[28] SOD ACTIVITY IN Drosophila 287

mononuclear cells, nonstimulated and stimulated by PMA, and also shows the superoxide dimutase effect. Nonstimulated mononuclear cells reach an equilibrium with external H202 at about 0.25/zM H202, and after stimulation by PMA the level is increased to about 0.80/zM H202. Figure 2B shows the kinetics of H202 release from thymocytes. The basal situation indicates an intracellular concentration of about 35 nM H202, which is increased to about 45 and 75 nM H202 after apoptosis stimulation by methylprednisole and thapsigargin, respectively. The addition of superoxide dismutase to the reaction medium usually has no significant effect on cells other than the phagocytic cells, which release significant amounts of 02- to the suspending medium.

[28] Biochemical Assay of Superoxide Dismutase Activity in Drosophila

By ROBIN J. MOCKETT, ANNE-CI~CILE V. BAYNE, BARBARA H. SOHAL,

and RAJINDAR S. SOHAL

I n t r o d u c t i o n

A variety of indirect assays of superoxide dismutase (SOD) activity have been described. 1 These assays use chemical or enzymatic systems to generate superox- ide anion radicals (02"-), which then react with an indicator molecule. Superoxide dismutase activity decreases the supply of 02"-, and is measured on the basis of the degree of inhibition of the indicator reaction. Validation of these methods requires the exclusion of numerous potential interferences. Potential sources of error include direct inhibition or amplification of either 02"- production or the indicator reaction by other enzymes, metal ions, or other substances in tissue samples.

One convenient method uses xanthine-xanthine oxidase to generate 02"-, and reduction of nitroblue tetrazolium (NBT) to a blue formazan as the indicator reac- tion. The NBT assay was originally used to determine SOD activity either spec- trophotometdcally or in polyacrylamide gels. 2 The method was later modified to suppress various interferences occurring in crude mammalian tissue homogenates. 3 The introduction of metal chelators that also inhibit the electron transport chain was particularly advantageous. In our laboratory, this assay has been adapted slightly for measurement of SOD activity in Drosophila homogenates.

1 L. Floh4 and E 0tting, Methods Enzymol. 10S, 93 (1984). 2 C. Beauchamp and I. Fridovich, Anal. Biochem. 44, 276 (1971). 3 D. R. Spitz and L. W. Oberley, Anal. Biochem. 179, 8 0989).

Copyright 2002, Elsevier Science (USA). All rights reserved.

METHODS 1N ENZYMOLOGY, VOL. 349 0076-6879/02 $35.00

288 In Vivo SOURCES, CELL SIGNALING [28]

Supe rox ide D i s m u t a s e Activity A s s a y

Principle

Xanthine-xanthine oxidase is used to generate 02'- , which reduces NBT to a blue formazan. Inhibition of this reaction by SOD is monitored spectrophotometri- cally at 560 rim. Treatment of samples with cyanide permits direct measurement of MnSOD activity. 3 Pretreatment with sodium dodecyl sulfate (SDS) permits direct measurement of Cu,ZnSOD. 4 Total SOD activity is measured in untreated samples.

Reagents

Potassium phosphate buffer (50 mM), pH 7.8: All other reagents are dissolved in this buffer except SDS and KC1.

Xanthine (1.8 mM): NaOH is added to dissolve the xanthine. Nitroblue tetrazolium (NBT; 2.24 mM): Prepare fresh daily and shield from

light. Diethylenetriaminepentaacetic acid (DTPA; 1.33 mM). Catalase (40 units/ml) Bathocuproine disulfonic acid, disodium salt (BCS; 10 mM). NaCN (0.33 M): Prepare fresh daily. Bovine serum albumin (BSA, defatted; 0.20 mg/ml) in potassium phosphate

buffer containing 1.33 mM DTPA: Referred to in working solution as potassium phosphate-DTPA-BSA

Xanthine oxidase (about 0.025 units/ml) in potassium phosphate buffer con- taining 1.33 mM DTPA and 0.20 mg/ml BSA; the amount is adjusted until the absorbance change in working solution is 0.02-0.03/min in the absence of SOD.

Sodium dodecyl sulfate (SDS; 10%, w/v). KC1 (3 M) Working solution: The reagents listed above are mixed in the following ratio

and shielded from light: 132 ml of potassium phosphate-DTPA-BSA, 5 ml of catalase, 5 ml of NBT, 17 ml of xanthine, and 1 ml of BCS (for assay of MnSOD, add 3 ml of NaCN and subtract 3 ml of potassium phosphate- DTPA-BSA).

Sample Preparation

Whole-body homogenates (5%, w/v) of fresh or frozen flies are prepared in 50 mM potassium phosphate buffer, pH 7.8, and centrifuged 10 min at 10,000g, 4 °. For total SOD activity, the supernatants are diluted 8 : 11 with the same buffer (80/zl

4 B. L. Geller and D. R. Winge, Anal. Biochem. 128, 86 (1983).

[28] SOD ACTIVITY IN Drosophila 289

of supernatant is combined with 30 ~1 of buffer), and recentrifuged for 10 min, 20,000g, 4 °. For MnSOD activity, the 20,000g supernatant is then incubated with 5 mM NaCN (final concentration) for at least 45 min at room temperature. For Cu,ZnSOD activity, the 10,000g supernatant is mixed with 10% (w/v) SDS (20/zl of SDS is combined with 80 ~1 of supernatant), incubated for 30 min at 37 °, and then chilled for 5 min on ice. The mixture is treated next with 3 M KC1 (10/zl KC1 per 100 #1), chilled for 30 rain on ice, and then centrifuged for 10 min, 20,000g, 4 °. The supernatants are retained for measurement of activity.

Procedure

1. The absorbance change is initially measured in one cuvette, containing 800 #1 of working solution, 100 #1 of potassium phosphate, and 100 #1 of xanthine oxidase, for 4 -6 min at 560 nm, 25 °, to check the dilution of xanthine oxidase and the linearity of the reaction. If the reaction becomes nonlinear after less than 4 min, fresh xanthine oxidase should be prepared.

2. Measurements of SOD-containing samples are made simultaneously in six cuvettes. Mix 800/~1 of working solution, 100/zl of potassium phosphate plus diluted sample, and 100 gl of xanthine oxidase per cuvette. Include one cuvette with no sample to obtain the maximum, uninhibited rate. Add varying amounts of samples to the other cuvettes. A typical sample is diluted about 8x for measurement of total SOD, 6x for Cu,ZnSOD, and 3x for MnSOD. Typical sample volumes with these dilutions are 15, 20, 30, 40, and 60/zl. Ideally, the dilution is such that one SOD sample inhibits the reaction by less than 50%, another inhibits by about 50%, and the other three inhibit by more than 50%. Some deviation from this guideline is permissible.

3. A blank reaction with the maximum concentration of sample and either no xanthine or no xanthine oxidase will normally have a rate close to 0, but this assumption should be checked occasionally.

4. The protein concentration is determined separately for the samples with and without SDS pretreatment. The Lowry and bicinchoninic acid (BCA) assays are both suitable for this purpose.

Calculation of Activity

The unit of SOD activity is defined as the amount that inhibits NBT reduction half-maximally under the conditions described above. Note that the unit is con- ventionally defined as half-maximal inhibition in a 3-ml reaction volume, whereas the reaction volume has been reduced to 1 ml here, so a conversion factor of 3 is introduced in comparisons with some other work.

The slope divided by the intercept (ordinate) of double-reciprocal plots, that is, plots of 1/% inhibition of NBT reduction vs.1/protein content, gives the amount

290 In Vivo SOURCES, CELL SIGNALING [28]

of sample protein that contains 1 unit of SOD activity. 5 The reciprocal of this number is the number of units of SOD activity per unit protein. Although this calculation gives a reasonable approximation of the SOD activity in most samples, it also tends to amplify the impact of pipetting errors or residual interferences in the homogenates. More consistent activity results are obtained by determining the reciprocal of the IC50, using Eq. (1) (provided in the GraFit 4 software package6):

NBT reduction rate = range

(protein content) s 1 + (ICs0)s

+ background (1)

Use of Eq. (1) requires the choice of reasonable starting estimates for the four parameters in the estimatrix. The estimatrix value of the background is almost always zero, except in the special case of flies with Cu,ZnSOD gene mutations, discussed below. The range is estimated as the absorbance change in the uninhibited reaction, minus any background. The starting estimate for the slope factor, s, is normally 1.0, and the estimate for the IC50 depends on the expected level of SOD activity in the sample. If the initial estimate differs from the final result by more than 1 unit, the procedure can be repeated with new starting values in the estimatrix to ensure that the iterative calculation converges on a reasonable final result. The reasonableness of the result is inferred from the fit of the curve to the data points in the plot of NBT reduction rate versus protein content (protein content is plotted on a logarithmic scale).

Activity is determined three or four times for both total SOD and either MnSOD or Cu,ZnSOD (via NaCN treatment or SDS pretreatment, respectively). The re- maining quantity may then be calculated by subtraction of the other two, but for some samples this is less accurate than direct measurement of all three quantities.

D i s c u s s i o n

To confirm that NBT reduction by Drosophila tissue samples is due to SOD activity, homogenates prepared as described for the spectrophotometric assay were also separated on native polyacrylamide gels. These gels were soaked in NBT, followed by riboftavin-N,N,N',N'-tetramethylethylenediamine (TEMED), and SOD activity was detected on the basis of its inhibition of OK--mediated NBT photoreduction. 2 Using homogenates of flies containing a wild-type complement of SOD alleles prepared for the assay of total SOD, three bands were observed. The uppermost band, identified putatively as MnSOD, was always eliminated by pretreatment of samples with SDS. The two lower bands, identified putatively as Cu,ZnSOD, were both totally absent in samples from SOD x39/SOD x16 flies,

5 j. E. Bell and E. T. Bell, "Proteins and Enzymes." Prentice-Hall, Englewood Cliffs, New Jersey, 1988. 6 R. J. Leatherbarrow, "GraFit," version 4.0. Erithacus Software, Staines, U.K., 1998.

[28] SOD ACTIVITY IN Drosophila 291

which contain deletions overlapping part of the coding region of both copies of the Cu,ZnSOD gene. 7 Bovine erythrocyte SOD, used as a positive control, also migrated as a double band on native gels, an observation that has also been made with purified SOD from other sources.l'2

These identifications were confirmed immunologically, using rabbit anti- Drosophila MnSOD and Cu,ZnSOD antibodies. 8 After SDS-PAGE and immuno- blot analysis, strong signals were detected from wild-type samples with both antibodies. The SDS pretreatment essentially abolished the signal detected with the MnSOD antibody, without decreasing the signal with the Cu,ZnSOD antibody. Conversely, in the SOD x39/SOD x16 sample, there was no reaction with the Cu,ZnSOD antibody, but a strong signal was detected with the MnSOD antibody. Pretreatment of SOD x39/SOD x16 samples with SDS abolished SOD activity on activity gels, and abolished the reaction of the denatured sample with MnSOD an- tibody in immunoblot analysis. Regrettably, these antibodies are unreactive with the native proteins, and SOD activity is lost on SDS-containing gels, so a direct link cannot be made between the bands on activity gels and immunoblots.

In homogenates of Drosophila samples with wild-type or higher levels of Cu,ZnSOD and MnSOD activity, the sum of the SOD activities after SDS pretreat- ment (Cu,ZnSOD) and NaCN treatment (MnSOD) approximates the total SOD activity fairly. The background level of NBT reduction in the ICs0 calculation is zero, that is, half-maximal inhibition of NBT reduction is the same as 50% inhibi- tion. In the case of flies with Cu,ZnSOD gene deletions, the ICs0 background is still zero in the calculation of total SOD activity, because of the presence of MnSOD. However, after pretreatment of such samples with SDS, where no Cu,ZnSOD ac- tivity is detectable on activity gels, there remains a low level of inhibition of NBT reduction in the spectrophotometric assay. The ICs0 calculation does not then con- verge on a reasonable result, that is, there is no fit to the data points, unless up to 65% of the NBT reduction is treated as background in the estimatrix. The absorbance maximum at the end of the reaction is shifted from 545-550 to 520- 525 nm, and there is a plateau in NBT reduction, so that the maximum percent inhibition is 35-60%, not 100%. With large volumes of undiluted samples, the plateau is followed eventually by a further decrease in NBT reduction, possibly through direct inhibition of O2"- production.

It is likely that most or all of the "activity" detected in the SDS-pretreated, Cu,ZnSOD null samples is spurious, reflecting residual interferences in the assay that are drowned out in samples with wild-type levels of SOD activity. Owing to the high background in the ICs0 calculation, there is a large discrepancy between 50% inhibition and half-maximal inhibition of NBT reduction. For such samples,

7 j. p. Phillips, J. A. Tainer, E. D. Getzoff, G. L. Boulianne, K. Kirby, and A. J. Hilliker, Proc. Natl. Acad. Sci. U.S.A. 92, 8574 (1995).

8 V. I. Klichko, S. N. Radyuk, and W. C. Orr, Neurobiol. Aging 20, 537 (1999).

292 In V]vo SOURCES, CELL SIGNALING [28]

an alternative calculation can be made by taking the point closest to 50% inhibition, calculating the actual protein content and percent inhibition, and then making a linear extrapolation to 50% inhibition. Although this estimate is not accurate, be- cause the assumption of a linear relationship between protein content and percent inhibition is false, the activity gel results are much more consistent with this crude approximation than with the IC50 calculation. The results of the two calculations are almost identical when the estimatrix background is zero, but they diverge by up to 5-fold when both copies of the Cu,ZnSOD gene contain deletions. When only one Cu,ZnSOD gene is expressed, as a transgene in a Cu,ZnSOD null back- ground, the estimatrix background may be zero or there may be a low background and a divergence between the calculations, depending on the level of transgene expression.

C o n c l u s i o n s

The conclusions drawn from these experiments are that Drosophila Cu,ZnSOD and MnSOD inhibit the reduction of NBT by O2"-, and that, under most conditions, most or all of the activity detected is attributable to the two SOD enzymes. The Cu,ZnSOD activity can be measured by pretreating the samples with SDS and the MnSOD activity can be measured by treatment with NaCN. Where the level of SOD activity is low, residual interferences in the assay become apparent, and the activity can be estimated only on the basis of rough approximations.

A c k n o w l e d g m e n t s

The authors thank S. N. Radyuk and W. C. Orr for the Drosophila Cu,ZnSOD and MnSOD anti- bodies and L. W. Oberley for advice about the SOD assay.