Vanadium Effect on the Activity of Horseradish Peroxidase, Catalase, Glu~thione Peroxidase, and Superoxide Dismutase In Vitro Miguel Angel Serra, Alessandro FIntar,* Luigi CaseJla, and Enrico Sabbioni Commission of the European Communities, Joint Research Centre (Ispra Site), Environment Institute, Ispra, (Varese), Itaiy.-AP, LC. Department of Inorganic and Metal~organicChemistry CNR, University of Milano, Milano, Italy
MAS, ES.
ABSTRACT The eifect of vanadium (V) on the activity of horseradish peroxidase, catalase, glutathioue peroxidase, and superoxide dismutase bas been studied. A competitive inhibition pattern was evident for vanadate ions on the activity of horseradish peroxidase (Ki = 41.2 PM). No significant inhibitory effects were found when V(V) was tested with cat&se and when either V(W) or V(v) were assayed with glutathione peroxidase. For the latter, the effect of V on tbe different components of the reaction system was investigated. V(V) did not significantly affect SOD activity when assayed with the sulfite method, which is devoid of interferences with V(V); however, there was an apparent ~bito~ dose-response pattern for either V(IV) or V(V) using the pyrogallol assay, owing to an interference of pyrogallol with tbe metal. Besides, no significant binding of V(W) or V(V) to the enzyme could be demonstrated. The lack of a direct ~bito~ effect of V on the activity of the main ~tio~ enzymes suggests that many biological and toxicological effects of V may be medii more by oxidative reactions of the metal or of its complexes with physiologically relevant biomoiecules than by a direct modulation of enzymatic activities. ABBREVIATIONS XRP, horseradish peroxiW, GSH-Px, gh~tathioneperoxidase, GSSG-R, glutathioue reductase; SOD, Cu-Zn superoxide dismutase; DFO, deferoxamine; guaiacol, methylcatbecoI; pyrogallol, 1,2,3-t&y-
Address reprint requests and correspondence to: Dr. Enrico Sabbioni, Commission of the European Cafe, Joint Research Centre (Ispra Site), Env~~t Institute, Life Scii Unit, T.P. 460, I-21020 ISPRA (Varese), Italy. *Present address: Bracco Industria chimica S.P.A., Via E. Folli, SO, I-20133 Miiano, Italy. JournalofInorganic B&&em&y, 46, 161- 174 (1992) 161 @ 1992 Elswier Science Publishing Co., Inc., 655 Avenueof the Americas.NY, NY 10010 0162-0134/92/S5.00
162
M. A. Serra et al.
droxybemene; radical.
UOD, units of optical density; NAA, neutron activation analysis; OF, superoxide
INTRODUCTION The study of the enzymes dealing with the elimination of oxygen active species (antioxidant enzymes) is of considerable interest because they constitute an essential defense against the well-demonstrated toxicity of those compounds [ 11. Oxy-radicals and other oxygen derived species have been proposed to be involved in several human diseases, including cancer, multiple sclerosis, Parkinson’s disease, autoimmune disease, and senile dementia, although their roles as causative agents are not clear [2]. Undoubtedly, the finding of specific inhibitors of antioxidant enzymes is of great relevance in understanding the physiological consequences of their deficit. However, not all these enzymes are easy to assay; while peroxidases and catalases can always be tested directly, others such as glutathione peroxidases and superoxide dismutases are frequently tested by coupled or indirect assays, making their study considerably more complicated [3, 41. Badly performed assays or absence of the adequate controls when testing possible inhibitors have led many authors to mistaken or confusing conclusions, as has been shown elsewhere [3-61. Moreover, the research on the role of trace metals in biological oxidations (oxy-radicals and lipid peroxidation) and hence the use of metal chelators to ascertain and/or prevent their effects, is a field of growing interest [2]. Several studies have been published on how these processes can be affected by metal ions such as Fe [‘?I, Fe + Al [8], Fe + Al + Pb [9], Al [lo, 111, and V [12, 131. Many of these metals are environmental pollutants and can accumulate in the human body [ 14- 161, so they may be involved in the biology of oxygen active species. Particularly, V is involved in many oxidative reactions which may account, at least in part, for its physiological or toxicological effects in vivo [12]. On the other hand, comprehensive studies of the effects of V on the activity of the main antioxidant enzymes are not so abundant. In this work we present the resuhs of a thorough kinetic study of the vanadate inhibition of HRP and the results obtained with catalase, GSH-Px and Cu-Zn SOD. MATERIALS AND METHODS Apparatus A x2 Perkin-Elmer thermostat&d spectrophotometer was used for the determination of enzyme activity, protein, and hydrogen peroxide concentration. EPR measurements were made in a Varian E-109 at X-band frequency by means of a variable temperature-control V/4000 apparatus. 48V radioactivity was measured with a Philips Automatic Gamma Counter PW4800 apparatus. Enzymes Horseradish peroxidase (EC 1.11.1.7), beef liver catalase (EC 1.11.1.6) (specific activity 260,000 U/ml), glutathione reductase (from yeast) (EC 1.6.4.2), glutathione peroxidase (EC 1.11.1.9), and Cu-Zn superoxide dismutase (EC 1.15.1.1), from bovine erythrocytes, were analytical grade and purchased from Boehringer (Mannheim, F.R.G.), and were used without further purification. Protein concentrations were determined spectrophotometrically at 403 nm (E = 89.5 mM-’ cm-‘) for HRP
VANADIUM EFFECT ON ANTIOXIDANT ENZYMES
163
[17], at 405 nm (~ = 324 mM -~ cm -~) for cataiase [18], at 280 nm (e = 63 mM -1 cm -~) for GSH-Px [19], and at 259 nm (e = 9.84 mM - l cm -~) for SOD [20]. Chemicals, Substrates, and Radiochemicals Deionized Mfllipore water (18 M t2 cm-3) and deionized water-rinsed glassware and plasticware were used in all experiments. Hydrogen peroxide was obtained from BDH Chemicals Ltd. (U.K.). It was used in diluted solutions which were prepared from a 30% stock solution immediately before use; HaO 2 concentration was determined spectropbotometrically at 240 nm (~ = 0.0394 mM -I cm - l ) [21]. Guaiacol, EDTA, NADPH, and cumene hydroperoxide were purchased from Sigma (U.S.); pyrogailol, ammonium acetate, and phosphate buffer were obtained from Merck (F.R.G.); GSH, sodium sulfite, ammonium vanadate, and vanadyl sulfate (prepared in acidic conditions or in the presence of GSH, as indicated in each case, to prevent air-oxidation), were purchased from Fluka (F.R.G.); Tris buffer was from Serva (Heidelberg, F.R.G.). Sephadex G25 was from Pharmacia (Uppsala, Sweden) and deferoxamine (Deferoxamine B mesylate, Desferal ®) was a gift from CIBAGEIGY (Basel, Switzerland). 4SV radiotracer was prepared as described elsewhere [13]. Enzymatic Assays Peroxidase activity was determined following guaiacol oxidation at 470 nm [21] with minor modifications [13] in 10 mM phosphate buffer, pH 7.0. Catalase activity was determined following the disappearance of H 2 0 2 at 240 nm [23] in 50 mM Tris, pH 7.8 or in 50 mM phosphate, pH 7.0. GSH-Px was coupled to NADPH via glutathione reductase and the rate of NADPH oxidation was measured at 340 nm [3] in 50 mM Tris buffer, pH 7.8; the hydroperoxide-independent oxidation of NADPH was substracted in every assay [3]. SOD activity (20~ + 2 H + ~ O 2 + H202) [24] was measured as inhibition of sulfite oxidation at 235 nm in 50 mM phosphate buffer, pH 7.0 [25] or as inhibition of pyrogallol autoxidation at 420 nm in 50 mM Tris buffer, pH 7.8 [26]. Sulfite was prepared under N 2 atmosphere and kept in a sealed tube with a fitted syringe, with which aliquots of the solution could be withdrawn without significant sulfite oxidation, while pyrogallol was prepared in 10 mM HC1 in order to prevent its autoxidation. All assays were performed at 25"C. Enzymatic activities were calculated from initial velocities as estimated from the slope of progress curves and were expressed as indicated for each enzyme. SOD activity was expressed as percentage inhibition of sulfite or pyrogallol oxidation. The effect of V in the above-mentioned systems was tested at the metal concentrations indicated. When necessary, metal chelators EDTA and/or DFO were used. The effect of any product(s) on SOD activity was tested by taking the rate in the presence of a given level of that product(s) as the baseline rate, and measuring the effect of SOD in terms of decrease of this rate [5]. In this way, paired-assays were always carded out so that meaningful SOD activity values could be obtained. These values were compared with SOD activities calculated from control paired-assays without the tested products. Gel-Filtration Chromatographies A possible binding of V to SOD was investigated by two different methods. For V(IV), 0.6 ml of a solution containing 0.1 mM 4SV-V(IV) (prepared in the presence of GSH) and 10 t~M SOD, or 0.6 ml of a solution containing the same components
164
M, A.
&ma et al.
plus 0.2 mM pyrogallol, always in 50 mM Tris buffer, pH 7.8, were pre-incubated for 30 mitt at 25°C and then applied to Sephadex G25 mini-columns (1 x 5 cm), previously equilibrated with 50 mM Tris buffer, and eluted with the same buffer. 4sV radioactivity and SOD concentration were measured for collected fractions and for the cohnnn gel. For V(V), ~~~ of 3.6 pM of SOD or of 4.4 FM of SOD plus 0.85 mM ammonium vanadate, both in 50 mM ammonium acetate, pH 7.0, were pre-incubated for 30 min at 37”C, and then applied to a Sephadex G25 column, previously equilibrated with 50 mM ammonium acetate, and eluted with the same buffer. The protein concentration was measured in collected fractions and the NAA of the joint protein fractions was carried ant as described elsewhere [ 121. EPR studim Samples of 0.2-0.3 ml containing V(W) or V(V) and pyrogallol were prepared, mixed, and incubated for 10 min under N, atmosphere (in order to rnini~~ 0, concentrations), then transferred to EPR-quartz tubes and froxen using liquid nitrogen. First-derivative X-band RPR spectra were recorded as indicated elsewhere [ 121. The conditions used allowed us to determine V concentrations in the order of 10 ~$4 in our samples. Statistics Student’s t test was used to compare e~y~c activities in the presence and in the absence of a given product at the appropriate significance level (p). Differences between means were considered significant for, at least, a confidence limit of 95% (p < 0.05). RESULTS Figure 1 shows the dose-response curve for the effect of increasing concentrations of vanadate on the activity of HRP; no increases in the inhibitory effect were observed when the metal was pre-incubated with the enzyme (data not shown). Figure 2 displays the double-reciprocal plot of an experiment in which the inhibitory effect of
0
0.5
nf+?l 6ml
1
PIGURJZ1. L&e-response plot of the el%ct of V(V) on the activity of HRP. Concentrations are 1 n&i guakol, 0.1 m&fH,O,, and 1.17 n&i HRP, in 10 m&fphosphatebuffer, pH 7.0.
VANADIUM EFFECT ON ABOUT
0
1
5
I
IO lI(HI%l
ENZYMES
8
I
lb
20
165
mw
FIGURE 2. Double-reciprocal plot of the V(V)-mediated inhibition of HRP activity at a fixed guaiaccl coucentration (1 rnM) and variable HzO, concentrations for different V(V) concentrations: (a) 40 &i (O), (b) 30 PM (O), (c) 20 pM (A),and(d) 0 CM (+). HRP ~~n~ti~ is 0.75 nM; assays were carried out in 10 r&f plro@a& buffer, pH 7.0, HRP activity (v) is expressed as units of specific activity and is given in nun01 ruin-’ pm01 enzyme- ‘. Straight lines were calculated using a lii regression program.
V(V) on HRP was tested with a fixed guaiacol co~~~ti~ and variable H,O, concentrations. Vanadate concentrations are in the order of magnitude of Km (H,O,), 12.6 gM [12]. The lines intercept by the Y-axis, which is indicative of a competitive inhibition pattern. The graphically estimated value of Ki [27], is found to be 41.2 FM. Table 1 shows the results of the effect of 0.1 mM V(V) on the activity of catalase in Tris and phosphate buffers. No significant differences, as compared with control assays w&ho@V(V), are found in any case. Table 2 displays the results of the effect of 0.1 mM V(W) or 0.1 mM V(V) on the GSH-Px system in the presence and in the absence of 0.1 mM EDTA. The rate of NADPH oxidation was calculated for the whole system as well as for three subsystems which may account for different partial reactions. So, more exact values
TABLE 1. VanadiumEffect on Cat&se Activity* compound 1 mM H,O, lmMH,O,+O.lmMV(V) 5 mM H,Oz 5 mA4H,Oz + 0.1 mM V(V)
TliS
Phosphate
1.72 (0.03) 1.82 (O.a2) 118 7.86 (0.12) 8.28 (0.08) ns
2.59 (0.0s) 2.52 (0.35) us 11.7’7(0.83) 11.66 (1.01) ns
* Buffers are SO mM Tris, pH 7.8 or SO mM phoqhte, pH 7.0. Cat&se is 0.04 nM. Activity is expressed in mUOD/min, and is the mean of at least three determinations. Standard &vibms are indicatedinpprenthesar.Thccomparison of the activities in the presence of V(V) with controls in its absence is also indi& ns means not signihnt.
166
M. A . Serra et al.
TABLE 2. Vanadium Effect on Glutathione Peroxidase Activity Conditions a'b A B C D A-B
Control without V EDTA No EDTA
0.1 mM V(IV) c EDTA No EDTA
0.1 mM V(V) EDTA No EDTA
11.38 (0.34) 3.45 (0.17) 0.400 (0.010) nd
12.39 (0.62) 3.76 (0.19) 0.044 (0.011) nd
7.93 (0.29)
8.63 (0.43)
13.77 (0.84) 6.88 (0.06) 2.29 (0.09) 0.065 (0.004) 6.89 ns (0.50)
11.67 (1.83) 4.90 (0.04) 0.086 (0.004) 0.037 (0.007) 6.77 ns (0.67)
18.91 (1.96) 11.00 (0.71) 6.18 (0.86) 0.133 (0.004) 7.91 ns (0.68)
15.01 (0.53) 5.68 (0.20) 0.25 (0.09) 0.044 (0.004) 9.33 ns (0.33)
aAll assays were carried out in 50 mM Tris buffer, pH 7.8, in the following conditions: D: 0.12 mM NADPH; C: D + 0.25 mM GSH and 0.2 mM cumene hydroperoxide; B: C + 0.21 /zM GSSG-R; A: B + 50 aM GSH-Px. b Values correspond to nmoles NADPH oxidized vain- 1, and are the mean of at least three determinations; nd means not detectable. Standard deviations are shown in parentheses. The comparison of the values of GSH-Px activity (A-B) in the presence of V with controls in its absence, with or without 0.1 mM EDTA, is indicated; ns means not significant. c V(IV) was prepared in 10 mM HCI to avoid air-oxidation.
o f G S H - P x activity w e r e obtained (listed under the heading A-B), w h i c h s h o w that there are not significant differences b e t w e e n V(IV) or V ( V ) samples and the control ones, either with or without E D T A . H o w e v e r , there are significant differences b e t w e e n the subsystems studied. In all cases the rates in the absence o f E D T A are higher than in its presence. Table 3 displays the results o f the effect o f 0.1 m M V ( V ) on S O D activity, tested by the sulfite method. This assay seems d e v o i d o f interferences with V(V). It can be seen that there are no significant differences b e t w e e n controls and V samples, and that oxidation rates are c o m p a r a b l e in both cases, which is also indicative o f the inexistence o f interferences. Metal chelators, E D T A and D F O , w e r e also tested but sulfite oxidation rates w e r e too l o w to estimate S O D activity correctly. F i g u r e 3 displays the d o s e - r e s p o n s e c u r v e for the effect o f V(IV) (prepared in the presence o f G S H , which does not affect the pyrogallol assay at the concentrations w e
TABLE 3. Vanadate Effect on SOD Activity a Compound Tested b
Oxidation Rate c (mUOD/min)
SO3 = SO3 = +SOD SO3 ffi +V(V) SO3 = +V(V) + SOD
6.60 (1.47) 4.29 (0.74) 6.93 (1.17) 4.42 (0.79)
SOD Activity d 34.38 (4.27) 36.27 (3.28) ns
a Assays were carded out in 50 mM phosphate buffer, pH 7.0. b Concentrations are: 1 mM SO3 = , 0.29/zM SOD, and 0.1 mM V(V). c Oxidation rate expresses SO3ffi oxidation under different experimental conditions and are the mean of at least four determinations. Standard errors arc shown in parentheses. d SOD activity is expressed as percentage inhibition of SO3= oxidation. Standard errors are indicated in parentheses. The comparison of the activity in the presence of V(V) with controls in its absence is also indicated; ns means not significant.
VANADIUM EFFECT ON ANTIOXIDANT ENZYMES
0:
I
I
I
0.2
0.3
0.4
I
0.1
167
Vanadium (mM)
FIGURE 3. Dose-response plot of the effect of V(V) (0) or V(IV) (0) on SOD activity. Concentrations are 0.2 mM pyrogallol, 0.1 pM SOD for V(V), and 0.2 PM SOD for V(IV) in 50 mM Tris buffer, pH 7.8. V(IV) was prepared iu the presence of GSH to prevent air-oxidation.
employ [28]) or V(V) on the activity of SOD in a wide range of V concentrations.
An apparent decrease of SOD activity was observed when V concentrations increase, which is more pronounced for V(V) than for V(W). The addition of metal chelators, EDTA and DFO, partially reverts this effect (data not shown). On the other hand, pyrogallol autoxidation rates in the presence of V are higher than in its absence (data not shown), indicating that the oxidative process is accelerated by the metal. Figure 4 displays the EPR spectra of mixtures containing 0.2 mM pyrogallol plus 0.1 mM V(IV) or 0.1 mM V(V), under N, atmosphere. Similar EPR patterns are observed for both samples; as V(V) is EPR-silent the appearance of a signal indicates
I
2.6
I
I.
2.8
I.,,
3.0
I.
3.2
3.4
Magnetic
FIGURE 4.
t,,.n.:.,.,.
1.
3.8
2.6 Field
2.8
3.0
3.2
3.4
3.6
(KGaur)
X-band EPR spectra of samples of 0.2 mM pyrogallol with (A) 0.1 mM V(V) or (B) 0.1 mM V(IV) (without GSH) that was prepared, mixed, and kept under N, atmosphere in 50 mM Tris buffer, pH 7.8. Recording conditions were: time constant, 0.064 s; modulation amplitude, 8 G; modulation frequency, 100 kHz; microwave power, 5 mW; microwave frequency, 9.000 GHz; receiver gain 2.5;103 (A), or 1.6i103 (B). Sainples were kept and recorded at - 150°C.
19
M. A. Serra et al.
that V(IV) is the oxidation form present in reaction mixtures owing to V(V) reduction by pyrogallol. SOD itself does not alter this process (data not shown). Table 4 displays the results of the NAA of the joint protein fraction of a sample of SOD and of a mixture of SOD + V(V) after gel filtration; there does not appear to be a significant amount of V in the protein fraction, so it can be concluded that there is no binding of V(V) to SOD under the studied conditions. In addition, the metal constituents of the protein, Cu and Zn, were not affected by the presence of V. Figure 5 shows the results of the gel filtration experiments of mixtures of SOD + 48V-V(IV) and of SOD +48V-V(IV) + pyrogallol in Tris buffer; no significant binding (V/SOD ratio 0.17:1) of V(IV) to the enzyme was demonstrated and the presence of pyrogallol did not significantly alter this behavior. It can be then concluded that neither V(IV) nor V(V) are bound to SOD in the studied conditions. Besides, there were no significant changes in the UV spectra of SOD samples when recorded in the presence of V (data not shown). DISCUSSION EffectofVonHRP The results presented here confirm the vanadate inhibitory effect of HRP activity found elsewhere [ 131. There is a competitive inhibition pattern, with Km (H,O,) and Ki (V(V)) of the same order of magnitude, 12.6 FM and 41.2 PM, respectively. However, the exact inhibitory mechanism is difficult to establish. An interference in the formation of Compound I (HRP + H,O, -+Compound I) may be possible. Vanadium binds to HRP in stoichiometric amounts and this binding is favored in the presence of H,O, [ 131, perhaps because the binding of the latter to the enzyme can make some hydrophilic groups more’ available for V binding to the enzyme. The metal should bind then to a site close to or overlapping with the H,O, binding site, being just a partiahy competitive inhibition. On the other hand the functional inhibitory species bound could be some type of peroxyvanadium complex, which have been postulated to exist also at neutral pH values [29]. Effect of V on Catalase There is not a V(V)-mediated inhibition of catalase activity, neither in Tris nor in phosphate buffers. Although V-peroxy complexes can also be formed in these conditions [29], they do not seem to be rate-limiting as no significant inhibition is TABLE
4. Neutron Activation Analysis of SOD” Metal
Cu Zn V Mn AU As
Amount found by NAA (g atom/mole enzyme, MW = 32600) No pre-incubation with V 2.11 2.02 0.004 0.006 0.006
After prc-incubation with V 2.06 2.04 0.06 0.10 -
’ NM of the joint protein fraction obtained after applying samples of SOD with (4.42 PM) or without (3.6 CM) pre-incubation(for 30 min) with 0.85 mM V(V) to Sephadex G25 columns, using 50 mM ammoniumacetate buffer, pH 7.0, at 37% For more details, see Materials and Mahods.
VANADIUM EFFECT ON ANTIOXIDANT ENZYMES
169
FIGURE 5. SephadexG25 gel titration chromatography of 33 PM SOD incubated with 0.1 mM “V-V(W) in the absence (0) and in the presence (A) of 0.2 mM pyrogallol in 50 m&l Tris buffer, pH 7.8. Proteii (----)w~~~at~S~ei~rin~e~~r(O)~in~ latter (A) conditions. The total remvery of ABVwas 82.4% and 86.796, respectively.
obtained, in contrast with the case of HRP. That result is not in agreement with a vanadate inhibition of cat&se described elsewhere [30]. However, they use a decavanadate solution that is slowly transformed into a orthovanadate/metavanadate one [31], while in our case a stable ova solution was employed. On the other hand, they carry out catalase activity measurements at a somehow elevated hydrogen peroxide concentration (15 n&f), as compared to what has been recommended (10 n&i) in order to avoid apparent higher initial rates [23] that can lead to an over-estimation of catalase activity. Effect of V on GSH-Px Vanadium effect in the GSH-Px system merits a detailed analysis. Owing to known reactions of the metal with some components of the system, we calcnlated the NADPH oxidation rates of different subsystems. In the following scheme we represent the whole system, which indicates the overall reaction: reaction (1) is catalyzed by GSH-Px and reaction (2) is catalyzed by GSSG-R:
Vanadium does not inhibit GSH-Px directly, as shown by the detailed analysis of the four subsystems studied (Table 2):
170
M. A. Serra et aL
i) Subsystem D. VOV) seems capable of oxidizing NADPH at an appreciable rate (0.133) more than V(V) (0.044), as compared to the control, probably through the production of superoxide radical by means of V(IV)-air-oxidation and subsequent peroxovanadyl oxidation of NADPH [12, 32]. This process is mediated by the O~radical and should be inhibited by SOD, as it readily happens (90% inhibition of NADPH oxidation rates when 0.79/zM SOD was added).
ii) Subsystem C. When cumene hydroperoxide and GSH are present, NADPH oxidation increases markedly as compared to the control; V(V) can be reduced to V(IV) by GSH [32, 33], which should favor the above-explained reactions. However, the final NADPH oxidation rate will be smaller than for the V(IV) subsystem C, which produces the O~- radical promptly and not by a two-step process, as in the V(V) subsystem C. There can also be considered two other reactions which can account for increased NADPH oxidation rates in addition to the peroxovanadyl pathway. First, the superoxide radical generated can oxidize GSH to GSSG, being regenerated at the end [34]; and second, it can also react with the hydroperoxide producing an alkoxyl radical ( R O . ) capable of initiating a chain of peroxidative reactions [12, 35]. For this subsystem also, there was a significant (around 80%) inhibition of NADPH oxidation rates when 0.79/zM SOD was added, confirming the involvement of the superoxide radical. iii) Subsystem B. When GSSG-R is added the high amounts already available of its specific substrate, GSSG, generated by the above-mentioned reactions (GSH oxidation by O2- and by alkoxyl radical-initiated peroxidative reactions), will accelerate further NADPH oxidation, more for the V(IV) than for the V(V) subsystem and for this than the control. A direct V(V) oxidation of NADPH, catalyzed by GSSG-R, has recently been described [36], and could also account for increased NADPH oxidation rates for the V(V) subsystem B. iv) Subsystem A. The addition of GSH-Px, completing the whole system, provides a faster NADPH oxidation by accelerating the curnene hydroperoxide reduction by GSH, either for V(IV) or V(V) or the control. When the effect of V in the above-explained subsystems is substracted from the whole system (A-B), we are trying to obtain a more exact determination of the metal's effect on GSX-Px itself; the comparison of the V(IV) or V(V) (A-B)values with the control ones indicates that the differences are not significant in any case, meaning that neither V(IV) nor V(V) are directly affecting GSH-Px activity. The role of the metal in the different oxidative reactions of the studied system is also evidenced by the fact that addition of EDTA always decrease NADPH oxidation rates [31], our output for all these oxidative processes. On the other hand, in vivo experiments show contradictory results [37, 38], which is not surprising due to the complexity of the system. Effect of V on SOD V(V) does not affect SOD activity when assayed with the sulfite method (Table 3), which seems devoid of interferences, so it is likely that vanadate is not a true inhibitor of the enzyme. On the other hand, there was an apparent dose-response inhibition of SOD activity for either V(IV) or V(V) using the pyrogallol assay (Fig. 3). In this case, however, it was due to an interference of the superoxide radical source with the metal: pyrogallol can reduce V(V) to V(IV) and complex it [39-41]; that reduction has been confirmed by our EPR spectra of pyrogaUol with V (Fig. 4). This could explain why SOD activity is more decreased for V(V) than for V(IV):
VANADIUM EFFECT ON ANTIOXIDANT ENZYMES
171
pyrogahol is oxidized not only via Oi- radicals but also by the metal, a non-superoxide radical-mediated mechanism which is obviously not affected by SOD. This means that there is not a true V-mediated inhibition of SOD but just a less responsive assay, confirming what the employment of the sulfite method shows readily. However, we think that it is interesting to point out this potentially misleading information using the pyrogahol method with a very common SOD assay also used in V studies [42]; perhaps it is not the most appropriate one for testing this metal. Moreover, we have not found a binding of V(IV) (Fig. 4) nor V(V) (Table 3) to SOD, even using somewhat high (0.1 mM-0.85 mM) V concentrations in order to emphasize any eventual V-protein complex that could be formed. A possible replacement of the protein metal constituents, Cu and Zn, by V can also be discarded because their stoichiometric amounts are maintained unchanged in spite of SOD pre-incubation with V, so the metal moieties of the enzyme seem not to be affected by V. Caution should be taken with apparent SOD inhibitors [5, 461, particularly when looking for a possible transition metal-mediated inhibition of SOD, because redox reactive metals can catalyse the autoxidation of indicating scavengers by inner sphere electron transfer, a mechanism that does not involve oxygen free radicals and is not inhibitable by SOD, rendering the assay less responsive to the enzyme and giving the false appearance of a metal-mediated inhibition. This is the case, for instance, for 6-hydroxydopamine [47] and for pyrogallol [26], as we describe in this work. However, any metal ligand, such as typical metal chelators (i.e., EDTA and DFO) or proteins (including SOD) should diminish the ability of metal ions to exchange electrons by an inner sphere mechanism, rendering autoxidation mainly dependent again on propagation by Oi- radicals and therefore inhibitable by SOD. This is the reason why addition of EDTA and DFO increased apparent SOD activities, also in our experiments (data not shown). Other authors testing transition metal ions as possible SOD inhibitors are warned against this potentially misleading information, as well as against interferences based on interactions of metal ions with reaction components of the SOD assay used. This could be the case of two recent works. A vanadate-mediated inhibition of SOD activity at physiological pH employing the xanthine/xanthine oxidase-nitroblue tetrazolium SOD assay has been published [43]. However, it has been shown that V competitively inhibits xanthine oxidase (Ki = 0.17 mM) at pH 7.4-7.6 [44, 451, which suggests that the xanthine oxidase-mediated production of superoxide radicals could be decreased by its vanadate-mediated inhibition more than by a direct effect of V on SOD, which would only find less Oi- radicals. On the other hand, an apparent vanadate-mediated inhibition of SOD using the 6-hydroxydopamine method [48] has not taken into account that cathecols and cathecolamines are metal reductants and chelators, also of V [39-41, 49, 501. Besides, their application of the 6-hydroxydopamine assay [51] is not fully convincing, as has been discussed elsewhere [ 111. In both cases we may have less responsive SOD assays instead of true V-mediated SOD inhibitions. It should therefore be advisable to test any supposed SOD inhibitor by, at least, two independent and interference-free methods, because a true inhibitor should be effective regardless of the SOD assay employed. Physiological Implications In conclusion we have described a direct inhibition of V(V) of an oxidoreductase, HRP, possibly by complex formation of V with hydrogen peroxide. On the other
172
M. A. Serra et al.
hand, the lack of V inhibition of the three main antioxidant enzymes (catalase, GSH-Px, and SOD) and the redox reactions in which V seems to be involved in some of these enzymatic systems indicates that many biological and toxicological effects of the metal, particularly V(V), may be mediated by oxidative reactions of V or V complexes more than by a direct effect on the enzyme itself [33, 52-551. All these findings, however, do not unequivocally ascertain a clear biological role for V, which needs further investigation. NOTE ADDED IN PROOF While this work was in press, an article by Wittenkeller et al. (J. Am. Chern. Sot. 113:7872, (1991)) was published reporting a significant and specific strong binding of V(V) to Cu-Zn SOD (8 atoms of V per molecule of enzyme) using “V NMR spectroscopy, in contrast with the absence of binding we show in Table 4. We have carried out exhaustive dialysis of 2 mM ammonium vanadate incubated in the presence of increasing SOD concentrations either in their (Hepes) or our (acetate) conditions, as well as dialysis of 0.1 mM SOD incubated with or without 2 mM ammonium vanadate against 2 mM ammonium vanadate in Hepes or acetate buffers. GFAAS analysis of all samples confirmed that there is no significant binding of V(V) to SOD in the former experiment and a small (2 g at of V/mol of SOD) but statistically significant binding in the latter one, which is, however, reverted by simple dialysis, an indication that it is a labile binding. Moreover, SOD activity in dialysis samples remains unaffected. Also, we have checked the purity of our SOD preparation by SDS-PAGE, which showed a single band, discarding the presence of contaminating proteins. The discrepancy between our results and the ones reported by Wittenkeller et al. could arise from the different source of the enzyme, erythrocytes, and liver, respectively. However, these authors agree with us in that the catalytically important Cu@) is unaffected by V(V) and also cannot demonstrate a V(V)-mediated inhibition of SOD activity. In any case, such a binding should be negligible in physiological conditions where V concentrations are too low to produce enough putative binding species and where V(V) is reduced to V(IV) inside cells. The authors would like to thank M. Mane0 and F. J. Ruiz for their technical asstitance.
REFERENCES 1. I. Fridovich. in Developments in cardiovascular medicine: oxygen radicals in the pathophysiology of heart disease, P. K. Singal, Ed., Kluwer Academic Publishers, Boston, 1988, Chap. 1, pp. l-11. 2. B. Halliwell and J. M. C. Gutteridge, Arch. B&hem. Biophys. 246, 501 (1986). 3. L. Fl& and W. A. Gbuler, in Methods in Ewmology, L. Packer, Ed., Academic Press, New York, 1984, Vol. 105, Chap. 12, pp. 114-121. 4. L. FlohC and F. 0ting, in Methods in Enzymology, L. Packer, Ed., Academic Press, New York, 1984, Vol. 105, Chap. 10, pp. 93-104. 5. H. M. Hassan, H. Dougherty, and I. Fridovich, Arch. B&hem. Biophys. 199,349 (1980). 6. W. F. Beyer and I. Fridovich, Anal. Biochem. 161,559 (1987). 7. S. E. Fridovich and N. A. Porter, J. Biol. Chem. 256, 260 (1981).
V A N A D I U M E F F E C T ON A N T I O X I D A N T E N Z Y M E S
173
8. J. M. C. Gutteridge, G. J. Quinlan, I. Clark, and B. Halliwell, Biochim. Biophys. Acta 835, 441 (1984). 9. G. L Quinhm, B. HalliweU, C. P. Moorhouse, and J. M. C. Gutteridge, Biochim. Biophys. Acta 962, 196 (1988). 10. M. Ohtawa, M. Seko, and F. Takayama, Chem. Pharm. Bull. 31, 1415 (1983). 11. M. A. Serra, V. Barassi, C. Canavese, and E. Sabbioni, Biol. Trace El. Res. 31, 79 (1991). 12. S. I. Liochev and I. Fridovich, Arch. Biochem. Biophys. 279, 1 (1990). 13. M. A. Serra, E. Sabbioni, A. Marchesini, A. Pintar, and M. Valoti, Biol. Trace El. Res. 23, 151 (1990). 14. A. C. Alfrey, Adv. Clin. Chem. 23, 69 (1983). 15. K. Tsuchiya, in Handbook on the toxicology of metals L. Friberg, G. F. Nordberg, and V. Vouk, Eds., Elsevier, Amsterdam, 1986, Vol. IT, Chap. 14, pp. 298-353. 16. B. Lagerkvist, G. F. Nordberg, and V. Vouk, in Handbook on the toxicology o f metals, L. Friberg, G. F. Nordberg, and V. Vouk, Eds., 1986, Vol. II, Chap. 27, pp. 638-663. 17. A. C. Maehly, in Methods in Enzymology, S. P. Colowick and N. O. Kaplan, Eds., Academic Press, New York, 1955, Vol. H, Chap. 143, pp. 801-813. 18. T. Samejima and J. T. Yang, J. Biol. Chem. 238, 3256 (1963). 19. L. Floh~, B. Eisele, and A. Wendel, Hoppe-Seyler's Z. Physiol. Chem. 352, 151 (1971). 20. U. Weser, G. Barth, C. Djerassi, H. J. Hartmarm, P. Krauss, G. Voelcker, W. Voelter, and W. Voetsch, Biochim. Biophys. Acta 278, 28 (1972). 21. D. P. Nelson and L. A. Kiesow, Anal. Biochem. 49, 474 (1972). 22. B. Chance and A. C. Maehly, in Methods in Enzymology, S. P. Colowick and N. O. Kaplan, Eds., Academic Press, New York, 1955, Vol. II, Chap. 136, pp. 764-775. 23. H. Aebi, in Methods in Enzymology, L. Packer, Ed., Academic Press, New York, 1984, Vol. 105, Chap. 13, pp. 121-126. 24. J. M. McCord and I. Fridovich, J. Biol. Chem. 244, 6049 (1969). 25. J. M. McCord and I. Fridovich, J. Biol. Chem. 244, 6056 (1969). 26. S. Marklund and G. Marklund, Eur. J. Biochem. 47, 469 (1974). 27. M. Dixon and E. C. Wehh, Enzymes, Longman, London, 1979, pp. 354-355. 28. E. F. Roth and H. S. Gilbert, Anal. Biochem. 137, 50 (1984). 29. O. W. Howarth and J. R. Hunt, J. Chem. Soc. Dalton, Trans., 1388, (1979). 30. M. Ran, M. ,S. Patole, C. Vijaya, C. K. Ramakrishna Kurup, and T. Ramasarma, Mol. Cell. Biochem. 75, 151 (1987). 31. D. Dart and I. Fridovich, Arch. Biochem. Biophys. 243, 220 (1985). 32. P. J. Stankiewicz, A. Stem, and A. J. Davison, Arch. Biochem. Biophys. 287, 8 (1991). 33. I. G. Macara, K. Kustin, and L. C. Cantley, Biochim. Biophys. Acta 629, 95 (1980). 34. R. Munday and C. C. Winterbourn, Biochem. Pharmacol. 38, 4349 (1989). 35. M. W. Sutherland and J. M. Gebicki, Arch. Biochem. Biophys. 214, 1 (1990). 36. X. Shi and N. S. Dalai, Arch. Biochem. Biophys. 275, 288 (1990). 37. S. Kacew, M. R. Parulekar, and Z. Meraii, Toxicol. Lett. 11, 119 (1982). 38. M. D. Cohen and C. I. Wei, J. Leukocyte Biol. 44, 122 (1988). 39. S. Ya Shnalderman, G. N. Prokof'eva, A. N. Demidovskaya, and E. P. Klimenko, Zh. Obshch. Khim. 42, 24 (1972). 40. J. Zelinka, M. Bartu~ek, and A. Ok~, Collection Czechoslov. Chem. Commun. 39, 83 (1974). 41. S. Ya Shnaiderman, E. P. Klimenko, and A. N. Demidovskaya, Ukr. Khim. Zh. 36, 8 (1970). 42. M. Elfant and C. L. Keen, Biol. Trace El. Res. 14, 193 (1982). 43. M. C. ApeUa, S. N. Gonz~lez, and E. J. Baran, Biol. Trace El. Res. 18, 123 (1988).
174
M. A . Serra et al.
C. S. Ramadoss, Z. Naturforsch. 35e, 702 (1980). A. M. Ghe, C. Stefanelli, and D. C. Carati, Talanta 31, 241 (1984). G. E. Eldred and J. R. Hoffert, Anal. Biochem. 110, 137 (1981). B. Bandy and A. J. Davison, Arch. Biochem. Biophys. 259, 305 (1987). R. Shainkin-Kestenbaum, C. Caruso, and G. M. Bedyne, Trace El. Med. 8, 6 (1991). R. K. Bogges and R. B. Martin, J. Am. Chem. Soc. 97, 3076 (1975). E. Mentasti, E. Pelizzetti, and C. Baioicchi, J. Inorg. Nucl. Chem. 38, 2017 (1976). R. E. Heikkila and F. Cabbat, Anal. Biochem. 75, 356 (1976). N. D. Chasteen, Struct. Bond. 53, 107 (1983). J. E. Benabe, L. A. Echegoyen, B. Pastrana, and M. MartJnez-Maldonado, J. Biol. Chem. 262, 9555 (1987). 54. J. A. Bonadies and C. J. Carrano, J. Am. Chem. Soc. 108, 4088 (1986). 55. H. V. Strout, P. P. Vicario, R. Saperstein, and E. E. Slater, Endocrinology 124, 1918 (1989). 44. 45. 46. 47. 48. 49. 50. 51. 52. 53.
Received September 4, 1991; accepted November 3, 1991
MAS, ES.
ABSTRACT The eifect of vanadium (V) on the activity of horseradish peroxidase, catalase, glutathioue peroxidase, and superoxide dismutase bas been studied. A competitive inhibition pattern was evident for vanadate ions on the activity of horseradish peroxidase (Ki = 41.2 PM). No significant inhibitory effects were found when V(V) was tested with cat&se and when either V(W) or V(v) were assayed with glutathione peroxidase. For the latter, the effect of V on tbe different components of the reaction system was investigated. V(V) did not significantly affect SOD activity when assayed with the sulfite method, which is devoid of interferences with V(V); however, there was an apparent ~bito~ dose-response pattern for either V(IV) or V(V) using the pyrogallol assay, owing to an interference of pyrogallol with tbe metal. Besides, no significant binding of V(W) or V(V) to the enzyme could be demonstrated. The lack of a direct ~bito~ effect of V on the activity of the main ~tio~ enzymes suggests that many biological and toxicological effects of V may be medii more by oxidative reactions of the metal or of its complexes with physiologically relevant biomoiecules than by a direct modulation of enzymatic activities. ABBREVIATIONS XRP, horseradish peroxiW, GSH-Px, gh~tathioneperoxidase, GSSG-R, glutathioue reductase; SOD, Cu-Zn superoxide dismutase; DFO, deferoxamine; guaiacol, methylcatbecoI; pyrogallol, 1,2,3-t&y-
Address reprint requests and correspondence to: Dr. Enrico Sabbioni, Commission of the European Cafe, Joint Research Centre (Ispra Site), Env~~t Institute, Life Scii Unit, T.P. 460, I-21020 ISPRA (Varese), Italy. *Present address: Bracco Industria chimica S.P.A., Via E. Folli, SO, I-20133 Miiano, Italy. JournalofInorganic B&&em&y, 46, 161- 174 (1992) 161 @ 1992 Elswier Science Publishing Co., Inc., 655 Avenueof the Americas.NY, NY 10010 0162-0134/92/S5.00
162
M. A. Serra et al.
droxybemene; radical.
UOD, units of optical density; NAA, neutron activation analysis; OF, superoxide
INTRODUCTION The study of the enzymes dealing with the elimination of oxygen active species (antioxidant enzymes) is of considerable interest because they constitute an essential defense against the well-demonstrated toxicity of those compounds [ 11. Oxy-radicals and other oxygen derived species have been proposed to be involved in several human diseases, including cancer, multiple sclerosis, Parkinson’s disease, autoimmune disease, and senile dementia, although their roles as causative agents are not clear [2]. Undoubtedly, the finding of specific inhibitors of antioxidant enzymes is of great relevance in understanding the physiological consequences of their deficit. However, not all these enzymes are easy to assay; while peroxidases and catalases can always be tested directly, others such as glutathione peroxidases and superoxide dismutases are frequently tested by coupled or indirect assays, making their study considerably more complicated [3, 41. Badly performed assays or absence of the adequate controls when testing possible inhibitors have led many authors to mistaken or confusing conclusions, as has been shown elsewhere [3-61. Moreover, the research on the role of trace metals in biological oxidations (oxy-radicals and lipid peroxidation) and hence the use of metal chelators to ascertain and/or prevent their effects, is a field of growing interest [2]. Several studies have been published on how these processes can be affected by metal ions such as Fe [‘?I, Fe + Al [8], Fe + Al + Pb [9], Al [lo, 111, and V [12, 131. Many of these metals are environmental pollutants and can accumulate in the human body [ 14- 161, so they may be involved in the biology of oxygen active species. Particularly, V is involved in many oxidative reactions which may account, at least in part, for its physiological or toxicological effects in vivo [12]. On the other hand, comprehensive studies of the effects of V on the activity of the main antioxidant enzymes are not so abundant. In this work we present the resuhs of a thorough kinetic study of the vanadate inhibition of HRP and the results obtained with catalase, GSH-Px and Cu-Zn SOD. MATERIALS AND METHODS Apparatus A x2 Perkin-Elmer thermostat&d spectrophotometer was used for the determination of enzyme activity, protein, and hydrogen peroxide concentration. EPR measurements were made in a Varian E-109 at X-band frequency by means of a variable temperature-control V/4000 apparatus. 48V radioactivity was measured with a Philips Automatic Gamma Counter PW4800 apparatus. Enzymes Horseradish peroxidase (EC 1.11.1.7), beef liver catalase (EC 1.11.1.6) (specific activity 260,000 U/ml), glutathione reductase (from yeast) (EC 1.6.4.2), glutathione peroxidase (EC 1.11.1.9), and Cu-Zn superoxide dismutase (EC 1.15.1.1), from bovine erythrocytes, were analytical grade and purchased from Boehringer (Mannheim, F.R.G.), and were used without further purification. Protein concentrations were determined spectrophotometrically at 403 nm (E = 89.5 mM-’ cm-‘) for HRP
VANADIUM EFFECT ON ANTIOXIDANT ENZYMES
163
[17], at 405 nm (~ = 324 mM -~ cm -~) for cataiase [18], at 280 nm (e = 63 mM -1 cm -~) for GSH-Px [19], and at 259 nm (e = 9.84 mM - l cm -~) for SOD [20]. Chemicals, Substrates, and Radiochemicals Deionized Mfllipore water (18 M t2 cm-3) and deionized water-rinsed glassware and plasticware were used in all experiments. Hydrogen peroxide was obtained from BDH Chemicals Ltd. (U.K.). It was used in diluted solutions which were prepared from a 30% stock solution immediately before use; HaO 2 concentration was determined spectropbotometrically at 240 nm (~ = 0.0394 mM -I cm - l ) [21]. Guaiacol, EDTA, NADPH, and cumene hydroperoxide were purchased from Sigma (U.S.); pyrogailol, ammonium acetate, and phosphate buffer were obtained from Merck (F.R.G.); GSH, sodium sulfite, ammonium vanadate, and vanadyl sulfate (prepared in acidic conditions or in the presence of GSH, as indicated in each case, to prevent air-oxidation), were purchased from Fluka (F.R.G.); Tris buffer was from Serva (Heidelberg, F.R.G.). Sephadex G25 was from Pharmacia (Uppsala, Sweden) and deferoxamine (Deferoxamine B mesylate, Desferal ®) was a gift from CIBAGEIGY (Basel, Switzerland). 4SV radiotracer was prepared as described elsewhere [13]. Enzymatic Assays Peroxidase activity was determined following guaiacol oxidation at 470 nm [21] with minor modifications [13] in 10 mM phosphate buffer, pH 7.0. Catalase activity was determined following the disappearance of H 2 0 2 at 240 nm [23] in 50 mM Tris, pH 7.8 or in 50 mM phosphate, pH 7.0. GSH-Px was coupled to NADPH via glutathione reductase and the rate of NADPH oxidation was measured at 340 nm [3] in 50 mM Tris buffer, pH 7.8; the hydroperoxide-independent oxidation of NADPH was substracted in every assay [3]. SOD activity (20~ + 2 H + ~ O 2 + H202) [24] was measured as inhibition of sulfite oxidation at 235 nm in 50 mM phosphate buffer, pH 7.0 [25] or as inhibition of pyrogallol autoxidation at 420 nm in 50 mM Tris buffer, pH 7.8 [26]. Sulfite was prepared under N 2 atmosphere and kept in a sealed tube with a fitted syringe, with which aliquots of the solution could be withdrawn without significant sulfite oxidation, while pyrogallol was prepared in 10 mM HC1 in order to prevent its autoxidation. All assays were performed at 25"C. Enzymatic activities were calculated from initial velocities as estimated from the slope of progress curves and were expressed as indicated for each enzyme. SOD activity was expressed as percentage inhibition of sulfite or pyrogallol oxidation. The effect of V in the above-mentioned systems was tested at the metal concentrations indicated. When necessary, metal chelators EDTA and/or DFO were used. The effect of any product(s) on SOD activity was tested by taking the rate in the presence of a given level of that product(s) as the baseline rate, and measuring the effect of SOD in terms of decrease of this rate [5]. In this way, paired-assays were always carded out so that meaningful SOD activity values could be obtained. These values were compared with SOD activities calculated from control paired-assays without the tested products. Gel-Filtration Chromatographies A possible binding of V to SOD was investigated by two different methods. For V(IV), 0.6 ml of a solution containing 0.1 mM 4SV-V(IV) (prepared in the presence of GSH) and 10 t~M SOD, or 0.6 ml of a solution containing the same components
164
M, A.
&ma et al.
plus 0.2 mM pyrogallol, always in 50 mM Tris buffer, pH 7.8, were pre-incubated for 30 mitt at 25°C and then applied to Sephadex G25 mini-columns (1 x 5 cm), previously equilibrated with 50 mM Tris buffer, and eluted with the same buffer. 4sV radioactivity and SOD concentration were measured for collected fractions and for the cohnnn gel. For V(V), ~~~ of 3.6 pM of SOD or of 4.4 FM of SOD plus 0.85 mM ammonium vanadate, both in 50 mM ammonium acetate, pH 7.0, were pre-incubated for 30 min at 37”C, and then applied to a Sephadex G25 column, previously equilibrated with 50 mM ammonium acetate, and eluted with the same buffer. The protein concentration was measured in collected fractions and the NAA of the joint protein fractions was carried ant as described elsewhere [ 121. EPR studim Samples of 0.2-0.3 ml containing V(W) or V(V) and pyrogallol were prepared, mixed, and incubated for 10 min under N, atmosphere (in order to rnini~~ 0, concentrations), then transferred to EPR-quartz tubes and froxen using liquid nitrogen. First-derivative X-band RPR spectra were recorded as indicated elsewhere [ 121. The conditions used allowed us to determine V concentrations in the order of 10 ~$4 in our samples. Statistics Student’s t test was used to compare e~y~c activities in the presence and in the absence of a given product at the appropriate significance level (p). Differences between means were considered significant for, at least, a confidence limit of 95% (p < 0.05). RESULTS Figure 1 shows the dose-response curve for the effect of increasing concentrations of vanadate on the activity of HRP; no increases in the inhibitory effect were observed when the metal was pre-incubated with the enzyme (data not shown). Figure 2 displays the double-reciprocal plot of an experiment in which the inhibitory effect of
0
0.5
nf+?l 6ml
1
PIGURJZ1. L&e-response plot of the el%ct of V(V) on the activity of HRP. Concentrations are 1 n&i guakol, 0.1 m&fH,O,, and 1.17 n&i HRP, in 10 m&fphosphatebuffer, pH 7.0.
VANADIUM EFFECT ON ABOUT
0
1
5
I
IO lI(HI%l
ENZYMES
8
I
lb
20
165
mw
FIGURE 2. Double-reciprocal plot of the V(V)-mediated inhibition of HRP activity at a fixed guaiaccl coucentration (1 rnM) and variable HzO, concentrations for different V(V) concentrations: (a) 40 &i (O), (b) 30 PM (O), (c) 20 pM (A),and(d) 0 CM (+). HRP ~~n~ti~ is 0.75 nM; assays were carried out in 10 r&f plro@a& buffer, pH 7.0, HRP activity (v) is expressed as units of specific activity and is given in nun01 ruin-’ pm01 enzyme- ‘. Straight lines were calculated using a lii regression program.
V(V) on HRP was tested with a fixed guaiacol co~~~ti~ and variable H,O, concentrations. Vanadate concentrations are in the order of magnitude of Km (H,O,), 12.6 gM [12]. The lines intercept by the Y-axis, which is indicative of a competitive inhibition pattern. The graphically estimated value of Ki [27], is found to be 41.2 FM. Table 1 shows the results of the effect of 0.1 mM V(V) on the activity of catalase in Tris and phosphate buffers. No significant differences, as compared with control assays w&ho@V(V), are found in any case. Table 2 displays the results of the effect of 0.1 mM V(W) or 0.1 mM V(V) on the GSH-Px system in the presence and in the absence of 0.1 mM EDTA. The rate of NADPH oxidation was calculated for the whole system as well as for three subsystems which may account for different partial reactions. So, more exact values
TABLE 1. VanadiumEffect on Cat&se Activity* compound 1 mM H,O, lmMH,O,+O.lmMV(V) 5 mM H,Oz 5 mA4H,Oz + 0.1 mM V(V)
TliS
Phosphate
1.72 (0.03) 1.82 (O.a2) 118 7.86 (0.12) 8.28 (0.08) ns
2.59 (0.0s) 2.52 (0.35) us 11.7’7(0.83) 11.66 (1.01) ns
* Buffers are SO mM Tris, pH 7.8 or SO mM phoqhte, pH 7.0. Cat&se is 0.04 nM. Activity is expressed in mUOD/min, and is the mean of at least three determinations. Standard &vibms are indicatedinpprenthesar.Thccomparison of the activities in the presence of V(V) with controls in its absence is also indi& ns means not signihnt.
166
M. A . Serra et al.
TABLE 2. Vanadium Effect on Glutathione Peroxidase Activity Conditions a'b A B C D A-B
Control without V EDTA No EDTA
0.1 mM V(IV) c EDTA No EDTA
0.1 mM V(V) EDTA No EDTA
11.38 (0.34) 3.45 (0.17) 0.400 (0.010) nd
12.39 (0.62) 3.76 (0.19) 0.044 (0.011) nd
7.93 (0.29)
8.63 (0.43)
13.77 (0.84) 6.88 (0.06) 2.29 (0.09) 0.065 (0.004) 6.89 ns (0.50)
11.67 (1.83) 4.90 (0.04) 0.086 (0.004) 0.037 (0.007) 6.77 ns (0.67)
18.91 (1.96) 11.00 (0.71) 6.18 (0.86) 0.133 (0.004) 7.91 ns (0.68)
15.01 (0.53) 5.68 (0.20) 0.25 (0.09) 0.044 (0.004) 9.33 ns (0.33)
aAll assays were carried out in 50 mM Tris buffer, pH 7.8, in the following conditions: D: 0.12 mM NADPH; C: D + 0.25 mM GSH and 0.2 mM cumene hydroperoxide; B: C + 0.21 /zM GSSG-R; A: B + 50 aM GSH-Px. b Values correspond to nmoles NADPH oxidized vain- 1, and are the mean of at least three determinations; nd means not detectable. Standard deviations are shown in parentheses. The comparison of the values of GSH-Px activity (A-B) in the presence of V with controls in its absence, with or without 0.1 mM EDTA, is indicated; ns means not significant. c V(IV) was prepared in 10 mM HCI to avoid air-oxidation.
o f G S H - P x activity w e r e obtained (listed under the heading A-B), w h i c h s h o w that there are not significant differences b e t w e e n V(IV) or V ( V ) samples and the control ones, either with or without E D T A . H o w e v e r , there are significant differences b e t w e e n the subsystems studied. In all cases the rates in the absence o f E D T A are higher than in its presence. Table 3 displays the results o f the effect o f 0.1 m M V ( V ) on S O D activity, tested by the sulfite method. This assay seems d e v o i d o f interferences with V(V). It can be seen that there are no significant differences b e t w e e n controls and V samples, and that oxidation rates are c o m p a r a b l e in both cases, which is also indicative o f the inexistence o f interferences. Metal chelators, E D T A and D F O , w e r e also tested but sulfite oxidation rates w e r e too l o w to estimate S O D activity correctly. F i g u r e 3 displays the d o s e - r e s p o n s e c u r v e for the effect o f V(IV) (prepared in the presence o f G S H , which does not affect the pyrogallol assay at the concentrations w e
TABLE 3. Vanadate Effect on SOD Activity a Compound Tested b
Oxidation Rate c (mUOD/min)
SO3 = SO3 = +SOD SO3 ffi +V(V) SO3 = +V(V) + SOD
6.60 (1.47) 4.29 (0.74) 6.93 (1.17) 4.42 (0.79)
SOD Activity d 34.38 (4.27) 36.27 (3.28) ns
a Assays were carded out in 50 mM phosphate buffer, pH 7.0. b Concentrations are: 1 mM SO3 = , 0.29/zM SOD, and 0.1 mM V(V). c Oxidation rate expresses SO3ffi oxidation under different experimental conditions and are the mean of at least four determinations. Standard errors arc shown in parentheses. d SOD activity is expressed as percentage inhibition of SO3= oxidation. Standard errors are indicated in parentheses. The comparison of the activity in the presence of V(V) with controls in its absence is also indicated; ns means not significant.
VANADIUM EFFECT ON ANTIOXIDANT ENZYMES
0:
I
I
I
0.2
0.3
0.4
I
0.1
167
Vanadium (mM)
FIGURE 3. Dose-response plot of the effect of V(V) (0) or V(IV) (0) on SOD activity. Concentrations are 0.2 mM pyrogallol, 0.1 pM SOD for V(V), and 0.2 PM SOD for V(IV) in 50 mM Tris buffer, pH 7.8. V(IV) was prepared iu the presence of GSH to prevent air-oxidation.
employ [28]) or V(V) on the activity of SOD in a wide range of V concentrations.
An apparent decrease of SOD activity was observed when V concentrations increase, which is more pronounced for V(V) than for V(W). The addition of metal chelators, EDTA and DFO, partially reverts this effect (data not shown). On the other hand, pyrogallol autoxidation rates in the presence of V are higher than in its absence (data not shown), indicating that the oxidative process is accelerated by the metal. Figure 4 displays the EPR spectra of mixtures containing 0.2 mM pyrogallol plus 0.1 mM V(IV) or 0.1 mM V(V), under N, atmosphere. Similar EPR patterns are observed for both samples; as V(V) is EPR-silent the appearance of a signal indicates
I
2.6
I
I.
2.8
I.,,
3.0
I.
3.2
3.4
Magnetic
FIGURE 4.
t,,.n.:.,.,.
1.
3.8
2.6 Field
2.8
3.0
3.2
3.4
3.6
(KGaur)
X-band EPR spectra of samples of 0.2 mM pyrogallol with (A) 0.1 mM V(V) or (B) 0.1 mM V(IV) (without GSH) that was prepared, mixed, and kept under N, atmosphere in 50 mM Tris buffer, pH 7.8. Recording conditions were: time constant, 0.064 s; modulation amplitude, 8 G; modulation frequency, 100 kHz; microwave power, 5 mW; microwave frequency, 9.000 GHz; receiver gain 2.5;103 (A), or 1.6i103 (B). Sainples were kept and recorded at - 150°C.
19
M. A. Serra et al.
that V(IV) is the oxidation form present in reaction mixtures owing to V(V) reduction by pyrogallol. SOD itself does not alter this process (data not shown). Table 4 displays the results of the NAA of the joint protein fraction of a sample of SOD and of a mixture of SOD + V(V) after gel filtration; there does not appear to be a significant amount of V in the protein fraction, so it can be concluded that there is no binding of V(V) to SOD under the studied conditions. In addition, the metal constituents of the protein, Cu and Zn, were not affected by the presence of V. Figure 5 shows the results of the gel filtration experiments of mixtures of SOD + 48V-V(IV) and of SOD +48V-V(IV) + pyrogallol in Tris buffer; no significant binding (V/SOD ratio 0.17:1) of V(IV) to the enzyme was demonstrated and the presence of pyrogallol did not significantly alter this behavior. It can be then concluded that neither V(IV) nor V(V) are bound to SOD in the studied conditions. Besides, there were no significant changes in the UV spectra of SOD samples when recorded in the presence of V (data not shown). DISCUSSION EffectofVonHRP The results presented here confirm the vanadate inhibitory effect of HRP activity found elsewhere [ 131. There is a competitive inhibition pattern, with Km (H,O,) and Ki (V(V)) of the same order of magnitude, 12.6 FM and 41.2 PM, respectively. However, the exact inhibitory mechanism is difficult to establish. An interference in the formation of Compound I (HRP + H,O, -+Compound I) may be possible. Vanadium binds to HRP in stoichiometric amounts and this binding is favored in the presence of H,O, [ 131, perhaps because the binding of the latter to the enzyme can make some hydrophilic groups more’ available for V binding to the enzyme. The metal should bind then to a site close to or overlapping with the H,O, binding site, being just a partiahy competitive inhibition. On the other hand the functional inhibitory species bound could be some type of peroxyvanadium complex, which have been postulated to exist also at neutral pH values [29]. Effect of V on Catalase There is not a V(V)-mediated inhibition of catalase activity, neither in Tris nor in phosphate buffers. Although V-peroxy complexes can also be formed in these conditions [29], they do not seem to be rate-limiting as no significant inhibition is TABLE
4. Neutron Activation Analysis of SOD” Metal
Cu Zn V Mn AU As
Amount found by NAA (g atom/mole enzyme, MW = 32600) No pre-incubation with V 2.11 2.02 0.004 0.006 0.006
After prc-incubation with V 2.06 2.04 0.06 0.10 -
’ NM of the joint protein fraction obtained after applying samples of SOD with (4.42 PM) or without (3.6 CM) pre-incubation(for 30 min) with 0.85 mM V(V) to Sephadex G25 columns, using 50 mM ammoniumacetate buffer, pH 7.0, at 37% For more details, see Materials and Mahods.
VANADIUM EFFECT ON ANTIOXIDANT ENZYMES
169
FIGURE 5. SephadexG25 gel titration chromatography of 33 PM SOD incubated with 0.1 mM “V-V(W) in the absence (0) and in the presence (A) of 0.2 mM pyrogallol in 50 m&l Tris buffer, pH 7.8. Proteii (----)w~~~at~S~ei~rin~e~~r(O)~in~ latter (A) conditions. The total remvery of ABVwas 82.4% and 86.796, respectively.
obtained, in contrast with the case of HRP. That result is not in agreement with a vanadate inhibition of cat&se described elsewhere [30]. However, they use a decavanadate solution that is slowly transformed into a orthovanadate/metavanadate one [31], while in our case a stable ova solution was employed. On the other hand, they carry out catalase activity measurements at a somehow elevated hydrogen peroxide concentration (15 n&f), as compared to what has been recommended (10 n&i) in order to avoid apparent higher initial rates [23] that can lead to an over-estimation of catalase activity. Effect of V on GSH-Px Vanadium effect in the GSH-Px system merits a detailed analysis. Owing to known reactions of the metal with some components of the system, we calcnlated the NADPH oxidation rates of different subsystems. In the following scheme we represent the whole system, which indicates the overall reaction: reaction (1) is catalyzed by GSH-Px and reaction (2) is catalyzed by GSSG-R:
Vanadium does not inhibit GSH-Px directly, as shown by the detailed analysis of the four subsystems studied (Table 2):
170
M. A. Serra et aL
i) Subsystem D. VOV) seems capable of oxidizing NADPH at an appreciable rate (0.133) more than V(V) (0.044), as compared to the control, probably through the production of superoxide radical by means of V(IV)-air-oxidation and subsequent peroxovanadyl oxidation of NADPH [12, 32]. This process is mediated by the O~radical and should be inhibited by SOD, as it readily happens (90% inhibition of NADPH oxidation rates when 0.79/zM SOD was added).
ii) Subsystem C. When cumene hydroperoxide and GSH are present, NADPH oxidation increases markedly as compared to the control; V(V) can be reduced to V(IV) by GSH [32, 33], which should favor the above-explained reactions. However, the final NADPH oxidation rate will be smaller than for the V(IV) subsystem C, which produces the O~- radical promptly and not by a two-step process, as in the V(V) subsystem C. There can also be considered two other reactions which can account for increased NADPH oxidation rates in addition to the peroxovanadyl pathway. First, the superoxide radical generated can oxidize GSH to GSSG, being regenerated at the end [34]; and second, it can also react with the hydroperoxide producing an alkoxyl radical ( R O . ) capable of initiating a chain of peroxidative reactions [12, 35]. For this subsystem also, there was a significant (around 80%) inhibition of NADPH oxidation rates when 0.79/zM SOD was added, confirming the involvement of the superoxide radical. iii) Subsystem B. When GSSG-R is added the high amounts already available of its specific substrate, GSSG, generated by the above-mentioned reactions (GSH oxidation by O2- and by alkoxyl radical-initiated peroxidative reactions), will accelerate further NADPH oxidation, more for the V(IV) than for the V(V) subsystem and for this than the control. A direct V(V) oxidation of NADPH, catalyzed by GSSG-R, has recently been described [36], and could also account for increased NADPH oxidation rates for the V(V) subsystem B. iv) Subsystem A. The addition of GSH-Px, completing the whole system, provides a faster NADPH oxidation by accelerating the curnene hydroperoxide reduction by GSH, either for V(IV) or V(V) or the control. When the effect of V in the above-explained subsystems is substracted from the whole system (A-B), we are trying to obtain a more exact determination of the metal's effect on GSX-Px itself; the comparison of the V(IV) or V(V) (A-B)values with the control ones indicates that the differences are not significant in any case, meaning that neither V(IV) nor V(V) are directly affecting GSH-Px activity. The role of the metal in the different oxidative reactions of the studied system is also evidenced by the fact that addition of EDTA always decrease NADPH oxidation rates [31], our output for all these oxidative processes. On the other hand, in vivo experiments show contradictory results [37, 38], which is not surprising due to the complexity of the system. Effect of V on SOD V(V) does not affect SOD activity when assayed with the sulfite method (Table 3), which seems devoid of interferences, so it is likely that vanadate is not a true inhibitor of the enzyme. On the other hand, there was an apparent dose-response inhibition of SOD activity for either V(IV) or V(V) using the pyrogallol assay (Fig. 3). In this case, however, it was due to an interference of the superoxide radical source with the metal: pyrogallol can reduce V(V) to V(IV) and complex it [39-41]; that reduction has been confirmed by our EPR spectra of pyrogaUol with V (Fig. 4). This could explain why SOD activity is more decreased for V(V) than for V(IV):
VANADIUM EFFECT ON ANTIOXIDANT ENZYMES
171
pyrogahol is oxidized not only via Oi- radicals but also by the metal, a non-superoxide radical-mediated mechanism which is obviously not affected by SOD. This means that there is not a true V-mediated inhibition of SOD but just a less responsive assay, confirming what the employment of the sulfite method shows readily. However, we think that it is interesting to point out this potentially misleading information using the pyrogahol method with a very common SOD assay also used in V studies [42]; perhaps it is not the most appropriate one for testing this metal. Moreover, we have not found a binding of V(IV) (Fig. 4) nor V(V) (Table 3) to SOD, even using somewhat high (0.1 mM-0.85 mM) V concentrations in order to emphasize any eventual V-protein complex that could be formed. A possible replacement of the protein metal constituents, Cu and Zn, by V can also be discarded because their stoichiometric amounts are maintained unchanged in spite of SOD pre-incubation with V, so the metal moieties of the enzyme seem not to be affected by V. Caution should be taken with apparent SOD inhibitors [5, 461, particularly when looking for a possible transition metal-mediated inhibition of SOD, because redox reactive metals can catalyse the autoxidation of indicating scavengers by inner sphere electron transfer, a mechanism that does not involve oxygen free radicals and is not inhibitable by SOD, rendering the assay less responsive to the enzyme and giving the false appearance of a metal-mediated inhibition. This is the case, for instance, for 6-hydroxydopamine [47] and for pyrogallol [26], as we describe in this work. However, any metal ligand, such as typical metal chelators (i.e., EDTA and DFO) or proteins (including SOD) should diminish the ability of metal ions to exchange electrons by an inner sphere mechanism, rendering autoxidation mainly dependent again on propagation by Oi- radicals and therefore inhibitable by SOD. This is the reason why addition of EDTA and DFO increased apparent SOD activities, also in our experiments (data not shown). Other authors testing transition metal ions as possible SOD inhibitors are warned against this potentially misleading information, as well as against interferences based on interactions of metal ions with reaction components of the SOD assay used. This could be the case of two recent works. A vanadate-mediated inhibition of SOD activity at physiological pH employing the xanthine/xanthine oxidase-nitroblue tetrazolium SOD assay has been published [43]. However, it has been shown that V competitively inhibits xanthine oxidase (Ki = 0.17 mM) at pH 7.4-7.6 [44, 451, which suggests that the xanthine oxidase-mediated production of superoxide radicals could be decreased by its vanadate-mediated inhibition more than by a direct effect of V on SOD, which would only find less Oi- radicals. On the other hand, an apparent vanadate-mediated inhibition of SOD using the 6-hydroxydopamine method [48] has not taken into account that cathecols and cathecolamines are metal reductants and chelators, also of V [39-41, 49, 501. Besides, their application of the 6-hydroxydopamine assay [51] is not fully convincing, as has been discussed elsewhere [ 111. In both cases we may have less responsive SOD assays instead of true V-mediated SOD inhibitions. It should therefore be advisable to test any supposed SOD inhibitor by, at least, two independent and interference-free methods, because a true inhibitor should be effective regardless of the SOD assay employed. Physiological Implications In conclusion we have described a direct inhibition of V(V) of an oxidoreductase, HRP, possibly by complex formation of V with hydrogen peroxide. On the other
172
M. A. Serra et al.
hand, the lack of V inhibition of the three main antioxidant enzymes (catalase, GSH-Px, and SOD) and the redox reactions in which V seems to be involved in some of these enzymatic systems indicates that many biological and toxicological effects of the metal, particularly V(V), may be mediated by oxidative reactions of V or V complexes more than by a direct effect on the enzyme itself [33, 52-551. All these findings, however, do not unequivocally ascertain a clear biological role for V, which needs further investigation. NOTE ADDED IN PROOF While this work was in press, an article by Wittenkeller et al. (J. Am. Chern. Sot. 113:7872, (1991)) was published reporting a significant and specific strong binding of V(V) to Cu-Zn SOD (8 atoms of V per molecule of enzyme) using “V NMR spectroscopy, in contrast with the absence of binding we show in Table 4. We have carried out exhaustive dialysis of 2 mM ammonium vanadate incubated in the presence of increasing SOD concentrations either in their (Hepes) or our (acetate) conditions, as well as dialysis of 0.1 mM SOD incubated with or without 2 mM ammonium vanadate against 2 mM ammonium vanadate in Hepes or acetate buffers. GFAAS analysis of all samples confirmed that there is no significant binding of V(V) to SOD in the former experiment and a small (2 g at of V/mol of SOD) but statistically significant binding in the latter one, which is, however, reverted by simple dialysis, an indication that it is a labile binding. Moreover, SOD activity in dialysis samples remains unaffected. Also, we have checked the purity of our SOD preparation by SDS-PAGE, which showed a single band, discarding the presence of contaminating proteins. The discrepancy between our results and the ones reported by Wittenkeller et al. could arise from the different source of the enzyme, erythrocytes, and liver, respectively. However, these authors agree with us in that the catalytically important Cu@) is unaffected by V(V) and also cannot demonstrate a V(V)-mediated inhibition of SOD activity. In any case, such a binding should be negligible in physiological conditions where V concentrations are too low to produce enough putative binding species and where V(V) is reduced to V(IV) inside cells. The authors would like to thank M. Mane0 and F. J. Ruiz for their technical asstitance.
REFERENCES 1. I. Fridovich. in Developments in cardiovascular medicine: oxygen radicals in the pathophysiology of heart disease, P. K. Singal, Ed., Kluwer Academic Publishers, Boston, 1988, Chap. 1, pp. l-11. 2. B. Halliwell and J. M. C. Gutteridge, Arch. B&hem. Biophys. 246, 501 (1986). 3. L. Fl& and W. A. Gbuler, in Methods in Ewmology, L. Packer, Ed., Academic Press, New York, 1984, Vol. 105, Chap. 12, pp. 114-121. 4. L. FlohC and F. 0ting, in Methods in Enzymology, L. Packer, Ed., Academic Press, New York, 1984, Vol. 105, Chap. 10, pp. 93-104. 5. H. M. Hassan, H. Dougherty, and I. Fridovich, Arch. B&hem. Biophys. 199,349 (1980). 6. W. F. Beyer and I. Fridovich, Anal. Biochem. 161,559 (1987). 7. S. E. Fridovich and N. A. Porter, J. Biol. Chem. 256, 260 (1981).
V A N A D I U M E F F E C T ON A N T I O X I D A N T E N Z Y M E S
173
8. J. M. C. Gutteridge, G. J. Quinlan, I. Clark, and B. Halliwell, Biochim. Biophys. Acta 835, 441 (1984). 9. G. L Quinhm, B. HalliweU, C. P. Moorhouse, and J. M. C. Gutteridge, Biochim. Biophys. Acta 962, 196 (1988). 10. M. Ohtawa, M. Seko, and F. Takayama, Chem. Pharm. Bull. 31, 1415 (1983). 11. M. A. Serra, V. Barassi, C. Canavese, and E. Sabbioni, Biol. Trace El. Res. 31, 79 (1991). 12. S. I. Liochev and I. Fridovich, Arch. Biochem. Biophys. 279, 1 (1990). 13. M. A. Serra, E. Sabbioni, A. Marchesini, A. Pintar, and M. Valoti, Biol. Trace El. Res. 23, 151 (1990). 14. A. C. Alfrey, Adv. Clin. Chem. 23, 69 (1983). 15. K. Tsuchiya, in Handbook on the toxicology of metals L. Friberg, G. F. Nordberg, and V. Vouk, Eds., Elsevier, Amsterdam, 1986, Vol. IT, Chap. 14, pp. 298-353. 16. B. Lagerkvist, G. F. Nordberg, and V. Vouk, in Handbook on the toxicology o f metals, L. Friberg, G. F. Nordberg, and V. Vouk, Eds., 1986, Vol. II, Chap. 27, pp. 638-663. 17. A. C. Maehly, in Methods in Enzymology, S. P. Colowick and N. O. Kaplan, Eds., Academic Press, New York, 1955, Vol. H, Chap. 143, pp. 801-813. 18. T. Samejima and J. T. Yang, J. Biol. Chem. 238, 3256 (1963). 19. L. Floh~, B. Eisele, and A. Wendel, Hoppe-Seyler's Z. Physiol. Chem. 352, 151 (1971). 20. U. Weser, G. Barth, C. Djerassi, H. J. Hartmarm, P. Krauss, G. Voelcker, W. Voelter, and W. Voetsch, Biochim. Biophys. Acta 278, 28 (1972). 21. D. P. Nelson and L. A. Kiesow, Anal. Biochem. 49, 474 (1972). 22. B. Chance and A. C. Maehly, in Methods in Enzymology, S. P. Colowick and N. O. Kaplan, Eds., Academic Press, New York, 1955, Vol. II, Chap. 136, pp. 764-775. 23. H. Aebi, in Methods in Enzymology, L. Packer, Ed., Academic Press, New York, 1984, Vol. 105, Chap. 13, pp. 121-126. 24. J. M. McCord and I. Fridovich, J. Biol. Chem. 244, 6049 (1969). 25. J. M. McCord and I. Fridovich, J. Biol. Chem. 244, 6056 (1969). 26. S. Marklund and G. Marklund, Eur. J. Biochem. 47, 469 (1974). 27. M. Dixon and E. C. Wehh, Enzymes, Longman, London, 1979, pp. 354-355. 28. E. F. Roth and H. S. Gilbert, Anal. Biochem. 137, 50 (1984). 29. O. W. Howarth and J. R. Hunt, J. Chem. Soc. Dalton, Trans., 1388, (1979). 30. M. Ran, M. ,S. Patole, C. Vijaya, C. K. Ramakrishna Kurup, and T. Ramasarma, Mol. Cell. Biochem. 75, 151 (1987). 31. D. Dart and I. Fridovich, Arch. Biochem. Biophys. 243, 220 (1985). 32. P. J. Stankiewicz, A. Stem, and A. J. Davison, Arch. Biochem. Biophys. 287, 8 (1991). 33. I. G. Macara, K. Kustin, and L. C. Cantley, Biochim. Biophys. Acta 629, 95 (1980). 34. R. Munday and C. C. Winterbourn, Biochem. Pharmacol. 38, 4349 (1989). 35. M. W. Sutherland and J. M. Gebicki, Arch. Biochem. Biophys. 214, 1 (1990). 36. X. Shi and N. S. Dalai, Arch. Biochem. Biophys. 275, 288 (1990). 37. S. Kacew, M. R. Parulekar, and Z. Meraii, Toxicol. Lett. 11, 119 (1982). 38. M. D. Cohen and C. I. Wei, J. Leukocyte Biol. 44, 122 (1988). 39. S. Ya Shnalderman, G. N. Prokof'eva, A. N. Demidovskaya, and E. P. Klimenko, Zh. Obshch. Khim. 42, 24 (1972). 40. J. Zelinka, M. Bartu~ek, and A. Ok~, Collection Czechoslov. Chem. Commun. 39, 83 (1974). 41. S. Ya Shnaiderman, E. P. Klimenko, and A. N. Demidovskaya, Ukr. Khim. Zh. 36, 8 (1970). 42. M. Elfant and C. L. Keen, Biol. Trace El. Res. 14, 193 (1982). 43. M. C. ApeUa, S. N. Gonz~lez, and E. J. Baran, Biol. Trace El. Res. 18, 123 (1988).
174
M. A . Serra et al.
C. S. Ramadoss, Z. Naturforsch. 35e, 702 (1980). A. M. Ghe, C. Stefanelli, and D. C. Carati, Talanta 31, 241 (1984). G. E. Eldred and J. R. Hoffert, Anal. Biochem. 110, 137 (1981). B. Bandy and A. J. Davison, Arch. Biochem. Biophys. 259, 305 (1987). R. Shainkin-Kestenbaum, C. Caruso, and G. M. Bedyne, Trace El. Med. 8, 6 (1991). R. K. Bogges and R. B. Martin, J. Am. Chem. Soc. 97, 3076 (1975). E. Mentasti, E. Pelizzetti, and C. Baioicchi, J. Inorg. Nucl. Chem. 38, 2017 (1976). R. E. Heikkila and F. Cabbat, Anal. Biochem. 75, 356 (1976). N. D. Chasteen, Struct. Bond. 53, 107 (1983). J. E. Benabe, L. A. Echegoyen, B. Pastrana, and M. MartJnez-Maldonado, J. Biol. Chem. 262, 9555 (1987). 54. J. A. Bonadies and C. J. Carrano, J. Am. Chem. Soc. 108, 4088 (1986). 55. H. V. Strout, P. P. Vicario, R. Saperstein, and E. E. Slater, Endocrinology 124, 1918 (1989). 44. 45. 46. 47. 48. 49. 50. 51. 52. 53.
Received September 4, 1991; accepted November 3, 1991
