- Email: [email protected]
Two hypotheses for the peroxidase activity of Mn-superoxide dismutase
Free Radical Biology and Medicine 65 (2013) 1533
Contents lists available at ScienceDirect
Free Radical Biology and Medicine journal homepage: www.elsevier.com/locate/freeradbiomed
Letter to the Editor
Two hypotheses for the peroxidase activity of Mn-superoxide dismutase To the Editor We have recently proposed that Mn-superoxide dismutase (MnSOD) has a traditional peroxidase activity based, in part, on the H2O2-dependent oxidation of Amplex red, which was followed photometrically [1]. Liochev and Fridovich [3] found this surprising in view of our report describing a photochemical artifact inherent in assays that use Amplex red [2]. In this work, we found that modest illumination of the resorufin impurity in Amplex red solutions was observed to cause additional resorufin formation by a process forming Od2 and H2O2. Cu,ZnSOD was seen to increase this conversion of Amplex red to resorufin [2]. Although we gave no explanation for this effect on the photochemistry, Liochev and Fridovich proposed that MnSOD, by lowering the steady-state level of Od2 , displaces a quasi-equilibrium in a reaction of the Amplex red radical with oxygen and in that way increases the formation of resorufin. Liochev and Fridovich go on to say that ‘‘For some reason the authors of reference [3] failed to consider or even refer to the artifact described in their reference [4] that explains the observations reported in their reference [3].’’ Before we consider the merit of this statement, we would like to point out that the references cited have different authors and are, in fact, contributions by different research groups. Now, going back to the statement, the explanation is that Liochev and Fridovich are comparing two very different experiments. The experiments on the photochemistry of Amplex red used an FL3-22 spectrofluorimeter with a 450-W xenon lamp and a 10-nm slit width for excitation [2], whereas our experiments on MnSOD used a Varian Cary spectrophotometer with a 55-W lamp (basically a lamp light bulb) and a 0.5-nm slit width [1]. In retrospect, we should have investigated the effect of instrumental light in our absorbance experiments because we knew that 30 min of room light could affect Amplex red-based assays [2]. We did not imagine that significant photochemistry would occur in the course of a normal absorbance experiment. To test the effect of the instrumental light from a Varian Cary spectrophotometer (55-W lamp), control experiments incubated in the dark were performed (see the Supplementary material). Amplex red (100 mM), H2O2 (2 mM), and MnSOD (1 mg/ml) in 100 mM phosphate buffer with 25 mM diethylenetriaminepentaacetic acid, pH 7.4, were continuously exposed to the 55-W instrumental light (absorbance at 571 nm with a 0.5-nm slit width) and compared to a single final absorbance measured in samples incubated in the dark. It is clear from the results that regardless of whether the samples were incubated in the dark or exposed to the 55-W instrumental light, the oxidation of Amplex red by MnSOD was dependent on the presence of H2O2, which is the fundamental characteristic of a peroxidase. The Amplex red 0891-5849/$ - see front matter & 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.freeradbiomed.2013.02.013
oxidation under the 55-W instrumental light in the presence of H2O2 but in the absence of MnSOD was almost negligible. The Amplex red oxidation by MnSOD in the absence of H2O2 was similarly negligible. If Liochev and Fridovich were correct, then, the oxidation of Amplex red by MnSOD would not depend on H2O2, which it does. As shown in the Supplementary material, some artifactual oxidation of Amplex red by the spectrophotometer 55-W light does occur, but it accounts for approximately 20% of the overall response and does not affect the conclusions of our work, because the oxidation is totally dependent on both MnSOD and H2O2 [1]. Most critically, the oxidation of Amplex red still occurs in the dark with a brief exposure to light to measure the absorbance. Nonetheless, once resorufin is formed, light will form additional resorufin from Amplex red. In all probability, the light effect is present in all published works with Amplex red detected by photometric methods. The mechanism of the relatively weak H2O2-dependent oxidation of Amplex red by MnSOD is unknown and needs to be further investigated, but this oxidation is not due to a light-dependent artifact. Furthermore, the demonstrated peroxidase activity of MnSOD occurs at nonphysiological hydrogen peroxide concentrations and is apparently due to the ‘‘presence of a high excess of H2O2 [that] potentiates the enzyme’s peroxidase activity, possibly by oxidatively stabilizing/modifying the MnSOD structure’’ as we have indicated [1].
Appendix A.
Supplementary Information
Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.freeradbiomed.2013. 02.013.
References [1] Ansenberger-Fricano, K.; Ganini, D.; Mao, M.; Chatterjee, S.; Dallas, S.; Mason, R. P.; Stadler, K.; Santos, J. H.; Bonini, M. G. The peroxidase activity of mitochondrial superoxide dismutase. Free Radic. Biol. Med. 54:116–124; 2013. [2] Zhao, B.; Summers, F. A.; Mason, R. P. Photooxidation of Amplex red to resorufin: implications of exposing the Amplex red assay to light. Free Radic. Biol. Med. 53:1080–1087; 2012. [3] Liochev, Fridovich. Peroxidase activity by MnSOD?, http://dx.doi.org/10.1016/ j.freeradbiomed.2012.09.034.
Ronald P. Mason n Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA E-mail address: [email protected]
Available online 19 February 2013
Contents lists available at ScienceDirect
Free Radical Biology and Medicine journal homepage: www.elsevier.com/locate/freeradbiomed
Letter to the Editor
Two hypotheses for the peroxidase activity of Mn-superoxide dismutase To the Editor We have recently proposed that Mn-superoxide dismutase (MnSOD) has a traditional peroxidase activity based, in part, on the H2O2-dependent oxidation of Amplex red, which was followed photometrically [1]. Liochev and Fridovich [3] found this surprising in view of our report describing a photochemical artifact inherent in assays that use Amplex red [2]. In this work, we found that modest illumination of the resorufin impurity in Amplex red solutions was observed to cause additional resorufin formation by a process forming Od2 and H2O2. Cu,ZnSOD was seen to increase this conversion of Amplex red to resorufin [2]. Although we gave no explanation for this effect on the photochemistry, Liochev and Fridovich proposed that MnSOD, by lowering the steady-state level of Od2 , displaces a quasi-equilibrium in a reaction of the Amplex red radical with oxygen and in that way increases the formation of resorufin. Liochev and Fridovich go on to say that ‘‘For some reason the authors of reference [3] failed to consider or even refer to the artifact described in their reference [4] that explains the observations reported in their reference [3].’’ Before we consider the merit of this statement, we would like to point out that the references cited have different authors and are, in fact, contributions by different research groups. Now, going back to the statement, the explanation is that Liochev and Fridovich are comparing two very different experiments. The experiments on the photochemistry of Amplex red used an FL3-22 spectrofluorimeter with a 450-W xenon lamp and a 10-nm slit width for excitation [2], whereas our experiments on MnSOD used a Varian Cary spectrophotometer with a 55-W lamp (basically a lamp light bulb) and a 0.5-nm slit width [1]. In retrospect, we should have investigated the effect of instrumental light in our absorbance experiments because we knew that 30 min of room light could affect Amplex red-based assays [2]. We did not imagine that significant photochemistry would occur in the course of a normal absorbance experiment. To test the effect of the instrumental light from a Varian Cary spectrophotometer (55-W lamp), control experiments incubated in the dark were performed (see the Supplementary material). Amplex red (100 mM), H2O2 (2 mM), and MnSOD (1 mg/ml) in 100 mM phosphate buffer with 25 mM diethylenetriaminepentaacetic acid, pH 7.4, were continuously exposed to the 55-W instrumental light (absorbance at 571 nm with a 0.5-nm slit width) and compared to a single final absorbance measured in samples incubated in the dark. It is clear from the results that regardless of whether the samples were incubated in the dark or exposed to the 55-W instrumental light, the oxidation of Amplex red by MnSOD was dependent on the presence of H2O2, which is the fundamental characteristic of a peroxidase. The Amplex red 0891-5849/$ - see front matter & 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.freeradbiomed.2013.02.013
oxidation under the 55-W instrumental light in the presence of H2O2 but in the absence of MnSOD was almost negligible. The Amplex red oxidation by MnSOD in the absence of H2O2 was similarly negligible. If Liochev and Fridovich were correct, then, the oxidation of Amplex red by MnSOD would not depend on H2O2, which it does. As shown in the Supplementary material, some artifactual oxidation of Amplex red by the spectrophotometer 55-W light does occur, but it accounts for approximately 20% of the overall response and does not affect the conclusions of our work, because the oxidation is totally dependent on both MnSOD and H2O2 [1]. Most critically, the oxidation of Amplex red still occurs in the dark with a brief exposure to light to measure the absorbance. Nonetheless, once resorufin is formed, light will form additional resorufin from Amplex red. In all probability, the light effect is present in all published works with Amplex red detected by photometric methods. The mechanism of the relatively weak H2O2-dependent oxidation of Amplex red by MnSOD is unknown and needs to be further investigated, but this oxidation is not due to a light-dependent artifact. Furthermore, the demonstrated peroxidase activity of MnSOD occurs at nonphysiological hydrogen peroxide concentrations and is apparently due to the ‘‘presence of a high excess of H2O2 [that] potentiates the enzyme’s peroxidase activity, possibly by oxidatively stabilizing/modifying the MnSOD structure’’ as we have indicated [1].
Appendix A.
Supplementary Information
Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.freeradbiomed.2013. 02.013.
References [1] Ansenberger-Fricano, K.; Ganini, D.; Mao, M.; Chatterjee, S.; Dallas, S.; Mason, R. P.; Stadler, K.; Santos, J. H.; Bonini, M. G. The peroxidase activity of mitochondrial superoxide dismutase. Free Radic. Biol. Med. 54:116–124; 2013. [2] Zhao, B.; Summers, F. A.; Mason, R. P. Photooxidation of Amplex red to resorufin: implications of exposing the Amplex red assay to light. Free Radic. Biol. Med. 53:1080–1087; 2012. [3] Liochev, Fridovich. Peroxidase activity by MnSOD?, http://dx.doi.org/10.1016/ j.freeradbiomed.2012.09.034.
Ronald P. Mason n Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA E-mail address: [email protected]
Available online 19 February 2013