was found to be 6 to 10 times more sensitive and more consistent than that measured at 480 nm.

Superoxide dismutase (SOD)* (EC 1.15.1.1.) is suspect of being present in all oxygen-consuming living cells (1,2). It has a crucial function in pro- tecting living organisms against unrestrained reactivity of the superoxide radical (O,-.) which is generated in the univalent reduction of oxygen either chemically or enzymatically (2). For this reason, there is a growing concern and interest in this enzyme system among many biologists and biochemists.

In recent years, attempts have been made to find a simple, reliable, and quantitative method to assay this unique enzyme under different conditions (2,3). A simple and rapid spectrophotometric assay for SOD was reported by Misra and Fridovich (4). This method is based on the ability of SOD to inhibit the autoxidation of epinephrine at alkaline pH. Since the oxidation of epinephrine leads to the production of pink-colored adrenochrome, the rate of increase of the absorbance at 480 nm, which represents the rate of autoxidation of epinephrine, can be conveniently followed. SOD was found to inhibit this radical-mediated chain process.

Although this assay method is sensitive and convenient, it also has several inherent defects. For example, the absorption at 480 nm (A& of adrenochrome corresponds to a relatively weak and broad absorption band with an extinction coefficient of about 4000 (5). Second, adrenochrome is not stable, particularly at alkaline pH conditions, as we will demonstrate below. Third, epinephrine is not the direct precursor of adrenochrome,

r This work was supported by NIH Grant No. EY 00459. 2 Abbreviations used: SOD, superoxide dismutase.

81 0003-2697/78/0901-0081$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction m any form reserved.

82 SUN AND ZIGMAN

as several intermediates are known to be involved in the autoxidative conversion of epinephrine to adrenochrome (6,7).

In the present report, we have examined the stability of adrenochrome. A more sensitive and stable region of absorption at 310-320 nm was em- ployed to assay for SOD activity. In addition to the higher sensitivity at- tained by measuring at 320 rather than at 480 nm, the measurements in this near-uv region take into account the other possible derivatives of epinephrine which do absorb in this region and which are generated during the same autoxidative process. Significant improvements in the time dependency of the reaction and the consistency of the measurement over A48,, measurements were demonstrated.

METHODS AND MATERIALS

L-Epinephrine, superoxide dismutase [purified from bovine blood; 2880 units/mg (8)], and adrenochrome were purchased from the Sigma Chem- ical Company. Stock solutions of 20 and 50 mM epinephrine were made in 0.1 N HCl and aliquots were stored in a freezer (-20°C) until use. Superoxide dismutase stock solution was made up at 1 mg/ml in 50 mM Tris-HCl buffer, pH 7.2. Adrenochrome (20 mM stock solution) was made fresh each time in deionized distilled water. Absorption spectra were recorded using a Cary 14 recording spectrophotometer, and absorption readings at individual wavelengths were made using a Beckman DU spectrophotometer.

To assay for SOD activity, l-20 ~1 of SOD stock solution was first added to 50 mM Tris-HCl or 50 mM sodium carbonate buffer at the desired pH, and the epinephrine was added at time zero. The final assay mixture contained I-20 pg of SOD and 0.5 to 2 x 10e4 M epinephrine in a total of 2 ml of buffer medium. Matched controls at the same pH but with- out SOD were measured at time intervals in parallel with those of the experimental sample. Activity of SOD was expressed as the percentage of inhibition of the control absorption at 320 or 480 nm. Calf retina cytosomal fraction was prepared in 50 mrvt Tris-HCl buffer, pH 7.4. Fresh calf eyes were obtained from a local slaughterhouse. Aldolase, chymotrypsinogen A, and ribonuclease A were from Worthington Biochemical Corporation.

RESULTS

Absorption spectra of epinephrine and adrenochrome are presented in Fig. 1. Sample solutions were in 50 mM Tris-HCl buffer at 0.1 mM concentration. Epinephrine exhibits two absorption peaks at 222 and 280 nm. Adrenochrome has an absorption band at 302 nm besides the one in the visible region. The intensity of absorption in the near-uv region is roughly three times that in the visible region, and its extinction coefficient is

SUPEROXIDE DISMUTASE/IMPROVED ASSAY 83

0.8 -

0.6 -

Q

d 0.4-

650 600 550 500 450 400 350 300 250

WAVELENGTH, nm

FIG. 1. Absorption spectra of epinephrine and adrenochrome at 0.1 mM in 50 mM Tris- HCI buffer, pH 7.2.

approximately three times that of epinephrine in the same wavelength region.

The course of epinephrine conversion at pH 10.2 in 50 mM sodium carbonate buffer is shown in Fig. 2. The spectral scanning time from

d d

0.8

0.6

0.4

0.2

0 530 490 450 410 370 330 290 250

WAVELENGTH, nm

FIG. 2. Absorption spectral change during epinephrine autoxidation. Concentration of epinephrine is 0.1 mM in 50 mM sodium carbonate buffer, pH 10.1. Time indicated on the figure represents the starting scanning time for the particular spectrum.

84 SUN AND ZIGMAN

230 to 530 nm was 6 min (10 A/S; chart speed: 2 in./min). Thus, the absorption at any point between 230 and 530 nm of the spectrum was recorded at the finite time equal to:

T + (A - 230) x k (minutes),

where T is the starting time of scanning for the particular spectrum as indicated on the figure and A is the wavelength at any point. It is clear that at time intervals from 0 to 15 min the 480-nm absorption has already peaked. The spectral changes observed in this figure revealed a further conversion of adrenochrome to a more stable product. Also, Fig. 2 shows a progressive shift of the absorption maximum at 290 nm to the near-uv region, accompanied by an enhancement of absorptivity. After 30 min of standing, the absorption spectrum displayed a clear absorption maximum at 300 nm, with a prominent shoulder at 330 nm. This shoulder of absorption corresponds to the dominant absorption band of the final product(s).

Figure 3 shows that conversion of adrenochrome to unknown product(s) at alkaline pH is marked by the disappearance of its pink color at 480 nm.

0.6

0 550 450 350 250

WAVELENGTH, nm

FIG. 3. Absorption spectral change during adrenochrome autoxidation. Adrenochrome concentration is 0.05 mM in 50 rnM sodium carbonate buffer at pH 9.76. Times indicated in the figure are the starting times of spectral scanning.

SUPEROXIDE DISMUTASEiIMPROVED ASSAY 85

Adrenochrome is less stable in media with pH values higher than 9.7. At pH 5.0 and O-SC, adrenochrome is stable for days.

Spectral changes of adrenochrome were followed at pH 9.76. A clear isobestic point is seen at 418 nm, indicating that there is only one absorb- ing product from adrenochrome conversion. This product has a higher absorptivity at 302 nm than adrenochrome, and it has a unique shoulder at 350 nm. Also a noticeable feature of these spectra is an inflection point at 320 nm. At this wavelength the intensities of the absorption of different time courses show only very small changes.

SOD activity was assayed according to Misra and Fridovich (4) utilizing the enzyme’s ability to inhibit the conversion of epinephrine to adrenochrome. Thus the enzyme activity should be proportional to AAdsO, (AEpi

- AEpi+SOD at 480 nm, where AEpi is the optical density measured in the absence of SOD, and AEpi+SOD is that in the presence of SOD). The percentage of inhibition of adrenochrome formation could be calculated from AA4SdA480,Epi. Figure 4 shows SOD concentration and time-dependent activities in a pH 10.2 buffer. Several features are to be noted: (i) At 2 pg/ml SOD concentration, the activity has already reached the plateau; (ii) A480 measured at 5 min appeared to reach the same level as that at 10 min; at 20-min intervals, the Ada0 of the experimental sample

SOD CONCENTRATION, UG/ML

FIG. 4. Time- and concentration-dependent SOD activity, expressed as AA, i.e., AEP,

-A EDI+SOD. Epinephrine concentration is 0.1 mM in 50 mM sodium carbonate buffer, pH 10.0. Circles, AA values measured 5 min after the addition of epinephrine; triangles, at 10 mitt; squares, at 20 min. Solid symbols, A.&,, values; open symbols, A,&, values. At the plateau and/or maximum of 5-min measurements, the inhibitions were 89.5% for AA,,, and 80.2% for AA,,,.

86 SUN AND ZIGMAN

exceeded that of the control, indicating that for such prolonged incubation periods, A,,, could not be used to represent the SOD activity; (iii) in contrast to the Ads,, course, A,,, increases proportionally with time as shown in this figure at 5- and IO-min intervals; and (iv) absorption change at 320 nm is about 6 to 10 times greater than that at 480 nm.

Figure 5 shows the time courses of SOD inhibition of epinephrine oxidation at different pH values as represented by AA at 320 nm. At pH 10.0, AASZO values between the control and experimental samples were close to linear with time. At more alkaline conditions, the enzyme time course shows a sigmoid shape, and as a result, the activity expressed as percentage of inhibition (AA/A,pi) at these pH values varied greatly with time. Thus, we found that the enzyme activity at pH 11 .O, after standing for 10 min. is lower than that at pH 10.0.

lOOr

80-

8 20-In

0 /it

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, , , , # , , , (

0 2 4 6 8 IO

1 012345678 9 IO

MINUTES

FIG. 5. Time course of SOD inhibition of autoxidation of epinephrine at pH 10.0 (A), 10.5 (O), 10.8 (U), and 11.1 (0). SOD at 10 &ml was added to 2 x lo-’ M epinephrine in sodium carbonate buffer media at indicated pH values. The enzyme activities (bottom) expressed as AA,,, (i.e., AEP, - AEPi+SOD ) and the percentage of inhibition (top) calculated from AA32dAEP, are shown.


Figure 6 presents the pH-dependent absorptions at 320 nm in the presence and absence of 10 pg/ml SOD. The largest gap between the two curves appeared between pH 9.5 and 10.8. Also, Fig. 6 shows that the enzyme activity could be measured at a pH below 8.0. The rate of AszO increase during the first 5 min was about 0.01 unit/min. In the presence of enzyme this rate is decreased by about half at this pH.

Table 1 shows the SOD activity in the calf retina cytosomal fraction. The enzyme activity was completely abolished after the enzyme solution was heated to 80°C for 5 min. Aldolase, chymotrypsinogen A, and ribonuclease A possess no SOD activity.

DISCUSSION

There are two regions in the absorption spectrum of adrenochrome which are distinguishable from the absorption spectrum of epinephrine (Fig. 1). One is in the near-uv region and the other is in the visible region. The change in the near-uv absorption region was marked by two maxima (at 300 and 320 nm, respectively) arising during the course of epinephrine autoxidation (Fig. 2). It is clear that the enhancement in the near-uv region

70 8.0 9.0 100 11.0

PH

FIG. 6. pH dependence of AEp, and AEP,+S,,D at 320 nm. SOD concentration is 10 pg/ml. Epinephrine concentration is 0.1 mM in 50 mM Tris-HCI or sodium carbonate buffer. (0) A,,,, IO-min measurement; (0) A,,,+soo, IO-min measurement; (A) A,,,, 5-min measurement; (4 AE,,+soo, 5-min measurement.

88 SUN AND ZIGMAN

TABLE 1

SOD ACTIVITY EXPRESSED AS PERCENTAGE OF INHIBITION OF

THE AUTOXIDATION OF EPINEPHRINE

Enzyme source

0 SOD (Sigma), 5 pg Retina cytosomal (calf), 0.4 mg Retina cytosomal (calf), 0.8 mg Retina cytosomal (heat-treated),

1.0 mg Aldolase, 50 pg Chymotrypsinogen A, 50 pg Nuclease A, 50 pg

A 320 Inhibition (10 min)” (%o)

1.86 - 0.34 82 0.80 57 0.52 72

1.80 3 1.85 0 1.83 1.6 1.88 0

a AszO was followed for the initial 5 min in 50 mM sodium carbonate buffer at pH 9.8. Epinephrine concentration was 0.1 mM.

is much greater than that in the visible region. The maximum at 300 nm probably pertains to adrenochrome and/or its derivative (Fig. 3). How- ever, these may not be the only products contributing to the absorption in this region; comparison of the intensity of the absorption peak at 300 nm with that at 480 nm shows that A3JAd8,, is greater than the ratio of the extinction coefficients (&,J&,,) of adrenochrome at these two wavelengths (Fig. 1). The absorption maximum at 320 nm is probably due to a different derivative than adrenochrome. Since SOD leads to an inhibition of its formation, we can safely assume that this is also a product resulting from the radical chain-propagating process. Thus, assay of SOD at 320 nm not only enhances the sensitivity but is more inclusive of the total radical chain-propagating reaction.

One of the other defects in the Aas,, assay measurement is that adrenochrome is unstable, particularly at alkaline pH. The further conversion from adrenochrome to a more stable product is marked by a decrease in the AJ8,, peak and a simultaneous increase in As00 absorption (Fig. 3). This oxidative conversion is also catalyzed by O,- (4). Misra and Fridovich (4) reported a decrease of 0.017 absorption units/min for the adrenochrome oxidation at 480 nm in pH 10.2 buffer medium. They also found that SOD could inhibit up to 25-30% of this process at concentrations ranging from 3 to 20 pg/ml at the same pH (4). Because of this double role played by O,- in adrenochrome formation and conversion, and because both O,--mediated processes are subject to SOD inhibition, the two opposing effects of SOD on AA,,, will partially cancel each other. As a result, the observed SOD activity at AdBO, p articularly at times after the initial period, is always lower than the true value.


Indeed, SOD time course studies at Ah*,, show a large deviation from linearity (Fig. 4). The absorption of adrenochrome started to diminish after the initial 15 min (Fig. 2, at 480 nm). In contrast with A,,, measurement, Asso shows about the same increment from 0 to 5 minutes and from 5 to 10 min (Fig. 4). The time courses (Fig. 5) at different pH values indicate a continuing increase of As2,, during the initial O-12 min at pH 10.

The pH-dependent SOD activities measured at As2,, were presented in Fig. 6. Because of the greater sensitivity of the measurement at 320 nm than that at 480 nm, we were able to measure the SOD inhibition at pH values lower than 8.0, although the baseline rate is much lower than that at pH 10.2. In addition, since the measurement at 320 nm is not hindered by adrenochrome oxidation, we could measure the SOD activity even at pH 10.8 to 11.1. At such alkaline pH conditions the enzyme could be in- activated and would thus decline in activity with time. In conclusion, we have demonstrated that both epinephrine and adrenochrome are unstable at alkaline pH. The SOD assay based on Ad8,, measurement as a function of adrenochrome formation is not as sensitive, stable, or consistent as the measurement at As*,,.

REFERENCES

1. McCord, J. M., Keele, B. B., Jr., and Fridovich, I. (1971) Proc. Nat. Acad. Sci. USA 68, 1024.

2. Fridovich, I. (1974) Advan. Enzymol. 40, 35-97. 3. Misra, H. P., and Fridovich, I. (1977) Anal. Biochem. 79, 553-560.

4. Misra, H. P., and Fridovich, I. (1972) J. Biol. Chem. 247, 3170-3175. 5. Green, S., Maxur, A., and Shorr, J. (1956) J. Biol. Chem. 220, 237-255.

6. Hawley, M. D., Tatawawadi, S. V., Piekarski, S., and Adams, R. N. (1967) J. Amer. Chem. Sot. 89, 447-450.

7. Ball, E. G., and Chen, T. T. (1933)J. Biol. Chem. 102, 691-719. 8. McCord, J. M., and Fridovich, I. (1969) J. Biol. Chem. 244, 6049-6055.

9. Nagatsu, T. (1973) Biochemistry of Catecholamines, Univ. of Tokyo Press, Tokyo, Japan.

Documents

An improved spectrophotometric assay for superoxide dismutase based on epinephrine autoxidation