New method for the differentiation of ribonucleotides from deoxyribonucleotides

ANALYTICAL

New

BIOCHEMISTRY

Method

29,

for

the

from HSIAO

210-222

(1969)

Differentiation

of

Ribonucleotides

Deoxyribonucleotidesl

MAY

WU

AND

S. ARTHUR

CONTI

Section of Hematology, Division of Medicine, and Department of Biochemistry, Presbyterian-St. Luke’s Hospital, Chicago, Illinois 60612, and Department of Biological Chemistry, University of Il1inoi.s College of Medicine, Chicago, Illinois 60612

Received August 9, 1968

Polyethyleneimine (PEI) anion-exchange cellulose was first synthesized and utilized by Randerath (1) for thin-layer chromatographic separation of nucleotides; in his investigatiohs, he used solutions of electrolytes as developing solvents (1, 2). A mixture of ammonia and isobutyric acid (AmBu), pH 3.63.8, was formulated independently by Magasanik et al. (3) and Zetterstrom and Ljunggren (4), and modified by Krebs and Hems (5) for paper chromatographic study of tissue nucleotides ; the pH of the modified AmBu, according to Thomson (6)) is 4.6. In this study, the two systems were combined for the analysis of nucleotides and their precursors. In addition to the Rf values, nucleoside mono-, di-, and triphosphates were further identified by a calorimetric method ; the color reagent, consisting of an acidic solution of ammonium molybdate, was described by Hanes and Isherwood (7)) modified by Bandurski and Axelrod (8) and by Burrows et al. (9), and detailed by Thomson (6). After initial color formation, the plate was exposed to fumes of ‘NH, (6, 7) and we found that, in ammonia, some blue spots remained blue while the others became yellow; the primary objective of this report is to present this phenomenon and its application to differentiate 16 nucleotides and 2 inorganic phosphates. MATERIALS

AND

METHODS

Sorbents. MN-cellulose 300 PEI (polyethyleneimine) and MN-cellulose 300 (standard cellulose), containing no binders, were obtained from 1 This study was supported by U. S. Public Health Service research grant CA 04144 from the National Cancer Institute to Frank E. Trobaugh, Jr., Director, Section of Hematology, Presbyterian-St. Luke’s Hospital, Chicago, Illinois. 210

DIFFERENTIATION

OF

NUCLEOTIDES

211

Brinkmann Instruments, Inc., Westbury, N. Y.; the two celluloses were used to make laboratory-coated thin-layer glass plates. Precoated PEI cellulose plastic plates, Polygram Cel 300 PEI, were also purchased from Brinkmann. Precoated standard cellulose plastic plates, Chromagram Sheet 6064, were obtained from Eastman Kodak Co., Rochester, N. Y. Preparation and Treatment of Thin-Layer Plates. 8 gm PEI cellulose plus 2 gm standard cellulose, or 10 gm standard cellulose, was well mixed with 55 ml of H,O; this was applied on five 20 X 20 cm glass plates using a Brinkmann template and an applicator; the thickness of the layer was 250 mp. The plates were then allowed to dry in the air. Glass or plastic plate containing PEI anion exchanger was predeveloped in H,O to full length up the plate so that any UV-positive contaminants which were water soluble were removed from critical zones; the plate was again dried in the air (2). Preparation of Developing Solvent (Am&). A mixture of 500 ml isobutyric acid, 275 ml H,O, and 8 ml 0.1 M Versene (disodium) was first titrated to pH 4.5 with NH,OH (28--300/a NH,) using a Beckman Zeromatic pH meter. The solution was then allowed to cool to room temperature and titrated to pH 4.6 with an additional 1.5 ml NH,OH (total volume 51 ml). We found that, for thin-layer chromatography of nucleotides, AmBu acts not only as an excellent solvent but also has several practical features: (a) It can be used repeatedly without affecting the chromatographic behavior of a nucleotide. (5) The pH remained constant after repeated use. (c) Different batches can be mixed for reuse. (d) If isolat’ion and rechromatography are not required, isobutyric acid of economic grade may also be used. Preparation of Known Compounds. Each compound was dissolved in 0.1 M HCl except guanine, guanosine, and deoxyguanine, which were solubilized in 0.2 M NaOH. A concentration of 10 mM of individual compound was then prepared. Routinely, 2 ,LL~was applied on the thin layer; or the equivalent of 20 mpmole per spot. The sources of all compounds can be found in the footnotes of Tables 1,2, and 3. Application of Sample. A deep horizontal line was made 10 cm above the origins of application so that the distance of migration was kept constant. Samples were applied under a stream of warm air using micropipets guided with a Plexiglas template. Development of Plate. The plate was developed in a closed tank containing 200 ml AmBu solvent at room temperature in a hood. Flexible plastic plate was support,ed on a clean glass plate of the same size with the upper corners joined together by bulldog paper clamps; after placing in the solvent, the lower portion of the plastic was tapped gently so that it was firmly attached to the glass. Developing time varied from 3 to 5

212

WU

AND

CONTI

hours depending on sorbents; for the same sorbent, a longer time was required to develop precoated than laboratory-coated plate. Visualization of Sample. The dried plate was exposed to fumes of HCl for 5 minutes and viewed under a short-wave (253.7 rnp) UV lamp, Mineralight UVS 12 (Ultra-Violet Products, Inc., San Gabriel, Cal.). Spots having deep blue fluorescence were recorded; guanine derivatives gave bright blue fluorescence. The plate was then sprayed with a mixture of ammonium molybdate, concentrated HCl and HClO, dispersed in either acetone (6, 9) or methanol. Two short-wave UV lamps were mounted opposite each other on lamp stands; the sprayed plate was exposed 12 cm from the light sources for $ hour until blue color was formed. Nucleotides,

ortho- and pyrophos hates on thin layers of PEI and standard celluloses deve Poped in AmBu solvent. (1) Expose to HCl fumes 5 min. I (2) Observe under UV light. All compounds show deep blue fluorescence; guanine derivatives show bright blue. (3) Spray with mixt. of: (NH&MoOa, aq.; HCIOI, 720/,; HCl, cont. in acetone or MeOH. I (4) Expose to UV light, 253.7 rnp, 30 min. All compounds reduce to give a blue color; if not, hold plate over steam for 2 min. 1 (5) Expose to fumes of NH,

Blue color remains: dAMP, dGMP, dTDP, dTTP, ADP, ATP, Pi, PPi.

l-2 min.

Blue color disap ears: AMP(5’) CMP(%) UMP(5’) GMP(S’), AMP(2’,3’), CMI$2’,3’), UMP(2’,3’), GMP(2’,3’), dTMP, dCMP. (6) Expose to HCl fumes, overnight

Most blue spots are faded. (7) Let stand at room temp. 0.5-l hr or steam 2 min. 1 The

rate at which each compound becomes blue is as follows: ATP, dTTP, ADP, PPi, Pi > dTDP > dAMLIP, dGMP > dTMP > dCMP > AMP(5’), GMP(5’) > AMP(2’,3’), GMP(2’,3’) > UMP(5’), CMP(5’) > UMP(2’,3’), CMP(2’,3’). (8) Continue to stand at room temp. 4 hr or continue to steam 8-10 min. I

Blue : dAMP,

dGMP, dTMP, dTDP, dTTP, ADP, ATP, Pi, PPi.

Yellow

(or with a little blue center) : All ribonucleotides and dCMP.

1

(9) Repeat steps (5) to (8) twice if no steaming required, more if steaming is necessary. I All compounds are blue. FIG. 1. Development of stable blue complex of nucleotides molybdate on thin layers of PEI and standard celluloses.

with acidic ammonium

DIFFERENTIATION

OF

213

NUCLEOTIDES

When the relative humidity of the laboratory environment fell below 10, the blue complexes did not form regardless of the length of time exposed to UV light (up to 2 hours). If, however, after l/z hour exposure, the plate was held over an 86” water bath so that steam escaped but the water did not spatter (see water mark in Figure 4)) the blue color formed within 2 minutes. Further exposure to steam led to a change of blue complex to yellow complex depending upon the nature of the nucleotide. Procedures which facilitate nucleotide identification are outlined in Figure 1; steps 1 to 4 were never repeated; steps 5 to 8 were repeated 3 to 6 times; step 9 is the final step. Steps required for steaming are indicatecl in Figure 1. Photograph of Developed Plate. A Polaroid-Land camera 1lOA (Polaroid Corp., Cambridge, Mass.) was used. Following the first and second steps, the plate was photographed under short UV light (3 lamps) using Polaroid type 47 film, 3000 ASA, F32, time 2.5 second. Colored print was made after step 4, 5, 8, or 9 under fluorescent white light using Polaroid color film type 48 75ASA, F32, time 30 second. In this report, colored photo was reproduced in black and white under fluorescent white light using Polaroid type 47 film, 3000 ASA, F32, time l/15 second.

Rligratory

Rates

of Bases

TABLE 1 and Nucleosides on Two in AmBu Solvent

Cellulose

Thin

Layers

Developeci

Rr X 100 PEI Compound’

Adenine Cytosine Uracil Guanine Thymine 5-Methylcytosine Adenosine Cytidine Uridine Guanosine Deoxyadenosine Deoxycytosine Thymidine Deoxyguanosine

(1)

8.1~. S.F. 77 68 89 90 89 88 72 66 S.F. S.F. 89 78

Standard

. --___

(2)

(1)

9.5 86 69 66 86 88 88 75 56 50 95 x4 81 65

78 64 64 78 64 86 90 88 74 77 no 89 70

--

(2) 97 91 67 63 79 92 84 78 63 61 95 85 77 69

S.F. = solvent front, (1) = laboratory-coated plates, (2) = precoated plates. a All bases are from Nutritional Biochemical Corp., Cleveland, Ohio, except B-methylcytosine, which is from Sigma Chemical Co., St. Louis, MO. All nucleosides are from California Foundation for Biochemical Research, Los Angeles, Cal., except thymid&, which is from Sigma.

214

WU

AND

CONTI

RESULTS

The migratory rates of purines, pyrimidines and nucleosides are shown in Table 1; those of nucleoside monophosphates are presented in Table 22; those of nucleoside di- and triphosphates and inorganic ortho- and pyrophosphates are shown in Table 3.2 On the same sorbent, the Rf values of a compound migrated on laboratory-coated and precoated plates are not always identical, but the relative order of migration remains the same: the derivatives of adenine (A) migrate fastest, then the cytosine (C), the uracil (U), and the thymine (T), and finally guanine (G). Migratory

TABLE 2 Rates of Nucleoside Monophosphates on Two Cellulose Thin Layers Developed in AmBu Solvent Rf X 100 PEI

Compounds

AMP(5’) CMP(5’) UMP(5’) GMP(5’) AMP(2’,3’) CMP(2’,3’) UMP(2’,3’) GMP(2’,3’) dAMP(5’) dCMP(5’) dTMP(5’) dGMP(5’)

(1)

53 39 63 31 69 64 55 38

Standard (3)

54 44 34 25 64 44 36 31 63 52 47 32

(1)

(2)

-

70 63 50 4.5 66 60 46 40 69 63 56 49

58 41 69

42 72 65 56 48

(1) = laboratory-coated plates, (2) = precoated plates. ,z CMP(5’), UMP(B’), dAMP(S’), dCMP(5’), and dTMP(5’) are from Calbiochem, Los Angeles, Cal. AMP(2’,3’) and GMP(2’,3’) are from California Foundation for Biochemical Research, Los Angeles, Cal. dGMP(5’), AMP(5’), CMP(2’,3’), GMP(2’,3’), and UMP(2’,3’) are from General Biochemicals, Chagrin Falls, Ohio.

From Tables mono-, di-, and cellulose, since same Rf value

2 and 3, it can be seen that the separation of nucleoside triphosphates is more effective on PEI than on standard on the former no nucleoside di- or triphosphate has the as that of monophosphates. Therefore, in a mixture of

‘The mono-, di-, and triphosphates of ribosyl adenine, guanine, hypoxanthine, cytosine, and uracil: AMP, GMP, IMP, CMP, UMP; ADP, GDP, IDP, CDP, UDP; ATP, GTP, ITP, CTP, UTP. The monophosphat,es of 2’-deoxyribosyl adenine, guanine, cytosine, and thymine: dAMP, dGMP, dCMP, dTMP. The di- and triphosphates of 2’-deoxyribosyl adenine and thymine : dADP, dTDP; dATP, dTTP. The inorganic ortho- and pyrophosphates: Pi and PPi.

DIFFERENTIATION

OF

215

NUCLEOTIDES

TABLE 3 Migratory Rates of Nucleoside Di- and Triphosphates and Inorganic Ortho- and Pyrophosphates onTwo Cellulose Thin Layers Developed in AmBu Solvent RI

Xl00

PEI

Standard

Compound*

(1)

(2)

(1)

(2)

ADP(5’) CDP(5’) IDP(5’) UDP(5’) GDP(5’) ATP(5’) CTP(5’) ITP(5’) UTP(5’) GTP(5’) dTDP(5’) dTTP(5’)

20 16 9 7

18 12 7 5

.57 50 32 33

51 44 26 ‘27

1

1

SO

‘25

2 I I (I 0 4 0 17

46 :3X

40 :i:i

‘2 1

1x

Pi

4 ‘2 I I 0 :; 1 20

20 I!1 44 38 50

PPi

0

0

I9 17 41 3) 48 40

38

(1) = laboratory-coated plate, (2) = precoated plates. QAll nucleoside triphosphates are from Nutritional Biochemical Corp., Cleveland, Ohio. ADP is from C. F. Boehringer und Soehne GmbH, Mannheim, Germany; the other nucleoside diphosphates are present in corresponding nucleoside triphosphates. dTDP and dTTP are from Sigma Chemical Co., St. Louis, MO. nucleoti,des, such as a tissue extract, once the nucleoside monophosphates are separated and removed frown di- and triphosphates on PEI cellulose,

the polyphosphates can be eluted as individual group, rechromatographed, and resolved into single component on standard cellulose. The separation and differentiation of 12 nucleotides of a plate of laboratory-coated PEI cellulose are presented in Figure 2. In this study, 8 nucleoside monophosphates, 2 diphosphates, and 2 triphosphates are included. The formation of yellow complex for 6 mononucleotides, excluding dAMP, dGMP, and nucleoside di- and triphosphates, can be seen clearly in column 3, or step 8-l. The rate at which each compound becomes permanently blue is apparent and has been outlined in Figure 1. A photograph taken under UV light, of 16 nucleotides, Pi, and PP, separated on a plate of precoated PEI cellulose is shown in Figure 3. Figure 4 is a serial photograph of the same plate after being sprayed with acidic ammonium molybdate and processed according to the procedure outlined in Figure 1. The study shown in Figure 4 was made when the relative humidity in our laboratory read 6, therefore steaming was necessary to bring out the coloration, which demonstrates that moisture is

216

WU

AND

CONTI

FIG. 2. TLC of 12 nucleotides (concentration 20 mymole/spot) and their differcntial rates in forming a stable blue complex with acidic ammonium molybdate on PEI cellulose. Numbers on left vertical bar denote migration distance in cm. Of each column, spots on the right consist of 6 ribonucleotides: AMP(2’,3’) > CMP(5’) > UMP(5’) > GMP(2’,3’) > ADP(5’) > ATP(5’) ; and on the left, 6 deoxyribonucleotides: dAMP(5’) > dCMP(5’) > dTMP(5’) > dGMP(5’) > dTDP(5’) > dTTP(5’). The symbol ” > ” means “migrating faster than.” Below each column, the first number in a circle represents steps indicated in Figure 1; the number in the same circle following the hyphen denotes the original run (number 1) or repeated run of steps 5 through 8 (number 2 or 3). Step 9 is the final step.

required to complete the reaction between a nucleotidc and an acidic solution of ammonium molybdate on cellulose layer. The arc in the lower portion of the plate is a water mark that may be disregarded. Figure 4 reveals that orthophosphate is present in many of the nucleotides chromatographed : at position 4 with CMP (5’) ; 5 with GMP (5’) ; 6 with AMP(2’,3’) ; 10 with dAMP(50 ; 12 with dTDP (5’) ; and 13 with dGMP(5’). The rate at which each compound becomes blue has been outlined in Figure 1 and is the same as that presented in Figure 2. In Figure 2, AMP(5’), GMP (5’)) UMP (2’,3’), CMP(2’,3’), Pi, and PPi were not included. Figure 4 also shows that the initial blue color formed for CMP(2’,3’) at position 7 and for UMP(2’,3’) at position 8, once lost by contact with fumes of NH,, was not recovered even after six repeated runs in the fumes of HCI and NH, (therefore, a photograph of step 9, the final step, was not taken). This implies that the blue color formed by pyrimidine ribonucleotide having PO, at carbon-2’ and carbon-3’ of ribose is extremely unstable, and the rate at which each compound becomesblue is closely related to the ease with which a nucleotide releases its phosphate group. That inorganic phosphate or nucleoside di- or triphosphate

DIFFERENTIATION

OF

NUCLEOTIDIW

217

FIG. 3. TLC of 16 nucleotides, Pi, and PPI photographed under UV light, 253.7 m/l. Number 2-l in the circle on the upper right refers to step 2 indicated in Figure 1 and the original run (number 11, respectively. The individual compound is identified in Figure 4. The bright UV spot at position 1 with (RI X 100) = 65 is deolryguanosine.

readily forms a blue complex with acidic ammonium molybdate indicates an absolute requirement of free Pi for t’his reaction; nucleoside polyphosphates have labile phosphate groups. In general, deoxyribonucleotides (excqt dCMP) give blue coloration sooner than all ribonucleotides, purine ribonucleotides sooner than pyrimidine ribonucleotides, 5’-ribonucleotides sooner than 2’,3’-ribonucleotides. Between the pyrimidine derivatives, the color reaction of CMP(5’) is identical to that of UMP(5’) and CMP(2’,3’) to that of UMP(2’,3”) ; but this is not so for dTMP and dCMP-dTMP reacts more like a purine deoxynucleotide. DISCUSSION

The reaction of phosphate with molybdate in an acid solution is often used for the detection of inorganic as well as organic phosphates. The influences of acids, acidity, and reducing agents on color formation in chemical analyses have been reported by Hurst (lo), and by Kuttner and Cohen (11). The effects of acids, moisture, heat, UV light, and other reducing agents on color formation on paper chromatograms were discussed by Hanes and Isherwood (7)) Bandurski and Axelrod (g), and Burrows et al. (9). According to Hurst (lo), the reaction depends upon the reduction of a yellow to blue complex of phosphomolybdate; under the present conditions of experiment, the conversion of the two complexes appears to have been made reversible before a stable blue complex is fully

2-5

1 2

:

12

6- 0 10-13

Position

> GMP

( 5') -dTMP

>dTDP

>dTTP

(2’,3’)-AMP ’ CMP ’ UMP ’ GMP (5’) -dAMP > dCMP >dTMP > dGMP

Pi > PPi (5') - AMP > ADP ’ ATP (5’) -AMP > CMP >lJMP

1311

9

7

5

3

1

FIG. 4. TLC of 16 nucleotides, Pi and PPi (concentration 20 mpmole/spot) and their differential rates in forming a stable blue complex with acidic ammonium molybdate on PEI cellulose. Numbers on left vertical bar denote migrat.ion distance in cm. Horizontal numbers indicate posit.ion of applications; compound (or compounds) applied at each position is shown at upper right block at top of photograph. The symbol ” > ” means “migrating faster than.” The first number in a circle of each serial photo represents step indicated in Figure 1; the number in the same circle following the hyphen denotes the original run (number 1) or repeated run of steps 5 through 8 (numbers 3 or 6). Final step 9 is not photographed.

i

220

WU

AND

CONTI

developed (7) ; however, once a stable blue complex is formed, the conversion becomes irreversible. In their study, Hanes and Isherwood (7) mentioned that the free Pi released from hydrolyzed organic phosphoric ester is responsible for the formation of a blue color with ammonium molybdate. Our study shows that one of the most important factors controlling Pi release is the moisture content of the thin layer which tends to equilibrate with that of ambient humidity; if there is not enough moisture in the air, it must be supplied by other means-in the present case, by steaming. The acid hydrolytic property of nucleoside di- or triphosphates appears to depend upon the phosphate-phosphate bond only, since, regardless of the moiety of purine, pyrimidine, or sugar, a polyphosphate readily forms a stable blue complex with the color reagent under short UV light; thus, they react like ortho-and pyrophosphates. Hill and Morales (12) attribute the immediate release of the phosphate group to electrostatic repulsion between the adjacent phosphate groups from the polyphosphate. On the other hand, the acid hydrolytic property of nucleoside monophosphates is intimately related to the nature of the base and sugar; it therefore involves the stability of the N-,deoxyribonucleosidic bond and of the N-ribonucleosidic bond as well as the sugar-phosphate bond; the position of the phosphate in the sugar is also important. The present datum shows that the relative rate of formation of blue complex, thus the relative ease of hydrolysis, of 12 nucleoside monophosphates on a thin layer of cellulose is: dAMP (5’)) dGMP (5’) > dTMP (5’) > dCMP(5’) > AMP(5’), GMP(5’), AMP(2’,3’), GMP(2’,3’) > UMP(5’), CMP(5’), > UMP(2’,3’), CMP(2’,3’). In short, under acidic condition, deoxyribonucleotides release their PO, with greatest ease followed by both 5’- and 2’,3’-purine ribonucleotides, 5’-pyrimidine ribonucleotides, and finally 2’,3*-pyrimidine ribonucleotides. An indirect evidence as to the lability of the PO, group in deoxyribonucleotides may be inferred by the discussion of Overend and Stacey (13) on the lability of 2-deoxy-n-ribose l-phosphate; further, Anderson et al. (14) showed that the purine deoxyribonucleotide derived from DNA is more sensitive to acid than the phosphodiester group in polymeric DNA. According to Schmidt (15), (a) the N-deoxyribonucleosidic bond in a polynucleotide is more sensitive to acid than the N-ribonucleosidic bond; (b) the N-ribonucleosidic or the phosphomonoester bond of purine ribonucleotide is more acid-labile than that of pyrimidine ribonucleotide; and (c) the phosphodiester group of a ribonucleotide is more acid labile than the phosphomonoester formed from it. Some of Schmidt’s observations have been discussed by Loring (16) and by Millar and Springall (17). We have shown that pyrimidine ribonucleotides having 2’,3’-phosphate

DIFFERENTIATION

OF

221

NUCLEOTIDES

groups is slow to free its phosphate as Pi. Cyclization of this group between C-2’ and C-3’ in the ribose probably contributes to this stability under acidic conditions. Khorana (18) has reviewed in detail the property of cyclization of various 2’,3’-phosphate esters including ribonucleotides in acid solution, and Brown et al. (19) have shown that the cyclic phosphates are stable in an aqueous medium between pH 4 and 9. In conclusion, differentiation was accomplished for the following compounds: (CL) ribonucleotide from deoxyribonucleotide; (b) purine derivative from pyrimidine derivative; (c) 5’-ribonucleotide from 2’,3’-ribonucleotide; (d) mononucleotides (e.g., AMP-5’, CMP-5’, UMP-5’, and CMP-5’) from each other; and (e) nucleoside mono-, di-, and triphosphates (e.g., AMP, ADP, and ATP) from each other. Finally, it should be mentioned that this method is particularly useful for analyzing nucleotide contents of tissue extracts, hydrolyzates of oligonucleotides, polynucleotides, or nucleic acids, and fractions of column chromatography where the concentration of individual nucleoside or nucleotide may be minute. We have used this procedure and found that, in the spleens of mice, the pool of ribonucleotides far exceeded that of deoxyribonucleotides, and the pool of thymidine was almost negligible. SUMMARY

Nucleotides were separated on thin layer of PEI or standard cellulose developed in buffered aqueous ammonium isobutyrate at pH 4.6. In addition to Rf values, nucleoside mono-, di-, and triphosphates were further differentiated according to the rate at which a nucleotide forms a stable blue complex with ammonium molybdate. The blue color formation, in turn, depends upon the ease at which a nucleotide liberates its Pi; this property appears to be closely related to the nature of the phosphatephosphate bond, the sugar-phosphate bond, and the base-sugar bond. Differentiation was made between: (a) ribonucleotide and deoxyribonucleotide, (5) purine derivative and pyrimidine derivative, (c) 5’ribonucleotide and 2’,3’-ribonucleotide, (d) nucleoside monophosphates (e.g., 5’-AMP, 5’-CMP, 5’-UMP, and 5’-GMP), and (e) nucleoside mono-, di-, and triphosphates (e.g., AMP, ADP, and ATP). ACKNOWLEDGMENT We Luke’s

wish to Hospital,

thank Dr. Patricia Chicago, Illinois,

McFate, Division of for her helpful editorial

Medicine, review

Presbyterian-St. of the manuscript.

ed., pp.

22, 38. New

REFERENCES 1. RANDERATH, 2. R.ANDER.~TH, 1966.

K, Biochim. K., “Thin

Biophys. Acta 61,852 Layer Chromatography,”

(1962). 2nd

York,

222

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AND

CONTl

B., VISCHER, E., DONIQER, R., ELSON, D., AND CHARGAFF, E., J. Biol. Chem. 186, 37 (1950). 4. ZETTERSTR~M, R., AND LJUNGGREN, M., Acta Chem. &and. 5, 291 (1951). 5. KREBS, H. A., AND HEMS, R., &&him. Biophys. Acta 12, 172 (1953). 6. THOMSON, R. Y., in “Chromatographic and Electrophoretic Techniques,” Vol. 1, “Chromatography” (I. Smith, ed.), pp. 235, 241. Interscience, New York, 1960. 7. HANES, C. S., AND ISHERWOOD, F. A., Nature 164, 1107 (1949). 8. BANDURSKI, R. S., AND AXELROD, B., J. Bid. Chem. 193,405 (1951). 9. BORROWS,S., GRYLIS, F. S. M., AND HARRISON, J. S., Nature 170, 800 (1952). 10. HURST, R. O., Can. J. Biochem. 42,287 (1964). 11. KUTTNF,R, T., AND COHEN, H. R., J. Biol. Chem. 75, 517 (1927). 12. HILL, T. L., AND MORALES, M. F., J. Am. Chem. Sot. 73, 1656 (1951). 13. OVEREND, W. G., AND STACEY, M., in “The Nucleic Acids” (E. Chargaff and J. N. Davidson, eds.), Vol. I, p. 58. Academic Press, New York, 1955. 14. ANDERSON, W., DEKKER, C. A., AND TODD, A. R., J. Chem. Sot. 1952,272l. 15. SCHMIDT, G., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. III, p. 752. Academic Press, New York, 1957. 16. LORING, S., in “The Nucleic Acids” (E. Chargaff and J. N. Davidson, eds.), Vol. I, p. 194. Academic Press, New York, 1955. 17. MILLAR, I. T., AND SPRINGALL, H. D., in “Sidgwick’s Organic Chemistry of Nitrogen,” 3rd ed., p. 840. Clarendon Press, Oxford, 1966. 18. KHORANA, H. G., “Some Recent Developments in the Chemistry of Phosphatr Esters of Biological Interest,” p. 63. Wiley, New York, 1961. 19. BROWN, D. M., MAGRATH, D. I., AND TODD, A. E., J. Chem. Sot. 1952, 2078. 3. MAGASANIK,