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The isolation of a 4-thiouridine-containing disulfide from oxidized Escherichia coli tRNA is not artifactual
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 187, No. 1, April 15, pp. 272-273, 1978
SHORT
COMMUNICATIONS
The Isolation of a 4-Thiouridine-Containing Disulfide from Oxidized tRNA Is Not Artifactual
Escherichia
co/i
Iodine-oxidized Escherichia coli tRNA yields an oxidized 4-tbiouridine dinucleoside after enzymatic digestion and dephosphorylation. The original isolation has been questioned on the basis of an observed instability of 4-thiouridine disulfide in Tria-Cl buffer and a persistent oxidizing capacity of some resins after exposure to iodine. It is here pointed out that the original isolation specifically negated both of these objections. The fragment recently isolated by Kaiser (Arch. B&hem. Biophys. 183, 421-431, 1977) appears to be very similar to the one previously reported, although probably neither one is the symmetrical 4-thiouridine disulfide. A recent paper by Kaiser (1) corroborates a report published a decade ago from this laboratory (2) on the isolation of an oxidized 4-thiouridine dinucleotide fragment from enzymatic hydrolysates of iodine-oxidized tRNA from Escherichia coli. However, Kaiser also states that our earlier isolation was based on artifact, a statement which is incorrect. Solutions of I-thiouridine (s’U),’ a minor nucleotide in E. coli tRNA, can be easily oxidized to the disulfide stage by KI IZ solutions and subsequently reduced back to the thiol by thiosulfate or other mild reducing agents (3). These changes can be followed spectrally, since the absorption maximum shifts from 331 nm in the thiol to 310 nm in the disulfide, with accompanying hypochromicity. The treatment of intact tRNA with KI. Iz produces similar changes in the s4U region of the absorption spectrum. The dinucleotides isolated by both laboratories from enzyme hydrolysates of iodine-oxidized tRNA exhibit .cPU-disulfide spectra and are reducible to s4U. In my paper, further purification of this fragment indicated that, on reduction, the spectrum was that of pure s4U, with no additional absorption at 280 nm, which might indicate that the disulfide had been asymmetrical. Certain other reactions in alkali also corresponded with those of L&Jdisulfide, and the fragment was accordingly identified as symmetrical s4U-disulfide. Kaiser presents two relevam observations. First, he finds that his s4U-disulfide is urn able in 0.2 M Tris-Cl, pH 7.3, and decomposes into s4U nd other fragments. Since Tris-Cl was the buffer I USI 1 during enzymatic hydrolysis of my oxidized tRNA, he concludes that any s4U in my preparation must have been converted to the reduced form during hydrolysis. The published spectra of my preparation, however, indicate that both before and after hydrolysis, the s4U remains disulfide in form and is reducible in both cases [Fig. 3 in Ref.
REFERENCES
WI. Second, Kaiser reports that Bio-Gel which has once been exposed to KI Iz retains an oxidizing capacity or r Abbreviation
“memory,” and that s4U subsequently contacting such Bio-Gel becomes oxidized. However, Sephadex resin does not behave in this fashion, and this was the material I used, as stated, to separate oxidized tRNA from excess KI ‘1~. Neutral water and salt solutions of s4U-disulfide are somewhat unstable, but the instability does not approach the level reported by Kaiser. I suspect that part of the discrepancy may lie in the fact that he uses oxidizing columns of Bio-Gel to produce his s4U-disulfide standard and his oxidized tRNAs. It may be that Bio-Gel oxidation proceeds beyond the disulfide to an oxidation stats that is more labile in solution. He remarks that the yields of s4U on reduction are substantially lower with Bio-Gel-oxidized preparations than with preparations produced by short treatment with cold KI ‘12. Since the original description of the s’U-dinucleotide fragment, it has become increasingly improbable that it is indeed the symmetrical s4U-disulfide. As more tRNA sequences appear, the scarcity of species containing more than one s4U is striking, and efforts to demonstrate tRNA dimers after oxidation have failed. However, the lack of non-s4U absorption around 280 MI in the “disulfide” points to some unusual bonding partner for s’U. The putative disulfide cannot be the s4U-C photodimer isolated by Favre et al. (4), since that product is desulfurated and not reversible on reduction. The moderate instability of known s4U-disulfide during prolonged incubation in Tris buffer also argues against the previous identification of the fragment as symmetrical s4U-disulfide, but it does not make the isolation of a s4U-containing fragment artifactual. Kaiser himself has contirrned that such a piece can bc isolated.
1. KAISER, I. I. (1977) Arch. Biochem. Biophys. 183, 421-431. 2. LIPSETT, M. N. (1967) J. Biol. Chem. 242, 4067-4071.
used: SW, 4-thiouridine.
272
COMMUNICATIONS: 3. MILLER, W. H., R~BLIN, WOOD, E. B. (1945) J. 2201- 2204. 4. FAVRE, A., MICHELSON, (1971) J. Mol. Biol. 58,
ISOLATION
OF A 4-THIOURIDINE-CONTAINING
R. O., JR., AND ASTAmer. Chem. Sot. 67, A. M., AND YANIV, 367-379.
M.
MARIE N. LIPSETT
DISULFIDE
27 3
Laboratory of Biochemistry and Metabolism National Institute of Arthritis, Metabolism, and Digestive Diseases National Institutes of Health Bethesda, Maryland 20014 Received November 7, 1977; revised December II, 1977
SHORT
COMMUNICATIONS
The Isolation of a 4-Thiouridine-Containing Disulfide from Oxidized tRNA Is Not Artifactual
Escherichia
co/i
Iodine-oxidized Escherichia coli tRNA yields an oxidized 4-tbiouridine dinucleoside after enzymatic digestion and dephosphorylation. The original isolation has been questioned on the basis of an observed instability of 4-thiouridine disulfide in Tria-Cl buffer and a persistent oxidizing capacity of some resins after exposure to iodine. It is here pointed out that the original isolation specifically negated both of these objections. The fragment recently isolated by Kaiser (Arch. B&hem. Biophys. 183, 421-431, 1977) appears to be very similar to the one previously reported, although probably neither one is the symmetrical 4-thiouridine disulfide. A recent paper by Kaiser (1) corroborates a report published a decade ago from this laboratory (2) on the isolation of an oxidized 4-thiouridine dinucleotide fragment from enzymatic hydrolysates of iodine-oxidized tRNA from Escherichia coli. However, Kaiser also states that our earlier isolation was based on artifact, a statement which is incorrect. Solutions of I-thiouridine (s’U),’ a minor nucleotide in E. coli tRNA, can be easily oxidized to the disulfide stage by KI IZ solutions and subsequently reduced back to the thiol by thiosulfate or other mild reducing agents (3). These changes can be followed spectrally, since the absorption maximum shifts from 331 nm in the thiol to 310 nm in the disulfide, with accompanying hypochromicity. The treatment of intact tRNA with KI. Iz produces similar changes in the s4U region of the absorption spectrum. The dinucleotides isolated by both laboratories from enzyme hydrolysates of iodine-oxidized tRNA exhibit .cPU-disulfide spectra and are reducible to s4U. In my paper, further purification of this fragment indicated that, on reduction, the spectrum was that of pure s4U, with no additional absorption at 280 nm, which might indicate that the disulfide had been asymmetrical. Certain other reactions in alkali also corresponded with those of L&Jdisulfide, and the fragment was accordingly identified as symmetrical s4U-disulfide. Kaiser presents two relevam observations. First, he finds that his s4U-disulfide is urn able in 0.2 M Tris-Cl, pH 7.3, and decomposes into s4U nd other fragments. Since Tris-Cl was the buffer I USI 1 during enzymatic hydrolysis of my oxidized tRNA, he concludes that any s4U in my preparation must have been converted to the reduced form during hydrolysis. The published spectra of my preparation, however, indicate that both before and after hydrolysis, the s4U remains disulfide in form and is reducible in both cases [Fig. 3 in Ref.
REFERENCES
WI. Second, Kaiser reports that Bio-Gel which has once been exposed to KI Iz retains an oxidizing capacity or r Abbreviation
“memory,” and that s4U subsequently contacting such Bio-Gel becomes oxidized. However, Sephadex resin does not behave in this fashion, and this was the material I used, as stated, to separate oxidized tRNA from excess KI ‘1~. Neutral water and salt solutions of s4U-disulfide are somewhat unstable, but the instability does not approach the level reported by Kaiser. I suspect that part of the discrepancy may lie in the fact that he uses oxidizing columns of Bio-Gel to produce his s4U-disulfide standard and his oxidized tRNAs. It may be that Bio-Gel oxidation proceeds beyond the disulfide to an oxidation stats that is more labile in solution. He remarks that the yields of s4U on reduction are substantially lower with Bio-Gel-oxidized preparations than with preparations produced by short treatment with cold KI ‘12. Since the original description of the s’U-dinucleotide fragment, it has become increasingly improbable that it is indeed the symmetrical s4U-disulfide. As more tRNA sequences appear, the scarcity of species containing more than one s4U is striking, and efforts to demonstrate tRNA dimers after oxidation have failed. However, the lack of non-s4U absorption around 280 MI in the “disulfide” points to some unusual bonding partner for s’U. The putative disulfide cannot be the s4U-C photodimer isolated by Favre et al. (4), since that product is desulfurated and not reversible on reduction. The moderate instability of known s4U-disulfide during prolonged incubation in Tris buffer also argues against the previous identification of the fragment as symmetrical s4U-disulfide, but it does not make the isolation of a s4U-containing fragment artifactual. Kaiser himself has contirrned that such a piece can bc isolated.
1. KAISER, I. I. (1977) Arch. Biochem. Biophys. 183, 421-431. 2. LIPSETT, M. N. (1967) J. Biol. Chem. 242, 4067-4071.
used: SW, 4-thiouridine.
272
COMMUNICATIONS: 3. MILLER, W. H., R~BLIN, WOOD, E. B. (1945) J. 2201- 2204. 4. FAVRE, A., MICHELSON, (1971) J. Mol. Biol. 58,
ISOLATION
OF A 4-THIOURIDINE-CONTAINING
R. O., JR., AND ASTAmer. Chem. Sot. 67, A. M., AND YANIV, 367-379.
M.
MARIE N. LIPSETT
DISULFIDE
27 3
Laboratory of Biochemistry and Metabolism National Institute of Arthritis, Metabolism, and Digestive Diseases National Institutes of Health Bethesda, Maryland 20014 Received November 7, 1977; revised December II, 1977