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The separation of nucleic acids on a modified methylated albumin-kieselguhr (MAK) column
SHORT COMMUNICATIONS
477
loadings of catechols and phenolic substances is shown in Figure 2. For each substance the area under the peak can be related to its concentration, this provides a useful check on the recovery of substances applied to the chromatogram. When specific fluorometric methods were employed for the estimation of the recovery irom the columns, it was found to be 95100% for loadings of 5 pg or mort (1). The method presented in this communication suggests that virtually quantitat,ive recovery can be obtained with 0.2 pg and possibly with less. REFEREKCES R. J., AND OFFERMAN, J., Biochem. J. 99, 538 (1966). R. J., “Proceeding of the 1965 Technicon Symposium, Automation in Analytical Chemistry.” Technicon Instruments Co. Ltd., Chertsey, Surrey, England.
1. MERRILLS, 2. MERRRILLS,
R. J. MERRILLS J. P. FARRIER Department of Biochemical Pfizer Limited Sandwich, Kent, England Received July 2.4, 1967
Pharmacology
The Separation of Nucleic Acids on a Modified Methylated Albumin-Kieselguhr (MAK) Column
The use of MAK columns for the separation of nucleic acids was first introduced by Lermann (1). The met’hod was further developed by Mandell and Hershey (2), then improved by Hershey and Burgi (3) and Sueoka and Cheng (4). Essential criteria for the separation are the size of the molecule and, according to Sueoka, the composition of the bases, the number of hydrogen bonds in the DNA, and the guanine-cytosine content. Recent studies have shown that newly synthesized DNA, which still has proteins attached to it, is eluted after the main part of the DNA (5). The separation method used by many authors is disadvantageous because the columns are easily blocked and it is necessary to apply pressure for the elution; this often leads to irregularities in the separation. To avoid this we chose a granulated, acid-washed, heated kieselguhr to absorb the methylated albumin. This kieselguhr is normally used to fill
-1 -L -L -L -L . i
1 1 71
1
FIG. 1. Separation of nucleic acids from a carcinoma by phenol treatment (6). A linear NaCl gradient was used for the elution: 200 ml of 0.2 M NaCl and 200 ml of 1.5M NaCl. 1. Low molecular nucleic acid precursors (0.23 M NaCl). 2. sRNA (0.40 Al NaCl). 3. DNA (0.57 M NaCl). 4. rRNA (0.74 M NaCl). Fractions to 3.3 ml. :: 0 * 2 -4 6 6 5ar
LOO-
300.
200.
100.
10 20 30 4a 50 60 FRAC JlO!VS FIG. 2. Separation of nucleic acids from rat livers by phenol treatment (6). A linear NaCl gradient was used for the elution: 200 ml of 0.2 M NaCl and 200 ml of 1.5 M NaCl. 1. Low molecular nucleic acid precursors (0.23 M NaCl). 2. sRNA (0.40 M NaCl). 3. DNA (0.57 M NaCl). 4. rRNA (0.74 M KaCl). Fractions to 3.0 ml.
478
SHORT
479
COMMUNICATIONS
gas chromatographic columns and is known as Gasahrom P (SO-SO mesh) ; it was obtained from Serva, Heidelberg (we are indebted to Dr. 0. Klamerth for this valuable information). The Gaschrom P was added to the methylated albumin at a temperature of 4°C. It was suspended several times in distilled water and then left to settle. The solvent was decanted off and the residue mixed with an equal volume of 0.1 M NaCl solution in 0.05 Tris buffer, pH 6.7; 10 ml
3 8 i -1.4 13 5
12 . 1.1
100
. 10 . 0.9 . 0.8
3
.a7 0.6 . 0.5
200
. a4 03 100
. a? . 0.1
1 10
20
30
40
50
60
FRACTIONS.
FIG. 3. Separation of nucleic acids from Chlorella pyrenoidosa by the method of Richter and Senger (7). A linear NaCl gradient was used for the elution: 250 ml of 0.2 M NaCl and 250 ml of 1.5 M NaC1. 1. Low molecular nucleic acid precursors (0.26 M NaCI). 2. sRN.4 (0.42 M NaCI). 3. DNA (0.65 M NaCI). 4. z-RNA (0.76 or 0.83 M XaCl). Fractions to 5.5 ml.
of an aqueous 1% methylated albumin solution was added per 50 gm Gaschrom P. This solution was stirred for 60 min and the NaCl concentration then brought up to 0.2M. After stirring for a further 30 min, the supernatent was decanted off and the MAK was washed several times with 0.2 M NaCl (pH 6.7) to remove surplus methylated albumin. The MAK suspension was then placed in a 300 X 15 mm column and washed until the extinction of the eluate at 280 nm reached ,zero: The nucleic acids should be applied in dilute solution and the amount should not exceed 2.5 mg in columns of this dimension. We used a linear NaCl gradient for elution: 200 ml 0.2 M and 200 ml
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-
477
loadings of catechols and phenolic substances is shown in Figure 2. For each substance the area under the peak can be related to its concentration, this provides a useful check on the recovery of substances applied to the chromatogram. When specific fluorometric methods were employed for the estimation of the recovery irom the columns, it was found to be 95100% for loadings of 5 pg or mort (1). The method presented in this communication suggests that virtually quantitat,ive recovery can be obtained with 0.2 pg and possibly with less. REFEREKCES R. J., AND OFFERMAN, J., Biochem. J. 99, 538 (1966). R. J., “Proceeding of the 1965 Technicon Symposium, Automation in Analytical Chemistry.” Technicon Instruments Co. Ltd., Chertsey, Surrey, England.
1. MERRILLS, 2. MERRRILLS,
R. J. MERRILLS J. P. FARRIER Department of Biochemical Pfizer Limited Sandwich, Kent, England Received July 2.4, 1967
Pharmacology
The Separation of Nucleic Acids on a Modified Methylated Albumin-Kieselguhr (MAK) Column
The use of MAK columns for the separation of nucleic acids was first introduced by Lermann (1). The met’hod was further developed by Mandell and Hershey (2), then improved by Hershey and Burgi (3) and Sueoka and Cheng (4). Essential criteria for the separation are the size of the molecule and, according to Sueoka, the composition of the bases, the number of hydrogen bonds in the DNA, and the guanine-cytosine content. Recent studies have shown that newly synthesized DNA, which still has proteins attached to it, is eluted after the main part of the DNA (5). The separation method used by many authors is disadvantageous because the columns are easily blocked and it is necessary to apply pressure for the elution; this often leads to irregularities in the separation. To avoid this we chose a granulated, acid-washed, heated kieselguhr to absorb the methylated albumin. This kieselguhr is normally used to fill
-1 -L -L -L -L . i
1 1 71
1
FIG. 1. Separation of nucleic acids from a carcinoma by phenol treatment (6). A linear NaCl gradient was used for the elution: 200 ml of 0.2 M NaCl and 200 ml of 1.5M NaCl. 1. Low molecular nucleic acid precursors (0.23 M NaCl). 2. sRNA (0.40 Al NaCl). 3. DNA (0.57 M NaCl). 4. rRNA (0.74 M NaCl). Fractions to 3.3 ml. :: 0 * 2 -4 6 6 5ar
LOO-
300.
200.
100.
10 20 30 4a 50 60 FRAC JlO!VS FIG. 2. Separation of nucleic acids from rat livers by phenol treatment (6). A linear NaCl gradient was used for the elution: 200 ml of 0.2 M NaCl and 200 ml of 1.5 M NaCl. 1. Low molecular nucleic acid precursors (0.23 M NaCl). 2. sRNA (0.40 M NaCl). 3. DNA (0.57 M NaCl). 4. rRNA (0.74 M KaCl). Fractions to 3.0 ml.
478
SHORT
479
COMMUNICATIONS
gas chromatographic columns and is known as Gasahrom P (SO-SO mesh) ; it was obtained from Serva, Heidelberg (we are indebted to Dr. 0. Klamerth for this valuable information). The Gaschrom P was added to the methylated albumin at a temperature of 4°C. It was suspended several times in distilled water and then left to settle. The solvent was decanted off and the residue mixed with an equal volume of 0.1 M NaCl solution in 0.05 Tris buffer, pH 6.7; 10 ml
3 8 i -1.4 13 5
12 . 1.1
100
. 10 . 0.9 . 0.8
3
.a7 0.6 . 0.5
200
. a4 03 100
. a? . 0.1
1 10
20
30
40
50
60
FRACTIONS.
FIG. 3. Separation of nucleic acids from Chlorella pyrenoidosa by the method of Richter and Senger (7). A linear NaCl gradient was used for the elution: 250 ml of 0.2 M NaCl and 250 ml of 1.5 M NaC1. 1. Low molecular nucleic acid precursors (0.26 M NaCI). 2. sRN.4 (0.42 M NaCI). 3. DNA (0.65 M NaCI). 4. z-RNA (0.76 or 0.83 M XaCl). Fractions to 5.5 ml.
of an aqueous 1% methylated albumin solution was added per 50 gm Gaschrom P. This solution was stirred for 60 min and the NaCl concentration then brought up to 0.2M. After stirring for a further 30 min, the supernatent was decanted off and the MAK was washed several times with 0.2 M NaCl (pH 6.7) to remove surplus methylated albumin. The MAK suspension was then placed in a 300 X 15 mm column and washed until the extinction of the eluate at 280 nm reached ,zero: The nucleic acids should be applied in dilute solution and the amount should not exceed 2.5 mg in columns of this dimension. We used a linear NaCl gradient for elution: 200 ml 0.2 M and 200 ml
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-