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Direct demonstration that ferrous ion complexes of di- and triphosphate nucleotides catalyze hydroxyl free radical formation from hydrogen peroxide
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 225, No. 1, August, pp. 263-2'70, 1983
Direct Demonstration that Ferrous Ion Complexes of Di- and Triphosphate Nucleotides Catalyze Hydroxyl Free Radical Formation from Hydrogen Peroxide ROBERT A. FLOYD Biomembrane Research Ldoratory, Oklahoma Medical Research Foundation, and Department of Biochemistry and Molecular Bio&g, University of Oklahoma Health Sciences Center, Oklahoma City, Ok&xnm 75104 Received February
22, 1983
Utilizing an electron paramagnetic resonance (EPR) spin-trapping technique it was demonstrated that the di- and triphosphate nucleotides of adenosine, cytidine, thymidine, and guanosine in the presence of Fe(I1) catalyze hydroxyl free radical formation from Hz02. The triphosphate nucleotides in general were about 20% more effective than the diphosphate nucleotides. The amount of OH produced from HzOz as a function of nucleotide level tended to increase in a sigmoidal fashion beginning at a nucleotide/Fe(II) ratio of 2 but then rose rapidly up to a ratio of 5 at which point the increase became more gradual. The monophosphate nucleotides did not cause an increase in the amount of hydroxyl free radical produced from HzOz over the low level obtained in the buffer system only. The cations, Me and Ca2+,even at much higher than physiological levels and much higher than the level of added Fe(U), did not cause a-substantial diminution of the Fe(II)-nucleotide-catalyzed breakdown of H20z to yield OH. A study of the time course of the effectiveness of Fe(II)-nucleotide-mediated OH formation from HzOz demonstrated that Fe(I1) in the presence of nucleotides remained in an effective catalytic state with a halftime of about 160 s whereas in the absence of the nucleotides the halftime was 7.5 s. All observations indicate that Fe(I1) ligates with di- and triphosphate nucleotides and remains in the ferrous state which is then capable of catalyzing CH formation from H202; but with time, oxidation of the metal ion to the ferric state occurs, which either ligated to the nucleotide or to buffer ions, is ineffective in H20z catalysis to yield OH. Iron-nucleotide complexes may be of importance in mediating oxygen free radical damage to biological systems. The observations presented here indicate that hydroxyl free radicals will be produced when HzOz is present with ferrousnucleotide complexes.
A major portion of the biological consumption of molecular oxygen occurs during reduction to water via oxidative phosphorylation. However, a small portion of the total oxygen consumed is reduced in a univalent pathway yielding superoxide, hydroxyl free radical, and hydrogen peroxide, all of which are potentially damaging to biological systems (1). The protective enzymes superoxide dismutase,
catalase, and glutathione peroxidase usually prevent excessive oxidative damage unless the flux of univalent oxygen reduction becomes so large as to override the capacity of the enzymes (1). Oxidative damage in biological systems can occur from the presence of more than normal amounts of 6,- and/or H202. The iron-catalyzed Haber-Weiss reaction may be responsible for oxidative damage. Thus, 263
0003-9861183 $3.00 Copyright 0 1993by AcademicPress,Inc. All rights of reproductionin any form reserved.
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6,- and HzOz in the presence of a catalytic amount of iron yields 6H which is a very strong oxidant, i.e., 6,- + HzOz ‘2 6H + OH- + Oz.
[l]
Reaction [l] can be written as the sum of the two following reactions; Cz- + Fe(II1) - Fe(I1) + Oz,
[2]
H202 + Fe(I1) Fe(II1) + CH + OH-.
[3]
It is clear that iron plays an essential role in these reactions cycling from the ferric to ferrous oxidation state. Certain chelators will ligate iron ions in a manner such that both reactions 1 and 2 will occur but other chelators prohibit either reaction 2 or 3. Lately, we have focused our attention upon trying to understand if there are natural ligands of iron which interact with the ferrous ion in a manner such as to allow reaction 3 to occur. In these studies we have taken advantage of the spin-trapping technique (2) to measure the amount of OH produced in reaction 3. The spin-trap DMPO’ reacts very rapidly (3.2 X 10’ M-’ s-l, Ref. (3)) with 6H to yield an EPR-detectable unique spectrum consisting of a 1:2:2:1 pattern where AN = ABH = 14.92 gauss (4, 5). Iron in biological systems is very precisely regulated such that there is very little, if any “free” iron present. We have recently shown that ADP and ATP, but not AMP, will ligate Fe(I1) in a manner such that 6H is produced from HzOz in large yield (6). In the present report, results are presented which extend our previous results, and thus demonstrate that in genera1 di- and triphosphate nucleotides ligate Fe(I1) such that this metal ion catalyzes H202 breakdown to yield large amounts of 6H. In addition, results are presented which show that Mgz+, even in much higher than normal levels, doesn’t prevent Fe(II)nucleotide-catalyzed HzOz breakdown to yield OH. i Abbreviations used: DMPO, 5,bdimethyl pyrroline-l-oxide; EPR, electron paramagnetic resonance.
A. FLOYD EXPERIMENTAL
PROCEDURES
The nucleotides, as the sodium salts of the highest grade of purity available, were purchased from Sigma Chemical Company and Boehringer-Mannheim GMBH. They were used usually within a month of receipt. This was because it was noted in initial experiments that results with some nucleotides were variable as the length of storage time increased. This can be explained by the known slow hydrolysis rate of some nucleotides. The spin trap, DMPO, was synthesized, made up in stock solutions, and stored under a nitrogen atmosphere at 4°C as described before (6,7). In a typical experiment, the nucleotide would be made up fresh as a 20 mM solution in water, kept on ice, and used within a 2- to 4-h period. No evidence of loss of activity was observed within this time period. Solutions of less concentration than 20 mM were prepared by dilution immediately (2 min) before use. Ferrous chloride solutions (1 mM) were prepared in 0.0012 N HCI at the start of each day. Loss of activity was not evident until 4 to 6 h after preparation of the 1 mM solutions from a 0.1 M stock solution in 0.0012 N HCl as described before (6). Typically an experiment would be carried out with careful attention to detail as such: to a 50-p] solution of buffer (100 mM NaCl-25 mM NaHC03, pH 6.7) 20 ~1 of 780 mM DMPO is added and then 10 ~1 of nucleotide solution; after mixing, then 10 ~1 of 1 mM FeCI, is added, mixed immediately, and then placed in a 37°C shaking water bath for 30 s at which time 10 ~1 of 0.3% HzOz is added, the solution mixed rapidly followed by 30 s incubation at 37°C. At the end of this period, a portion of the sample is then added to a sealed transfer pipet and an EPR spectrum started. The time elapsed before starting the EPR spectrum after HzOz addition is about 1 min. The EPR spectra were obtained on a Varian E-109 X-band instrument typically with the following parameters: 100 KHz field modulation with 2 gauss amplitude, 9.14 GHz microwave frequency, scan rate 100 gauss/S min, time constant 0.3 s, temperature 25°C incident microwave power 25 mW, and usually the gain was 6.3 X 102. Comparisons of the efficacy of each nucleotideFe(I1) solution as to the yield of 6H from HzOz was accomplished by utilizing a specific standard batch of ADP. The standard batch of 20 mM ADP (Sigma grade X, sodium salt from equine muscle) was prepared and frozen in 200~pl aliquots. At the start of an experiment, a freshly thawed aliquot of ADP was utilized to obtain a signal height under well-defined experimental conditions maintained constant from day to day. The standard ADP solution showed no signs of loss of activity during 40 days of storage at -20°C. In fact, on a day to day basis, the maximum signal height variation was less than 10%. The amount of spin-trapped hydroxyl free radical present in the various experiments was determined by the peak to
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range of tenfold lower concentration of peroxide (6). Ferrous ion was the limiting component as was demonstrated by a linear increase in OH trapped as a function of added Fe(I1) (6). The amount of 6H spin trapped at 2 mM ADP was found to be 21 PM which is about one-fifth of the amount of Fe(D) added to the system (6). The amount of OH spin trapped using the cytidine nucleotides as ligating agents for Fe(I1) is shown in Fig. 2. At ail concentrations of CMP utilized, there was no enhancement of 6H formed over the values obtained in the complete absence of nuRESULTS cleotide. This is exactly as was observed Figure 1 presents results illustrating the with AMP (6). On the other hand, increasamount of hydroxyl free radical spin ing concentrations of CDP caused a sigtrapped as a function of increasing con- moidal increase in OH spin trapped in almost a similar fashion as that obtained centration of ADP. As ADP concentration increased the amount of hydroxyl free with ADP. In fact the amount of 6H spin radical spin trapped by DMPO from HzOz trapped at 2 mM CDP, was 6% higher than increased in a sigmoidal fashion as we that obtained with 2 mM ADP. Increasing noted before (6). As demonstrated previ- CTP concentrations enhanced the amount ously, the reaction of ADP-Fe(I1) catalyzed of 6H spin trapped over that obtained with breakdown of HzOzto yield OH was carried CDP. At 2 InM CTP, there was a 26% inout such that H202 was in large excess such crease in the amount of OH spin trapped that the amount of OH spin trapped was over that obtained with 2 mM ADP. The independent of HzOz concentration over a increase obtained with CTP over CDP was
peak height of one transition in the EPR spectrum. The peak to peak height was related to the total spins present as obtained by hand integration of the first derivative spectrum utilizing the Reimann sum method (6, 8). The standard used was the stable nitroxide 2,2,6,6-tetra-methylpiperidinol-l-oxy as described before (6, 8). A 2 mM ADP stock solution yielded about 21 pM spin-trapped hydroxyl free radical when 100 pM Fe& was present and HzOz was added to 0.03% with the incubations and EPR measurement conducted as described earlier. The pH of the incubation solution after DMPO, nucleotide, FeClz, and H,Oz additions was no different than the buffer itself.
ADP (mhl)
FIG. 1. The amount of hydroxyl free radical spin trapped as a function of ADP concentration. The ADP utilized was purchased from two different companies (different symbols) as described in the text and illustrates the reproducibility of the results. The experiments were carried out with careful attention to detail as described under Experimental Procedures. The signal height is normalized relative to that obtained with a standard solution of 2 mM ADP as described in the text which was approximately 21 pM spin-trapped hydroxyl free radical.
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ROBERT A. FLOYD
Nucleotide
( mM 1
FIG. 2. The amount of hydroxyl free radical spin trapped as a function of cytidine nucleotide concentration. The experiments were carried out with careful attention to detail as described in Fig. 1. The triangles, squares, and circles refer to CTP, CDP, and CMP, respectively.
very similar to that obtained with ATP over ADP (6). Figure 3 shows the results obtained with the guanosine nucleotides. As with AMP (6) and CMP (Fig. 2), addition of GMP did not enhance the amount of OH produced from HzOz over that obtained by Fe(I1) in the presence of buffer only. GDP, and even more so GTP, enhanced OH production in a sigmoidal fashion as the concentration of nucleotides were increased. But, the amount of 6H produced with GDP was not as much as that obtained in the presence of equivalent amounts of ADP. In fact GDP was about 40% less effective at 2 InM than ADP as the same concentration. GTP was only slightly (10%) more effective than ADP at 2 mM, but in the 0.1 to 1 mM region was much more effective than ADP. GTP was less effective at 2 mM than CTP (Fig. 2) or ATP (6). Figure 4 shows the results obtained with the thymidine nucleotides. As with the other monophosphate nucleotides, TMP was without effect on the amount of 6H spin trapped. However, TDP, and even
more so TTP, enhancement was very similar to ATP (6) in the amount of 6H spin trapped as a function of increasing nucleotide concentration, but TDP was less effective than ADP (Fig. 1). Since it has been presumed that most of the nucleotides within a cell are associated with M8+, it was necessary to determine if Me would interfere with Fe(I1) ligation with nucleotides to catalyze H202 breakdown to yield 6H. Figure 5 presents the results of studies conducted to test this idea. Figure 5 (top trace) shows the 1:2:2:1 EPR spectrum of the DMPO spin-trapped 6H free radical obtained with 2 mM ATP and 100 PM Fe(I1) to which 0.03% HzOz had been added as described under Experimental Procedures. The second trace down shows an EPR spectrum of an experiment carried out as the experiment pertaining to the top trace except in this case, 2 mM MgClz was present. Magnesium ions at 2 mM, which is the same concentration as that of ATP but 20 times the concentration of the added Fe(II), caused an 8% decrease in the amount of OH spin trapped. If Mg2+
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COMPLEXES
0.0 El El
GDP
a6
El
0.4
Nucleotii
( mM )
FIG. 3. The amount of hydroxyl free radical as a function of guanosine nucleotide concentration. The experiments were carried out as in Fig. 2. The triangles, squares, and circles refer to GTP, GDP, and GMP, respectively.
but no Fe(I1) was present, then very little signal was obtained as shown in the lowest trace. The amount of spin-trapped CH as a function of the concentration of added MgCl, is not shown but decreased in a linear fashion such that at 2 mM final concentration the signal had decreased 8%. This high concentration of Me is much higher than would be observed in the cell, but 2 mM ATP is near that which would be expected in a cell. I have also tested Ca’+, another divalent cation that may be present under natural conditions, and have found that 2 mM CaCl, caused a 27% decrease in signal height in the presence of 2 mM ATP and 100 pM Fe(I1) to which 0.03% HzOzhad been added. This high level of Ca2+is on the order of at least lo3 times higher concentration than that which would be expected to be present in a cell. Figure 6 demonstrates the amount of OH spin trapped as a function of time after Fe(I1) has been added before the addition of HzOz. If ADP is present, then ferrous ion is in a statesuch that it has the capacity of generating OH from HzOz for a considerable length of time as compared to the
situation in the absence of ADP. The zero time points were taken by the addition of H202 immediately (approximately 10 s) prior to the addition of Fe(I1). The zero time point in the presence of ADP is smaller than subsequent time points until about the 2-min time point. This result has been observed many times. On the other hand the zero time value in the absence of ADP is higher than any value obtained in the presence or absence of ADP, but if Fe(I1) remains in the buffer in the absence of ADP for 15 s and then HzOz added, the yield of 6H is less than one-sixth of the zero time point. The time required for the amount of OH spin trapped to fall to onehalf of its original value (zero time value) in the absence of ADP is about 7.5 s, whereas the time required in the presence of ADP is about 160 s to decrease to onehalf of the highest values obtained. DISCUSSION
The data presented demonstrate that if Fe(I1) is added to a solution containing diand triphosphate nucleotides then this metal ion apparently ligates with the nu-
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ROBERT
A. FLOYD
0
TTP
m TDP
n
El
0.5
1.0
I.5
TMP
2.0
Nucleotide ( mM 1 FIG. 4. The amount of hydroxyl free radical as a function of thymidine nucleotide concentration. The experiments were carried out as in Fig. 2. The circles, triangles, and squares refer to TTP, TDP, and TMP, respectively.
cleotides in a manner such that it is capable of catalyzing OH formation from H202 in high yield. This observation may be useful in understanding oxidative damage to biological systems. The reasons for this statement are as follows. It has been largely
regarded that oxidative damage in biological systems involves the so-called ironcatalyzed Haber-Weiss reaction (9, lo), which is the resultant of the reaction of superoxide with H202 in the presence of a catalytic amount of iron to produce a
FIG. 5. Electron paramagnetic resonance spectra of the Fe(D)-ADP-catalyzed hydroxyl free radical adduct of DMPO from H202. The top spectrum was taken in the absence of MgClz, whereas the experiment represented by the second spectrum down contained 2 mrd MgClz, in the ADPbuffer solution before Fe(II)Clz was added. The lowest spectrum is that obtained for a solution containing ADP and MgClz but no Fe(I1) was added. The experiments were carried out with careful attention to detail as described under Experimental Procedure with the exception of the modifications described above. The instrument gain in this experiment was 4 X 102, but the remaining instrument parameters were as described in the text.
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t (sec.) FIG. 6. The amount of hydroxyl free radical spin trapped in a solution containing 2 mM ADP or in the absence of the nucleotide to which 0.1 mM FeCla had been added and allowed to incubate at 37°C for the indicated time period before HaOa was added. The zero time point was obtained by adding Hz02 immediately before adding FeCla as described in the text.
strong oxidant, the hydroxyl free radical. The presence of iron and specifically the nature of the ligation state of iron is of prime importance. The present work demonstrates an important point, namely, that the di- and triphosphate nucleotides, but not the monophosphate nucleotides, will ligate with Fe(I1) to catalyze an important portion of the iron-catalyzed Haber-Weiss reaction. The di- and triphosphate nucleotides are prevalent in biological systems, i.e., ATP is usually present at about 2 mM. Even though it is generally considered that the nucleotides in biological systems exist as the Mgz+ complex, the present work demonstrates that even in the presence of this cation at much higher than physiological levels it does not prevent Fe(I1) from ligating with nucleotides to catalyze HzOz breakdown to yield OH. Thus, the availability of Fe(I1) may be the important limiting component in initiation of oxidative damage. The importance of Fe-nucleotide complexes in mediating membrane lipid peroxidation has been known since the original work of Hockstein and Ernster (11) and has been repeatedly emphasized and elaborated by the work of Aust (12, 13). It is hard to relate the present results
to the Fe-nucleotide-mediated membrane lipid peroxidation. For instance, Tien et al. (13) demonstrated that with certain chelators in the presence of iron ions OH appeared to be involved in phospholipid liposome peroxidation, but this appeared not to be the case when ADP was the iron ligand. Gutteridge (14) also recently concluded that 6H was not involved in brain liposome peroxidation and the same conclusion was also reached by Morehouse et al. (15) with a natural membrane system. It is important to note that Fe-nucleotide complexes have been found in biological systems (see Ref. (16)) and it has been demonstrated that the nucleotides mediate transport of iron from transferrin to mitochondria (17, 18). ATP and ADP were effective in this regard but AMP was not (17). The data presented here suggest that Fe(I1) forms a complex with di- and triphosphate nucleotides. Most likely the nucleotide complex with ferrous ion slows down oxidation to the ferric ion which is catalytically much less active (6) in forming OH from H202. Based on the timecourse study presented here and the previously demonstrated effect of oxygen on
270
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the system (6) this is the most reasonable interpretation. The recent demonstration by Tien et al. (19) of only a slow oxygen consumption from ADP-Fe(I1) complexes supports this interpretation. The nature of the Fe(II)-nucleotide complex is not known. Apparently no systematic study of the Fe(I1) association constants with nucleotides has been done. This is probably because of the difficulty of maintaining ferrous ion in its reduced state. In regard to the nature of the ironnucleotide complexes, we have not been able to obtain a 77°K electron paramagnetic resonance spectrum of Fe(III)-nucleotide complexes (6): The observed sigmoidal dependence of OH production as a function of increasing nucleotide levels suggests there may be cooperativity of the nucleotides in forming catalytically active complexes. The nature of the Fe(II)-nucleotide complexes and their participation in oxidative damage in biological systems needs further study. ACKNOWLEDGMENTS This work was supported in part by a Department of Health and Human Services Grant AG02599. The author thanks Mrs. Anita Hill for excellent secretarial services and Mrs. S. K. Nank for technical help. REFERENCES 1. FRIDOVICH, I. (1978) Science 201.875-880. 2. JANZEN, E. G. (1971) Aw. Chemiud R~s 4,31-40.
A. FLOYD 3. FINKELSTEIN, E., ROSEN, G. M., AND RAUCKMAN, E. J. (1980) J. Amer. Chem. Sot 102.4994-4999. 4. HA-OUR, J. R., CHOW, V., AND BOLTON, J. R. (1974) Can& J. Chem 52,3549-3553. 5. FLOYD, R. A., SOONG, L. M., STUART, M. A., AND REIGH, D. L. (1978) Photochem. Photobid 28, 857-862. 6. FLOYD, R. A., AND LEWIS, C. A. (1983) Biochemtit?-fj 22,2645-2649. 7. FLOYD, R. A., AND WISEMAN, B. B. (1979) Biochim. Biophgs. A& 586, 196-207. 8. FLOYD, R. A., SOONG, L. M., STUART, M. A., AND REIGH, D. L. (1978) Arch Biochim. Biophys. 185, 450-457.
9. MCCORD, J. M., AND DAY, E. D. JR. (1978) FEBS z&t. 86, 139-142. 10. GUTTERIDGE, J. M. C., ROWLEY, D. A., AND HALLIWELL, B. (1982) Bbchem J. 206,605-609. 11. HOCHSTEIN, P., AND ERNSTER, L. (1963) Biochem Biqphys. Res. Commun 12, 388-394. 12. SVINGE, B. A., BUEGE, J. A., O’NEAL, F. O., AND AUST, S. D. (1979) J. Biol Chem. 254,5892-5899. 13. TIEN, M., SVINGE, B. A., AND AUST, S. D. (1982) Arch. Biochem Biophys. 216,142-151. 14. GUTTERIDGE, J. M. C. (1982) FEBS L&t. 150,454458. 15. MOREHOUSE, L. A., TIEN, M., BUCHER, J.R., AND AUST, S. D. (1983) Biochem Pharmud 32,123127. 16. HOCHSTEIN, P. (1981) Israel J. Chem 21, 52-53. 17. KONOPKA, K. (1978) FEBS I.&t. 92, 308-312. 18. KONOPKA, K., AND RoMALO, I. (1980) Eur. .I Biochem 107.433-439. 19. TIEN, M., MOREHOUSE, L. A., BUCHER, J. R., AND AUST, S. A. (1982) Arch. Biochem Biophys. 218. 450-458.
Direct Demonstration that Ferrous Ion Complexes of Di- and Triphosphate Nucleotides Catalyze Hydroxyl Free Radical Formation from Hydrogen Peroxide ROBERT A. FLOYD Biomembrane Research Ldoratory, Oklahoma Medical Research Foundation, and Department of Biochemistry and Molecular Bio&g, University of Oklahoma Health Sciences Center, Oklahoma City, Ok&xnm 75104 Received February
22, 1983
Utilizing an electron paramagnetic resonance (EPR) spin-trapping technique it was demonstrated that the di- and triphosphate nucleotides of adenosine, cytidine, thymidine, and guanosine in the presence of Fe(I1) catalyze hydroxyl free radical formation from Hz02. The triphosphate nucleotides in general were about 20% more effective than the diphosphate nucleotides. The amount of OH produced from HzOz as a function of nucleotide level tended to increase in a sigmoidal fashion beginning at a nucleotide/Fe(II) ratio of 2 but then rose rapidly up to a ratio of 5 at which point the increase became more gradual. The monophosphate nucleotides did not cause an increase in the amount of hydroxyl free radical produced from HzOz over the low level obtained in the buffer system only. The cations, Me and Ca2+,even at much higher than physiological levels and much higher than the level of added Fe(U), did not cause a-substantial diminution of the Fe(II)-nucleotide-catalyzed breakdown of H20z to yield OH. A study of the time course of the effectiveness of Fe(II)-nucleotide-mediated OH formation from HzOz demonstrated that Fe(I1) in the presence of nucleotides remained in an effective catalytic state with a halftime of about 160 s whereas in the absence of the nucleotides the halftime was 7.5 s. All observations indicate that Fe(I1) ligates with di- and triphosphate nucleotides and remains in the ferrous state which is then capable of catalyzing CH formation from H202; but with time, oxidation of the metal ion to the ferric state occurs, which either ligated to the nucleotide or to buffer ions, is ineffective in H20z catalysis to yield OH. Iron-nucleotide complexes may be of importance in mediating oxygen free radical damage to biological systems. The observations presented here indicate that hydroxyl free radicals will be produced when HzOz is present with ferrousnucleotide complexes.
A major portion of the biological consumption of molecular oxygen occurs during reduction to water via oxidative phosphorylation. However, a small portion of the total oxygen consumed is reduced in a univalent pathway yielding superoxide, hydroxyl free radical, and hydrogen peroxide, all of which are potentially damaging to biological systems (1). The protective enzymes superoxide dismutase,
catalase, and glutathione peroxidase usually prevent excessive oxidative damage unless the flux of univalent oxygen reduction becomes so large as to override the capacity of the enzymes (1). Oxidative damage in biological systems can occur from the presence of more than normal amounts of 6,- and/or H202. The iron-catalyzed Haber-Weiss reaction may be responsible for oxidative damage. Thus, 263
0003-9861183 $3.00 Copyright 0 1993by AcademicPress,Inc. All rights of reproductionin any form reserved.
264
ROBERT
6,- and HzOz in the presence of a catalytic amount of iron yields 6H which is a very strong oxidant, i.e., 6,- + HzOz ‘2 6H + OH- + Oz.
[l]
Reaction [l] can be written as the sum of the two following reactions; Cz- + Fe(II1) - Fe(I1) + Oz,
[2]
H202 + Fe(I1) Fe(II1) + CH + OH-.
[3]
It is clear that iron plays an essential role in these reactions cycling from the ferric to ferrous oxidation state. Certain chelators will ligate iron ions in a manner such that both reactions 1 and 2 will occur but other chelators prohibit either reaction 2 or 3. Lately, we have focused our attention upon trying to understand if there are natural ligands of iron which interact with the ferrous ion in a manner such as to allow reaction 3 to occur. In these studies we have taken advantage of the spin-trapping technique (2) to measure the amount of OH produced in reaction 3. The spin-trap DMPO’ reacts very rapidly (3.2 X 10’ M-’ s-l, Ref. (3)) with 6H to yield an EPR-detectable unique spectrum consisting of a 1:2:2:1 pattern where AN = ABH = 14.92 gauss (4, 5). Iron in biological systems is very precisely regulated such that there is very little, if any “free” iron present. We have recently shown that ADP and ATP, but not AMP, will ligate Fe(I1) in a manner such that 6H is produced from HzOz in large yield (6). In the present report, results are presented which extend our previous results, and thus demonstrate that in genera1 di- and triphosphate nucleotides ligate Fe(I1) such that this metal ion catalyzes H202 breakdown to yield large amounts of 6H. In addition, results are presented which show that Mgz+, even in much higher than normal levels, doesn’t prevent Fe(II)nucleotide-catalyzed HzOz breakdown to yield OH. i Abbreviations used: DMPO, 5,bdimethyl pyrroline-l-oxide; EPR, electron paramagnetic resonance.
A. FLOYD EXPERIMENTAL
PROCEDURES
The nucleotides, as the sodium salts of the highest grade of purity available, were purchased from Sigma Chemical Company and Boehringer-Mannheim GMBH. They were used usually within a month of receipt. This was because it was noted in initial experiments that results with some nucleotides were variable as the length of storage time increased. This can be explained by the known slow hydrolysis rate of some nucleotides. The spin trap, DMPO, was synthesized, made up in stock solutions, and stored under a nitrogen atmosphere at 4°C as described before (6,7). In a typical experiment, the nucleotide would be made up fresh as a 20 mM solution in water, kept on ice, and used within a 2- to 4-h period. No evidence of loss of activity was observed within this time period. Solutions of less concentration than 20 mM were prepared by dilution immediately (2 min) before use. Ferrous chloride solutions (1 mM) were prepared in 0.0012 N HCI at the start of each day. Loss of activity was not evident until 4 to 6 h after preparation of the 1 mM solutions from a 0.1 M stock solution in 0.0012 N HCl as described before (6). Typically an experiment would be carried out with careful attention to detail as such: to a 50-p] solution of buffer (100 mM NaCl-25 mM NaHC03, pH 6.7) 20 ~1 of 780 mM DMPO is added and then 10 ~1 of nucleotide solution; after mixing, then 10 ~1 of 1 mM FeCI, is added, mixed immediately, and then placed in a 37°C shaking water bath for 30 s at which time 10 ~1 of 0.3% HzOz is added, the solution mixed rapidly followed by 30 s incubation at 37°C. At the end of this period, a portion of the sample is then added to a sealed transfer pipet and an EPR spectrum started. The time elapsed before starting the EPR spectrum after HzOz addition is about 1 min. The EPR spectra were obtained on a Varian E-109 X-band instrument typically with the following parameters: 100 KHz field modulation with 2 gauss amplitude, 9.14 GHz microwave frequency, scan rate 100 gauss/S min, time constant 0.3 s, temperature 25°C incident microwave power 25 mW, and usually the gain was 6.3 X 102. Comparisons of the efficacy of each nucleotideFe(I1) solution as to the yield of 6H from HzOz was accomplished by utilizing a specific standard batch of ADP. The standard batch of 20 mM ADP (Sigma grade X, sodium salt from equine muscle) was prepared and frozen in 200~pl aliquots. At the start of an experiment, a freshly thawed aliquot of ADP was utilized to obtain a signal height under well-defined experimental conditions maintained constant from day to day. The standard ADP solution showed no signs of loss of activity during 40 days of storage at -20°C. In fact, on a day to day basis, the maximum signal height variation was less than 10%. The amount of spin-trapped hydroxyl free radical present in the various experiments was determined by the peak to
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range of tenfold lower concentration of peroxide (6). Ferrous ion was the limiting component as was demonstrated by a linear increase in OH trapped as a function of added Fe(I1) (6). The amount of 6H spin trapped at 2 mM ADP was found to be 21 PM which is about one-fifth of the amount of Fe(D) added to the system (6). The amount of OH spin trapped using the cytidine nucleotides as ligating agents for Fe(I1) is shown in Fig. 2. At ail concentrations of CMP utilized, there was no enhancement of 6H formed over the values obtained in the complete absence of nuRESULTS cleotide. This is exactly as was observed Figure 1 presents results illustrating the with AMP (6). On the other hand, increasamount of hydroxyl free radical spin ing concentrations of CDP caused a sigtrapped as a function of increasing con- moidal increase in OH spin trapped in almost a similar fashion as that obtained centration of ADP. As ADP concentration increased the amount of hydroxyl free with ADP. In fact the amount of 6H spin radical spin trapped by DMPO from HzOz trapped at 2 mM CDP, was 6% higher than increased in a sigmoidal fashion as we that obtained with 2 mM ADP. Increasing noted before (6). As demonstrated previ- CTP concentrations enhanced the amount ously, the reaction of ADP-Fe(I1) catalyzed of 6H spin trapped over that obtained with breakdown of HzOzto yield OH was carried CDP. At 2 InM CTP, there was a 26% inout such that H202 was in large excess such crease in the amount of OH spin trapped that the amount of OH spin trapped was over that obtained with 2 mM ADP. The independent of HzOz concentration over a increase obtained with CTP over CDP was
peak height of one transition in the EPR spectrum. The peak to peak height was related to the total spins present as obtained by hand integration of the first derivative spectrum utilizing the Reimann sum method (6, 8). The standard used was the stable nitroxide 2,2,6,6-tetra-methylpiperidinol-l-oxy as described before (6, 8). A 2 mM ADP stock solution yielded about 21 pM spin-trapped hydroxyl free radical when 100 pM Fe& was present and HzOz was added to 0.03% with the incubations and EPR measurement conducted as described earlier. The pH of the incubation solution after DMPO, nucleotide, FeClz, and H,Oz additions was no different than the buffer itself.
ADP (mhl)
FIG. 1. The amount of hydroxyl free radical spin trapped as a function of ADP concentration. The ADP utilized was purchased from two different companies (different symbols) as described in the text and illustrates the reproducibility of the results. The experiments were carried out with careful attention to detail as described under Experimental Procedures. The signal height is normalized relative to that obtained with a standard solution of 2 mM ADP as described in the text which was approximately 21 pM spin-trapped hydroxyl free radical.
266
ROBERT A. FLOYD
Nucleotide
( mM 1
FIG. 2. The amount of hydroxyl free radical spin trapped as a function of cytidine nucleotide concentration. The experiments were carried out with careful attention to detail as described in Fig. 1. The triangles, squares, and circles refer to CTP, CDP, and CMP, respectively.
very similar to that obtained with ATP over ADP (6). Figure 3 shows the results obtained with the guanosine nucleotides. As with AMP (6) and CMP (Fig. 2), addition of GMP did not enhance the amount of OH produced from HzOz over that obtained by Fe(I1) in the presence of buffer only. GDP, and even more so GTP, enhanced OH production in a sigmoidal fashion as the concentration of nucleotides were increased. But, the amount of 6H produced with GDP was not as much as that obtained in the presence of equivalent amounts of ADP. In fact GDP was about 40% less effective at 2 InM than ADP as the same concentration. GTP was only slightly (10%) more effective than ADP at 2 mM, but in the 0.1 to 1 mM region was much more effective than ADP. GTP was less effective at 2 mM than CTP (Fig. 2) or ATP (6). Figure 4 shows the results obtained with the thymidine nucleotides. As with the other monophosphate nucleotides, TMP was without effect on the amount of 6H spin trapped. However, TDP, and even
more so TTP, enhancement was very similar to ATP (6) in the amount of 6H spin trapped as a function of increasing nucleotide concentration, but TDP was less effective than ADP (Fig. 1). Since it has been presumed that most of the nucleotides within a cell are associated with M8+, it was necessary to determine if Me would interfere with Fe(I1) ligation with nucleotides to catalyze H202 breakdown to yield 6H. Figure 5 presents the results of studies conducted to test this idea. Figure 5 (top trace) shows the 1:2:2:1 EPR spectrum of the DMPO spin-trapped 6H free radical obtained with 2 mM ATP and 100 PM Fe(I1) to which 0.03% HzOz had been added as described under Experimental Procedures. The second trace down shows an EPR spectrum of an experiment carried out as the experiment pertaining to the top trace except in this case, 2 mM MgClz was present. Magnesium ions at 2 mM, which is the same concentration as that of ATP but 20 times the concentration of the added Fe(II), caused an 8% decrease in the amount of OH spin trapped. If Mg2+
HYDROXYL
RADICALS
AND
IRON-NUCLEOTIDE
267
COMPLEXES
0.0 El El
GDP
a6
El
0.4
Nucleotii
( mM )
FIG. 3. The amount of hydroxyl free radical as a function of guanosine nucleotide concentration. The experiments were carried out as in Fig. 2. The triangles, squares, and circles refer to GTP, GDP, and GMP, respectively.
but no Fe(I1) was present, then very little signal was obtained as shown in the lowest trace. The amount of spin-trapped CH as a function of the concentration of added MgCl, is not shown but decreased in a linear fashion such that at 2 mM final concentration the signal had decreased 8%. This high concentration of Me is much higher than would be observed in the cell, but 2 mM ATP is near that which would be expected in a cell. I have also tested Ca’+, another divalent cation that may be present under natural conditions, and have found that 2 mM CaCl, caused a 27% decrease in signal height in the presence of 2 mM ATP and 100 pM Fe(I1) to which 0.03% HzOzhad been added. This high level of Ca2+is on the order of at least lo3 times higher concentration than that which would be expected to be present in a cell. Figure 6 demonstrates the amount of OH spin trapped as a function of time after Fe(I1) has been added before the addition of HzOz. If ADP is present, then ferrous ion is in a statesuch that it has the capacity of generating OH from HzOz for a considerable length of time as compared to the
situation in the absence of ADP. The zero time points were taken by the addition of H202 immediately (approximately 10 s) prior to the addition of Fe(I1). The zero time point in the presence of ADP is smaller than subsequent time points until about the 2-min time point. This result has been observed many times. On the other hand the zero time value in the absence of ADP is higher than any value obtained in the presence or absence of ADP, but if Fe(I1) remains in the buffer in the absence of ADP for 15 s and then HzOz added, the yield of 6H is less than one-sixth of the zero time point. The time required for the amount of OH spin trapped to fall to onehalf of its original value (zero time value) in the absence of ADP is about 7.5 s, whereas the time required in the presence of ADP is about 160 s to decrease to onehalf of the highest values obtained. DISCUSSION
The data presented demonstrate that if Fe(I1) is added to a solution containing diand triphosphate nucleotides then this metal ion apparently ligates with the nu-
268
ROBERT
A. FLOYD
0
TTP
m TDP
n
El
0.5
1.0
I.5
TMP
2.0
Nucleotide ( mM 1 FIG. 4. The amount of hydroxyl free radical as a function of thymidine nucleotide concentration. The experiments were carried out as in Fig. 2. The circles, triangles, and squares refer to TTP, TDP, and TMP, respectively.
cleotides in a manner such that it is capable of catalyzing OH formation from H202 in high yield. This observation may be useful in understanding oxidative damage to biological systems. The reasons for this statement are as follows. It has been largely
regarded that oxidative damage in biological systems involves the so-called ironcatalyzed Haber-Weiss reaction (9, lo), which is the resultant of the reaction of superoxide with H202 in the presence of a catalytic amount of iron to produce a
FIG. 5. Electron paramagnetic resonance spectra of the Fe(D)-ADP-catalyzed hydroxyl free radical adduct of DMPO from H202. The top spectrum was taken in the absence of MgClz, whereas the experiment represented by the second spectrum down contained 2 mrd MgClz, in the ADPbuffer solution before Fe(II)Clz was added. The lowest spectrum is that obtained for a solution containing ADP and MgClz but no Fe(I1) was added. The experiments were carried out with careful attention to detail as described under Experimental Procedure with the exception of the modifications described above. The instrument gain in this experiment was 4 X 102, but the remaining instrument parameters were as described in the text.
HYDROXYL
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COMPLEXES
269
t (sec.) FIG. 6. The amount of hydroxyl free radical spin trapped in a solution containing 2 mM ADP or in the absence of the nucleotide to which 0.1 mM FeCla had been added and allowed to incubate at 37°C for the indicated time period before HaOa was added. The zero time point was obtained by adding Hz02 immediately before adding FeCla as described in the text.
strong oxidant, the hydroxyl free radical. The presence of iron and specifically the nature of the ligation state of iron is of prime importance. The present work demonstrates an important point, namely, that the di- and triphosphate nucleotides, but not the monophosphate nucleotides, will ligate with Fe(I1) to catalyze an important portion of the iron-catalyzed Haber-Weiss reaction. The di- and triphosphate nucleotides are prevalent in biological systems, i.e., ATP is usually present at about 2 mM. Even though it is generally considered that the nucleotides in biological systems exist as the Mgz+ complex, the present work demonstrates that even in the presence of this cation at much higher than physiological levels it does not prevent Fe(I1) from ligating with nucleotides to catalyze HzOz breakdown to yield OH. Thus, the availability of Fe(I1) may be the important limiting component in initiation of oxidative damage. The importance of Fe-nucleotide complexes in mediating membrane lipid peroxidation has been known since the original work of Hockstein and Ernster (11) and has been repeatedly emphasized and elaborated by the work of Aust (12, 13). It is hard to relate the present results
to the Fe-nucleotide-mediated membrane lipid peroxidation. For instance, Tien et al. (13) demonstrated that with certain chelators in the presence of iron ions OH appeared to be involved in phospholipid liposome peroxidation, but this appeared not to be the case when ADP was the iron ligand. Gutteridge (14) also recently concluded that 6H was not involved in brain liposome peroxidation and the same conclusion was also reached by Morehouse et al. (15) with a natural membrane system. It is important to note that Fe-nucleotide complexes have been found in biological systems (see Ref. (16)) and it has been demonstrated that the nucleotides mediate transport of iron from transferrin to mitochondria (17, 18). ATP and ADP were effective in this regard but AMP was not (17). The data presented here suggest that Fe(I1) forms a complex with di- and triphosphate nucleotides. Most likely the nucleotide complex with ferrous ion slows down oxidation to the ferric ion which is catalytically much less active (6) in forming OH from H202. Based on the timecourse study presented here and the previously demonstrated effect of oxygen on
270
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the system (6) this is the most reasonable interpretation. The recent demonstration by Tien et al. (19) of only a slow oxygen consumption from ADP-Fe(I1) complexes supports this interpretation. The nature of the Fe(II)-nucleotide complex is not known. Apparently no systematic study of the Fe(I1) association constants with nucleotides has been done. This is probably because of the difficulty of maintaining ferrous ion in its reduced state. In regard to the nature of the ironnucleotide complexes, we have not been able to obtain a 77°K electron paramagnetic resonance spectrum of Fe(III)-nucleotide complexes (6): The observed sigmoidal dependence of OH production as a function of increasing nucleotide levels suggests there may be cooperativity of the nucleotides in forming catalytically active complexes. The nature of the Fe(II)-nucleotide complexes and their participation in oxidative damage in biological systems needs further study. ACKNOWLEDGMENTS This work was supported in part by a Department of Health and Human Services Grant AG02599. The author thanks Mrs. Anita Hill for excellent secretarial services and Mrs. S. K. Nank for technical help. REFERENCES 1. FRIDOVICH, I. (1978) Science 201.875-880. 2. JANZEN, E. G. (1971) Aw. Chemiud R~s 4,31-40.
A. FLOYD 3. FINKELSTEIN, E., ROSEN, G. M., AND RAUCKMAN, E. J. (1980) J. Amer. Chem. Sot 102.4994-4999. 4. HA-OUR, J. R., CHOW, V., AND BOLTON, J. R. (1974) Can& J. Chem 52,3549-3553. 5. FLOYD, R. A., SOONG, L. M., STUART, M. A., AND REIGH, D. L. (1978) Photochem. Photobid 28, 857-862. 6. FLOYD, R. A., AND LEWIS, C. A. (1983) Biochemtit?-fj 22,2645-2649. 7. FLOYD, R. A., AND WISEMAN, B. B. (1979) Biochim. Biophgs. A& 586, 196-207. 8. FLOYD, R. A., SOONG, L. M., STUART, M. A., AND REIGH, D. L. (1978) Arch Biochim. Biophys. 185, 450-457.
9. MCCORD, J. M., AND DAY, E. D. JR. (1978) FEBS z&t. 86, 139-142. 10. GUTTERIDGE, J. M. C., ROWLEY, D. A., AND HALLIWELL, B. (1982) Bbchem J. 206,605-609. 11. HOCHSTEIN, P., AND ERNSTER, L. (1963) Biochem Biqphys. Res. Commun 12, 388-394. 12. SVINGE, B. A., BUEGE, J. A., O’NEAL, F. O., AND AUST, S. D. (1979) J. Biol Chem. 254,5892-5899. 13. TIEN, M., SVINGE, B. A., AND AUST, S. D. (1982) Arch. Biochem Biophys. 216,142-151. 14. GUTTERIDGE, J. M. C. (1982) FEBS L&t. 150,454458. 15. MOREHOUSE, L. A., TIEN, M., BUCHER, J.R., AND AUST, S. D. (1983) Biochem Pharmud 32,123127. 16. HOCHSTEIN, P. (1981) Israel J. Chem 21, 52-53. 17. KONOPKA, K. (1978) FEBS I.&t. 92, 308-312. 18. KONOPKA, K., AND RoMALO, I. (1980) Eur. .I Biochem 107.433-439. 19. TIEN, M., MOREHOUSE, L. A., BUCHER, J. R., AND AUST, S. A. (1982) Arch. Biochem Biophys. 218. 450-458.