THE JOURNAL OF BIOLOGICAL CHEMISTRY (cl 1984 by The American Society of Biological Chemists, Inc. Vol. 259, No. 20, Issue of October 25, pp. 12678-12684,1984

Printed in U. S. A.

Inhibition of Superoxide Dismutase by Nitroprusside and Electron Spin Resonance Observations on the Formation of a Superoxide- mediated Nitroprusside Nitroxyl Free Radical*

(Received for publication, April 2, 1984)

Hara P. Misra From the Biomembrane and Cardiovascular Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104

Nitroprusside appears to inhibit the known types of superoxide dismutases irrespective of their metal pros- thetic group and regardless of the source from which the enzymes were isolated. Thus the copper-zinc en- zyme from bovine erythrocyte or Neurospora crwsa behaved identically as did the manganese enzymes from Escherichia coli or red alga and the iron enzyme from E. coli and a blue-green alga. The inhibition was dose dependent with a Ki = 2.5 X lo-‘ for nitroprus- side. Nitroprusside does not bind to the copper moiety of copper-zinc enzyme and seems to compete with 0, for superoxide dismutase. These inhibitions by nitro- prusside, which were elicited not only in purified en- zymes but also in crude soluble extracts of biological samples, were rapidly reversible.

Nitroprusside was found to react with 0; to form a paramagnetic species with three absorption lines of equal width with a separation AN = 15.0 G and a g value of 2.028. The spin adduct appears to be a nitrox- ide radical and was stable for several minutes.

Superoxide dismutases, which appear to be an essential defense against the toxicity of oxygen, have been isolated from a wide range of organisms. Thus far, only three grossly dissimilar forms of this enzyme have been found, i.e. the copper and zinc-, or iron-, or manganese-containing enzymes. The literature describing these enzymes has been reviewed (1-5). Since these enzymes are present in all oxygen-metab- olizing cells and in mitochondria, a specific inhibitor of these enzymes would be very helpful in monitoring superoxide anion production inside cells and subcellular organelles. Cyanide selectively inhibits copper-zinc superoxide dismutase (6) and is thought to ligate to the copper (7-9). Diethyldithiocarba- mate, an effective copper chelator, also has been reported to inhibit copper-zinc superoxide dismutase (10-13). HzOz plus EDTA has been used to inactivate the iron and the copper- zinc superoxide dismutases without affecting the manganese enzymes. Azide inhibits the known types of superoxide dis- mutases to some degree but inhibits the iron-containing en- zymes most strongly (14).

Sodium nitroprusside (hydrated sodium nitrosyl pentacy- anoferrate; Na2[Fe(CN),NO]. HzO), an effective hypotensive drug (15-20), has been reported to be broken down by eryth- rocytes to cyanide (21, 22), probably by the interaction of the iron atoms with the sulfhydryl groups of erythrocytes, and by

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

liver to thiocyanate (23) leading to cyanide intoxication (24- 28). The chemical and physical properties of the nitroferri- cyanide ion [Fe(CN)5N0]2- have been investigated (29). The chemistry of this ion is dominated by the nitrosyl moiety. This moiety can undergo numerous substitution and addition reactions with various reagents. Photoirradiation of nitrofer- ricyanide in dimethylformamide yields an ESR spectrum which is essentially identical to that obtained by electrolytic reduction of nitroferricyanide in dimethylformamide and has been attributed to nitroferrocyanide (30-32). We have inves- tigated the effects of nitroprusside on superoxide dismutase and have observed that nitroprusside inhibits all superoxide dismutases to the same degree, irrespective of their metal prosthetic group and regardless of the organism from which the enzyme was isolated. Also, we report here an investigation of the reaction of nitroprusside with superoxide anion (Og), in aqueous solution, yielding a nitrogen-centered spin adduct.

MATERIALS AND METHODS

The copper-zinc-superoxide dismutases were prepared from bovine erythrocytes (33) and Neurospora crassa (34). The Mn-containing superoxide dismutases were isolated from Escherichia coli (35) and Porphyridium cruentum (36). The Fe-containing superoxide dismu- tases were purified from E. coli (37) and Plectonema boryanurn (38). These enzymes were routinely assayed either in terms of their ability to prevent the reduction of cytochrome3+ c by 0, (33) or to augment riboflavin-sensitized photooxidation of dianisidine (39). Xanthine oxidase was isolated from cream and was assayed in terms of the conversion of xanthine to urate a t 295 nm (40). Sodium nitroprusside, cytochrome3+ c (type III), and dianisidine were purchased from Sigma, while riboflavin was obtained from Eastman.

Spectrophotometric assays were performed in a Gilford model 2000 absorbance recorder at 25 “C. The rate of cytochrome3+ c reduction was calculated assuming that between the oxidized and reduced states is 2.1 X lo4 M” cm” (41). EPR spectra were obtained with a Varian model E-109 X-band instrument.

RESULTS

inhibition of Superoxide Dismutases by Nitroprusside in the Cytochrome c Assay-The classical assay of superoxide dis- mutase depends upon the generation of 0, by the xanthine oxidase reaction, the reduction of cytochrome3+ c by this O;, and finally upon the competition between superoxide dismu- tase and cytochrome3+ c for the flux of 0; (33, 42). The net effect is that superoxide dismutase inhibits the 0;-dependent reduction of cytochrome3+ c and is quantitated on this basis. Nitroprusside, at 0.01, 0.03, and 3.0 mM, did not inhibit the oxidation of xanthine by xanthine oxidase as measured by the formation of urate at 295 nm (40). As shown in Fig. 1, nitroprusside up to 30 FM did not inhibit the reduction of cytochrome3+ c. Nitroprusside did, however, inhibit the reduc- tion of cytochrome”+ c at higher concentrations. Thus, 0.8 and

12678

Nitroprusside, Og, and Superoxide Dismutase 12679

Nitroprusside, PM FIG. 1. Inhibition of superoxide dismutase by nitroprusside

in cytochrome c reduction assay. Inhibiton of various superoxide dismutases was measured in 50 mM potassium phosphate buffer, pH 7.8, in the presence of 0.1 mM EDTA in the standard xanthine oxidase-cytochrome c assay (33) as a function of the concentration of nitroprusside. Addition of 30 p~ nitroprusside in the absence of superoxide dismutase (*). copper-zinc superoxide dismutases from bovine erythrocytes (a ) and neurospora crmsa (*); manganese super- oxide dismutase from E. coli (0) and the red alga Porphyridiurn cruenturn (A); and iron superoxide dismutase from E. coli (0) and from the blue-green alga Plectonurna boryanurn (0). Inhibition of superoxide dismutase was determined as described in the text.

1.2 mM nitroprusside inhibited cytochrome3+ c reduction by 50 and 84%, respectively.

The effects of nitroprusside on several superoxide dismu- tases were investigated. The results are presented in Fig. 1. It is apparent that nitroprusside inhibits all types of superoxide dismutases in a dose-dependent manner. The sensitivity of superoxide dismutase toward nitroprusside was independent of the metal prosthetic groups and the source of the enzyme.

At all levels of nitroprusside, the rate of cytochrome3+ c reduction in the absence of superoxide dismutase was taken as the uninhibited rate, and the amount of superoxide dis- mutase needed to cause 50% inhibition of that rate was assumed to be one unit of this activity. In this way, if x is the amount of enzyme needed to cause 50% inhibition in the absence of nitroprusside and y is the corresponding amount in its presence, then the inhibition of the enzyme by nitro- prusside is given by (y - x ) 1OO/y. Thus, the concentration of nitroprusside required to cause 50% inhibition of superoxide dismutase activity was found to be 10 pM. In Fig. 1 (inset) these data are presented on reciprocal coordinates to show that the inhibitions were in all cases kinetically simple. Cell- free extracts of rat liver responded to nitroprusside as did the purified superoxide dismutases.

Inhibition of Superoxide Dismutmes by Nitroprusside in the Photochemical Augmentation Assay-If the inhibitions by ni- troprusside of various superoxide dismutases, as shown in Fig. 1, are truly a reflection of interaction of nitroprusside with the enzymes rather than some interference with the assay, then identical results should be obtained with a differ- ent assay. That this was the case is illustrated by the data in Fig. 2 where a different assay, a photochemical augmentation assay (39), was adapted. In this assay superoxide dismutases increased the rate of the aerobic photooxidation of dianisidine sensitized by riboflavin. As shown in Fig. 2 A , nitroprusside inhibited the various concentrations of bovine erythrocyte superoxide dismutase in a dose-dependent manner. Inhibition by nitroprusside was, in all cases, effective immediately, and

0 1 2 3 4 Superoxide Dismutase,

Units I ml

B

FIG. 2. Effect of nitroprusside on superoxide dismutase ac- tivity in photochemical augmentation assay (39). In A the effect of varying concentrations of nitroprusside on superoxide dismutase activity is shown as a function of [superoxide dismutase]. Line 1 (W), no nitroprusside; line 2 (*---*), 3 pM nitroprusside; line 3 (A-A), 10 pM nitroprusside; and line 4 (u) 20 p M nitro- prusside. Line 5 (- - -), 20 p M nitroprusside, was preincubated with varying concentrations of superoxide dismutase and was subsequently diluted to 1.2 p~ nitroprusside before adding to the reaction mixture. Bovine erythrocyte superoxide dismutase was used for these studies. In B the per cent of inhibition of superoxide dismutase as a function of nitroprusside is presented. Inhibition of superoxide dismutase was determined as described in the text. In these studies 0.4 pg/ml of bovine erythrocyte superoxide dismutase (W) or 0.4 pg/ml of E. coli Mn-superoxide dismutase (*---a) was used, and the reaction mixtures were illuminated for 6 min as described previously (39).

as shown in line 5 in Fig. 2 A , was rapidly reversed by dilution. Again, 10 p~ nitroprusside inhibited 50% of the superoxide dismutase activity (Fig. 2B) when Cu-Zn-superoxide dismu- tase from bovine erythrocytes or Mn-superoxide dismutase

12680 Nitroprusside, OT, and Superoxide Dismutase

from E. coli was used. This excellent agreement among results obtained by these very different assays leaves little doubt that the inhibition by nitroprusside relates to its interaction with the enzymes rather than to any effect on the assays.

Effects of Nitroprusside on Superoxide Dismutase Activity on Polyacrylamide Gel Electropherograms-Polyacrylamide gel electropherograms soaked in riboflavin plus dianisidine and subsequently illuminated develop stable brown bands a t positions bearing superoxide dismutase activity (43). As shown in Fig. 3 nitroprusside suppressed the appearance of superoxide dismutase activity bands. Thus, the activity bands of both bovine erythrocyte Cu-Zn-enzyme and E. coli Mn- enzyme were suppressed when the gels were presoaked in nitroprusside solution and then soaked in developing reagents or were soaked in the developing reagents along with nitro- prusside.

Effects of Nitroprusside on Superoxide Dismutase Activity in Pyrogallol Autooxidation Assay-Although nitroprusside inhibited the three varieties of superoxide dismutases, at least in two different assays, there is still reason to believe that the product of the nitroprusside-0; reaction could reduce

TABLE I Effect of nitroprusside on superoxide dismutase activity in pyrogallol

autooxidation assay The control rate was obtained by adding 0.2 mM pyrogallol to 50

mM Tris-cacodylic acid buffer, pH 8.2, containing 1 mM diethylene- triaminepentaacetic acid (56). Bovine erythrocyte superoxide dismu- tase (SOD) was used in these studies. NP, nitroprusside.

Additions A4 at 420 nm/min

Control +50 ng/ml SOD +I00 ng/ml SOD +200 ng/ml SOD

+10 pM N P +20 p M N P +40 pM N P +lo0 pM N P

+lo0 ng/ml SOD + 40 p~ N P +lo0 ng/ml SOD + 100 p~ N P +200 ng/ml SOD + 40 p~ N P +200 ng/ml SOD + 100 p~ N P

FIG. 3. Effects of nitroprusside on superoxide dismutase activity as ap- plied on polyacrylamide gel electro- pherograms. 0.3 pg each of bovine erythrocyte superoxide dismutase or E. coli Mn-superoxide dismutase was ap- plied to 7.5% polyacrylamide gels and stained for superoxide dismutase activity as described previously (39). Gels from left to right: control bovine erythrocyte enzyme; plus M nitroprusside in re- action mixture; 10" M nitroprusside in reaction mixture; M nitroprusside in reaction mixture; gels were presoaked in

M nitroprusside, washed once in water, and then soaked in the reaction mixture; control Mn-enzyme from E. coli; Mn-enzyme + M nitroprusside in reaction mixture; Mn-enzyme + nitroprusside in reaction mixture. Gels were soaked for 30 min in reaction mix- ture, washed once with water, and ex- posed for 10 min to fluorescent light as described (39). The colored bands at the bottom of each gel are bromphenol blue dye indicating the electrophoretic front.

0.022 0.018 0.01 1 0.006

0.023 0.022 0.023 0.024

0.013 0.017 0.008 0.01 1

cytochrome3' c and could also reduce the dianisidine radical. Nitroprusside would then lower the steady-state level of 0; and appear to inhibit superoxide dismutase, competitive with O;, yet would not affect the assay in the absence of superoxide dismutase. Such evidence is present in the literature where pamoic acid appeared to inhibit superoxide dismutases by diminishing the sensitivity of assays (55). In order to lessen the likelihood of this subtle artifact, we have investigated the effect of nitroprusside on superoxide dismutase activity in an assay in which 0; acted as an oxidant, not as a reductant. Table I shows the effects of nitroprusside on superoxide dismutase activity in one such assay, i.e. the pyrogallol au- tooxidation assay (56). As shown in this table, 100 ng/ml of superoxide dismutase inhibited the pyrogallol autooxidation by 50% and nitroprusside at 40 and 100 PM concentrations reduced this inhibition in a dose-dependent manner.

Effect of Nitroprusside on Mn(II)-dependent Inhibition of Cytochrome c Reduction Catalyzed by Xanthine Oxidase-If

TABLE I1 Effect of nitroprusside on Mn2+-dependent inhibition of cytochrome3+

c reduction catalyzed by xanthine oxidase The condition of this study was as in Fig. 1 except that 5 mM

HEPES buffer, pH 7.4, was used in place of potassium phosphate buffer. The reduction of cytochrome c was followed at 550 nm using &50nm = 2.1 X lo4 m" cm-' (41). NP, nitroprusside.

Additions A550 nm/min" AFemytochrome c

M X loelmin None 0.024 f 0.001 1.143 f 0.048

+0.5 p~ MnCI2 0.015 f 0.001 0.714 f 0.048 +1.0 pM MnCI2 0.012 f 0.001 0.571 f 0.048 +2.0 p~ MnCI2 0.007 f 0.001 0.333 f 0.048

+10 pM N P 0.023 & 0.001 1.095 f 0.048 +15 pM N P 0.024 f 0.001 1.143 f 0.048 +20 p M N P 0.025 f 0.001 1.190 f 0.048 +lo0 pM N P 0.021 f 0.001 1.000 f 0.048

+I PM MnCI2 + 10 pM N P 0.012 f 0.001 0.571 f 0.048 +1 p~ MnC12 + 15 p M N P 0.013 f 0.001 0.619 f 0.048 +1 pM MnC12 + 20 pM N P 0.014 f 0.002 0.714 f 0.096 +1 p~ MnC12 + 100 pM N P 0.011 f 0.001 0.524 f 0.048

a Mean of three assays f S.D. .- . .

' S F

Nitroprusside, Og, and Superoxide Dismutase 12681

nitroprusside appears to inhibit superoxide dismutase by in- terfering in the enzyme assay, then it should inhibit the effects of some nonenzymic scavengers of 0; in that assay. That this was not the case is illustrated by the following experiment.

Manganese(I1) is known to scavenge 0, and has been known to inhibit the reduction of cytochrome3+ c by xanthine oxidase (57) . As shown in Table 11,l p~ MnCL inhibited the rate of cytochrome3+ c reduction by 50% and 10, 15, 20, and 100 p~ nitroprusside had no significant effect in reducing the inhibitory effects of Mn(I1).

Competition between Nitroprusside and 0; for Superoxide Disrnutase-In a typical superoxide dismutase assay (33), the aerobic action of xanthine oxidase on xanthine generates OZ, which in turn either spontaneously dismutes

20; + 2H+ - 02 + HZ02 k, (1)

or reacts with cytochrome3' c ,

0; + cyt c3+ - o2 + cyt cz+. kc (2)

Superoxide dismutase inhibits the reduction of cytochrome3+ c by scavenging the O;,

0; + 0; + 2H+ - 02 + H202. (3)

Since there exists a competition between cytochrome3+ c and superoxide dismutase for the available O;, the higher the concentration of cytochrome3+ c the more enzyme will be required to produce a given degree of inhibition. At saturating concentrations of cytochrome3+ c the spontaneous dismuta- tion (Equation 1) is negligible with respect to the cyto- chrome3+ c reaction (Equation 2 ) .

Fig. 4, line 1, shows the relationship between the initial rate of reduction of cytochrome3+ c and its concentration. Thus, at 1.5 x M cytochrome3+ c the rate of spontaneous dismutation of 0; (V,) would be negligible compared with the rate of 0, generation ( V ) by xanthine oxidase, and V would be equivalent to the initial rate of reduction of cytochrome3+ c (V,) where

V, = kJcyt3'c][O;]. (4)

The rate of reduction of cytochrome3+ c, at saturating concen- tration, decreases with increased levels of superoxide dismu- tase. Therefore, at steady state,

ksoD

v = v, + VSOD (5)

where VsoD is assumed to be VSOD = kso~[SODl[O~l. (6)

Fig. 4, line 3, shows that superoxide dismutase did inhibit the rate of cytochrome3+ c reduction, and the per cent of inhibition was greater at the lower concentrations of cytochrome3' c. Thus as expected, 0.1 pg/ml of superoxide dismutase inhibited the cytochrome3+ c reduction by 80, 50, and 40% a t 1, 10, and 20 p~ cytochrome3+ c, respectively. Nitroprusside at 10 p~ concentration inhibited the activity of superoxide dismutase at any given concentration of cytochrome3+ c (line 2 in Fig. 4).

Equation 6 shows the dependence of VsoD on the concen- tration of 0;. The second-order rate constant for the reaction between enzyme and substrate has already been reported (44- 48). We have used different concentrations of xanthine oxi- dase (4-32 nM) t o generate varying rates of 01. As shown in Fig. 5A, increased levels of xanthine oxidase increased V,, and addition of 10, 20, or 30 p~ nitroprusside to the reaction mixture had no detectable effects on the rate of cytochrome3+

t 1

1 I I

0 .5 1.0 1-5 2.0 3.0 ' /w

[~ytochrornch] x 1 6 M FIG. 4. Yeduction rate of cytochrome'+ c as a function of its

Concentration. The initial rate of formation of cytochrome3+ c was observed at 550 nm. Reaction mixtures contained 50 p~ xanthine, 8 nM xanthine oxidase, and various amounts of cytochrome3+ c in 50 mM potassium phosphate buffer, pH 7.8, with 0.1 mM EDTA at 25 "C. Reaction was started by the addition of xanthine oxidase. Line 1, control; line 2, control + 0.1 pg/ml bovine erythrocyte superoxide dismutase + 10 pM nitroprusside; l i n e 3, control + 0.1 pg/ml super- oxide dismutase.

c reduction (line 1 in Fig. 5A) . Again, 0.1 pg/ml of superoxide dismutase inhibited the rate of cytochrome3+ c reduction at any given concentration of 0, flux (line 4 in Fig. 5A) , and this action of superoxide dismutase was inhibited by nitro- prusside in a dose-dependent manner (lines 3 and 2 in Fig. 5A) .

The concentrations of 0, could be calculated from V, with the known value of kc, 1.2 X lo4 M" s" at pH 7.8 (48), using Equation 4. Fig. 5B, line I , shows a plot of VsoD against the concentration of 0,. VsoD was calculated from Equation 5 at a given V. As shown in line 1 in Fig. 5B the reaction of superoxide dismutase appears to be first order with respect to 0; under these conditions. This is in accord with Sawada and Yamazaki (48) who reported that under physiological condi- tions and at low concentrations of 0; (below 1 pM) the reaction of superoxide dismutase was first order with respect to its substrate. Lines 2 and 3 in Fig. 5B show, again, the dose-dependent inhibition of VSOD by nitroprusside. The ob- served Vso0 in the presence of 0, 10, and 30 p~ nitroprusside is also presented in reciprocal coordinates as a function of [Or] in the inset of Fig. 5B. This figure illustrates the com- petitive behavior of nitroprusside with 0, for superoxide dismutase with an apparent V,,, of 2 x M s-', apparent K, of 2 X 1O"j M for OH, and a K, of 2.5 X M for nitroprusside.

Binding of Nitroprusside to Superoxide Dismutase-The inhibitory action of nitroprusside on superoxide dismutase was quickly reversed by dilution. This was true when the enzyme was assayed by cytochrome3+ c reduction assay (33) or by photochemical augmentation assay (39). Since cyanide is known to inhibit Cu-Zn-superoxide dismutases and to ligate the Cu2+ (6-9) and since nitroprusside was shown to have a competitive inhibitory effect on the enzyme, it appeared pos- sible that nitroprusside or a product of nitroprusside-0; might ligate to the Cu2+ of Cu-Zn-superoxide dismutase. As

12682 Nitroprusside, Og, and Superoxide Dismutase

I B

0 a 16 24 32 X a n t h i n e o x i d a s e , n M

FIG. 5. Effects of the concentration of superoxide anion on the reaction rate of superoxide dismutase (VSOD). Initial rate of cytochrome3+ c reduction was measured at 550 nm in reaction mixtures containing 50 ,AM xanthine, 15 ,AM cytochrome3+ e, and 50 mM potassium phosphate plus 0.1 mM EDTA, pH 7.8, a t 25 "C. Steady- state concentrations of 0; were generated by using varying concentrations of xanthine oxidase. In A : reduction rate of cytochrome3+ c (V,) as a function of [xanthine oxidase], line 1, control; line 2, 0.1 pg/ml superoxide disrnutase + 30 g M nitroprusside; line 3, 0.1 pg/ml superoxide dismutase + 10 p M nitroprusside; line 4, 0.1 pg/ml superoxide dismutase. Line 1 (a) in the presence of 10, 20, or 30 ,AM nitroprusside on control rate. In B: the reaction rate of superoxide dismutase ( VsOD) is plotted as a function of [ O;]. The estimation of VSOD and [ 0.3 is described in the text. Line 1, control; line 2, control + 10 p~ nitroprusside; line 3, control + 30 p~ nitroprusside. The data is presented both in linear and on reciprocal coordinates.

FIG. 6. The effect of nitroprusside and cyanide on the EPR spectrum of superoxide dismutase. Bovine erythrocyte superoxide dkmutase at 1 mM in 50 mM potassium phosphate a t pH 7.8 and at 25 "C was treated with 2 mM potassium cyanide and/or 4 mM nitro- prusside, and within 1 min samples were frozen in liquid nitrogen and EPR spectra were recorded. Nitroprusside-0; adduct was gen- erated by reacting 4 mM nitroprusside with the xanthine-xanthine oxidase system (similar to the conditions given in the legend to Fig. 7) for 4 min at room temperature. Line I, enzyme only; line 2, enzyme + 4 mM nitroprusside or enzyme + nitroprusside-0; adduct; line 3, enzyme + 4 mM nitroprusside and after 5 min at 25 "c, 2 mM cyanide was added; line 4 , enzyme + 2 mM cyanide. Conditions for EPR spectroscopy were: 9.108 GHz frequency, 25-milliwatt microwave power, 4 G modulation amplitude, 2500 receiver gain, 1.0 s time constant, 125 G min" scan rate, and the field was set at 3000 G.

evident from the EPR spectra (Fig. 6 ) , neither nitroprusside nor the nitroprusside-02 adduct bind to the copper moiety of the enzyme. We have further explored the possibility of the competitive behavior of cyanide and nitroprusside. Fig. 6, line 4, demonstrates that nitroprusside does not inhibit the liga- tion of cyanide to Cu2+ moiety (active site) of the enzyme. Thus, the changes imposed on the EPR spectra of the enzyme by 2 mM cyanide (Fig. 5 , line 3 ) were not inhibited by 4 mM nitroprusside. Therefore, it is possible that nitroprusside may

FIG. 7. EPR spectrum of nitroprusside nitroxyl free radical in aqueous solution at 25 "C. The spectrum was obtained by reacting 50 pl of 0.1 M nitroprusside, 100 pl of M xanthine, 100 pI of 0.05 M potassium phosphate + M EDTA, pH 7.8, and 10 pl of 3 ,AM xanthine oxidase. The spectrum was obtained 3 min after adding the xanthine oxidase. The conditions of EPR spectroscopy were: field set at 3240 G, scan rate 50 G rnin", microwave power 25 milliwatts, frequency 9.108 GHz, receiver gain 2000, modulation amplitude 2G, time constant 1 s.

not be binding to the Cu(I1) at the active site of the enzyme. Detection of a Nitroxide Spin Adduct-Bernd and Hockings

(30) have deduced from EPR data that pentacyanonitrosyl- ferrate ion should be considered as a molecular species with an unpaired electron delocalized between the Fe and the nitrogen atom of the NO+ group. It appeared possible that 0; and/or other oxyradicals might react with nitroprusside to form a stable spin adduct which could be detected by EPR spectroscopy. To explore this possibility nitroprusside was added to a 0; or OH. generating system and its effects were noted. Fig. 7 illustrates that a paramagnetic species is formed when nitroprusside reacts with 0;. Thus, three absorption

Nitroprusside, 0 2 , and

12/

lot

Eu8 -

6 -

E 4 E

- .- Y

Q 2 - 0 -

Y

I Y I Y

T c T

0 1 2 3 4 Minutes

FIG. 8. Effect of various agents on the kinetics of appear- ance of the free radical of nitroprusside. The EPR spectrometer was set at a field setting such as to monitor the height of the second line of the nitroxyl radical signal. The conditions of the EPR spec- troscopy were as in Fig. 7 except that the scan was off. The following agents were added to the reaction mixture before the addition of xanthine oxidase: 0, control; 0, 10 pg of bovine erythrocyte super- oxide dismutase; 0, 20 Kg of catalase; *, 12 mM mannitol; V, 10 pg of superoxide dismutase plus 20 pg of catalase; *, 25 pl of 1 mM cytochrome3+ c. Total volume was 260 pl.

lines of approximately equal width with a separation of 15.0 G and a g value of 2.028 were obtained when 0;) generated by the xanthine oxidase reaction, was allowed to react with nitroprusside. The g values and splitting constants of trapped free radicals were calculated using 2,2,6,6-tetramethyl-4-pi- peridinooxy as a standard.

The hydroxyl radicals, formed in a Fenton-type system (Fez+ + H202 .+ OH. + OH- + Fe3+), were not effective in forming paramagnetic species when reacted with nitroprus- side although a similar system yields a spin adduct with DMPO' (data not presented). Thus, a well characterized 1:2:2:1 pattern of DMPO-OH was obtained when 30 p1 of NaCl/NaHCO3 (pH 7.0) buffer (100 m ~ / 2 5 mM, respectively), 20 pl of 900 mM DMPO, and 10 p1 of 0.3% Hz02 were incubated with 10 pl of 1 mM freshly prepared FeCL in 0.0012 M HCl (49). The EPR signal of DMPO-OH was formed immediately and was stable for several minutes. However, in a similar system, when DMPO was replaced with nitroprus- side no EPR signal could be detected.

Further experiments were conducted to monitor the time course of the appearance of the free radical of nitroprusside, obtained by exposing nitroprusside to xanthine plus xanthine oxidase. The EPR spectrometer was set at a field setting so as to monitor the height of the second line of the spin adduct trio as a function of time after initiating the formation of 02:. Results of the experiments utilizing superoxide dismutase, catalase, mannitol, and cytochrome3+ c in this system are presented in Fig. 8. As shown in this figure, catalase and mannitol had trivial effects on the peak amplitude of the spectra indicating no role for Hz02 and OH. in the formation of the nitroxyl radical. Superoxide dismutase which removes 0; at a rapid rate also had little effect in this process. Since superoxide dismutase is quickly inactivated by nitroprusside the inability of superoxide dismutase to inhibit the formation of the spin adduct is, therefore, understandable. However, addition of cytochrome'+ c to the reaction mixture or removal

' The abbreviations used are: DMPO, 5,5-dimethyl-l-pyrroline-l- oxide; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.

Superoxide Dismutase 12683

of oxygen from the system completely suppressed the signal indicating a possible role of 02: in forming the spin adduct. Equimolar concentration of ferrocytochrome c was without effect in this system.

DISCUSSION

Nitroprusside inhibits the known types of superoxide dis- mutase irrespective of their metal prosthetic group and re- gardless of their source. The inhibition of nitroprusside in all cases was effective rapidly after mixing and reversed quickly by dilution of nitroprusside. I t appears likely that nitroprus- side does not bind to the copper moiety (active site) of the copper-zinc-enzyme. It appears more likely that nitroprusside competes with 02: for superoxide dismutase. The K, for nitro- prusside was found to be 2.5 X M.

The kinetic parameters reported here were obtained by relying on an enzyme-catalyzed alteration in steady-state concentrations of 02:. Under these conditions the reaction of superoxide dismutase was found to be first order with respect to 0;. This is in accord with Sawada and Yamazaki (48). The dependence of Vs&, on the concentration of 02: has been studied by optically monitoring the superoxide dismutase- catalyzed disappearance of electron pulse-generated 0: over a millisecond time scale (44, 45). These authors have con- cluded that the overall rate is governed by a second-order rate constant for reaction between enzyme and 02:. These results were obtained at M concentrations of 0;. That the kinetics of the dismutation reaction are not always simple second order has been discussed in some detail by Rabani and Nielson (50) and documented by Bielski and Chen (51). These authors have concluded that initially there is a second-order decay but as the concentration of 0; decreases a first-order component begins to dominate.

We have obtained kinetic data using a competitive reaction between V, and VsoD. At pH 7.8 the rate constant for the reduction of cytochrome3+ c by 0; was reported to be 1.2 x IO4 M-' s-l (48) while the enzyme's rate constant remains at 2 x IO9 M-' s-'. Under these conditions, one molecule of superoxide dismutase competes equally with 1.67 X lo5 mol- ecules of cytochrome3+ c. Therefore, even a t saturated concen- trations of cytochrome3+ c ( M), nanomolar concentrations of superoxide dismutase could compete effectively with cytochrome3+ c for the available 0;.

Nitroprusside seems to exert two discrete effects. It not only inhibits superoxide dismutase at micromolar concentra- tions of nitroprusside but also interacts with its substrate, 02, to form a spin adduct and thus inhibits the 0;-dependent reduction of cytochrome3+ c at higher (millimolar) concentra- tions. Furthermore, the observed inhibitory effects of nitro- prusside utilizing entirely different assays, the cytochrome3+ c reduction assay, the pyrogallol autooxidation assay, and the photochemical augmentation assay, leaves little doubt that the inhibition by nitroprusside relates to its interaction with the enzyme rather than to any effect on the assays.

The paramagnetic species formed by reaction of 02: with nitroprusside appears to be a nitroxide free radical. The spin adduct, so generated, was found to have an AN = 15.0 G and g value of 2.028 in close agreement with the results of Bernd and Hockings (30) and Raynor (31) who have observed similar spectra by either photoirradiation or electrolytic reduction of solutions of disodium pentacyanonitrosylferrate in N,N-di- methylformamide. These authors have concluded that irra- diated solutions of pentacyanonitrosylferrate in an electron- rich medium contain [Fe(CN)6N0]3-; the unpaired electron seems to be localized mainly on the metal in an orbital which overlaps an approximately sp2 nitrogen orbital. It also has

12684 Nitroprusside, Og, and Superoxide Dismutase

been shown by EPR studies (52) that two paramagnetic species are obtained by dithionite reduction of pentacyano- nitrosoferrate which are in a protonation-deprotonation equi- librium.

The spin-trapping chemistry of OH. and 0; or HO; has been reviewed (53, 54). DMPO has been used extensively to identify the oxyradicals, OG, OOH and OH., since the EPR spectra of DMPO spin adduct of 0; and OH. are readily distinguishable. Since nitroprusside forms a nitroxyl free rad- ical with reducing agents, e.g. dithionite, ascorbate (data not presented), and 02-, but not with oxidizing agents, e.g. ferri- cyanide (data not presented) and OH., it seems possible that the paramagnetic species we have observed is a single elec- tron-reduced product of nitroprusside. The biological signifi- cance of nitroprusside as an inhibitor of superoxide dismutase or spin trap of 0; and correlation of these effects with its effect as therapeutic agent (antihypertensive agent) remains to be determined. A specific inhibitor of both cytoplasmic (Cu-Zn-enzyme) and mitochondrial (Mn-enzyme) superoxide dismutases, however, would be useful in both experimental and clinical situations. The observation that one can inhibit these enzymes at micromolar concentrations of nitroprusside raises the likelihood that one can now accurately monitor the low levels of 0, produced in both metabolizing cells and subcellular organelles without the interference of the endog- enous superoxide dismutases.

Acknowledgments-I would like to thank the following people for their support and advice in completing this study: Drs. Paul B. McCay, William B. Weglicki, Robert A. Floyd, and J. Lee Poyer for constructive comments and for critical review of the manuscript; Dr. Irwin Fridovich, Duke University, for his valuable advice and initia- tion of this work in his laboratory; and Rashid Abdulla for his expert assistance in performing some of the spectrophotometric analysis.

1. 2. 3. 4. 5. 6.

7. 8.

9.

10. 11.

12. 13. 14.

15.

16. 17. 18.

19.

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