- Email: [email protected]
Ion-exchange thin-layer chromatography
480
SHORT COMMUNICATIONS
1.5 M solution in 0.05 M Tris buffer (pH 6.7). The elution rate was approximately 1 ml/min. Although the material is very expensive, the high quality of the separation, and the fact that it is possible to use the columns once more after bringing the MAK up to 0.2 M NaCl again, makes this method advantageous. There is no difference in the quality of separations with new and once-used MAK. However, there is one drawback to this method-that singlestrand DNA, which can normally be eluted only with a very alkaline solution, adheres to the material when the NaCl concentration described here is employed. REFERENCES 1. LERMANN, 2.
3. 4. 5.
6. 7.
L. S., Biochim. Biophvs. Acta 18, 132 (1955). MANDELL, J. D., AND HERSHEY, A. D., Anal. Biochem. 1, 66 (1960). HERSHEY, A. D., AND BUROI, E. J., J. Mol. BioZ. 2, 143 (1960). SUEOKA, N., AND CHENG, T. Y., J. Mol. BioZ. 4, 161 (1962). HOLOUBEK, V., Anal. Biochem. 18, 375 (1967). COLTER, J. S., BROWN, R. A., AND ELLEN, K. A. O., Biochim. Biophys. 31 (1962). RICHTER, G., AND SENGER, H., Biochim. Biophys. Acta 87, 502 (1964).
Acta
55,
H. ALTMANN I. DOLEJS F. FETTER Institute for Biology and Agriculture Reaktorzentrum Seibersdorf, Austria Received August 15, 1967
Ion-Exchange XVIII.
Detection
Thin-Layer
of Purine Derivatives by Phosphorescence
Chromatography in the Nanogram at 77°K
Range
Steele and Saent-Gyijrgyi (1) first described the low-temperature phosphorescence of adenine and some of its derivatives. More recently, the luminescent spectra of nucleic acid bases (2), nucleosides (2), mononucleotides (2, 3), dinucleotides (3), and polynucleotides (4-6) have been examined in detail. The data of Longworth et al. (2) indicate that adenine, guanine, and their respective derivatives fluoresce and phos-
SHORT
481
COMMUNICATIONS
phoresce in neutral and basic ethylene glycol-water glasses at 77”K, the temperature of liquid nitrogen. Upon protonation (pH < 4)) the phosphorescence of adenine nucleotides is completely quenched (1, 2). The pyrimidines uracil, thymine, and cytosine phosphoresce at 77°K only after they have lost a proton (pH = 11) (2). The pyrimidine nucleosides and nucleotides behave similarly to the parent bases, with the exception of cytosine derivatives, in which pentose substitutes for the N, proton, which ionizes in cytosine. Cytidine and its derivatives therefore do not phosphoresce at 77°K (2). Quantum yields of phosphorescence are considerably lower for most pyrimidine compounds than for purine compounds (2). Low-temperature fluorescence and phosphorescence have been occasionally used for the detection on chromatograms of various compounds (7-12), but it is not widely known that, for many aromatic and heterocyclic compounds, low-temperature fluorescence and/or phosphorescence are by far the most sensitive detection methods available. The present communication describes procedures for the detection and characterization of nucleotides by phosphorescence at 77’K on thin layers of PEIcellul0se.l Experimentad: PEI-cellulose thin layers were prepared on plastic sheets and washed by soaking in NaCl solution and water followed by ascending development with water (14). Nucleotides were applied 2 cm from the lower edge of the sheet and chromatographed by ascending irrigation with 1.0 M LiCl solution (15). After the layer had been dried in a current of cool air, it was treated in a darkroom with liquid nitrogen as follows. About 200 ml of liquid nitrogen was allowed to evaporate in a metal tray of suitable size, which was placed on a thick layer of insulating plastic material. After the tray had been precooled in this way, the chromatogram (layer side up) was put immediately in the tray and its center was held down with a bent spatula. Metal weights (150-200 gm), which had also been precooled with liquid nitrogen, were placed in the corners of the sheet. While the center of the sheet was still held down, liquid nitrogen was poured int,o the tray beside the sheet so that it formed a layer approximately 1 cm deep on top of the chromatogram. After a few seconds the spatula was removed from the center of the sheet. The layer was then illuminated briefly with the unfiltered radiation of a high-intensity ultraviolet lamp (Mineralight? model No. R 51) from a distance of about 10 cm (CAUTION: avoid looking into this light for prolonged periods of time). Bfter the ultraviolet lamp had been turned off, the ’ A cellulose anion-exchange lose with poly (ethyleneimine) ’ Ultraviolet Products, Inc.,
material obtained by (13). San Gabriel, California.
treating
chromatography
cellu-
482
SHORT
COMMUNICATIONS
phosphorescent afterglow was photographed (Polaroid” Copymaker model NO. 160, no filter, Polaroid 3000 speed, t’ype 47 film, exposure time l-3 set, exposure value = 10). Because of a rapidly decaying weak background phosphorescence, a brief delay of the exposure usually improved the contrast of the picture. Repeating illumination and photography several times had the same effect and increased the sensitivity of the procedure. Results: Figure IA is a photograph in short-ware ultraviolet light of a chromatogram of 5’-AMP, ADP, ATP, 5’-GMP, GDP, and GTP in amounts ranging from 8 ppmoles to 20 X IO” p,umoles. Figures 1B and IC represent pictures of the same chromatogram taken after illumination at 77°K with a short-wave ultraviolet light source. Figure 1C was obtained after the chromatogram had been sprayed t,horoughly with 0.2 M NH, citrate, pH 2.9, and dried. It can be seen that, whereas the limit of detection by ultraviolet light at room temperature is in the 100-200 ppmole range (Fig. IA), amounts as low as 8 pprnoles can be easily detected by low-temperature phosphorescence (Fig. 1B). Spraying with pH 2.9 citrate almost completely quenches the phosphorescence of the adenine nucleotides (Fig. 1C) and thus permits distinguishing between extremely small amounts of adenine and guanine derivatives. Phosphorescence of guanine nucleotides can be effectively quenched by spraying with 1 N HCl and subjecting the still wet layer to liquid nitrogen treatment. The residual phosphorescence is weak and decays much more rapidly t’han at neutral pH. In our experience, the limit of detection of adenine and guanine mononucleotides at neutral pH on one-dimensional chromatograms by the method described is between 1 and 3 ppmolcs (about 0.5-1.5 nanograms). The eye appears to be ten to twenty times less sensitive than the photographic emulsion. Sensitivity appears to be greatest immediately after the last visible trace of nitrogen has evaporated from the surface of the sheet. The intensity of phosphorescence depends strongly on the type of shortwave ultraviolet lamp used for irradiation. Removal of the filter from the lamp is required only for the detection of amounts below 2 p+oles under the conditions used. Figure 2 depicts a two-dimensional map on PEI-cellulose of di- and trinueleotides from a pancreatic ribonuclease digest of high molecular weight RNA (16) photographed after illumination at 77°K. The minor components W, X, and Y could not be made visible by ultraviolet absorption. No phosphorescence of hypoxanthine derivatives was observed under neutral or acidic conditions; inosine nucleotides did, however, phos‘Polaroid
Corp.,
Cambridge,
Massachusetts.
SHORT
COMMUNICATIONS
FIG. 1. PEI-cellulose thin-layer chromatogram of 5’-AMP, ADP, ATP, 5’-GMP, GDP, and GTP: (A) photography by short-wave ultraviolet light at room temperature, (B) photography of afterglow at. 77°K. CC) photography of afterglow at 77°K after the layer had been sprayed with 0.2 ill NH, citrate, pH 2.9. Development with l.OM LiCl up to 7 cm above line of application. Applied: 1 ~1 of solutions containing different concentrations of thca caomgounds. Amounts, in ppmoles, of each nucleotide (l-5, AMP + ADP + ATP: 6-10, GMP + GDP + GTP) : (1) 20 x lOa; (2) 2 X10’; (3) 200; (4) 20: (5) 8; (6) 20 x lff; (7) 2 x 103; (8) 200; (9) 20; (10) 8.
phoresce at 77’K after the layer had been exposed to ammonia fumes or Eprayed with dilute alkali (sensitivity about 100 ppmoles) . Thymidine derivatives also exhibited low-temperature phosphorescence on alkaline layers, whereas cytidine and uridine nucleotides did not phosphoresce at alkaline, neutral, or acidic pH. Conclusions: The combination of PEI-cellulose t,hin-layer chromatography and low-temperature phosphorescence appears to be the most
484
SHORT
COMMUNICATIONS
e U
Acn
‘,*: -,
00 GGC
GGU
FIG. 2. Two-dimensional map of di- and trinucleotitl:,a on a PEI-rcllulosc (16). First dimension, from right to left: second dimcneion, from bottom The chromatogram was cut 5 cm above the origin of the second dimension. tography of afterglow at 77”Ii. Abbw~~iatior~s~ . AC = ApCp, etc., as published issue’s of the J. BioI. ClJe,,r. W,S,T,Z, unknown compounds.
layer top. Phoin the
to
sensitive chromatographic method for unlabeled adenine and guanine derivatives available at present. It is 50-100 times more sensitive than detection by ultraviolet absorption on the same layer and over 2000 times more sensitive than detection by absorption on paper chromatograms.
SHORT
485
COMMUNICATIONS
The method presented allows one to distinguish between purine derivatives at concentrations too low for spectrophotometry. Because of its sensitivity it will facilitate the detection and tentative identification of minor components in biological extracts and in digest’s of nucleic acids. In addition, it appears that. low-temperature phosphorimetry can serve as a sensitive method for quantitative estimation of mono- and oligonucleotides, because luminescence techniques are usually considerably more sensitive than absorptiometric methods (cf. ref. 17). ACKNOWLEDGMENTS This work has been supported by grants-in-aid from the U. S. Atomic Energy commission (AT(30-l&2643) and the U. S. Public Health Service (CA 05018-11). This is publication No. 1309 of the Cancer Commission of Harvard University. REFERENCES 1. STJSELE,It. H., AND SZENT-GP~RGYI, A., Proc. N&l. Acad. Sci. U. S. 43, 477 (1957). 2. LONCIWOHTH. J. W., RAHS, R. O., AND SHULMAN, R. G., J. Chem. Phys. 45, 2930 (1966). 3. HB~ise, C., AND MICHELSON, A. M., Biochim. Biophgs. Acta 142, 12 (1967). 4. R~HN, R. O., Y~M.~NE, T., &SINGER, J., LONGWORTH, J. W., AND SHULMAN, R. G., J. Chem. Phys. 45, 2947 (1966). 5. R.4HN, R. O., SHULMAN, R. G., AND LQSG~ORTH, J. W., J. Chem. Phys. 45, 2955 (1966). 6. STEINER, R. F., MILLAR, D. B., AND HoERMAx, K. C., Arch. Biochem. Biophys. 120, 464 (1967). 7. SZENT-GY~RGYI, A., Science 126, 351 (1957). 8. ZANDER, M., Erddl und Kohle 15, 362 (1962). 9. GORDON, M. P., AND SOUTH, D., J. Chromatog. 10, 513 (1963). 10. SAWICKI, E., AND JOHNSON, H., Microchem. J. 8, 83 (1964). 11. ELUOTT, W. B., .~ND KLINGMAN, J. D., Nature 266, 1044 (1965). 12. CHOU. .J. S. T., .IND LAWRENCE, B. M., J. Chromatog. 27, 279 (1967). 13. RANDERATH, K., Angew. Chena. 74, 780 (1962) ; Intern. Ed. 1, 553 (1962). 14. R.~XDEHATH, K., AND RANDERATH, E., J. Chromatog. 22, 110 (1966). 15. RANDERATH, K., AND RANDERATH, E., J. Chromatog. 16, 111 (1964). 16. RANDER.\TH, K., AND RANDERATH, E., J. Chromatog., in press (1967). 17. WINEFORDXER, J. D., in “Fluorescence and Phosphorescence Analysis” (D. M. Herc.ulrs, rd.), p. 169. Interscience, New York, 1966. K. Joh?L Collins Warren Laboratories of the Huntington of Harvard University at the Massachusetts General Boston, Massachusetts 02114 Receiwd Jnly 10, 19fT7
Memorial Hospital
Hospital
RANDER4TH
SHORT COMMUNICATIONS
1.5 M solution in 0.05 M Tris buffer (pH 6.7). The elution rate was approximately 1 ml/min. Although the material is very expensive, the high quality of the separation, and the fact that it is possible to use the columns once more after bringing the MAK up to 0.2 M NaCl again, makes this method advantageous. There is no difference in the quality of separations with new and once-used MAK. However, there is one drawback to this method-that singlestrand DNA, which can normally be eluted only with a very alkaline solution, adheres to the material when the NaCl concentration described here is employed. REFERENCES 1. LERMANN, 2.
3. 4. 5.
6. 7.
L. S., Biochim. Biophvs. Acta 18, 132 (1955). MANDELL, J. D., AND HERSHEY, A. D., Anal. Biochem. 1, 66 (1960). HERSHEY, A. D., AND BUROI, E. J., J. Mol. BioZ. 2, 143 (1960). SUEOKA, N., AND CHENG, T. Y., J. Mol. BioZ. 4, 161 (1962). HOLOUBEK, V., Anal. Biochem. 18, 375 (1967). COLTER, J. S., BROWN, R. A., AND ELLEN, K. A. O., Biochim. Biophys. 31 (1962). RICHTER, G., AND SENGER, H., Biochim. Biophys. Acta 87, 502 (1964).
Acta
55,
H. ALTMANN I. DOLEJS F. FETTER Institute for Biology and Agriculture Reaktorzentrum Seibersdorf, Austria Received August 15, 1967
Ion-Exchange XVIII.
Detection
Thin-Layer
of Purine Derivatives by Phosphorescence
Chromatography in the Nanogram at 77°K
Range
Steele and Saent-Gyijrgyi (1) first described the low-temperature phosphorescence of adenine and some of its derivatives. More recently, the luminescent spectra of nucleic acid bases (2), nucleosides (2), mononucleotides (2, 3), dinucleotides (3), and polynucleotides (4-6) have been examined in detail. The data of Longworth et al. (2) indicate that adenine, guanine, and their respective derivatives fluoresce and phos-
SHORT
481
COMMUNICATIONS
phoresce in neutral and basic ethylene glycol-water glasses at 77”K, the temperature of liquid nitrogen. Upon protonation (pH < 4)) the phosphorescence of adenine nucleotides is completely quenched (1, 2). The pyrimidines uracil, thymine, and cytosine phosphoresce at 77°K only after they have lost a proton (pH = 11) (2). The pyrimidine nucleosides and nucleotides behave similarly to the parent bases, with the exception of cytosine derivatives, in which pentose substitutes for the N, proton, which ionizes in cytosine. Cytidine and its derivatives therefore do not phosphoresce at 77°K (2). Quantum yields of phosphorescence are considerably lower for most pyrimidine compounds than for purine compounds (2). Low-temperature fluorescence and phosphorescence have been occasionally used for the detection on chromatograms of various compounds (7-12), but it is not widely known that, for many aromatic and heterocyclic compounds, low-temperature fluorescence and/or phosphorescence are by far the most sensitive detection methods available. The present communication describes procedures for the detection and characterization of nucleotides by phosphorescence at 77’K on thin layers of PEIcellul0se.l Experimentad: PEI-cellulose thin layers were prepared on plastic sheets and washed by soaking in NaCl solution and water followed by ascending development with water (14). Nucleotides were applied 2 cm from the lower edge of the sheet and chromatographed by ascending irrigation with 1.0 M LiCl solution (15). After the layer had been dried in a current of cool air, it was treated in a darkroom with liquid nitrogen as follows. About 200 ml of liquid nitrogen was allowed to evaporate in a metal tray of suitable size, which was placed on a thick layer of insulating plastic material. After the tray had been precooled in this way, the chromatogram (layer side up) was put immediately in the tray and its center was held down with a bent spatula. Metal weights (150-200 gm), which had also been precooled with liquid nitrogen, were placed in the corners of the sheet. While the center of the sheet was still held down, liquid nitrogen was poured int,o the tray beside the sheet so that it formed a layer approximately 1 cm deep on top of the chromatogram. After a few seconds the spatula was removed from the center of the sheet. The layer was then illuminated briefly with the unfiltered radiation of a high-intensity ultraviolet lamp (Mineralight? model No. R 51) from a distance of about 10 cm (CAUTION: avoid looking into this light for prolonged periods of time). Bfter the ultraviolet lamp had been turned off, the ’ A cellulose anion-exchange lose with poly (ethyleneimine) ’ Ultraviolet Products, Inc.,
material obtained by (13). San Gabriel, California.
treating
chromatography
cellu-
482
SHORT
COMMUNICATIONS
phosphorescent afterglow was photographed (Polaroid” Copymaker model NO. 160, no filter, Polaroid 3000 speed, t’ype 47 film, exposure time l-3 set, exposure value = 10). Because of a rapidly decaying weak background phosphorescence, a brief delay of the exposure usually improved the contrast of the picture. Repeating illumination and photography several times had the same effect and increased the sensitivity of the procedure. Results: Figure IA is a photograph in short-ware ultraviolet light of a chromatogram of 5’-AMP, ADP, ATP, 5’-GMP, GDP, and GTP in amounts ranging from 8 ppmoles to 20 X IO” p,umoles. Figures 1B and IC represent pictures of the same chromatogram taken after illumination at 77°K with a short-wave ultraviolet light source. Figure 1C was obtained after the chromatogram had been sprayed t,horoughly with 0.2 M NH, citrate, pH 2.9, and dried. It can be seen that, whereas the limit of detection by ultraviolet light at room temperature is in the 100-200 ppmole range (Fig. IA), amounts as low as 8 pprnoles can be easily detected by low-temperature phosphorescence (Fig. 1B). Spraying with pH 2.9 citrate almost completely quenches the phosphorescence of the adenine nucleotides (Fig. 1C) and thus permits distinguishing between extremely small amounts of adenine and guanine derivatives. Phosphorescence of guanine nucleotides can be effectively quenched by spraying with 1 N HCl and subjecting the still wet layer to liquid nitrogen treatment. The residual phosphorescence is weak and decays much more rapidly t’han at neutral pH. In our experience, the limit of detection of adenine and guanine mononucleotides at neutral pH on one-dimensional chromatograms by the method described is between 1 and 3 ppmolcs (about 0.5-1.5 nanograms). The eye appears to be ten to twenty times less sensitive than the photographic emulsion. Sensitivity appears to be greatest immediately after the last visible trace of nitrogen has evaporated from the surface of the sheet. The intensity of phosphorescence depends strongly on the type of shortwave ultraviolet lamp used for irradiation. Removal of the filter from the lamp is required only for the detection of amounts below 2 p+oles under the conditions used. Figure 2 depicts a two-dimensional map on PEI-cellulose of di- and trinueleotides from a pancreatic ribonuclease digest of high molecular weight RNA (16) photographed after illumination at 77°K. The minor components W, X, and Y could not be made visible by ultraviolet absorption. No phosphorescence of hypoxanthine derivatives was observed under neutral or acidic conditions; inosine nucleotides did, however, phos‘Polaroid
Corp.,
Cambridge,
Massachusetts.
SHORT
COMMUNICATIONS
FIG. 1. PEI-cellulose thin-layer chromatogram of 5’-AMP, ADP, ATP, 5’-GMP, GDP, and GTP: (A) photography by short-wave ultraviolet light at room temperature, (B) photography of afterglow at. 77°K. CC) photography of afterglow at 77°K after the layer had been sprayed with 0.2 ill NH, citrate, pH 2.9. Development with l.OM LiCl up to 7 cm above line of application. Applied: 1 ~1 of solutions containing different concentrations of thca caomgounds. Amounts, in ppmoles, of each nucleotide (l-5, AMP + ADP + ATP: 6-10, GMP + GDP + GTP) : (1) 20 x lOa; (2) 2 X10’; (3) 200; (4) 20: (5) 8; (6) 20 x lff; (7) 2 x 103; (8) 200; (9) 20; (10) 8.
phoresce at 77’K after the layer had been exposed to ammonia fumes or Eprayed with dilute alkali (sensitivity about 100 ppmoles) . Thymidine derivatives also exhibited low-temperature phosphorescence on alkaline layers, whereas cytidine and uridine nucleotides did not phosphoresce at alkaline, neutral, or acidic pH. Conclusions: The combination of PEI-cellulose t,hin-layer chromatography and low-temperature phosphorescence appears to be the most
484
SHORT
COMMUNICATIONS
e U
Acn
‘,*: -,
00 GGC
GGU
FIG. 2. Two-dimensional map of di- and trinucleotitl:,a on a PEI-rcllulosc (16). First dimension, from right to left: second dimcneion, from bottom The chromatogram was cut 5 cm above the origin of the second dimension. tography of afterglow at 77”Ii. Abbw~~iatior~s~ . AC = ApCp, etc., as published issue’s of the J. BioI. ClJe,,r. W,S,T,Z, unknown compounds.
layer top. Phoin the
to
sensitive chromatographic method for unlabeled adenine and guanine derivatives available at present. It is 50-100 times more sensitive than detection by ultraviolet absorption on the same layer and over 2000 times more sensitive than detection by absorption on paper chromatograms.
SHORT
485
COMMUNICATIONS
The method presented allows one to distinguish between purine derivatives at concentrations too low for spectrophotometry. Because of its sensitivity it will facilitate the detection and tentative identification of minor components in biological extracts and in digest’s of nucleic acids. In addition, it appears that. low-temperature phosphorimetry can serve as a sensitive method for quantitative estimation of mono- and oligonucleotides, because luminescence techniques are usually considerably more sensitive than absorptiometric methods (cf. ref. 17). ACKNOWLEDGMENTS This work has been supported by grants-in-aid from the U. S. Atomic Energy commission (AT(30-l&2643) and the U. S. Public Health Service (CA 05018-11). This is publication No. 1309 of the Cancer Commission of Harvard University. REFERENCES 1. STJSELE,It. H., AND SZENT-GP~RGYI, A., Proc. N&l. Acad. Sci. U. S. 43, 477 (1957). 2. LONCIWOHTH. J. W., RAHS, R. O., AND SHULMAN, R. G., J. Chem. Phys. 45, 2930 (1966). 3. HB~ise, C., AND MICHELSON, A. M., Biochim. Biophgs. Acta 142, 12 (1967). 4. R~HN, R. O., Y~M.~NE, T., &SINGER, J., LONGWORTH, J. W., AND SHULMAN, R. G., J. Chem. Phys. 45, 2947 (1966). 5. R.4HN, R. O., SHULMAN, R. G., AND LQSG~ORTH, J. W., J. Chem. Phys. 45, 2955 (1966). 6. STEINER, R. F., MILLAR, D. B., AND HoERMAx, K. C., Arch. Biochem. Biophys. 120, 464 (1967). 7. SZENT-GY~RGYI, A., Science 126, 351 (1957). 8. ZANDER, M., Erddl und Kohle 15, 362 (1962). 9. GORDON, M. P., AND SOUTH, D., J. Chromatog. 10, 513 (1963). 10. SAWICKI, E., AND JOHNSON, H., Microchem. J. 8, 83 (1964). 11. ELUOTT, W. B., .~ND KLINGMAN, J. D., Nature 266, 1044 (1965). 12. CHOU. .J. S. T., .IND LAWRENCE, B. M., J. Chromatog. 27, 279 (1967). 13. RANDERATH, K., Angew. Chena. 74, 780 (1962) ; Intern. Ed. 1, 553 (1962). 14. R.~XDEHATH, K., AND RANDERATH, E., J. Chromatog. 22, 110 (1966). 15. RANDERATH, K., AND RANDERATH, E., J. Chromatog. 16, 111 (1964). 16. RANDER.\TH, K., AND RANDERATH, E., J. Chromatog., in press (1967). 17. WINEFORDXER, J. D., in “Fluorescence and Phosphorescence Analysis” (D. M. Herc.ulrs, rd.), p. 169. Interscience, New York, 1966. K. Joh?L Collins Warren Laboratories of the Huntington of Harvard University at the Massachusetts General Boston, Massachusetts 02114 Receiwd Jnly 10, 19fT7
Memorial Hospital
Hospital
RANDER4TH