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Vanadate enhancement of the oxidation of NADH by O2−: Effects of phosphate and chelating agents
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 243, No. 1, November 15, pp. 229-227,1985
Vanadate Enhancement of the Oxidation of NADH by 0;: Effects of Phosphate and Chelating Agents’ DOUGLAS Department of Biodmmistly, Received
DARR
AND
IRWIN
FRIDOVICH’
Duke University Medical Center, April
9,1985,
and in revised
form
Durham, July
North Carolina
27710
26,1985
Vanadate markedly stimulates the oxidation of NADH by 0;. Both phosphate and Tris are inhibitory, but phosphate diminishes the greater inhibitory effect of Tris and thus gives the appearance of stimulating when added to Tris-buffered reaction mixtures. Chelating agents moderately increased the oxidation of NADH but eliminated the much greater catalytic effect of vanadate. Desferal was the most effective of the chelating agents, and could be used to titrate vanadate spectrophotometrically or in terms of the diminution of its catalytic activity. This permitted the demonstration that metavanadate or orthovanadate could form 1:l complexes with desferal and that orthovanadate was the catalytically active species. Q I985 Academic Press, k.
Vanadate markedly stimulated the oxidation of NADH by plasma membranes from liver or erythrocytes and this effect was eliminated by superoxide dismutase (SOD)3 (1). It appeared possible that the plasma membranes were generating 02 in the presence of NAD(P)H and that vanadate might catalyze the oxidation of NAD(P)H by 0,. We tested this supposition by using the xanthine oxidase reaction, as a source of OS, and by showing that either vanadate or molybdate would enhance the oxidation of NAD(P)H and that SOD, but not catalase, eliminated this effect (2). Several aspects of the stimulation of NAD(P)H oxidation by vanadate appeared puzzling and in need of further exploration.
Thus, Vijaya et al. (3) reported that the only active form of vanadate was decavanadate and that phosphate was an essential component of the reaction mixture. Moreover, Goetz and Proctor (4) reported that chelating agents stimulated O;-dependent oxidations of NADH. We now describe experiments demonstrating that orthovanadate, rather than decavanadate, is the active form of vanadate; that phosphate is not needed for vanadate catalysis; and that chelating agents eliminate the catalytic effect of vanadate, while increasing the availability of 0; for reaction with NAD(P)H. MATERIALS
0603-9861/85
$3.90
8 1985 by Academic Prens. Inc. of reproduction in any form reserved.
METHODS
Xanthine oxidase, isolated from bovine cream (5), was kindly provided by R. D. Wiley and Dr. K. V. Rajagopalan. SOD was isolated from bovine erythrocytes as previously described (6). Vanadium pentoxide (V,O,) was purchased from either Alpha Products Inc. (reagent grade) or the Aldrich Chemical Company (ultrapure). Stock solutions (30 mM) were prepared by dissolving the VzOr, in 1.0 M NaOH, neutralizing to pH 7.8 with 1.0 M HCI, and bringing to the desired volume with glass-distilled H,O. This resulted in a deep yellow-orange solution characteristic of decavanadate (7) whose pH stabilized at 6.0. The
i This work was supported by research grants from the United States Army Research Office (Research Triangle Park, N. C.); the National Science Foundation (Washington, D. C.); the Council for Tobacco Research (New York, N. Y.); the Glenn Foundation; and the National Institutes of Health. 2 To whom correspondence should be addressed. ’ Abbreviations used: SOD, superoxide dismutase; desferal, desferrioxamine; DTPA, diethylenetriamine pentaacetate; EGTA, ethylene glycol his@-aminoethyl ether)-N,N’-tetraaeetic acid.
Copyright All rights
AND
226
VANADATE
ENHANCES
NADH
OXIDATION
221
idase reaction in the presence of vanadate. Omission of either xanthine or xanthine oxidase eliminated this oxidation of NADH. Results such as these led Vijaya et al. (3) to conclude that phosphate was essential for this reaction. In fact, when reaction mixtures were prepared in the absence of buffer salts and were adjusted to pH 7.8, the cooxidation of NADH was lofold faster than it had been in the phosphate-Tris mixture. Indeed, buffer salts such as Tris-HCl or potassium phosphate, or neutral salts such as or KCl, inhibited the cooxidation of NADH. The order of inhibitory potencies and the concentrations causing 50% inhibition were Tris-HCl (2.5 mM) > KzHPOl (10 mM) > K2S04 (85 mM) > KC1 (145 InM). Tris-HCl inhibited NADH oxidation more profoundly than did potassium phosphate. Moreover, the inhibition by Tris approached a limit of lOO%, whereas phosphate exhibited a maximum inhibition of 84% at 20 mM and less inhibition at higher RESULTS concentrations. The pH was 7.8 and SOD completely inhibited NADH oxidation in Efects of phosphate. As shown in Fig. 1, all cases. The inhibitory effects of Tris or addition of phosphate to a Tris-HCl-buffof ered reaction mixture increased the rate of of phosphate were not due to inhibition cooxidation of NADH by the xanthine ox- the xanthine oxidase reaction. Thus, as shown in Table I, the rate of conversion of xanthine to urate was nearly twice as fast in either Tris (lo-25 mM) or phosphate (100 mM) as it was in the unbuffered reaction mixtures. The apparent stimulation of NADH oxidation, shown in Fig. 1 and reported by Vijaya et al (3), can now be seen as a partial reversal, by phosphate, of an inhibition imposed by Tris. This conclusion was tested by comparing the rates of NADH oxidation at pH 7.8 + 1.0 mM potassium phosphate. The data in Table II show that phosphate did not stimulate and that SOD inhibited NADH oxidation, whether or not phos[Phosphate] mM phate was present. Eflects of chelating agents. Chelating FIG. 1. Effect of phosphate on the vanadate-stimagents were examined because they can ulated oxidation of NADH in the presence of Tris. form stable complexes with vanadate (8,9) Reaction mixtures contained 50 mM Tris-HCl, 100 #d and because they were reported to stimuNADH, 50 PM xanthine, 0.01 PM xanthine oxidase, the late the oxidation of NAD(P)H by 0; made indicated concentrations of potassium phosphate, and by the granule fraction of activated neu150 (line 1,O) or 300 PM V,O, (line 2,0). NADH oxitrophils or by the xanthine oxidase reaction dation at pH 7.8 and 25°C was followed in terms of the decrease in Am “,,,. (4). The data in Table III show that 0.1 mM
concentration of these stock solutions and dilutions thereof are expressed throughout in terms of V,O,. Ammonium metavanadate, DTPA, and NADH were purchased from Sigma. Desferal was purchased from Ciba Inc., while EDTA was from Mallinkrodt. When the possible effects of metal contaminants were being examined, a stock 50 mM potassium phosphate buffer at pH 7.8 was passed twice through a 2 X ‘7-cm column of Chelex-100 (Bio-Rad). Working buffers were then prepared by dilution of the stock with Chelex-treated, twice-glass-distilled water and were stored in new plastic bottles. All other reagents were stirred with Chelex-100 for a minimum of 1 h at 25°C and were then separated from the resin by decantation. Oxidation of NADH was followed at 340 nm, while 0; production was followed in terms of SOD-inhibitable reduction of cytochrome c at 550 nm (6). Xanthine oxidase activity was measured in terms of the rate of conversion of xanthine to urate followed at 293 nm. Unless other conditions are specified, reactions were performed in 1.0 mM potassium phosphate, 0.1 mM NADH, 0.1 mM VzOr,, 50 pM xanthine, and 0.01 PM xanthine oxidase, all at pH 7.8 and at 25°C. All absorption spectra were recorded with a Shimadzu UV240 recording spectrophotometer.
222
DARR TABLE
AND
I
EFFECTS OF SALTS IN THE PRESENCE OF VANADATE ON THE XANTHINE OXIDASE REACTION’
Salt added
CantroP 10 mM Tris-HCl 25 rnM Tris-HCl 20 mM KeHPOl 100 mM KsHPO, 200 mM KsHPO, 100 mM KC1 500 mM KC1 100 mM KpSOl 500 mrd K&30,
AA,., (min-I) loo (0.012) 162 170 ND 183 204 ND 94 ND 98
AA,., (mine’)
A&onmC (min-‘)
100 (0.032) 15 6 16 35 33 59 22 33 8
100 (0.023) ND 100 124 113 ND 104 74 104 52
“Reaction mixtures contained 50 PM xanthine, 100 NM NADH, 106 PM Ve06, 0.01 PM xanthine oxidase, and the indicated concentrations of the salts listed, all made up in glassdistilled H*O. In all cases the pH was adjusted to 7.8 and the reaction was run at 25°C. ND, Not determined. ‘Controls were identical to above conditions but contained none of the salts. Numbers are a percentage of the control value for the given experiment. Typical control values are given in parentheses. ‘These reaction mixtures contained 10 pM ferricytochrome e in addition to the components listed in a.
vanadate stimulated NADH cooxidation 24-fold whereas chelating agents exerted much more modest effects. Thus, DTPA increased NADH oxidation 3-fold, while EDTA and EGTA merely doubled the rate of this reaction and o-phenanthroline was without effect. The possibility that the chelating agents might be increasing the rate of production of 0; by the xanthine oxidase reaction was ruled out by measuring the rate of the SOD-inhibitable reduction of cytochrome c. Vanadate (0.1 mM) diminished this cytochrome c reduction by 28%, while the chelating agents had less effect. As a further control the rate of conversion of xanthine to urate was examined, and was found to be unaffected by the chelating agents. SOD completely inhibited NADH oxidation, whether chelating agents were present or not, a result in agreement with the report by Goetz and Proctor (4). It seems likely that the modest stimulation of NADH oxidation by chelating agents was due in part to their ability to eliminate the scavenging of 0; by trace metal contaminants which were able to compete
FRIDOVICH
more effectively with NADH for 02, than with cytochrome c. The profound stimulation of the cooxidation of NADH by vanadate was inhibited by chelating agents (Fig. 2). Among the compounds tested the order of effectiveness was desferal > EDTA > DTPA > EGTA. The role of chelation was examined by comparing o-phenanthroline, which is an effective chelating agent, with m-phenanthroline, which is not. As expected, ophenanthroline was a potent inhibitor of the vanadate-stimulated oxidation of NADH, while m-phenanthroline was without effect. The possibility that the effect of chelating agents reflected a catalytic role for metal impurities in the vanadate, was considered. Ultrapure vanadate was tested in a reaction mixture, all of whose components had been treated with Chelex-100, and it was slightly more effective in stimulating NADH oxidation than was the less pure material. It is clear that vanadate itself, rather than metal impurities, is the catalyst of NADH oxidation. The chelating agents must therefore inhibit by complexing vanadate. Desferal at 100 I.LM was able to suppress the stimulatory effect of up to 200 pM V205 (Fig. 2). When several different concentraTABLE
II
PHOSPHATE DOES NOT STIMULATE THE VANADATESTIMULATED OXIDATION OF NADH” A&a
KaHPO, (mM) 0 0 1.0 1.0
SOD (units/ml) 0 2 0 2
nm
(min-‘) Normalized* 100 (0.017) 0 (0) 87 0
a Reaction mixtures contained 50 PM xanthine, 100 NM NADH, 100 PM Vz05, 0.01 NM xanthine oxidase, and the indicated concentrations of KaHPO,, all made up in glass-distilled H20. Reactions were run at 25”C, pH 7.8. In these experiments, basal NADH oxidation by the xanthine + xanthine oxidase was recorded, after which the VzOs was added to the reaction mixture. bNumbers are a percentage of the control values for the given experiment. Typical control values are given in parentheses.
VANADATE
ENHANCES TABLE
NADH
223
OXIDATION
III
EFFECTS OF VANADATE AND OF CHELATING AGENTS ON NADH OXIDATION AND 0; PRODUCTION BY THE XANTHINE
VzOs or Chelator Controlb Control 100 yM v,o, 100 /AM vpob 100 FM EDTA 100 PM EGTA 100 PM EGTA 100 PM DTPA 100 jtM DTPA 100 PM Desferal 100 ELMo-Phenanthroline 100 PM m-Phenanthroline
OXIDASE
REACTION”
SOD
AA 293“rn (mini)
AA 340nm (mini)
AA 550run (mini)
0 1.0 pg/ml 0 1.0 fig/ml 0 0 1.0 pg/ml 0 1.0 pg/ml 0 0 0
100 (0.026) ND 89 ND 102 102 ND 104 ND 98 114 104
100 (0.034) 0 2400 0 195 207 0 319 0 120 100 100
100 (0.032) ND 72 ND 96 84 ND 82 ND 98 ND ND
‘Reaction mixtures contained 50 PM xanthine, 1 mM potassium phosphate, 100 PM NADH, 0.01 /IM xanthine oxidase, and the indicated amounts of vanadate, chelating agent, or SOD at pH 7.8 and at 25°C. When AAssonm was followed ferricytochrome c was also present at 10 PM. ND, Not determined. *Controls were identical to above conditions but contained none of the VzOs or chelators. Numbers are a percentage of the control values for the given experiment. Typical control values are given in parentheses.
tions of desferal were tested (Fig. 3), one molecule of desferal was consistently able to suppress the effect of two V205 or of four
100
200 [“2O5]
atoms of V(v). This result suggested that the desferal was inhibiting by complexing a tetravanadium form of vanadate. Metavanadate (V,O;;) is a colorless form of vanadate which is stable in the pH range 6.5-9.5, whereas decavanadate is orange in color and is stable below pH 6.5 (7). Vz05 was dissolved in NaOH and then neutralized (final concentration = 30 InM
300
PM
FIG. 2. Effects of chelating agents on the vanadate stimulation of the oxidation of NADH. Reaction mixtures contained 50 @Mxanthine, 100 &M NADH, 1.0 mM potassium phosphate, 0.01 ELMxanthine oxidase, and the indicated concentrations of vanadate, all at pH 7.8 and at 25°C. The chelating agents present were none (line 1,O); 109 pM EDTA (line 2, n); 109 PM DTPA (line 3, 0); 100 PM EGTA (line 4, 0); 100 PM desferal (line 5, A); 109 PM o-phenanthroline (A); and 100 JLM m-phenanthroline (e).
50
100
150
200
250
300
FIG. 3. Inhibition of vanadate stimulation of the oxidation of NADH by desferal. Reaction conditions were as in Fig. 2 except that the only chelating agent tested was desferal and it was present at none (line 1,O); 10 @i (line 2, 0); 50 ELM(line 3, Cl); and 100 PM (line 4, A).
224
DARR
AND
V205) to yield the orange-yellow solution described by Vijaya et al. (3). Addition of desferal to dilutions of this solution changed its optical spectrum as shown in Fig. 4. Increasing amounts of desferal caused a progressive increase in absorbance at 232 nm with a concomitant decrease in absorbance at wavelengths below 220 nm. There was a sharp isosbestic point at 220 nm. Spectrophotometric titration of the vanadate solution with desferal was feasible, and Fig. 5 presents the results of such titrations. Desferal clearly exhibits great affinity for vanadate, and one molecule of desferal complexed four atoms of pentavalent vanadium under these conditions. This indicates that an abundant form of vanadate in the 30 mM vanadate, prepared according to Vijaya et aL (3), was metavanadate. Moreover, since desferal, in complexing the metavanadate, inhibited the activity of this solution, the catalytically active species must have been metavanadate, or something in rapid equilibrium with metavanadate. When the 30 mM Vz05 solution was diluted to 3 mM and the pH was adjusted to 7.8 the orange-yellow color gradually vanished during several
‘.Or-----l
I 260 Wavelength
I’
I
I
310
360
(rim)
FIG. 4. Effect of desferal on the absorption spectrum of vanadate. A 30 mM stock solution of VrOh, pH 5.9, was diluted to 60 PM into 1.0 mM potassium phosphate, pH 7.8, containing the following concentrations of desferal: line 1.0; line 2,10 PM; line 3,20 pM, lines 46,30-60 PM. Absorption spectra were recorded within 5 min at 25°C. Desferal was in all cases present at the same concentration in the reference and sample cuvettes.
FRIDOVICH
0.25
2
[Desferag
E.~M
FIG. 5. Spectrophotometric titration of vanadate with desferal. A 30 mM stock solution of VrOs, pH 5.9, was diluted to 30 ).LLM(line 1,O); 60 PM (line 2, A); or 120 PM (line 3,O) in 1.0 mru potassium phosphate, pH 7.8, containing the indicated concentrations of desferal. Blank and sample cuvettes contained the same level of desferal. The increase in absorbance at 232 nm is plotted here as a function of the concentration of desferal.
days of incubation at 25”C, with a concomitant increase in catalytic activity. This is another indication that the colorless metavanadate, or something in rapid equilibrium with it, was the active species. It was clear that the conversion of decavanadate to metavanadate was a slow process under these conditions. The slow interconversion of decavanadate and metavanadate was followed spectrophotometrically. Thus the 30 InM V205 stock solution was diluted to 30 PM with 1.0 mM potassium phosphate, pH 7.8, and the ultraviolet absorption spectrum was followed as a function of time at 25°C. Figure 6 shows that the spectrum changed gradually on a time scale of hours, while Fig. 7 demonstrates a coincident increase in catalytic activity. We can again conclude that the slow interconversion of decavanadate to metavanadate was associated with an increase in catalytic activity and that metavanadate, or something in rapid equilibrium with it, was the active species. The orange-yellow 30 KIM stock solution of Vz05 was diluted to 3.0 mM with 1.0 mM phosphate, pH 7.8, and was incubated for 7 days at 25°C. During this time the solu-
VANADATE
ENHANCES
0. 25
0 190
260 Wavelength
360 (nm)
FIG. 6. Time-dependent changes in absorption following dilution and neutralization of vanadate solution. A 30 mM stock solution of VzOr,, pH 5.9, was diluted to 30 FM in 1.0 mM potassium phosphate, pH 7.8, at 25”C, and the absorption spectrum was recorded at t = 0 (line 1); t = 2 h (line 2); t = 5 h (line 3); and t = 48 h (line 4).
tion became colorless, changes in the ultraviolet reached a plateau, and catalytic activity had risen to a stable limit (data not shown). When this solution was then
NADH
225
OXIDATION
titrated with desferal in terms of changes in catalytic activity (Fig. 8) or in ultraviolet spectrum (Figs. 9 and lo), one desferal was seen to bind one atom of VCv,. The predominant form of vanadate in these solutions was therefore orthovanadate, which was catalytically active and which could be complexed by, and inactivated by, desferal. Finally, ammonium metavanadate from Sigma was diluted to 6.0 mM in terms of V(v) in 1.0 mM phosphate, pH 7.8. Its catalytic activity and its optical spectrum were virtually identical with those of the 3.0 mM V205 which had been allowed to equilibrate for 1 week in the phosphate buffer at pH 7.8. DISCUSSION
Tris, and to a lesser extent phosphate, diminished the ability of vanadate to catalyze the oxidation of NADH by 0,. Tris, phosphate, and a variety of chelating agents and ions should be able to ligate to vanadate, change its redox potential, and block or at least modify its interactions with 02 and NAD(P)H. The effect of phosphate was biphasic, progressively inhibitory in the range of O-20 mM and then less
25,
Tfme
(hours)
FIG. 7. Catalytic activity and absorbance change in parallel after dilution and neutralization of vanadate. A 30 mM stock solution of VzO, was diluted as described in Fig. 6. At intervals, aliquots were removed and their absorption at 208 nm (0) relative to the isosbestic point at 218 nm was measured, as was their ability to catalyze the oxidation of NADH by the 0; produced by 0.01 p~ xanthine oxidase acting on 50 PM xanthine in the 1.0 mM potassium phosphate buffer (Cl).
[“A’s] NJ FIG. 8. Desferal inhibition as a function of the vanadate stock solution. Vanadate from either a 3 mM colorless stock solution equilibrated at pH 7.8 (lines 1 and 2) or from a 30 rnM yellow stock solution at pH 5.9 (lines 3 and 4) was diluted into reaction mixtures containing 1.0 mM potassium phosphate, 100 PM NADH, 50 pM xanthine, and 0.01 PM xanthine oxidase, pH 7.8, at 25”C, and the oxidation of NADH was followed at 340 nm. Lines 1 and 3, no desferal; lines 2 and 4, 10 PM desferal.
226
DARR
B 20
260 Wavelength
AND
360 (nm)
FIG. 9. Effect of desferal on the absorption spectrum of vanadate. Vanadate was diluted to 30 PM VzOs into 1.0 rnre potassium phosphate, pH 7.8, at 25”C, containing the following concentrations of desferal: line 1, 0; line 2, 10 PM; line 3, 20 pMd; line 4, 40 fiM; line 5, 60 PM; line 6,80 PM; and line 7,100 FM. The vanadate stock was a colorless 3.0 mM VrO, solution equilibrated at pH 7.8 in 1.0 mM potassium phosphate. Desferal was at the same concentration in blank and sample cuvettes.
inhibitory in the range 20-100 mM. Phosphate should be expected to form heteropoly acids with vanadate, much as it does with molybdate (10). Phosphate could therefore affect the decavanadate S metavanadate c orthovanadate equilibrium. It is, in any case, clear that phosphate is not essential for the catalytic activity of vanadate, and only appears to stimulate when added to Tris-buffered solutions because of a diminution of the greater inhibition caused by Tris. Desferal was the most effective of the inhibitory chelating agents examined. It could be used to titrate vanadate either in terms of loss of catalytic activity (Figs. 3 and 8) or of changes in optical spectrum (Figs. 4 and 9). When a 30 mM Vz05 stock solution, pH 5.9, was used (Figs. 3 and 4) the ratio of vanadium atoms titrated per desferal added was 4:l. In contrast, when an equilibrated 3.0 mM Vz05 stock solution, pH 7.8, was used the corresponding ratio was 1:l. This can be explained in terms of a slow interconversion of decavanadate with metavanadate, followed by a rapid
FRIDOVICH
interconversion of metavanadate with orthovanadate, and the assumption that desferal can complex, on a one to one basis, with either metavanadate of orthovanadate. Thus, the 30 mM Vz05, pH 5.9, contained a mixture of decavanadate and metavanadate, and its catalytic activity was entirely dependent upon rapid dissociation of the metavanadate to orthovanadate upon dilution into the reaction mixtures. In that circumstance one molecule of desferal could, by ligating to metavanadate, prevent its dissociation and so eliminate the catalytic activity of four VCvj. The 3.0 mM VzOr,, pH 7.8, was mostly in the form of orthovanadate, in which case one desferal would be required per V(v). These data, plus the net increase in activity associated with the slow dissociation of decavanadate to metavanadate seen when the 30 mM Vz05, pH 5.9, was diluted to 3.0 mM Vz05, pH 7.8, establish that the catalytically active form of vanadate is orthovanadate rather that decavanadate. One must also conclude that the generally held opinion that desferal is specific for Fe+3 is overstated. By markedly increasing the rate of NADH oxidation by O;, orthovanadate can serve as an amplification device for the de-
.24-
FIG. 10. Spectrophotometric titration of vanadate with desferal. Vanadate at final concentrations of 15 pM VzOs (line 1, n ); 30 PM VrO, (line 2, 0), or 60 PM VaO, (line 3,O) was titrated with desferal as described in Fig. 9, and the absorbance change at 315 nm was recorded and plotted as a function of the concentration of desferal.
VANADATE
ENHANCES
tection of 0,. It thus appears likely that the stimulation by vanadate of the oxidation of NADH by plasma membranes (1,3) reflects the production of 0; by those membranes. The significance of this low level of production of 0; by plasma membranes in the presence of vanadate remains to be explored. It is possible that the toxicity of vanadate may in part derive from the reactions we have been discussing. Orthovanadate (VOi3) is an analog of phosphate and is the form of vanadate believed to most often influence biologically relevant reactions (11). We have now shown that it is also orthovanadate, rather than decavanadate, which catalyzes the oxidation of NADH by 0;. REFERENCES 1. RAMASARMA, T., MACKELLAR, W. C., AND CRANE, F. L. (1981) &o&m Biophys. Acta 646,88-98.
NADH
227
OXIDATION
2. DARR, D., AND FRIDOVICH,
I. (1984)
Arch Biochmn.
Biophys. 232,562-565. 3. VIJAYA, S., CRANE, F. L., AND RAMASARMA, (1984) Mel CeU Biochem 62,175-U%. 4. GOETZ, M. B., AND PROCTOR, R. A. (1984)
T.
Anal
Biochem 137,230-235. 5. WAUD, W. R., BRADY, F. O., WILEY, R. D., AND RAJAGOPALAN, K. V. (1975) Arch B&hem. Biophys. 169.695-701. 6. MCCORD, J. M., AND FRIDOVICH, I. (1969) J. BioL
Chem 244.6049-6055. 7. GARRELS, R. M., AND CHRIST, C. L. (1965) in Solutions, Minerals and Equilibria, p. 503, Freeman and Cooper, San Francisco. 8. PRZYBOROWSKI, L., SCHWARZENBACH, G., AND ZIMMERMAN, T. (1965) Helv. Chim Acta 48, 15561565. 9. AMOS, L. W., AND SAWYER, D. T. (1972) Inorg. Chem 11,2692-2698. 10. FISKE, C. H., AND SUBBAROW, Y. (1925) J. BioL Chem. 66,375-400. 11. CHASTEEN, N. D. (1983) Structure Bonding 53,105138.
Vanadate Enhancement of the Oxidation of NADH by 0;: Effects of Phosphate and Chelating Agents’ DOUGLAS Department of Biodmmistly, Received
DARR
AND
IRWIN
FRIDOVICH’
Duke University Medical Center, April
9,1985,
and in revised
form
Durham, July
North Carolina
27710
26,1985
Vanadate markedly stimulates the oxidation of NADH by 0;. Both phosphate and Tris are inhibitory, but phosphate diminishes the greater inhibitory effect of Tris and thus gives the appearance of stimulating when added to Tris-buffered reaction mixtures. Chelating agents moderately increased the oxidation of NADH but eliminated the much greater catalytic effect of vanadate. Desferal was the most effective of the chelating agents, and could be used to titrate vanadate spectrophotometrically or in terms of the diminution of its catalytic activity. This permitted the demonstration that metavanadate or orthovanadate could form 1:l complexes with desferal and that orthovanadate was the catalytically active species. Q I985 Academic Press, k.
Vanadate markedly stimulated the oxidation of NADH by plasma membranes from liver or erythrocytes and this effect was eliminated by superoxide dismutase (SOD)3 (1). It appeared possible that the plasma membranes were generating 02 in the presence of NAD(P)H and that vanadate might catalyze the oxidation of NAD(P)H by 0,. We tested this supposition by using the xanthine oxidase reaction, as a source of OS, and by showing that either vanadate or molybdate would enhance the oxidation of NAD(P)H and that SOD, but not catalase, eliminated this effect (2). Several aspects of the stimulation of NAD(P)H oxidation by vanadate appeared puzzling and in need of further exploration.
Thus, Vijaya et al. (3) reported that the only active form of vanadate was decavanadate and that phosphate was an essential component of the reaction mixture. Moreover, Goetz and Proctor (4) reported that chelating agents stimulated O;-dependent oxidations of NADH. We now describe experiments demonstrating that orthovanadate, rather than decavanadate, is the active form of vanadate; that phosphate is not needed for vanadate catalysis; and that chelating agents eliminate the catalytic effect of vanadate, while increasing the availability of 0; for reaction with NAD(P)H. MATERIALS
0603-9861/85
$3.90
8 1985 by Academic Prens. Inc. of reproduction in any form reserved.
METHODS
Xanthine oxidase, isolated from bovine cream (5), was kindly provided by R. D. Wiley and Dr. K. V. Rajagopalan. SOD was isolated from bovine erythrocytes as previously described (6). Vanadium pentoxide (V,O,) was purchased from either Alpha Products Inc. (reagent grade) or the Aldrich Chemical Company (ultrapure). Stock solutions (30 mM) were prepared by dissolving the VzOr, in 1.0 M NaOH, neutralizing to pH 7.8 with 1.0 M HCI, and bringing to the desired volume with glass-distilled H,O. This resulted in a deep yellow-orange solution characteristic of decavanadate (7) whose pH stabilized at 6.0. The
i This work was supported by research grants from the United States Army Research Office (Research Triangle Park, N. C.); the National Science Foundation (Washington, D. C.); the Council for Tobacco Research (New York, N. Y.); the Glenn Foundation; and the National Institutes of Health. 2 To whom correspondence should be addressed. ’ Abbreviations used: SOD, superoxide dismutase; desferal, desferrioxamine; DTPA, diethylenetriamine pentaacetate; EGTA, ethylene glycol his@-aminoethyl ether)-N,N’-tetraaeetic acid.
Copyright All rights
AND
226
VANADATE
ENHANCES
NADH
OXIDATION
221
idase reaction in the presence of vanadate. Omission of either xanthine or xanthine oxidase eliminated this oxidation of NADH. Results such as these led Vijaya et al. (3) to conclude that phosphate was essential for this reaction. In fact, when reaction mixtures were prepared in the absence of buffer salts and were adjusted to pH 7.8, the cooxidation of NADH was lofold faster than it had been in the phosphate-Tris mixture. Indeed, buffer salts such as Tris-HCl or potassium phosphate, or neutral salts such as or KCl, inhibited the cooxidation of NADH. The order of inhibitory potencies and the concentrations causing 50% inhibition were Tris-HCl (2.5 mM) > KzHPOl (10 mM) > K2S04 (85 mM) > KC1 (145 InM). Tris-HCl inhibited NADH oxidation more profoundly than did potassium phosphate. Moreover, the inhibition by Tris approached a limit of lOO%, whereas phosphate exhibited a maximum inhibition of 84% at 20 mM and less inhibition at higher RESULTS concentrations. The pH was 7.8 and SOD completely inhibited NADH oxidation in Efects of phosphate. As shown in Fig. 1, all cases. The inhibitory effects of Tris or addition of phosphate to a Tris-HCl-buffof ered reaction mixture increased the rate of of phosphate were not due to inhibition cooxidation of NADH by the xanthine ox- the xanthine oxidase reaction. Thus, as shown in Table I, the rate of conversion of xanthine to urate was nearly twice as fast in either Tris (lo-25 mM) or phosphate (100 mM) as it was in the unbuffered reaction mixtures. The apparent stimulation of NADH oxidation, shown in Fig. 1 and reported by Vijaya et al (3), can now be seen as a partial reversal, by phosphate, of an inhibition imposed by Tris. This conclusion was tested by comparing the rates of NADH oxidation at pH 7.8 + 1.0 mM potassium phosphate. The data in Table II show that phosphate did not stimulate and that SOD inhibited NADH oxidation, whether or not phos[Phosphate] mM phate was present. Eflects of chelating agents. Chelating FIG. 1. Effect of phosphate on the vanadate-stimagents were examined because they can ulated oxidation of NADH in the presence of Tris. form stable complexes with vanadate (8,9) Reaction mixtures contained 50 mM Tris-HCl, 100 #d and because they were reported to stimuNADH, 50 PM xanthine, 0.01 PM xanthine oxidase, the late the oxidation of NAD(P)H by 0; made indicated concentrations of potassium phosphate, and by the granule fraction of activated neu150 (line 1,O) or 300 PM V,O, (line 2,0). NADH oxitrophils or by the xanthine oxidase reaction dation at pH 7.8 and 25°C was followed in terms of the decrease in Am “,,,. (4). The data in Table III show that 0.1 mM
concentration of these stock solutions and dilutions thereof are expressed throughout in terms of V,O,. Ammonium metavanadate, DTPA, and NADH were purchased from Sigma. Desferal was purchased from Ciba Inc., while EDTA was from Mallinkrodt. When the possible effects of metal contaminants were being examined, a stock 50 mM potassium phosphate buffer at pH 7.8 was passed twice through a 2 X ‘7-cm column of Chelex-100 (Bio-Rad). Working buffers were then prepared by dilution of the stock with Chelex-treated, twice-glass-distilled water and were stored in new plastic bottles. All other reagents were stirred with Chelex-100 for a minimum of 1 h at 25°C and were then separated from the resin by decantation. Oxidation of NADH was followed at 340 nm, while 0; production was followed in terms of SOD-inhibitable reduction of cytochrome c at 550 nm (6). Xanthine oxidase activity was measured in terms of the rate of conversion of xanthine to urate followed at 293 nm. Unless other conditions are specified, reactions were performed in 1.0 mM potassium phosphate, 0.1 mM NADH, 0.1 mM VzOr,, 50 pM xanthine, and 0.01 PM xanthine oxidase, all at pH 7.8 and at 25°C. All absorption spectra were recorded with a Shimadzu UV240 recording spectrophotometer.
222
DARR TABLE
AND
I
EFFECTS OF SALTS IN THE PRESENCE OF VANADATE ON THE XANTHINE OXIDASE REACTION’
Salt added
CantroP 10 mM Tris-HCl 25 rnM Tris-HCl 20 mM KeHPOl 100 mM KsHPO, 200 mM KsHPO, 100 mM KC1 500 mM KC1 100 mM KpSOl 500 mrd K&30,
AA,., (min-I) loo (0.012) 162 170 ND 183 204 ND 94 ND 98
AA,., (mine’)
A&onmC (min-‘)
100 (0.032) 15 6 16 35 33 59 22 33 8
100 (0.023) ND 100 124 113 ND 104 74 104 52
“Reaction mixtures contained 50 PM xanthine, 100 NM NADH, 106 PM Ve06, 0.01 PM xanthine oxidase, and the indicated concentrations of the salts listed, all made up in glassdistilled H*O. In all cases the pH was adjusted to 7.8 and the reaction was run at 25°C. ND, Not determined. ‘Controls were identical to above conditions but contained none of the salts. Numbers are a percentage of the control value for the given experiment. Typical control values are given in parentheses. ‘These reaction mixtures contained 10 pM ferricytochrome e in addition to the components listed in a.
vanadate stimulated NADH cooxidation 24-fold whereas chelating agents exerted much more modest effects. Thus, DTPA increased NADH oxidation 3-fold, while EDTA and EGTA merely doubled the rate of this reaction and o-phenanthroline was without effect. The possibility that the chelating agents might be increasing the rate of production of 0; by the xanthine oxidase reaction was ruled out by measuring the rate of the SOD-inhibitable reduction of cytochrome c. Vanadate (0.1 mM) diminished this cytochrome c reduction by 28%, while the chelating agents had less effect. As a further control the rate of conversion of xanthine to urate was examined, and was found to be unaffected by the chelating agents. SOD completely inhibited NADH oxidation, whether chelating agents were present or not, a result in agreement with the report by Goetz and Proctor (4). It seems likely that the modest stimulation of NADH oxidation by chelating agents was due in part to their ability to eliminate the scavenging of 0; by trace metal contaminants which were able to compete
FRIDOVICH
more effectively with NADH for 02, than with cytochrome c. The profound stimulation of the cooxidation of NADH by vanadate was inhibited by chelating agents (Fig. 2). Among the compounds tested the order of effectiveness was desferal > EDTA > DTPA > EGTA. The role of chelation was examined by comparing o-phenanthroline, which is an effective chelating agent, with m-phenanthroline, which is not. As expected, ophenanthroline was a potent inhibitor of the vanadate-stimulated oxidation of NADH, while m-phenanthroline was without effect. The possibility that the effect of chelating agents reflected a catalytic role for metal impurities in the vanadate, was considered. Ultrapure vanadate was tested in a reaction mixture, all of whose components had been treated with Chelex-100, and it was slightly more effective in stimulating NADH oxidation than was the less pure material. It is clear that vanadate itself, rather than metal impurities, is the catalyst of NADH oxidation. The chelating agents must therefore inhibit by complexing vanadate. Desferal at 100 I.LM was able to suppress the stimulatory effect of up to 200 pM V205 (Fig. 2). When several different concentraTABLE
II
PHOSPHATE DOES NOT STIMULATE THE VANADATESTIMULATED OXIDATION OF NADH” A&a
KaHPO, (mM) 0 0 1.0 1.0
SOD (units/ml) 0 2 0 2
nm
(min-‘) Normalized* 100 (0.017) 0 (0) 87 0
a Reaction mixtures contained 50 PM xanthine, 100 NM NADH, 100 PM Vz05, 0.01 NM xanthine oxidase, and the indicated concentrations of KaHPO,, all made up in glass-distilled H20. Reactions were run at 25”C, pH 7.8. In these experiments, basal NADH oxidation by the xanthine + xanthine oxidase was recorded, after which the VzOs was added to the reaction mixture. bNumbers are a percentage of the control values for the given experiment. Typical control values are given in parentheses.
VANADATE
ENHANCES TABLE
NADH
223
OXIDATION
III
EFFECTS OF VANADATE AND OF CHELATING AGENTS ON NADH OXIDATION AND 0; PRODUCTION BY THE XANTHINE
VzOs or Chelator Controlb Control 100 yM v,o, 100 /AM vpob 100 FM EDTA 100 PM EGTA 100 PM EGTA 100 PM DTPA 100 jtM DTPA 100 PM Desferal 100 ELMo-Phenanthroline 100 PM m-Phenanthroline
OXIDASE
REACTION”
SOD
AA 293“rn (mini)
AA 340nm (mini)
AA 550run (mini)
0 1.0 pg/ml 0 1.0 fig/ml 0 0 1.0 pg/ml 0 1.0 pg/ml 0 0 0
100 (0.026) ND 89 ND 102 102 ND 104 ND 98 114 104
100 (0.034) 0 2400 0 195 207 0 319 0 120 100 100
100 (0.032) ND 72 ND 96 84 ND 82 ND 98 ND ND
‘Reaction mixtures contained 50 PM xanthine, 1 mM potassium phosphate, 100 PM NADH, 0.01 /IM xanthine oxidase, and the indicated amounts of vanadate, chelating agent, or SOD at pH 7.8 and at 25°C. When AAssonm was followed ferricytochrome c was also present at 10 PM. ND, Not determined. *Controls were identical to above conditions but contained none of the VzOs or chelators. Numbers are a percentage of the control values for the given experiment. Typical control values are given in parentheses.
tions of desferal were tested (Fig. 3), one molecule of desferal was consistently able to suppress the effect of two V205 or of four
100
200 [“2O5]
atoms of V(v). This result suggested that the desferal was inhibiting by complexing a tetravanadium form of vanadate. Metavanadate (V,O;;) is a colorless form of vanadate which is stable in the pH range 6.5-9.5, whereas decavanadate is orange in color and is stable below pH 6.5 (7). Vz05 was dissolved in NaOH and then neutralized (final concentration = 30 InM
300
PM
FIG. 2. Effects of chelating agents on the vanadate stimulation of the oxidation of NADH. Reaction mixtures contained 50 @Mxanthine, 100 &M NADH, 1.0 mM potassium phosphate, 0.01 ELMxanthine oxidase, and the indicated concentrations of vanadate, all at pH 7.8 and at 25°C. The chelating agents present were none (line 1,O); 109 pM EDTA (line 2, n); 109 PM DTPA (line 3, 0); 100 PM EGTA (line 4, 0); 100 PM desferal (line 5, A); 109 PM o-phenanthroline (A); and 100 JLM m-phenanthroline (e).
50
100
150
200
250
300
FIG. 3. Inhibition of vanadate stimulation of the oxidation of NADH by desferal. Reaction conditions were as in Fig. 2 except that the only chelating agent tested was desferal and it was present at none (line 1,O); 10 @i (line 2, 0); 50 ELM(line 3, Cl); and 100 PM (line 4, A).
224
DARR
AND
V205) to yield the orange-yellow solution described by Vijaya et al. (3). Addition of desferal to dilutions of this solution changed its optical spectrum as shown in Fig. 4. Increasing amounts of desferal caused a progressive increase in absorbance at 232 nm with a concomitant decrease in absorbance at wavelengths below 220 nm. There was a sharp isosbestic point at 220 nm. Spectrophotometric titration of the vanadate solution with desferal was feasible, and Fig. 5 presents the results of such titrations. Desferal clearly exhibits great affinity for vanadate, and one molecule of desferal complexed four atoms of pentavalent vanadium under these conditions. This indicates that an abundant form of vanadate in the 30 mM vanadate, prepared according to Vijaya et aL (3), was metavanadate. Moreover, since desferal, in complexing the metavanadate, inhibited the activity of this solution, the catalytically active species must have been metavanadate, or something in rapid equilibrium with metavanadate. When the 30 mM Vz05 solution was diluted to 3 mM and the pH was adjusted to 7.8 the orange-yellow color gradually vanished during several
‘.Or-----l
I 260 Wavelength
I’
I
I
310
360
(rim)
FIG. 4. Effect of desferal on the absorption spectrum of vanadate. A 30 mM stock solution of VrOh, pH 5.9, was diluted to 60 PM into 1.0 mM potassium phosphate, pH 7.8, containing the following concentrations of desferal: line 1.0; line 2,10 PM; line 3,20 pM, lines 46,30-60 PM. Absorption spectra were recorded within 5 min at 25°C. Desferal was in all cases present at the same concentration in the reference and sample cuvettes.
FRIDOVICH
0.25
2
[Desferag
E.~M
FIG. 5. Spectrophotometric titration of vanadate with desferal. A 30 mM stock solution of VrOs, pH 5.9, was diluted to 30 ).LLM(line 1,O); 60 PM (line 2, A); or 120 PM (line 3,O) in 1.0 mru potassium phosphate, pH 7.8, containing the indicated concentrations of desferal. Blank and sample cuvettes contained the same level of desferal. The increase in absorbance at 232 nm is plotted here as a function of the concentration of desferal.
days of incubation at 25”C, with a concomitant increase in catalytic activity. This is another indication that the colorless metavanadate, or something in rapid equilibrium with it, was the active species. It was clear that the conversion of decavanadate to metavanadate was a slow process under these conditions. The slow interconversion of decavanadate and metavanadate was followed spectrophotometrically. Thus the 30 InM V205 stock solution was diluted to 30 PM with 1.0 mM potassium phosphate, pH 7.8, and the ultraviolet absorption spectrum was followed as a function of time at 25°C. Figure 6 shows that the spectrum changed gradually on a time scale of hours, while Fig. 7 demonstrates a coincident increase in catalytic activity. We can again conclude that the slow interconversion of decavanadate to metavanadate was associated with an increase in catalytic activity and that metavanadate, or something in rapid equilibrium with it, was the active species. The orange-yellow 30 KIM stock solution of Vz05 was diluted to 3.0 mM with 1.0 mM phosphate, pH 7.8, and was incubated for 7 days at 25°C. During this time the solu-
VANADATE
ENHANCES
0. 25
0 190
260 Wavelength
360 (nm)
FIG. 6. Time-dependent changes in absorption following dilution and neutralization of vanadate solution. A 30 mM stock solution of VzOr,, pH 5.9, was diluted to 30 FM in 1.0 mM potassium phosphate, pH 7.8, at 25”C, and the absorption spectrum was recorded at t = 0 (line 1); t = 2 h (line 2); t = 5 h (line 3); and t = 48 h (line 4).
tion became colorless, changes in the ultraviolet reached a plateau, and catalytic activity had risen to a stable limit (data not shown). When this solution was then
NADH
225
OXIDATION
titrated with desferal in terms of changes in catalytic activity (Fig. 8) or in ultraviolet spectrum (Figs. 9 and lo), one desferal was seen to bind one atom of VCv,. The predominant form of vanadate in these solutions was therefore orthovanadate, which was catalytically active and which could be complexed by, and inactivated by, desferal. Finally, ammonium metavanadate from Sigma was diluted to 6.0 mM in terms of V(v) in 1.0 mM phosphate, pH 7.8. Its catalytic activity and its optical spectrum were virtually identical with those of the 3.0 mM V205 which had been allowed to equilibrate for 1 week in the phosphate buffer at pH 7.8. DISCUSSION
Tris, and to a lesser extent phosphate, diminished the ability of vanadate to catalyze the oxidation of NADH by 0,. Tris, phosphate, and a variety of chelating agents and ions should be able to ligate to vanadate, change its redox potential, and block or at least modify its interactions with 02 and NAD(P)H. The effect of phosphate was biphasic, progressively inhibitory in the range of O-20 mM and then less
25,
Tfme
(hours)
FIG. 7. Catalytic activity and absorbance change in parallel after dilution and neutralization of vanadate. A 30 mM stock solution of VzO, was diluted as described in Fig. 6. At intervals, aliquots were removed and their absorption at 208 nm (0) relative to the isosbestic point at 218 nm was measured, as was their ability to catalyze the oxidation of NADH by the 0; produced by 0.01 p~ xanthine oxidase acting on 50 PM xanthine in the 1.0 mM potassium phosphate buffer (Cl).
[“A’s] NJ FIG. 8. Desferal inhibition as a function of the vanadate stock solution. Vanadate from either a 3 mM colorless stock solution equilibrated at pH 7.8 (lines 1 and 2) or from a 30 rnM yellow stock solution at pH 5.9 (lines 3 and 4) was diluted into reaction mixtures containing 1.0 mM potassium phosphate, 100 PM NADH, 50 pM xanthine, and 0.01 PM xanthine oxidase, pH 7.8, at 25”C, and the oxidation of NADH was followed at 340 nm. Lines 1 and 3, no desferal; lines 2 and 4, 10 PM desferal.
226
DARR
B 20
260 Wavelength
AND
360 (nm)
FIG. 9. Effect of desferal on the absorption spectrum of vanadate. Vanadate was diluted to 30 PM VzOs into 1.0 rnre potassium phosphate, pH 7.8, at 25”C, containing the following concentrations of desferal: line 1, 0; line 2, 10 PM; line 3, 20 pMd; line 4, 40 fiM; line 5, 60 PM; line 6,80 PM; and line 7,100 FM. The vanadate stock was a colorless 3.0 mM VrO, solution equilibrated at pH 7.8 in 1.0 mM potassium phosphate. Desferal was at the same concentration in blank and sample cuvettes.
inhibitory in the range 20-100 mM. Phosphate should be expected to form heteropoly acids with vanadate, much as it does with molybdate (10). Phosphate could therefore affect the decavanadate S metavanadate c orthovanadate equilibrium. It is, in any case, clear that phosphate is not essential for the catalytic activity of vanadate, and only appears to stimulate when added to Tris-buffered solutions because of a diminution of the greater inhibition caused by Tris. Desferal was the most effective of the inhibitory chelating agents examined. It could be used to titrate vanadate either in terms of loss of catalytic activity (Figs. 3 and 8) or of changes in optical spectrum (Figs. 4 and 9). When a 30 mM Vz05 stock solution, pH 5.9, was used (Figs. 3 and 4) the ratio of vanadium atoms titrated per desferal added was 4:l. In contrast, when an equilibrated 3.0 mM Vz05 stock solution, pH 7.8, was used the corresponding ratio was 1:l. This can be explained in terms of a slow interconversion of decavanadate with metavanadate, followed by a rapid
FRIDOVICH
interconversion of metavanadate with orthovanadate, and the assumption that desferal can complex, on a one to one basis, with either metavanadate of orthovanadate. Thus, the 30 mM Vz05, pH 5.9, contained a mixture of decavanadate and metavanadate, and its catalytic activity was entirely dependent upon rapid dissociation of the metavanadate to orthovanadate upon dilution into the reaction mixtures. In that circumstance one molecule of desferal could, by ligating to metavanadate, prevent its dissociation and so eliminate the catalytic activity of four VCvj. The 3.0 mM VzOr,, pH 7.8, was mostly in the form of orthovanadate, in which case one desferal would be required per V(v). These data, plus the net increase in activity associated with the slow dissociation of decavanadate to metavanadate seen when the 30 mM Vz05, pH 5.9, was diluted to 3.0 mM Vz05, pH 7.8, establish that the catalytically active form of vanadate is orthovanadate rather that decavanadate. One must also conclude that the generally held opinion that desferal is specific for Fe+3 is overstated. By markedly increasing the rate of NADH oxidation by O;, orthovanadate can serve as an amplification device for the de-
.24-
FIG. 10. Spectrophotometric titration of vanadate with desferal. Vanadate at final concentrations of 15 pM VzOs (line 1, n ); 30 PM VrO, (line 2, 0), or 60 PM VaO, (line 3,O) was titrated with desferal as described in Fig. 9, and the absorbance change at 315 nm was recorded and plotted as a function of the concentration of desferal.
VANADATE
ENHANCES
tection of 0,. It thus appears likely that the stimulation by vanadate of the oxidation of NADH by plasma membranes (1,3) reflects the production of 0; by those membranes. The significance of this low level of production of 0; by plasma membranes in the presence of vanadate remains to be explored. It is possible that the toxicity of vanadate may in part derive from the reactions we have been discussing. Orthovanadate (VOi3) is an analog of phosphate and is the form of vanadate believed to most often influence biologically relevant reactions (11). We have now shown that it is also orthovanadate, rather than decavanadate, which catalyzes the oxidation of NADH by 0;. REFERENCES 1. RAMASARMA, T., MACKELLAR, W. C., AND CRANE, F. L. (1981) &o&m Biophys. Acta 646,88-98.
NADH
227
OXIDATION
2. DARR, D., AND FRIDOVICH,
I. (1984)
Arch Biochmn.
Biophys. 232,562-565. 3. VIJAYA, S., CRANE, F. L., AND RAMASARMA, (1984) Mel CeU Biochem 62,175-U%. 4. GOETZ, M. B., AND PROCTOR, R. A. (1984)
T.
Anal
Biochem 137,230-235. 5. WAUD, W. R., BRADY, F. O., WILEY, R. D., AND RAJAGOPALAN, K. V. (1975) Arch B&hem. Biophys. 169.695-701. 6. MCCORD, J. M., AND FRIDOVICH, I. (1969) J. BioL
Chem 244.6049-6055. 7. GARRELS, R. M., AND CHRIST, C. L. (1965) in Solutions, Minerals and Equilibria, p. 503, Freeman and Cooper, San Francisco. 8. PRZYBOROWSKI, L., SCHWARZENBACH, G., AND ZIMMERMAN, T. (1965) Helv. Chim Acta 48, 15561565. 9. AMOS, L. W., AND SAWYER, D. T. (1972) Inorg. Chem 11,2692-2698. 10. FISKE, C. H., AND SUBBAROW, Y. (1925) J. BioL Chem. 66,375-400. 11. CHASTEEN, N. D. (1983) Structure Bonding 53,105138.