Use of cyanide and diethyldithiocarbamate in the assay on superoxide dismutases

Free Radical Biology & Medicine. Vol. 10, pp. 69-77, 1991 0891-5849/91 $3.00 + .00 Printed in the USA. All rights reserved. Copyright ¢ 1991 Pergamon Press plc

- • " Original Contribution

USE OF CYANIDE AND DIETHYLDITHIOCARBAMATE IN THE ASSAY OF SUPEROXIDE DISMUTASES

JAWAID IQBAL* and PHILIP WHITNEYt

Calvin and Flavia Oak Asthma Research and Treatment Facility, Pulmonary Research Laboratories (R-120), Department of Medicine, University of Miami School of Medicine, P.O. Box 016960 Miami, FL 33136 and VA Medical Center, Miami, FL

33125

(Received 27 April 1990; Revised 24 August 1990; Accepted 16 October 1990)

Abstract--Eucaryotes have two major forms of superoxide dismutase (SOD). Cu,ZnSOD and MnSOD; in most tissues Cu,Zn- SOD is present in higher amounts than MnSOD. To assay MnSOD, Cu,ZnSOD can be inhibited selectively by millimolar concentrations of cyanide ion. However, calculation of MnSOD activity from the differential cyanide inhibition assay is complex and small experimental errors can cause large errors in the calculated MnSOD activity. We have assessed how interaction of cyanide and hydrogen peroxide with cytochrome c can lead to further errors in the xanthine oxidase--cytochrome c assay for SOD. Alternatively, Cu,ZnSOD can be completely inactivated by 50 mM diethyldithiocarbamate (DDC) at 30°(? for 1 h without affecting the activity of MnSOD. Since DDC reduces cytochrome c, the treated samples must be thoroughly dialyzed or desalted before assay. In the case of lung homogenates, dialysis is not an extra step since fresh, untreated samples must also be dialyzed or desalted before assaying by the cytochrome c method. Cu,ZnSOD activity is equal to the activity in the untreated sample minus the activity in the DDC- treated portion of the sample. Another copper chelator, triethylenetetramine, did not inactivate Cu,ZnSOD and could not be used instead of DDC. For accurate measurement of both enzymes in samples where MnSOD contributes only a small fraction of the total SOD activity, the DDC method has the advantage that it provides a direct measure of the MnSOD activity without interference by Cu,ZnSOD.

Keywords--Enzyme assay, Superoxide dismutase, Cyanide, Diethyldithiocarbamate, Cytochrome c, Hydrogen peroxide

INTRODUCTION

Eucaryotes possess two major forms of superoxide dismutase (SOD), Cu,ZnSOD and MnSOD; Cu,ZnSOD is mainly cytosolic and MnSOD is mitochondrial. ~ Meth- ods to measure the activities of these two forms selectively are based on differential responses of the enzymes to inhibitors, denaturants, or pH. The use of cyanide is based on the inhibition of Cu,ZnSOD but not MnSOD by the cyanide ion. 2 In tissues with high ratios of

Cu,ZnSOD to MnSOD, errors in the assay in high concentrations of cyanide will not have a great influence on the calculated activity for Cu,ZnSOD because the activity in high cyanide is small compared to the uninhibited SOD activity. However, errors in the measurements in high cyanide will directly affect the calculated activity for MnSOD. Furthermore, before the differential cyanide inhibition method can be used to quantitate Mn- SOD activity, one must demonstrate that the assay of

*Current address: Preclinical Science Building, Georgetown Univer- sity Medical Center, 3900 Reservoir Road N.W., Washington, DC 20007. tAuthor to whom correspondence should be addressed.

69

the solution in question is valid in high cyanide. Cya-

nide is not an innocuous component of the assay mixture. Cyanide complexes with cytochrome c, 3'4 and

inhibits cytochrome oxidase and cytochrome c per- oxidase; 5 it also catalyzes the oxidation of alpha-ketoal-

dehydes and alpha-ketoalcohols in a superoxide-mediated mechanism. 6 In this paper, we report several ways

cyanide can interfere with the SOD assay. In view of these complexities introduced by the use

of cyanide, we considered other methods to measure MnSOD activity. Sodium dodecyl sulfate has been used to inactivate MnSOD selectively, 7 but this assay is bet-

ter suited for samples with a low ratio of Cu,ZnSOD to MnSOD because MnSOD activity is calculated from the small difference in two large numbers representing total

SOD and Cu,ZnSOD activities. The differential pH assay is based on the observation that Cu,ZnSOD appears to be l0 times more active at pH 10 than at pH 7.8 whereas MnSOD appears to be only twice as active at the higher pH. 5 The differential pH method is also ill suited for measuring MnSOD because the fraction of the total SOD activity contributed by MnSOD will be even lower at pH 10 than at pH 7.8.

70 J. IQBAt_ and P. WHITNEY

The best method for assaying samples with high ratios of Cu,ZnSOD to MnSOD would inactivate the Cu,ZnSOD while maintaining the full activity of Mn- SOD and the validity of the assay system. Inactivation of Cu,ZnSOD can be achieved by treatment with diethyldithiocarbamate (DDC), a reagent that complexes and removes copper from Cu,ZnSOD. 8'9 In this report, we described conditions that permit the use of DDC to assay MnSOD without the use of high concentrations of cyanide. Some of these results have appeared in an abstract. ~o

METHODS

Male Sprague-Dawley rats (200-250 g) from Charles River Breeding Laboratories were maintained in the An- imal Care Facility at the University of Miami. We pur- chased bovine xanthine oxidase from Boehringer Mann- heim (Indianapolis, IN), bovine Cu,Zn superoxide dismutase from Diagnostic Data Co. (Mountain View, CA), hydroxyapatite and DEAE Bio Gel A from Bio Rad (Richmond, CA), and Sephadex G 100SF, xanthine, bathocuproinedisulfonic acid, diethylenetriaminepenta- acetic acid, triethylenetetramine tetrahydrochloride, diethyldithiocarbamate (DDC) and horse heart cytochrome c (Type VI) from Sigma Chemical Co. (St. Louis, MO) In some experiments, we used cytochrome c that had been passed through a 1.5 × 90 cm column of Sepha- dex G-100SF in 50 mM potassium phosphate (KPi) buffer (pH 7.8). Properties of the chromatographed cytochrome c were not distinguishably different than the unfractionated protein.

Tissue preparation

Tissue samples for SOD assays were obtained from rats that were killed by cutting the great vessels of the abdomen after anesthetization with sodium pentobarbi- tol ( - 5 0 mg/kg). Lungs were perfused with 0.9% NaC1. Each lung was homogenized in 10 mL 5 mM KPi buffer (pH 7.8) for 50 s with a Polytron Pl10/35 (Brinkman Instruments (Westbury, NY)) operated at highest speed. The homogenate was centrifuged at 27,000 g for 45 min. Rat liver homogenates were processed similarly, but centrifuged at 100,000 g for 60 min.

Purification of superoxide dismutases

Rat lung Cu,ZnSOD was purified as described by Crapo et al. 5 except DEAE Bio Gel A was substituted for DE-52. MnSOD was purified 20-fold and separated from Cu,ZnSOD by chromatography through a column of hydroxyapatite. ~

Assay of SOD activi~,

The xanthine oxidase-cytochrome c method was used for all assays of SOD activity. 5 A low concentration of sodium cyanide (0.015 mM) was used to inhibit cytochrome oxidase. A unit of SOD activity is the amount that halves the rate of reduction of cytochrome c. We only used 1 mL assay volume, so our unit of activity is three times that of Crapo et al. 5 Based on published equations 1213 and adding a correction for a blank measured in the absence of xanthine oxidase, a unit of activity can be calculated from eq. 1 where v o is the rate of reduction of cytochrome c (A55 o -0 .025 min - l ) in the presence of xanthine oxidase but without SOD, v is the rate with xanthine oxidase and SOD, and v' is the rate with SOD but without xanthine oxidase.

v0 U = v - 7 - 1 eq. 1

The value of v' was negligible for purified SOD samples or for total SOD in dialyzed or desalted lung homogenates.

The procedure utilizing diethyldithiocarbamate (DDC) to inactivate Cu,ZnSOD required two portions of each sample if they were to be assayed for both Cu,ZnSOD and MnSOD. One was untreated control and the other was incubated at pH 7.8 with 50 mM DDC at 30°C for 1 h. Both samples were then desalted or dialyzed. De- salting was accomplished using Bio Rad Econo-Pac 10DG columns equilibrated and eluted with 50 mM KPj buffer (pH 7.8) -0.1 mM EDTA. For dialysis, we used three changes of 400 volumes of 5 mM KP i buffer (pH 7.8) -0.1 mM EDTA. The SOD activity in the control was total SOD activity; the DDC-treated portion had only MnSOD activity. Cu,ZnSOD activity was obtained by subtracting the activity of the treated sample from that of the control. To assess recovery from desalting or dialysis, we measured protein concentrations by the method of Bradford. 14 (Unless the samples were first precipitated with trichloroacetic acid, the bichinchonic acid protein assay ~5 was not appropriate for this purpose because small molecular weight components in lung homogenates contributed about 20% of the color that de- veloped with the bichinchonic acid (BCA) reagent; as a result, the apparent recoveries determined with the BCA reagent were 20% lower than the actual protein recovery.) Since volume changes during dialysis were small, satisfactory corrections could also be made from the ratio of the total volume (or weight) of each sample before and after dialysis.

Other assays

Xanthine oxidase activity was assayed by measuring uric acid production as determined from the rate of in-

Pitfalls in assaying superoxide dismutases 71

crease in A293 in 50 mM KP i buffer (pH 7.8)-0.1 mM EDTA - 0.1 mM xanthine at 25°C. Catalase activity was assayed at 240 nm.16 Protein was analyzed by the method of Bradford 14 using bovine serum albumin as standard. Spectra were recorded with a Cary 14 spectrophotometer. Cytochrome c concentration and state of oxidation were determined from absorbances at 550 nm before and after reduction with - 1 mg sodium dithion- ite per 2.5 mL by using molar extinction coefficients given by Massay. 17

RESULTS AND DISCUSSION

The xanthine oxidase-cytochrome c assay for SOD ~ is based on the reactions in equations 2--4.

xanthine + O2

Cyt c 3+ + O2-

202- + 2H + + Cyt c 2+ + H202 Cyt c 3+ + CN-

xanthine oxidase > uric acid + 02 eq. 2

+ H202 > Cytc 2~ + 02 eq. 3

SOD > 02 + H202 eq. 4 > Cyt c 3+ q- H202 eq. 5 > Cyt c 3+ CN eq. 6

Equations 5 and 6 are reactions that can interfere with the assay under suboptimal conditions.

0 . 4 m

0 .2 - -

u.I ( J Z < ,.,n 0 m er O Of) nn <~

-0.2

I I I I 300 400 500 600

WAVELENGTH (nm)

Fig. 1. Difference spectrum of cyanide-ferricytochrome c versus ferricytochrome c. Ferricytochrome c (20 p.M) in 50 mM KP i buffer (pH 7.8)-0.1 mM EDTA was in sample and reference cuvettes for the baseline. A second spectrum was recorded 1.5 h after adding 1.5 mM NaCN + 1.4 mM HCI to the sample cuvette and an equal volume of buffer to the reference cuvette.

Influence of cyanide on the xanthine oxidase-cytochrome c system

We will use the term "cyanide" to include both its acid and salt forms. Hydrocyanic acid is a weak acid with a pK a above 9; addition of 1.5 mM NaCN to 50 mM KPi buffer at pH 7.8 resulted in almost complete conversion to HCN and required the addition of 1.4 mM HCI to maintain the proper pH. Without HC1, addition of 1.5 or 6 mM NaCN increased the pH of the 50 mM buffer to 7.94 or 8.6, respectively. Since even small changes in pH will alter the specific activity measured in the SOD assay, maintenance of a constant pH is important.

Cyanide has been widely employed in the xanthine oxidase-cytochrome c assay for SOD. Low concentrations of cyanide (5-50 ~M) have been used to inhibit peroxidases and cytochrome oxidase in crude samples. High concentrations of cyanide (1-2 mM) have been employed in a differential cyanide inhibition assay to inhibit Cu,ZnSOD and permit the quantitation of both Cu,ZnSOD and MnSOD. 2"18 These uses assume that cyanide does not influence the basic assay system in which superoxide, formed by xanthine oxidase during oxidation of xanthine to uric acid, is assayed by the rate it reduces ferricytochrome c. We have explored two in- teractions of cyanide with cytochrome c and assessed

how they can influence the correlation of superoxide concentration with the observed rate of reduction of cytochrome c.

The first interaction is the complex with ferricytochrome c in which cyanide displaces the methionine ligand to the heme iron and strongly inhibits the reduction of cytochrome C. 3'19 Binding of cyanide causes a shift in the visible spectrum leading to a large increase in absorbance at 417 nm, a large decrease at 403 nm, and several smaller changes including an increase at 550 nm (Fig. 1). The complex forms relatively slowly. We obtained a half-time of 10 min in 1.5 mM cyanide (Fig. 2); this is in good agreement with a half-time of 13 min calculated from the data of George and Tsou 3 that was obtained at a higher buffer concentration. For 1.5 mM cyanide plus 10 txM ferricytochrome c, the initial rate of increase in absorbance at 550 nm was less than 0.0005 min- ~, so it makes only a small contribution to the rate of change in absorbance as free ferricytochrome c is being reduced by superoxide in the SOD assay and would cause the SOD activity to be 4% lower than the true value. To correct for it, the 0.0005 min-1 should be subtracted from v 0 in eq. 1.

A more important consideration for the SOD assay is that the cyanide-cytochrome c complex is not reduced by superoxide, so complex formation decreases the ef-

72 J. IQBAL and P. WHn'NEY

70

50

z w

z "~ 30 L M 0:: I - z

20

I L l a,,

I I 1 10 20 30

TIME (min)

Fig. 2. Rate of cyanide inhibition of reduction of ferricytochrome c. Cyanide (1.5 raM) binding to 14 0.M cytochrome c in 50 mM KP, buffer (pH 8.0)~0.1 mM EDTA was followed at 417 nm (closed circles). Cyanide inhibition of the SOD assay was measured by adding 1.5 rnM NaCN to 10 ~M ferricytochrome c in 50 mM KP, buffer (pH 8.0) -0.1 mM EDTA - 100 ~M xanthine at designated times before initiating the assay with xanthine oxidase and measuring the initial rate of reduction of cytochrome c at 550 nm (open circles): each point in this curve is from a separate experiment with a different preincubation period.

fective concentration of cytochrome c. The other curve (open circles) in Fig. 2 shows how the initial rate of reduction of cytochrome c in SOD assay cocktail progres- sively decreases with increasing length of time for preincubation of cyanide with cytochrome c before initiating the assay with xanthine oxidase. The decrease in rate of reduction of cytochrome c in the assay system lags behind the rate of complex formation (Fig. 2) because cytochrome c in the assay medium is present at nearly saturating concentration. ]2 Consequently, a 5-min preincubation was required before there was a significant decrease in initial rate of reduction of cytochrome c in the SOD assay, and a relatively large fraction of cytochrome c had to be inactivated by cyanide before falling into the more linear region of the curve for the rate of reduction vs concentration of free cytochrome c. Inactivation of ferricytochrome c by 1.5 mM cyanide was not a problem if the cyanide was added immediately before initiating the reaction with xanthine oxidase.

Inactivation is more rapid and becomes a more seri- ous problem at higher concentrations of cyanide. An-

other consideration is that the rate of complex formation is very pH dependent with a peak at pH 9. 3 At pH 10 the rate was more than twice that at pH 7.83 The problem of cyanide inactivation of ferricytochrome c has been alluded to in the past 2"2° and some incorrect information has also appeared; 5 to our knowledge, this is the first published evaluation of its effect on the SOD assay.

A second effect of cyanide is to inhibit oxidation of ferrocytochrome c by H202. In a solution of partially reduced cytochrome c (8 ~M ferricytochrome c and 2 txM ferrocytochrome c), 2 ~M H202 completely oxidized the ferrocytochrome c within 3 rain. The oxidation was inhibited 50% by 4.5 p.M cyanide, 73% by 15 txM cyanide, and 97% by 150 ~zM cyanide. Sodium azide was a much weaker inhibitor 05o = 3 raM). Cat- alase (1500 units/mL assay medium) was nearly as effective as 150 I~M cyanide. Catalase is inhibited by cyanide (K i = 8 p.M at pH 7), so catalase should not be used along with cyanide. Addition of xanthine oxidase (in the absence of xanthine) did not influence the rate, so lactoperoxidase was not an impurity in xanthine oxidase.

The oxidation of ferrocytochrome c by H202 has a marked influence on the xanthine oxidase-cytochrome c system if the cytochrome c is not all in the oxidized form. When 20% of the cytochrome c was initially in the reduced form, the addition of xanthine oxidase brought about a brief phase of reduction of ferricytochrome c followed by a reversal that completely oxidized the cytochrome c (trace 1, Fig. 3) even though the xanthine oxidase was continuing to produce O~- . If cyanide was included in the assay medium, the oxidation reaction was inhibited and a linear trace was obtained for reduction of ferricytochrome c (trace 2, Fig. 3). If cyanide was added later in the assay, the oxidation was stopped at that point and a linear reduction trace obtained (trace 3, Fig. 3). An explanation of these results is that xanthine oxidase produces more H202 than O 2- at this pH 21 and 0 2- spontaneously dismutes to H202 and 0 2, thereby depleting 0 2 - and increasing H202. In the presence of cyanide, ferrocytochrome c is protected against oxidation by H202. The means of this protection is not known but is is likely due to a complex of cyanide with ferrocytochrome c. If such a complex is formed, it probably does not involve the heme, since no change in visible spectrum was noted when 100 txM NaCN was added to 40 p.M ferrocytochrome c.

In relating these results to the SOD assay, it should be noted that the initial slope in trace 1, Fig. 3 is only about 70% of that in trace 2 and it is very transient. When the assay was begun with ferricytochrome c that had no ferrocytochrome c, then a linear initial trace was obtained that was not enhanced by adding cyanide. The rate of uric acid production by xanthine oxidase was found to be independent of catalase or 3 mM cyanide.


~ = 0 . 0 1 1 min . ~

T i m e

Fig. 3. Cyanide inhibition of oxidation of ferricytochrome c by H202. Trace 1. The assay was initiated by adding xanthine oxidase to 8 i.tM ferricytochrome c/2 I.tM ferricytochrome c/100 p.M xanthine in 50 mM KP, buffer (pH 7.8)-0.1 mM EDTA. The absorbance at 550 am was recorded with time. Trace 2 is the same as trace l except 0.15 mM NaCN was included. Trace 3 is the same as trace 1 except 0.15 mM NaCN was added 2 min after the assay was begun.

Therefore, if cyanide alters the apparent rate of reduction of ferricytochrome c it is probably due to an effect on cytochrome c.

Our lot of Sigma type VI ferricytochrome c did not contain detectable amounts of ferrocytochrome c, but it has been reported that commercial ferricytochrome c may contain 5-20% ferrocytochrome c. 22 A sample that does contain significant levels of ferrocytochrome c can be used after it is oxidized with ferricyanide and then dialyzed or desalted. A second aspect of the problem is maintaining the cytochrome c in the oxidized form; significant levels of ferrocytochrome c were found in our assay cocktail of ferricytochrome c plus xanthine when it was kept at 25°C for several hours or at 4°C for several days.

A small fraction of ferrocytochrome c can be toler- ated in the assay of total SOD activity in the presence of 0.015 mM cyanide. However, if the objective is to compare initial rates at low and high concentrations of cyanide, the presence of ferrocytochrome c will compli- cate the assays because part of the effect of high cyanide will be more complete inhibition of oxidation by H20 2. In this case, the measured net rate of reduction of cytochrome c at lower cyanide concentration would be less than the actual rate of reduction, the measured rate would underestimate the concentration of 0 2 - , and a higher concentration of SOD would be required to

halve the measured rate of reduction of cytochrome c. We conclude that it is best to use fresh solutions with cytochrome c almost completely in the oxidized form.

Differential cyanide inhibition assay

The differential cyanide inhibition assay is based on the lack of inhibition of MnSOD by cyanide at concentrations that cause nearly complete inhibition of Cu,ZnSOD. 2 Cyanide inhibition of rat Cu,ZnSOD was rapid and completely reversible; after incubation of Cu,ZnSOD with 5 mM cyanide at pH 7.8 for 30 min, inhibition was reversed within the time it took ( - 5 s) to get the cuvette into the spectrophotometer. Since there was no irreversible inhibition, the inhibition was char- acterized by a K i. We found a Ki of 130 IxM for inhibition of rat lung Cu,ZnSOD by cyanide. This Ki is the same as that reported for human Cu,ZnSOD, 23 twice as high as the K i for the bovine enzyme, 23 and less than half that reported for the equine enzyme. 24 The xanthine oxidase-cytochrome c assay generally includes a low concentration of cyanide (0.015 mM) to inhibit cytochrome oxidase. That low concentration of cyanide inhibited Cu,ZnSOD only 7%. The unit of activity of Cu,ZnSOD was defined as that activity expressed in 0.015 mM cyanide. At high cyanide concentration (1.5 mM), purified rat Cu,ZnSOD was 92% inhibited compared to its activity in 0.015 mM cyanide. Defining I as the fraction of Cu,ZnSOD inhibited at high cyanide, then the activities of Cu,ZnSOD (C) and MnSOD (M) were calculated from activities in 0.015 mM cyanide (L) and 1.5 mM cyanide (H) with the following equations:

L - H C - I eq. 7

H - (1 - I)L eq. 8 M = I

Measurement of I requires a purified preparation of the same Cu,ZnSOD as is in the mixture to be assayed. If most of the SOD activity is due to Cu,ZnSOD, the ac- curacy of the assay for Cu,ZnSOD will not be greatly influenced by errors in H. However, the activity of Mn- SOD is strongly dependent on accurate values of H and I, both of which are measured in the presence of a high concentration of cyanide. A 1% error in I would produce more than a 10% error in MnSOD activity. Mea- surement of I may be further complicated because crude extracts of tissues may reduce the effective cyanide concentrations, s Although Cu,ZnSOD inhibition will be more complete at higher concentrations of cyanide, the problem of residual Cu,ZnSOD activity must still be addressed. For instance, if Cu,ZnSOD accounts for 90%

74 J. IQBAL and P. WHITNEY

0 . 0 1 0

'7

E

0.005 < <1

2.0

1.5 > - t ' - >

I . -

1.0 <

I , -

T~ D 0.5

_A ~ j n

o J o f ° f o ~ ° J

Y I I I I

250 500 750 1000 PROTEIN (/Jg/mL)

Fig. 4. Assay of rat lung MnSOD in the presence of low and high concentrations of cyanide. The soluble fraction from a homogenate of adult rat lung was treated with 50 mM DDC at 30°C for 1 h, dialyzed against 5 mM KP, buffer (pH 7.8)-1 mM EDTA, and diluted with 0.225 vols. 250 mM buffer to bring the final buffer concentration to 50 raM. In panel A, a blank rate was measured without added xanthine oxidase in the presence of 0.015 mM NaCN (open circlesl or 1.5 mM NaCN (open squares). In panel B, xanthine oxidase was added at zero time and the results are plotted without the blank correction (solid circle, 0.015 mM NaCN; solid square, 1.5 mM NaCN) or with the blank correction from A (open circles, 0.015 mM NaCN; open squares, 1.5 mM NaCN).

of the total SOD activity in 0.015 mM cyanide, the Cu,ZnSOD would be 97.4% inhibited but still contrib- ute about 20% of the total SOD activity in 5 mM cyanide. Obviously, corrections, as in eq. 8, still must be made before an accurate measure of MnSOD activity can be made.

For accurate assessment of MnSOD activity by this method it is also important to establish that cyanide does not compromise the validity of the assay. One way to assess the validity of an enzyme assay is to show that a plot of activity versus SOD concentration is a straight line that passes through the origin, t2. ~3,22 We found this

to be the case for MnSOD activity in a dialyzed soluble fraction from a rat lung homogenate that had been treated with DDC to inactivate Cu,ZnSOD (Fig. 4B). The lin- earity of the plot and the specific activity of MnSOD was the same in both concentrations of cyanide. This validates the assay conditions for the differential cyanide assay. The blank rates measured in the absence of xanthine oxidase (v' in eq. 1) were not negligible (Fig. 4A) and will be addressed further in the section on the SOD assay blank.

In our soluble fractions from rat liver and lung, we found 100 and 16 units total SOD activity/mg protein

w

5 < g o'} 25~

/" l . . . . . o . . . . . . . . . . . . . . . . . . . . . . . . . o . . . . . . . . . . . . . . . . . . . . . . . . . o - v ~ 10 30 50

DDC (mM)

Fig. 5. Inactivation of Cu,ZnSOD with DDC. Samples were treated with DDC for 1 h at 30°C, dialyzed against 5 mM KP, buffer (pH 7.8)-0.1 mM EDTA, and assayed for SOD activity. The samples were pure rat liver Cu,ZnSOD (circles), 20-fold purified rat liver MnSOD (squares~ and a 100,000 g supematant fraction from rat lung homogenate (triangles).

and 14 and 1.7 units MnSOD activity/mg protein in liver and lung, respectively. The values for MnSOD activity compare well with those obtained for the DDC treated samples (13 and 2.0 units/mg protein for liver and lung. respectively).

Use o f DDC to measure MnSOD

DDC interferes with the xanthine oxidase-cytochrome c assay by reducing the cytochrome c; 25 the concentration of DDC in the assay solution must be less than 2 txM to avoid interference with the SOD assay. (It was noted that the rate of reduction of cytochrome c by DDC was inhibited 20% by 0.015 mM cyanide and 80% by 1.5 mM cyanide even though the cyanide had been added to the assay mixture at the beginning of the assay and had not yet formed a significant amount of the ferricytochrome c-cyanide complex that was observed spectro- photometrically (Figs. 1 and 2). The inhibition of DDC reduction of cytochrome c by cyanide can be explained by rapid formation of an intermediate complex of cyanide with ferricytochrome c; in this complex, the cytochrome c is readily reduced by superoxide but not by DDC.)

It has been reported that DDC inhibits all types of SOD, 26 so we examined the stability of MnSOD activity upon treatment with DDC. We recovered an average of 98 --- 2% MnSOD activity in samples treated for one hour with 5, 10, 30, or 50 mM DDC at 25°C or with 0 or 50 mM DDC at 30°C (Fig. 5). We conclude that DDC treatment did not cause significant loss of MnSOD activity.


Purified rat and bovine Cu,ZnSOD were inactivated by more than 99% by treatment for l h at 30°C with as little as 5 mM DDC (Fig. 5). The SOD activity in a crude homogenate of rat lung was diminished by 90% after treatment with l0 mM DDC and this was not changed by increasing the concentration of DDC up to 50 mM (Fig. 5). We conclude that treatment with 50 mM DDC at 30°C for 1 h is sufficient to completely inactivate Cu,ZnSOD in pure or crude samples. Some of our results at room temperature were not so clear-cut, so we recommend the use of a controlled, elevated temperature for consistent results.

In addition to assaying for MnSOD, the method was also used to measure Cu,ZnSOD activity. Part of each sample was not treated with DDC and was dialyzed in parallel with the treated samples. The activity of both solutions was measured in 0.015 mM cyanide and the Cu,ZnSOD activity was the activity lost by DDC treatment. We measured Cu,ZnSOD activity in I 1 identical samples and got 21.5 _+ 1.0 units/mL activity with the differential cyanide inhibition assay and 23.0 __- 1.3 unit/mL with the DDC treatment method. We conclude that the DDC treatment method gives accurate results for Cu,ZnSOD as well as for MnSOD.

The SOD assay blank

An unusual contribution to the SOD assay blank was encountered when assaying a dialyzed supernatant fraction from a homogenate prepared from unperfused rat lungs that had been stored at -20°C for more than a year. Even without cytochrome c there was a significant initial slope at A55 o with 0.015 mM cyanide but not with 1.5 mM cyanide. The apparent explanation is that cyanide binding to methemoglobin gives large changes in A425 or -A404 and a small but significant increase in A55 o. The half-time of binding of 0.015 mM cyanide to rat methemoglobin under these conditions was 3 min whereas binding by 1.5 mM cyanide was essentially complete before the recording of A550 was begun. Therefore, in the presence of methemoglobin low cyanide would be expected to influence the initial slope of A550 but high cyanide would not.

As demonstrated in Fig. 4, use of v' in eq. 1 to correct for the blank reaction (measured in the absence of xanthine oxidase) can also be essential for samples from fresh lungs. Without the blank correction, the highest apparent activity reached only 0.6 units per assay. Ex- pressed as change in A55 o per rain normalized to a protein concentration of 0.1 mg/mL, the average blank in assay medium without xanthine oxidase was 0.0100 for the supernatant fluids obtained after centrifugation of homogenates of four perfused, adult rat lungs. For the same samples following desalting, the average blank was 0.0003; following DDC-treatment and desalting, the av-

erage blank was 0.0016. Using a protein concentration sufficient to give approximately one unit of SOD activity, the average blanks (v' in eq. I) were 0.0034, 0.0001, and 0.0045, respectively, for the untreated, desalted, and desalted-DDC-treated samples. A blank of only 0.0003 would make a 5% change in calculated activity, so the blanks for the untreated and DDC-treated samples are much too large to neglect.

The nature of the large blank for the untreated samples was explored briefly. It was somewhat labile; after storing samples at 4°C for 20 h the blank was about half of the original but there was still a large error in corrected SOD activity. Based on studies by Spitz and OberleyY we tried to reduce the blank by adding 0.05 mM bathocuproinedisulfonic acid or 1 mM diethylene- triaminepentaacetic acid; these did not influence the blank. Omitting xanthine from the assay medium was without effect, so the presence of endogenous xanthine oxidase was ruled out. Addition of 1000 units of bovine superoxide dismutase did not diminish the blank, so the reduction of cytochrome c was apparently not mediated by free superoxide anions. Reaction with 10 mM io- doacetamide to block sulfhydryls such as glutethione was also without effect.

Whatever the cause of the large blank for the untreated samples, subtracting it did not yield valid results for the assay of SOD activity in an untreated 27,000 g supernatant fraction from a homogenate of perfused adult rat lungs. In an experiment similar to the one in Fig. 4B, the graph of activity versus protein curbed upward; the specific SOD activity increased from 34 U/mg protein to 66 U/rag protein as the protein concentration increased from 0.013 mg/mL to 0.111 mg/mL. Following desalting, the specific activity was 19 U/mg protein and this must be considered to be the correct value. We conclude that the supernatant fraction from lung homogenates must by dialyzed or desalted before a valid SOD assay can be done using the xanthine oxidase-cytochrome c system. This advice has been given before. 5

As demonstrated in Fig. 4, use of the blank correction does yield a valid assay for the DDC-treated samples. The reason DDC treatment causes a five-fold increase in the blank may be because it is a reducing agent; a dithiothreitol-treated supernatant fraction was desalted and also found to have a high blank. Perhaps reduced macromolecular components are able to directly reduce ferricytochrome c.

The assay would be more accurate if the blank correction of DDC-treated samples were smaller, so we in- vestigated another copper chelator, triethylenetetramine, as an alternative to DDC. Trlethylenetetramine is not a reducing agent and 50 mM reagent did not interfere with the xanthine oxidase-cytochrome c assay. Although it has been reported that triethylenetetramine readily inac- tivates Cu,ZnSOD, 28 we found that the activity of purl-

76 J. IQBAL and P. WHrr~v

fled Cu,ZnSOD from rat lung was completely stable during treatment with triethylenetetramine at room temperature in 50 mM KP i -0.1 mM EDTA at pH 7.8. In the first experiment, we used triethylenetetramine tetrahydrochloride (Sigma) at a concentration of 20 mM for 2 h; in the second, we used triethylenetetramine free base (Eastman Kodak) at a concentration of 40 mM for 1 h. In both cases, the pH of the stock reagent solution was adjusted to pH 7.8 before it was added to the SOD sample. The previous investigators did not report the conditions for treatment with triethylenetetramine or the tissue source of the SOD, so we cannot explain the dis- parate results.

Precautions and comparisons of the DDC and differential cyanide inhibition methods

We have shown that with proper precautions both of these methods can yield valid results for the assay of MnSOD and Cu,ZnSOD in lung homogenates. Of the precautions we have evaluated, three must be taken with either procedure, i) The sample must be desalted or dialyzed before it is assayed. For the DDC assay, the control and DDC-treated portions must be desalted or dialyzed separately; if dialysis is employed the treated portion must be extensively dialyzed to remove DDC. ii) The ferricytochrome c in the assay cocktail must have little or no contamination with ferrocytochrome c; errors introduced by ferrocytochrome c are greater in the differential cyanide method than in the DDC method, iii) The results must be corrected for v' (see eq. 1), the blank reaction in the absence of xanthine oxidase. DDC-treated samples have higher values of v' than untreated samples, but valid results are obtained in both cases by use of eq. 1.

In addition to the above precautions, four others apply to the use of the differential cyanide inhibition method, iv) Acid must be added to correct for pH changes upon addition of 1.5 mM NaCN. v) The cyanide must be added immediately before initiating the assay; this is to limit formation of the inactive complex with ferricytochrome c. vi) The initial rate in the SOD assay should be corrected for the small change in A55 o that accompanies formation of the cyanide complex with ferricytochrome c. vii) The degree of inhibition of Cu,ZnSOD in 1.5 mM NaCN must be accurately assessed to apply eqs. 7 and 8 for calculating the separate activities of MnSOD and Cu,ZnSOD.

If one is only interested in Cu,ZnSOD activity, the differential cyanide inhibition method should give satisfactory results. If one needs accurate assessment of Mn- SOD activity in samples where MnSOD contributes only a small fraction of the total SOD activity, then we pre- fer the DDC method because it has fewer experimental pitfalls, and, especially, because the activity of the

treated sample gives MnSOD activity directly without the problems inherent in the use of eq. 8 where small errors can become a much larger error in the calculated MnSOD activity.

Acknowledgements--This work was supported by National Heart, Lung, and Blood Institute Grants HL-32109, HL-20633, HL-07283, and Veterans Administration Research Funds to Dr. D. Massaro.

We thank Dr. Donald Massaro for his support and helpful discus- sions, Dr. Irwin Fridovich for advice on the use of DDC and for re- viewing the manuscript prior to submission, and to Mrs. Martha Sanchez for preparing the manuscript.

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Use of cyanide and diethyldithiocarbamate in the assay on superoxide dismutases