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Purification of [3H]taurine of high specific activity
ANALYTICAL
BIOCHEMISTRY
79, 568-570 (1977)
Purification of PH]Taurine of High Specific Activity There has been recent interest in the possibility that taurine is an inhibitory neurotransmitter in the brain (1). Among the requirements necessary for a compound to be considered a neurotransmitter is that it should have a specific synaptic receptor, and that there should exist a mechanism for the termination of its action at the synapse, by either metabolism or sequestration. It appears unlikely that the action of taurine is terminated by metabolism, as the only metabolic reaction taurine is known to undergo in the brain is conversion to isethionic acid (2-hydroxyethanesulfonic acid). This reaction is extremely slow, and only a small percentage of the taurine in the brain is metabolized (7,lO). Attention has turned, therefore, to the search for a high-affinity transport system in brain slices and synaptosomes (pinced-off nerve endings), which would serve to terminate the synaptic actions of taurine (8,9,11,12). One of the requirements for the demonstration of a high-af8nity transport system is the availability of a substrate radiolabeled to a high specific activity. All reports to data have utilized commercially available taurine of relatively low specific activity (e.g., [35S]taurine at 0.047 Ci/mmol, or [14C]taurine at 0.002 Ci/mmol). With these specific activities, the highest affinity values reported for taurine to date have been found in the diencephalon, 21 PM (S), and cerebral cortex, 20 PM (11). Brain slices or synaptosomes prepared from other areas showed much lower affinities. With rH]taurine custom-labeled to a specific activity of 2.8 Ci/mmol, we have found a high-affinity transport system in synaptosomes, prepared from nuclei-free whole rat brain, with a K, value of 3-5 PM (4-6), in addition to a low-affinity uptake system with a K, value of about 400 FM. To study substrate-receptor interaction, substrates of high specific activities are also required. For example, in the demonstration of receptor binding of glycine, dopamine, norepinephrine, and acetylcholine, specific activities greater than 1 Ci/mmol have been employed (13). We report, therefore, a method we have used to purify rH]taurine of high specific activity. The availability of such material should be a useful biological research tool, with application to the study of high affinity transport mechanisms and the interactions of taurine with the postsynaptic receptor. Furthermore, the purification of [3H]taurine presented some unusual problems not immediately obvious. Tritiation of taurine. Taurine was recrystallized from water (three times). The recrystallized taurine (0.1 mmol) was custom tritiated by New England Nuclear by exchange with 10 Ci of tritium gas over a 568 Copyright 0 1977 by Academic Ress. Inc. All rights of reproduction in any form reserved.
ISSN 0003-2697
SHORTCOMMUNICATIONS
ruthenium-alumina catalyst. exchangeable tritium.
The product
contained
569 226 mCi of non-
Purification without dilution of specijc activity. An aliquot of the tritiated product (45 mCi), in water, was passed through an ionexchange column containing 5 x 0.7~cm AG l-X8 Cl- anion-exchange resin (Bio-Rad) layered over 11.5 x 0.7-cm AG 50-W-X8 H+ cationexchange resin (Bio-Rad). Fractions of approximately 1.5 ml were collected, and the activity was eluted in fractions 4 and 5, as did authentic taurine obtained from Sigma. This peak contained 80% (range on four procedures, 77.9-81.7%) of the radioactivity added to the column. Isotopic dilution analysis (see below) revealed that only 70.6-72.0% of the eluted activity was taurine. Radioactivity could not be removed from the eluent by chloroform extraction or by heating of a sample to 90°C for 2 hr (to evaporate volatile components). Passage through AG l-X8 OH- resin (10 x 0.7 cm; Bio-Rad) on a stepwise gradient (water, 0.01 N HCl, 0.05 N HCl) resulted in complete recovery of radioactivity, eluted in one peak by 0.05 N HCl (70% taurine by isotopic dilution). The contaminants must be zwitterionic, as they are retarded on an anion-exchange column, but pass through a dual-bed column. We considered hypotaurine, thiotaurine, and aminoethyl bisulfate as possible contaminants. Aminoethyl bisulfate was found not to be a contaminant by recrystallization of authentic aminoethyl bisulfate in the presence of the rH]taurine fraction. After three recrystallizations, only 0.4% of the added radioactivity remained in aminoethyl bisulfate. The presence of esters (i.e., sulfates, phosphates) was examined by hydrolysis at 100°C with 1 N NaOH for 20 min. All activity was retained in the taurine peak. To remove any thiotaurine or hypotaurine, a sample of [3H]taurine was boiled vigorously for 30 set and cooled, and two drops of hydrogen peroxide (30%) and one drop of saturated ammonium molybdate were added. This procedure converts both hypotaurine and thiotaurine to taurine. The sample was left overnight. Isotopic dilution analysis revealed that 80% of the activity was taurine. [3H]Taurine (0.5 ml of 80% purity) was further purified by chromatography on Eastman Chromatogram Cellulose tic plates, along with authentic unlabeled taurine. The solvent system used was acetone: formic acid:water (16:3:9). Two radioactive peaks were found when 15 l-cm chromatographic strips were counted. The R, value for taurine was 0.55 and for the contaminant, 0.80. The major peak, which cochromatographed with authentic taurine, contained 78-80% of the total radioactivity, whereas the second peak contained the remainder of the radioactivity. The radiolabeled portion which corresponded with authentic taurine was eluted from the tic plate with distilled water. Isotopic dilution analysis revealed that the rH]taurine was 100 + 1% pure. The chemical identity of the contaminant remains unknown.
570
SHORT COMMUNICATIONS
Determination of specijic activity. The concentration of the rH]taurine was determined by a fluorescamine assay procedure (2). A modification was made in the standard procedure by using 0.01 M instead of 0.2 M borate buffer. Radioactivity was determined by liquid scintillation spectrophotometry on a Nuclear Chicago Isocap 300 at a counting efficiency of 4%. These procedures revealed that the 100% pure [3H]taurine has a specific activity of 2.8 Wmmol. Procedure for isotopic dilution analysis. Authentic taurine (50 mg) in 1 ml of filtered water is mixed in a Craig tube (Cheronis, 1954) with a known quantity of radioactivity. The volume is reduced under a nitrogen stream, and one drop of ethanol is added to induce crystallization. The crystals are collected and dried in the Craig tube, and the specific activity is determined. The material is recrystallized to constant specific activity. In summary, we have obtained rH]taurine of specific activity of 2.8 Ci/mmol and 100% radiochemical purity. ACKNOWLEDGMENTS This work was supported by grants from the USPHS (HL 19394 and HL 20087 to RJH; MH 26967 to HIY). H. 1. Yamamura is the recipient of a Research Scientist Development Award from the National Institutes of Mental Health (MHOOO95).
REFERENCES 1. Barbeau, A., Tsukada, Y., and Inoue, N. (1976) in Taurine (Huxtable, R., and Barbeau, A., eds.), pp. 253-266, Raven Press, New York. 2. Bohlen, P., Stein, S., Dairman, W., and Udenfriend, S. (1973) Arch Biochem. Biophys. 155,213-220. 3. Cheronis, N. D. (1954) in Technique of Organic Chemistry (Weissberger, A., ed.), Vol. 6, pp. 49-54, Interscience, New York. 4. Hruska, R. E., Huxtable, R. J., Bressler, R., and Yamamura, H. I. (1976a) Fed. Proc. 35, 325. 5. Hruska, R. E., Huxtable, R. J., Bressler, R., and Yamamura, H. I. (1976b) Proc. West. Pharmacol. Sot. 19, 152-156. 6. Hruska, R. E., Bressler, R., and Yamamura, H. I. (1976~) Proc. Sot. Neurosci. 6, 583. 7. Huxtable, R., and Bressler, R. (1972) J. Nutr. 102, 805-814. 8. Lombardini, J. B. (1976) in Taurine (Huxtable, R., and Barbeau, A., eds.), pp. 311326, Raven Press, New York. 9. Oja, S. S., and Lalrdesmiiki, P. (1974) Med. Biol. 52, 138-143. 10. Peck, E. J., Jr., and Awapara, J. (1967) Biochim. Biophys. Acta 141,499-506. 11. Schmid, R., Sieghart, W., and Karobath, M. (1975) J. Neurochem. 25, 5-9. 12. Sieghart, W., and Karobath, M. (1974)J. Neurochem. 23, 911-915. 13. Snyder, S. H. (1975) Biochem. Pharmacol. 24, 1371-1374.
ROBERT E. HRUSKA RYAN J.HUXTABLE HENRY I. YAMAMURA Department of Pharmacology College of Medicine The University of Arizona Medical Center Tucson, Arizona 85724 Received March 29, 1976; accepted December 10, 1976
BIOCHEMISTRY
79, 568-570 (1977)
Purification of PH]Taurine of High Specific Activity There has been recent interest in the possibility that taurine is an inhibitory neurotransmitter in the brain (1). Among the requirements necessary for a compound to be considered a neurotransmitter is that it should have a specific synaptic receptor, and that there should exist a mechanism for the termination of its action at the synapse, by either metabolism or sequestration. It appears unlikely that the action of taurine is terminated by metabolism, as the only metabolic reaction taurine is known to undergo in the brain is conversion to isethionic acid (2-hydroxyethanesulfonic acid). This reaction is extremely slow, and only a small percentage of the taurine in the brain is metabolized (7,lO). Attention has turned, therefore, to the search for a high-affinity transport system in brain slices and synaptosomes (pinced-off nerve endings), which would serve to terminate the synaptic actions of taurine (8,9,11,12). One of the requirements for the demonstration of a high-af8nity transport system is the availability of a substrate radiolabeled to a high specific activity. All reports to data have utilized commercially available taurine of relatively low specific activity (e.g., [35S]taurine at 0.047 Ci/mmol, or [14C]taurine at 0.002 Ci/mmol). With these specific activities, the highest affinity values reported for taurine to date have been found in the diencephalon, 21 PM (S), and cerebral cortex, 20 PM (11). Brain slices or synaptosomes prepared from other areas showed much lower affinities. With rH]taurine custom-labeled to a specific activity of 2.8 Ci/mmol, we have found a high-affinity transport system in synaptosomes, prepared from nuclei-free whole rat brain, with a K, value of 3-5 PM (4-6), in addition to a low-affinity uptake system with a K, value of about 400 FM. To study substrate-receptor interaction, substrates of high specific activities are also required. For example, in the demonstration of receptor binding of glycine, dopamine, norepinephrine, and acetylcholine, specific activities greater than 1 Ci/mmol have been employed (13). We report, therefore, a method we have used to purify rH]taurine of high specific activity. The availability of such material should be a useful biological research tool, with application to the study of high affinity transport mechanisms and the interactions of taurine with the postsynaptic receptor. Furthermore, the purification of [3H]taurine presented some unusual problems not immediately obvious. Tritiation of taurine. Taurine was recrystallized from water (three times). The recrystallized taurine (0.1 mmol) was custom tritiated by New England Nuclear by exchange with 10 Ci of tritium gas over a 568 Copyright 0 1977 by Academic Ress. Inc. All rights of reproduction in any form reserved.
ISSN 0003-2697
SHORTCOMMUNICATIONS
ruthenium-alumina catalyst. exchangeable tritium.
The product
contained
569 226 mCi of non-
Purification without dilution of specijc activity. An aliquot of the tritiated product (45 mCi), in water, was passed through an ionexchange column containing 5 x 0.7~cm AG l-X8 Cl- anion-exchange resin (Bio-Rad) layered over 11.5 x 0.7-cm AG 50-W-X8 H+ cationexchange resin (Bio-Rad). Fractions of approximately 1.5 ml were collected, and the activity was eluted in fractions 4 and 5, as did authentic taurine obtained from Sigma. This peak contained 80% (range on four procedures, 77.9-81.7%) of the radioactivity added to the column. Isotopic dilution analysis (see below) revealed that only 70.6-72.0% of the eluted activity was taurine. Radioactivity could not be removed from the eluent by chloroform extraction or by heating of a sample to 90°C for 2 hr (to evaporate volatile components). Passage through AG l-X8 OH- resin (10 x 0.7 cm; Bio-Rad) on a stepwise gradient (water, 0.01 N HCl, 0.05 N HCl) resulted in complete recovery of radioactivity, eluted in one peak by 0.05 N HCl (70% taurine by isotopic dilution). The contaminants must be zwitterionic, as they are retarded on an anion-exchange column, but pass through a dual-bed column. We considered hypotaurine, thiotaurine, and aminoethyl bisulfate as possible contaminants. Aminoethyl bisulfate was found not to be a contaminant by recrystallization of authentic aminoethyl bisulfate in the presence of the rH]taurine fraction. After three recrystallizations, only 0.4% of the added radioactivity remained in aminoethyl bisulfate. The presence of esters (i.e., sulfates, phosphates) was examined by hydrolysis at 100°C with 1 N NaOH for 20 min. All activity was retained in the taurine peak. To remove any thiotaurine or hypotaurine, a sample of [3H]taurine was boiled vigorously for 30 set and cooled, and two drops of hydrogen peroxide (30%) and one drop of saturated ammonium molybdate were added. This procedure converts both hypotaurine and thiotaurine to taurine. The sample was left overnight. Isotopic dilution analysis revealed that 80% of the activity was taurine. [3H]Taurine (0.5 ml of 80% purity) was further purified by chromatography on Eastman Chromatogram Cellulose tic plates, along with authentic unlabeled taurine. The solvent system used was acetone: formic acid:water (16:3:9). Two radioactive peaks were found when 15 l-cm chromatographic strips were counted. The R, value for taurine was 0.55 and for the contaminant, 0.80. The major peak, which cochromatographed with authentic taurine, contained 78-80% of the total radioactivity, whereas the second peak contained the remainder of the radioactivity. The radiolabeled portion which corresponded with authentic taurine was eluted from the tic plate with distilled water. Isotopic dilution analysis revealed that the rH]taurine was 100 + 1% pure. The chemical identity of the contaminant remains unknown.
570
SHORT COMMUNICATIONS
Determination of specijic activity. The concentration of the rH]taurine was determined by a fluorescamine assay procedure (2). A modification was made in the standard procedure by using 0.01 M instead of 0.2 M borate buffer. Radioactivity was determined by liquid scintillation spectrophotometry on a Nuclear Chicago Isocap 300 at a counting efficiency of 4%. These procedures revealed that the 100% pure [3H]taurine has a specific activity of 2.8 Wmmol. Procedure for isotopic dilution analysis. Authentic taurine (50 mg) in 1 ml of filtered water is mixed in a Craig tube (Cheronis, 1954) with a known quantity of radioactivity. The volume is reduced under a nitrogen stream, and one drop of ethanol is added to induce crystallization. The crystals are collected and dried in the Craig tube, and the specific activity is determined. The material is recrystallized to constant specific activity. In summary, we have obtained rH]taurine of specific activity of 2.8 Ci/mmol and 100% radiochemical purity. ACKNOWLEDGMENTS This work was supported by grants from the USPHS (HL 19394 and HL 20087 to RJH; MH 26967 to HIY). H. 1. Yamamura is the recipient of a Research Scientist Development Award from the National Institutes of Mental Health (MHOOO95).
REFERENCES 1. Barbeau, A., Tsukada, Y., and Inoue, N. (1976) in Taurine (Huxtable, R., and Barbeau, A., eds.), pp. 253-266, Raven Press, New York. 2. Bohlen, P., Stein, S., Dairman, W., and Udenfriend, S. (1973) Arch Biochem. Biophys. 155,213-220. 3. Cheronis, N. D. (1954) in Technique of Organic Chemistry (Weissberger, A., ed.), Vol. 6, pp. 49-54, Interscience, New York. 4. Hruska, R. E., Huxtable, R. J., Bressler, R., and Yamamura, H. I. (1976a) Fed. Proc. 35, 325. 5. Hruska, R. E., Huxtable, R. J., Bressler, R., and Yamamura, H. I. (1976b) Proc. West. Pharmacol. Sot. 19, 152-156. 6. Hruska, R. E., Bressler, R., and Yamamura, H. I. (1976~) Proc. Sot. Neurosci. 6, 583. 7. Huxtable, R., and Bressler, R. (1972) J. Nutr. 102, 805-814. 8. Lombardini, J. B. (1976) in Taurine (Huxtable, R., and Barbeau, A., eds.), pp. 311326, Raven Press, New York. 9. Oja, S. S., and Lalrdesmiiki, P. (1974) Med. Biol. 52, 138-143. 10. Peck, E. J., Jr., and Awapara, J. (1967) Biochim. Biophys. Acta 141,499-506. 11. Schmid, R., Sieghart, W., and Karobath, M. (1975) J. Neurochem. 25, 5-9. 12. Sieghart, W., and Karobath, M. (1974)J. Neurochem. 23, 911-915. 13. Snyder, S. H. (1975) Biochem. Pharmacol. 24, 1371-1374.
ROBERT E. HRUSKA RYAN J.HUXTABLE HENRY I. YAMAMURA Department of Pharmacology College of Medicine The University of Arizona Medical Center Tucson, Arizona 85724 Received March 29, 1976; accepted December 10, 1976