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Quantitative two-dimensional thin-layer chromatography of deoxyribonucleic acid components
ANALYTICAL
BIOCHEMISTRY
Q,uantitative
94-99
(1964)
Two-Dimensional
raphy ZDZISLAW From
9,
the
Thin-Layer
of Deoxyribonucleic
Acid
F. CHMIELEWICZ
AND
Department of Medicinal Chemistry, State University of New York Buffalo, New York
Chromatog-
Components
MARGARET School
ACARA
of Pharmacy,
at Bufialo,
Received October 25, 1963
In studying the various parameters of normal and malignant mammalian tissues, it became apparent that there was a need for a rapid quantitative procedure for the determination of deoxyribosides and deoxyribotides. A new technique, thin-layer chromatography (1)) has shown great versatility in the separation of different types of chemical compounds. Application of this technique for the separation of various ribonucleosides and ribonucleotides has been recently reported by Randerath (2, 3), but his procedure does not give sufficient resolution for quantitative determination. We wish to report a two-dimensional thin-layer chromatographic method for the quantitative determination of deoxyribosides and their 5’-phosphates. PRELIMINARY
STUDIES
Three solvent systems were used in the preliminary single-dimensional chromatographic studies : (A) n-Butanol, water, 90% formic acid (60:30:10). The top layer, after equilibrating for 48 hr, was used for development of the chromatogram, while the bottom phase was used for equilibration of the plates as described below. (B) n-Butanol, ethanol, 5 N hydrochloric acid (4) (30:20:20). (C) n-Butanol, acetone, acetic acid, 5% aqueous ammonia, water (2) (3.5:2.5: 1.5: 1.5: 1.0). The plates were prepared in the usual manner using cellulose powder containing CaS04 as binder.l After drying for 30 min at room temperature, the plates were further dried at 105°C for 30 min and stored in a closed container over calcium chloride. ’ Excorna Zellulosepulver (with Inc., Great Neck, L. I., ?;. Y.
CaSO,) 94
obtained
from Brinkmann
Instruments,
After application of the samples containing the various deoxyribosides and their 5’-phosphates, individually or in mixture, the plates were equilibrated for 1 hr in appropriate chromatographic chambers. For solvent systems A or B, 20 X 20 cm plates were used, which were placed in a 7.5 X 27.5 X 26.5 cm glass chamber, and the solvent front was allowed to travel a distance of 14 cm from the starting line in approximately 2-2.5 hr. After removal of the plates from the chamber and evaporation of the solvent, the positions of the various components on the plate were located as fluorescent spots when observed in a viewing cabinet under ultraviolet light (2537 A). In the case of solvent A, complete separation of the four deoxyribosides was achieved; of the deoxyribotidcs, however, only thymidylic acid could be separated while the other three deoxyribotides migrated approximately equal distances. When solvent B was used, each deoxyriboside appeared to migrate with the same Rf value as its 5’-phosphate. Elution and analysis of the fluorescent areas indicated that the pyrimidine derivatives were unchanged but the purine deoxyribosides and deoxyribotides were quantitatively hydrolyzed by the highly acidic solvent B to their free bases. That the hydrolysis occurred quantitatively was shown by the absorbancies at x,,, and by the negative tests for deoxyribose and phosphorus of both acidic and basic eluates of the fluorescent areas corresponding to the purine derivatives. Thus, the four fluorescent spots obtained on chromatography with solvent B of a mixture containing all eight components, actually represent (1) thymidine plus thymidine 5’-phosphate, (2) deoxycytidine plus deoxycytidine 5’-phosphate, (3) adenine (derived from deoxyadenosine and dcoxyadenosine 5’-phosphate), and (4) guanine (derived from deoxyguanosine and deoxyguanosine 5’-phosphate). Attempts to effect separations on 20 X 20 cm plates with solvent C were unsuccessful ; however, when this solvent system was employed with 40 X 20 cm plates, using a 46 X 24.5 diameter glass jar, and the solvent front was allowed to move 27 cm, which required 5-6 hr, almost complete separation was attained. The results of the single-dimensional chromatography are given in Table 1. As can be seen from the R, values, all eight compounds could not be sufIiciently separated for quantitative determination with a single solvent system; however, when two of these solvent systems were employed in a two-dimensional chromatogram, complete separations were achieved. Using the solvent system pairs A and B, or C and B, respectively, two different equally effective chromatographic analysis procedures were developed.
96
CHMIELEWICZ
AND
ACARA
Solvent Compound”
A
dA dC dG dT dAMP dCMP dGMP dTMP
0.48 0.35 0.26 0.75 0.07 0.09 0.05
0.18
systema II
0.3-F 0.55 0.2id 0.8Y 0 .33” 0.57 0.2id 0.90
C
0.81 0.77 O.&l 0.84 0.54 0.49 0.33 0.58
(1See text for composition of solvent systems. b Abbreviations: dA = deoxyadenosine; dC = deoxycytosine; dG = deoxyguanosine; dT = thymidine; dAMP = deoxyadenylic acid; dCMP = deoxycytidylic acid; dGMP = deoxyguanylic acid; dTMP = thymidylic acid. c Adenine (see text). d Guanine (see text). PROCEDURES
In the first procedure, 20 X 20 cm plates were developed first with solvent A, then dried and subsequently developed with solvent B in the other dimension. In the second procedure, 40 x 20 cm plates were used. These were first developed in the long direction with solvent C, and then in the short direction with solvent B ; in the latter case, a 7.5 X 46 X 26 cm chamber was used which was constructed from plywood, lined with solvent-resistant, thermosetting, high-pressure laminate, and equipped with a viewing window of Thermopane glass. In both procedures, the sample, containing the individual compounds, or their mixture, was applied at a distance of 1.5 cm from the bottom as well as from one side of the plate. Each solvent system had to be equilibrated, in the manner described above for the single-dimension chromatograms, in order to obtain reproducible Rf values. Figures 1 and 2 illustrate the positions on the thin layer of the eight fluorescent spots corresponding to the eight nucleosides and nucleotides (i.e., their degradation products in the case of the purine derivatives), using the two procedures. Table 2 gives the numerical ‘LRfczJ” and “R f(Y) ” values, that is, the two Cartesian coordinates necessary to define the position of each spot on the two-dimensional chromatograms. These values were consistently reproducible and in good agreement with the corresponding single-dimensional RI values obtained with each solvent system separately (see Table 1).
TLC
AKALYSIS
Solvent
OF DrSA
B
97
COMPONENTI;
A
FIG. 1. Two-dimensional chromatography of deoxyribosides and deoxyribotides on 20 x 20 cm plates, with solvents A and B: 0 = origin, 1 = dGMP, 2 = dAMP, 3=dCMP, 4=dTMP, 5=dG, 6=dC, 7=dA, 8=dT.
TABLE Rfc,, AND tlrcrj
VALUES
dA dC dG dT dAMP dCMP dGMP dTMP
0.47 0.34 0.25 0.76 0.06 0.07 0.05 0.16
OBTAINED
2
IN TWO-DIMENSION.IL~CHROMATOGRAPHT
0.32 0.54 0.26 0.88 0.34 0.56 0.27 0.90
0.80 0.78 0.64 0.86 0.54 0.50 0.33 0.58
0.32 0.56 0.25 0.90 0.32 0.58 0.25 0.91
a See text for composition of solvent systems A, B, and C. b As originally present in mixture before chromatography (see Table 1 for abbreviations).
For the quantitative estimations, the areas of fluorescence under UV light were outlined and scraped from the plate. The compounds were eluted from the scrapings by occasional shaking with 0.1 N hydrochloric acid for 16 hr at room temperature. After filtration, the ultraviolet spectra of these eluates were determined and their absorbancies at X,,, were compared with those of standard solutions of the corresponding compounds. Since the purine deoxyribosides and their 5’-phosphates were
CHMIELEWICZ
AND
ACARA
0
Solvent
B
FIG. 2. Two-dimensional chromatography of deoxyribosides and deoxyribotides 40 X 20 cm plates, with solvents C and B. See Fig. 1 for symbols.
on
recovered from their respective fluorescent areas in the form of theoretically equimolar amounts of their free bases (see above), standard solutions of adenine and guanine were used for the quantitative estimation of these compounds. To compensate for the ultraviolet absorption of the acid-soluble materials originating from cellulose powder, appropriate nonfluorescent areas were also scraped, extracted with hydrochloric acid under the same conditions, and used as blanks. Using these procedures, recoveries of 95-102s were routinely achieved. It should be emphasized that the procedures presented in this paper are useful as analytical methods for the identification and quantitative determination of pyrimidine as well as purine deoxyribosides and deoxyribotides, but, because of the acidity of solvent B, they cannot be used for the isolation of the purine derivntives.
ACKNOWLEDGMEST The authors gratefully acknowledge the helpful advice of Dr. T. J. Bardos. This work was supported by a research grant (C&4-05522) from the Xational Cancer Institute, U. S. Public Health Service. REFERETCES E., Pharmazie 11, 633 (1956). K., Biochem. Biophys. Research Comlmuru. 6, 452 (1062). RANDERATH, K., Angew. Chem., Intern. Ed. Engl. 1, 435 (1962). VISCHER, E., CHARGAFF, E., J. Biol. Chem. 176, 703 (1948).
1. STAHL,
2. RANDERATH, 3. 4.
BIOCHEMISTRY
Q,uantitative
94-99
(1964)
Two-Dimensional
raphy ZDZISLAW From
9,
the
Thin-Layer
of Deoxyribonucleic
Acid
F. CHMIELEWICZ
AND
Department of Medicinal Chemistry, State University of New York Buffalo, New York
Chromatog-
Components
MARGARET School
ACARA
of Pharmacy,
at Bufialo,
Received October 25, 1963
In studying the various parameters of normal and malignant mammalian tissues, it became apparent that there was a need for a rapid quantitative procedure for the determination of deoxyribosides and deoxyribotides. A new technique, thin-layer chromatography (1)) has shown great versatility in the separation of different types of chemical compounds. Application of this technique for the separation of various ribonucleosides and ribonucleotides has been recently reported by Randerath (2, 3), but his procedure does not give sufficient resolution for quantitative determination. We wish to report a two-dimensional thin-layer chromatographic method for the quantitative determination of deoxyribosides and their 5’-phosphates. PRELIMINARY
STUDIES
Three solvent systems were used in the preliminary single-dimensional chromatographic studies : (A) n-Butanol, water, 90% formic acid (60:30:10). The top layer, after equilibrating for 48 hr, was used for development of the chromatogram, while the bottom phase was used for equilibration of the plates as described below. (B) n-Butanol, ethanol, 5 N hydrochloric acid (4) (30:20:20). (C) n-Butanol, acetone, acetic acid, 5% aqueous ammonia, water (2) (3.5:2.5: 1.5: 1.5: 1.0). The plates were prepared in the usual manner using cellulose powder containing CaS04 as binder.l After drying for 30 min at room temperature, the plates were further dried at 105°C for 30 min and stored in a closed container over calcium chloride. ’ Excorna Zellulosepulver (with Inc., Great Neck, L. I., ?;. Y.
CaSO,) 94
obtained
from Brinkmann
Instruments,
After application of the samples containing the various deoxyribosides and their 5’-phosphates, individually or in mixture, the plates were equilibrated for 1 hr in appropriate chromatographic chambers. For solvent systems A or B, 20 X 20 cm plates were used, which were placed in a 7.5 X 27.5 X 26.5 cm glass chamber, and the solvent front was allowed to travel a distance of 14 cm from the starting line in approximately 2-2.5 hr. After removal of the plates from the chamber and evaporation of the solvent, the positions of the various components on the plate were located as fluorescent spots when observed in a viewing cabinet under ultraviolet light (2537 A). In the case of solvent A, complete separation of the four deoxyribosides was achieved; of the deoxyribotidcs, however, only thymidylic acid could be separated while the other three deoxyribotides migrated approximately equal distances. When solvent B was used, each deoxyriboside appeared to migrate with the same Rf value as its 5’-phosphate. Elution and analysis of the fluorescent areas indicated that the pyrimidine derivatives were unchanged but the purine deoxyribosides and deoxyribotides were quantitatively hydrolyzed by the highly acidic solvent B to their free bases. That the hydrolysis occurred quantitatively was shown by the absorbancies at x,,, and by the negative tests for deoxyribose and phosphorus of both acidic and basic eluates of the fluorescent areas corresponding to the purine derivatives. Thus, the four fluorescent spots obtained on chromatography with solvent B of a mixture containing all eight components, actually represent (1) thymidine plus thymidine 5’-phosphate, (2) deoxycytidine plus deoxycytidine 5’-phosphate, (3) adenine (derived from deoxyadenosine and dcoxyadenosine 5’-phosphate), and (4) guanine (derived from deoxyguanosine and deoxyguanosine 5’-phosphate). Attempts to effect separations on 20 X 20 cm plates with solvent C were unsuccessful ; however, when this solvent system was employed with 40 X 20 cm plates, using a 46 X 24.5 diameter glass jar, and the solvent front was allowed to move 27 cm, which required 5-6 hr, almost complete separation was attained. The results of the single-dimensional chromatography are given in Table 1. As can be seen from the R, values, all eight compounds could not be sufIiciently separated for quantitative determination with a single solvent system; however, when two of these solvent systems were employed in a two-dimensional chromatogram, complete separations were achieved. Using the solvent system pairs A and B, or C and B, respectively, two different equally effective chromatographic analysis procedures were developed.
96
CHMIELEWICZ
AND
ACARA
Solvent Compound”
A
dA dC dG dT dAMP dCMP dGMP dTMP
0.48 0.35 0.26 0.75 0.07 0.09 0.05
0.18
systema II
0.3-F 0.55 0.2id 0.8Y 0 .33” 0.57 0.2id 0.90
C
0.81 0.77 O.&l 0.84 0.54 0.49 0.33 0.58
(1See text for composition of solvent systems. b Abbreviations: dA = deoxyadenosine; dC = deoxycytosine; dG = deoxyguanosine; dT = thymidine; dAMP = deoxyadenylic acid; dCMP = deoxycytidylic acid; dGMP = deoxyguanylic acid; dTMP = thymidylic acid. c Adenine (see text). d Guanine (see text). PROCEDURES
In the first procedure, 20 X 20 cm plates were developed first with solvent A, then dried and subsequently developed with solvent B in the other dimension. In the second procedure, 40 x 20 cm plates were used. These were first developed in the long direction with solvent C, and then in the short direction with solvent B ; in the latter case, a 7.5 X 46 X 26 cm chamber was used which was constructed from plywood, lined with solvent-resistant, thermosetting, high-pressure laminate, and equipped with a viewing window of Thermopane glass. In both procedures, the sample, containing the individual compounds, or their mixture, was applied at a distance of 1.5 cm from the bottom as well as from one side of the plate. Each solvent system had to be equilibrated, in the manner described above for the single-dimension chromatograms, in order to obtain reproducible Rf values. Figures 1 and 2 illustrate the positions on the thin layer of the eight fluorescent spots corresponding to the eight nucleosides and nucleotides (i.e., their degradation products in the case of the purine derivatives), using the two procedures. Table 2 gives the numerical ‘LRfczJ” and “R f(Y) ” values, that is, the two Cartesian coordinates necessary to define the position of each spot on the two-dimensional chromatograms. These values were consistently reproducible and in good agreement with the corresponding single-dimensional RI values obtained with each solvent system separately (see Table 1).
TLC
AKALYSIS
Solvent
OF DrSA
B
97
COMPONENTI;
A
FIG. 1. Two-dimensional chromatography of deoxyribosides and deoxyribotides on 20 x 20 cm plates, with solvents A and B: 0 = origin, 1 = dGMP, 2 = dAMP, 3=dCMP, 4=dTMP, 5=dG, 6=dC, 7=dA, 8=dT.
TABLE Rfc,, AND tlrcrj
VALUES
dA dC dG dT dAMP dCMP dGMP dTMP
0.47 0.34 0.25 0.76 0.06 0.07 0.05 0.16
OBTAINED
2
IN TWO-DIMENSION.IL~CHROMATOGRAPHT
0.32 0.54 0.26 0.88 0.34 0.56 0.27 0.90
0.80 0.78 0.64 0.86 0.54 0.50 0.33 0.58
0.32 0.56 0.25 0.90 0.32 0.58 0.25 0.91
a See text for composition of solvent systems A, B, and C. b As originally present in mixture before chromatography (see Table 1 for abbreviations).
For the quantitative estimations, the areas of fluorescence under UV light were outlined and scraped from the plate. The compounds were eluted from the scrapings by occasional shaking with 0.1 N hydrochloric acid for 16 hr at room temperature. After filtration, the ultraviolet spectra of these eluates were determined and their absorbancies at X,,, were compared with those of standard solutions of the corresponding compounds. Since the purine deoxyribosides and their 5’-phosphates were
CHMIELEWICZ
AND
ACARA
0
Solvent
B
FIG. 2. Two-dimensional chromatography of deoxyribosides and deoxyribotides 40 X 20 cm plates, with solvents C and B. See Fig. 1 for symbols.
on
recovered from their respective fluorescent areas in the form of theoretically equimolar amounts of their free bases (see above), standard solutions of adenine and guanine were used for the quantitative estimation of these compounds. To compensate for the ultraviolet absorption of the acid-soluble materials originating from cellulose powder, appropriate nonfluorescent areas were also scraped, extracted with hydrochloric acid under the same conditions, and used as blanks. Using these procedures, recoveries of 95-102s were routinely achieved. It should be emphasized that the procedures presented in this paper are useful as analytical methods for the identification and quantitative determination of pyrimidine as well as purine deoxyribosides and deoxyribotides, but, because of the acidity of solvent B, they cannot be used for the isolation of the purine derivntives.
ACKNOWLEDGMEST The authors gratefully acknowledge the helpful advice of Dr. T. J. Bardos. This work was supported by a research grant (C&4-05522) from the Xational Cancer Institute, U. S. Public Health Service. REFERETCES E., Pharmazie 11, 633 (1956). K., Biochem. Biophys. Research Comlmuru. 6, 452 (1062). RANDERATH, K., Angew. Chem., Intern. Ed. Engl. 1, 435 (1962). VISCHER, E., CHARGAFF, E., J. Biol. Chem. 176, 703 (1948).
1. STAHL,
2. RANDERATH, 3. 4.