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Peroxide oxidation of Tris to a free radical
ARCHIVES OF BIOCHEMISTRY
AND BIOPHYSICS
164, 776-777 (1974)
Peroxide Oxidation
of Tris to a Free Radical
II
Tris (tris(hydroxymethyl)aminomethane) is used Magnettc held 3330 G extensively in biochemical research as a buffer. During studies of the oxidation of some amino acids to nitroxides (1, 2), we observed that oxidation of the Tris buffer itself gave a strong well-defined EPR signal, indicative of the presence of a free radical. Reagents used were Tris (from Sigma, Trizma HCI and Sigma 7-9), sodium tungstate (Mallinckrodt), and 30% hydrogen peroxide (Mallinckrodt). Nine milliliters of 0.35 M Tris solution, 1 ml of 30% HIOO, and 30 mg sodium tungstate were mixed at room temperature, the mixture transferred to a flat cell, and the EPR spectral changes followed in an E-3 X-band EPR spectrometer (Varian). At intervals, samples were removed and spotted on silica gel plates (Eastman) for tic. The plates were developed with 95% ethanol for approximately 1 hr at room temperature and oven FIGURE 1 dried at 90°C for 5 min. Spots were visualized by their absorption at 365 nm (Chromati-vur, Model CC-20, were not effective. The observation of Van Vunakis et Ultra-Violet Products) and by development with 3% al. (3) that Tris is incorporated into DNA during ninhydrin. photooxidation may be explainable in terms of a The Tris EPR signal appeared only when both transient free radical similar to that described herein. peroxide and tungstate were present. At room temperIn a similar manner, EDTA also can be oxidized ature, the strength increased with time to a maximum with peroxide in the presence of tungstate to a in about 1 hr and thereafter decreased to a very small well-defined nitroxide-type signal (4). Other invessignal in 3 hr. tigators (5) have also observed free radical signals The results obtained by tic analysis (silica gel) of with oxidized EDTA (titanium catalyst) but with reaction mixtures that had first been adjusted to pH different spectral characteristics. These combined 7.0, 8.2, and 9.0 were as follows: at pH 7.0, one spot observations emphasize the need for careful studies of was found with Tris (I?, 0.69). After 1 hr of oxidation, the results of peroxide oxidation of simple nitrogenous an additional spot (R, 0.74) was seen, and a weak EPR reagents and the potential role that free radical signal was present. At pH 8.2, two spots were present intermediates might play in biochemical reactions in originally (R, 0.46, 0.69); after oxidation for 1 hr, a which the reagents used are presumably inert buffers third spot (R, 0.74) and a strong EPR signal had or chelating agents. developed. At pH 9.0, there was originally a single Summary. Tris is oxidized to a free radical by spot (R, 0.46). After 1 hr of oxidation, there were two spots (R, 0.46, 0.74) and a very strong EPR signal peroxide-tungstate, more readily at alkaline pH than (Fig. 1). At pH 8.2 and at pH 9.0, the slowest moving at neutrality. spot had decreased in size during 1 hr of oxidation. ACKNOWLEDGMENTS EPR signals were observable in aqueous extracts of the scraping from the spots with R, 0.74 but not from This work was supported by Sea Grant USDC the other spots. These observations suggest that the 235208, National Oceanic and Atmospheric Adminisfree base (R, 0.46) oxidized more readily than the tration, U. S. Department of Commerce. We thank J. protonated Tris (R, 0.69) to the free radical (R, 0.74). J. Windle for assistance in EPR spectra interpretaTris is thus readily oxidized to a free radical; tion. original structure not identified. After the signal had REFERENCES become weak, the residual peaks (not shown) had some nitmxide characteristics. No signal appeared in 1. VANDERVEEN, J., WEIL, J.T., KENNEDY, T.E., AND the absence of tungstate, but the specificity of the O~corr, H. S. (1970) Lipid 5, 509. catalyst is not known. Magnesium and mercuric ions 2. LIN, J. S., TOM, T. C., AND OLCO?T, H. S. (1974) J. 776
COMMUNICATIONS Ag. Food Chem. 22,528. 3. VAN VUNAKIS, H., SEAMAN, E., KAHAN, L., KAPPLER, J. L., AND LEVINE, L. (1966) Biochemistry 5, 3986. 4. WEIL, J. T. (1968) Ph.D. Thesis, University of California, Berkeley. 5. PAUPKO, R., LOEWENSTEIN, A., AND SILVER, B. L. (1970) J. Amer. Chem. Sot. 93, 580.
777 PATRICIA A. MURPHY JAMES S. LIN HAROLD S. OLCOTT
hstitute of Marine Resources Department of Food Science and Technology University of California Dauis, California 95616 Received July 1, 1974
AND BIOPHYSICS
164, 776-777 (1974)
Peroxide Oxidation
of Tris to a Free Radical
II
Tris (tris(hydroxymethyl)aminomethane) is used Magnettc held 3330 G extensively in biochemical research as a buffer. During studies of the oxidation of some amino acids to nitroxides (1, 2), we observed that oxidation of the Tris buffer itself gave a strong well-defined EPR signal, indicative of the presence of a free radical. Reagents used were Tris (from Sigma, Trizma HCI and Sigma 7-9), sodium tungstate (Mallinckrodt), and 30% hydrogen peroxide (Mallinckrodt). Nine milliliters of 0.35 M Tris solution, 1 ml of 30% HIOO, and 30 mg sodium tungstate were mixed at room temperature, the mixture transferred to a flat cell, and the EPR spectral changes followed in an E-3 X-band EPR spectrometer (Varian). At intervals, samples were removed and spotted on silica gel plates (Eastman) for tic. The plates were developed with 95% ethanol for approximately 1 hr at room temperature and oven FIGURE 1 dried at 90°C for 5 min. Spots were visualized by their absorption at 365 nm (Chromati-vur, Model CC-20, were not effective. The observation of Van Vunakis et Ultra-Violet Products) and by development with 3% al. (3) that Tris is incorporated into DNA during ninhydrin. photooxidation may be explainable in terms of a The Tris EPR signal appeared only when both transient free radical similar to that described herein. peroxide and tungstate were present. At room temperIn a similar manner, EDTA also can be oxidized ature, the strength increased with time to a maximum with peroxide in the presence of tungstate to a in about 1 hr and thereafter decreased to a very small well-defined nitroxide-type signal (4). Other invessignal in 3 hr. tigators (5) have also observed free radical signals The results obtained by tic analysis (silica gel) of with oxidized EDTA (titanium catalyst) but with reaction mixtures that had first been adjusted to pH different spectral characteristics. These combined 7.0, 8.2, and 9.0 were as follows: at pH 7.0, one spot observations emphasize the need for careful studies of was found with Tris (I?, 0.69). After 1 hr of oxidation, the results of peroxide oxidation of simple nitrogenous an additional spot (R, 0.74) was seen, and a weak EPR reagents and the potential role that free radical signal was present. At pH 8.2, two spots were present intermediates might play in biochemical reactions in originally (R, 0.46, 0.69); after oxidation for 1 hr, a which the reagents used are presumably inert buffers third spot (R, 0.74) and a strong EPR signal had or chelating agents. developed. At pH 9.0, there was originally a single Summary. Tris is oxidized to a free radical by spot (R, 0.46). After 1 hr of oxidation, there were two spots (R, 0.46, 0.74) and a very strong EPR signal peroxide-tungstate, more readily at alkaline pH than (Fig. 1). At pH 8.2 and at pH 9.0, the slowest moving at neutrality. spot had decreased in size during 1 hr of oxidation. ACKNOWLEDGMENTS EPR signals were observable in aqueous extracts of the scraping from the spots with R, 0.74 but not from This work was supported by Sea Grant USDC the other spots. These observations suggest that the 235208, National Oceanic and Atmospheric Adminisfree base (R, 0.46) oxidized more readily than the tration, U. S. Department of Commerce. We thank J. protonated Tris (R, 0.69) to the free radical (R, 0.74). J. Windle for assistance in EPR spectra interpretaTris is thus readily oxidized to a free radical; tion. original structure not identified. After the signal had REFERENCES become weak, the residual peaks (not shown) had some nitmxide characteristics. No signal appeared in 1. VANDERVEEN, J., WEIL, J.T., KENNEDY, T.E., AND the absence of tungstate, but the specificity of the O~corr, H. S. (1970) Lipid 5, 509. catalyst is not known. Magnesium and mercuric ions 2. LIN, J. S., TOM, T. C., AND OLCO?T, H. S. (1974) J. 776
COMMUNICATIONS Ag. Food Chem. 22,528. 3. VAN VUNAKIS, H., SEAMAN, E., KAHAN, L., KAPPLER, J. L., AND LEVINE, L. (1966) Biochemistry 5, 3986. 4. WEIL, J. T. (1968) Ph.D. Thesis, University of California, Berkeley. 5. PAUPKO, R., LOEWENSTEIN, A., AND SILVER, B. L. (1970) J. Amer. Chem. Sot. 93, 580.
777 PATRICIA A. MURPHY JAMES S. LIN HAROLD S. OLCOTT
hstitute of Marine Resources Department of Food Science and Technology University of California Dauis, California 95616 Received July 1, 1974