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ionic strength but this may have been due to the low charge of this protein. The binding to starch was not evident with any of the proteins studied in tris buffer at pH 8.6, I = 0.01. The obvious differences in the binding behavior of different proteins during electrophoresis just described suggests that use might be made of the ion-exchange properties of columns of starch gel particles at acid pH values and low ionic strengths for protein separations. At an ionic strength of 0.01 at pH 3.1, t.he capacity of starch gel for plasma albumin is in the region of 10 mg/gm dry starch. Lathe and Ruthven (6) have stressed the necessity of using high ionic strengths when using swollen potato starch for gel filtration. If mobility measurements are to be made from electrophoresis runs conducted at acid pH values, it is obviously necessary to consider the effect of buffer ionic strength. Where possible, high ionic strengths should be used. REFERENCES 1. SMITHIES, O., Nature 175, 307 (1955). 2. SMITHIES, O., Biochem. J. 71, 585 (1959). 3. WIEME, R. J., C&n. Chim. Acta 5, 150 (1959). 4. ROBINSON, J. C., AND PIERCE, J. E., Am. .I. Clin. Path. 40, 588 (1963). 5. KLAPPER, M. H., AND HACKETT, D. P., Biochim. Biophys. Acta 96, 6. LATHE, G. H., AND RUTHVEX, C. R. J., Biochem. J. 62, 665 (1956).

272 (1965)

J. W. LEE RHONDA

~UCIVER

Wheat Research Unit C. S. I. R. 0. North Ryde, N. S. W., Australia Received August 26, 1965

Ion-Exchange XIV.

Thin-Layer

Chromatography

Separation of Nucleotide Sugars and Nucleoside Monophosphates on PEI-Cellulose

Nucleoside diphosphate sugars and nucleoside monophosphates differing only with regard to their hexose or pentose moieties may be separated by partition (1, 2) or ion-exchange chroma,tography (3-5) if borate is incorporated into the solvent. Such separations may be obtained on columns (3), paper (1, 2) or thin layers (4, 5). Our own work (6, 7) had

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previously shown that nucleotide sugars may be separated according to the base and phosphate moieties on PEI-cellulose1 anion-exchange thin layers, and similar results were subsequently reported by Verachtert et al. (9) for PEI-paper. A one-dimensional ion-exchange t.hin-layer procedure for separating deoxyribonucleoside monophosphates from the corresponding ribo compounds has been described (5). Taking advantage of these previous results and of the high resolution obtained on PEIcellulose layers (6, 7)) we have developed a fast and sensitive chromatographic method capable of resolving complex mixtures of nucleotide sugars and nucleoside monophosphates. Experimental: (a) Preparation of PEf-Cellulose Thin-Layer Plates. The layers are prepared on glass plates (10) or plastic sheets (type” VSA 3310 Clear 31 Matte 06, 0.010 in.) (11). They are washed with NaCl solution and water (10). (b) Preparation of PEI-Papers. Sheets of Whatman No. 1 paper3 (19 X 45 cm) are soaked in a 2.5% poly (ethyleneimine) hydrochloride solution4 (12) and are dried in the air overnight. Prior to chromatography they are washed by descending irrigation with 10% NaCl solution for 15 min, followed by water without intermediate drying. After 6-8 hr, the papers are dried in the air and then washed a second time with water. (c) Chromatography. Compounds are applied 2 cm from the lower edge of the plate or paper. Ascending chromatography is carried out at 2225°C. Solvents: System 1. 1.0 N acetic acid is allowed to ascend up to 2 cm above the origin, followed, without intermediate drying, by l.ON acetic acid/3.0M LiCl (9:1, v/v) up to 15 cm. System d. A solution of 6 gm Na,B,07*10H,0, 3 gm H,BO,, and 25 ml ethylene glycol in 70 ml water is run up to 12-16 cm above the origin. For two-dimensional chromatography, System 1 is used in the first dimension, and System 2 in the second dimension.5 Prior to development with System 2, acetic acid and lithium chloride must be removed: The plate is dried for several minutes in a stream of cold air, then for 3 min in a stream of warm (60°C) air, and is laid in a flat dish (25 X 25 cm) containing a solution of 600 mg tris (hydroxymethyl) aminomethane (free base) in 500 ml anhydrous methanol. After 5 min, the plate is dried in a stream of cold air and is treated for 10 min with 500 ml anhydrous methanol. Solution is accelerated by agitating. 1 A cellulose anion-exchange material obtained by impregnating chromatography cellulose with poly(ethyleneimine) (8). ‘Union Carbide Corp., Cincinnati, Ohio. 3H. Reeve Angel, Clifton, N. J. 4A 50% solution of poly(ethyleneimine) in water was obtained from Chemirad Corp., East Brunswick, N. J. ‘The solvent front area of the first dimension should be excluded from further chromatography (7).

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Results: System 1 separates mainly according to the phosphate and base moieties of the nucleotides. The mobilities decrease as follows (see Fig. 1, first dimension) : monophosphates > nucleosidc diphosphate

FIG. 1. Two-dimensional separation of nucleotides. PEI-cellulose layer (0.5 mm). 100 pl of an aqueous solution containing 6-12 mpmoles of each nucleotide was applied to the starting spot (St) in 5ql portions without intermediate drying. Development as described in the text. First dimension, from right to left, 15 cm; second dimension, from bottom to top, 16 cm. Total chromatography time about 5 hr. 1 = CTP, 2 = GDP (impurity in the GDP-mannose preparation used), 3 = UDP-glucuronic acid, 4 = GDP-mannose, 5 = GDP-glucose, 6 = CDP, 7 = UDP-galactose, 8 = UDP10 = TDP-glucose, 11 = ADP-ribosr, 12 = glucose, 9 = UDP-N-acetylglucosamine, GMP, 13 = dGMP, 14 = ADP-glucose, 15 = IMP, 16 = VMP, 17 = CDP-glucose, 18 = dTMP; 19 = AMP, 20 = dAMP, 21 = CMP, itnd 22 = dCMP. Photographed by short-wave u.ltraviolet light.

sugars > diphosphates > triphosphates, and cytidine > adenosine > uridine (thymidine ) > inosine > guanosine derivatives of the same type. The borate system separates nccoiding to the sugar moiety: while System 1 hardly differentiates between VDP-glucose and UDP-galactose or between CMP and dCMP, these compounds are clearly separated by System 2. As shown in Fig. 1 (second dimension), nucleotide glucose pre-

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cedes nucleotide galactose, nucleotide mannose, and nucleotide ribose, and deoxyribonucleotides precede ribonucleotides of the same type. The mobility of each compound depends also upon the phosphate and the base moieties of the nucleotide (Fig. 1, second dimension). Di- and triphosphates migrate only a short distance or not at all with either system. A separation of nine monophosphates and ten nucleotide sugars is obtained by combining both systems on one plate (Fig. 1). TDP-glucose migrates with a second front. Resolution of the GDP-glucose/GDP-mannose pair can be improved by continuous flow development using System 2 (11). A comparison between PEI-cellulose thin-layer chromatography and

Pm. 2. Comparison between PEI-cellulose thin-layer chromatography (PEI-TLC) and PEI-paper chromatography (PEI-PC). 10, 5, and 1 ~1 of an aqueous solution containing 6-12 mpmoles/J each of UMP, UDP-N-acetylglucosamine, UDP-glucose, and UDP were applied to starting spots 1, 2, and 3, respectively. Both chromatograms were developed using System 1 up to 15 cm from the origin. Development times 121 min (PEI-TLC) and 58 min (PEI-PC). a = UMP, b = UDP-N-acetylglucosamine, c = UDP-glucose, d = UDP. A very small amount of an unknown impurity (i) in the mixture shows up only on thin layer, not on paper. Photographed by short-wave ultraviolet light.

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PEI-paper chromatography (Fig. 2) shows that, under identical conditions, substance zones on ion-exchange plates are more distinct than on ion-exchange paper. Mobilities are generally slightly greater on PEIpaper than on PEI-cellulose layers. Although a number of separations can be carried out on PEI-paper (9, 12), thin-layer procedures are preferable for separations requiring great sensitivity and/or a high degree of resolution. The procedures outlined in the present communication can be used to assay incubation mixtures and tissue extracts. Nucleotides are transferred quantit.atively from thin-layer plates to a paper wick and are determined spectrophotometrically after elution from the paper (10). Substance areas on paper chromatograms and on plastic plates are cut out, eluted with 0.7M MgCI,/B M tris hydrochloride, pH 7.4 (lOO:l, v/v), and nucleotides are determined spectrophotometrically (11). ACKNOWLEDGMENTS This work has been supported by Commission (AT(30-I)-2643), the U. National Science Foundation (22138), No. 1233 of the Cancer Commission of

grants-in-aid from S. Public Health and the Wellcome Harvard University.

the U. S. Atomic Energy Service (CA 5018-081, the Trust. This is publication

REFERENCES 1. KLENOW, H., AND LICHTLER, E., Biochim. Biophys. Acta 23, 6 (1957). 2. CARMINATTI, H., PASSERON, S., DANKERT, M., AND RECONDO, E., J. Chromatog. 18, 342 (1965). 3. COHN, W. E., AND BOLLUM, F. J., Biochim. Biophys. Acta 48, 588 (1961). 4. DIETRICH, C. P., DIETRICH, S. M. C., AND PONTIS, H. G., J. Chromatog. 15, 277 (1964). 5. RANDERATH, K., Biochim. Biophys. Acta 76, 622 (1963). 6. RANDERATH, K., AND RANDERATH, E., J. Chromatog. 16, 111 (1964). 7. RANDERATH, E., AND RANDERATH, K., J. Chromatog. 16, 126 (1964). 8. RANDERATH, K., Angew. Chem. 74, 780 (19622); Intern. Ed. 1, 553 (1962). 9. VERACHTERT, H., BASS, S. T., WILDER, J., AND HANSEN, R. G., Anal. Biochem. 11, 497 ( 1965). 10. RANDERATH, E., AND RANDERATH, K., Anal. Biochem. 12, 83 (1965). 11. RANDERATH, K., AND RANDERATH, E., in “Nucleic Acids” (L. Grossman and K. Moldave, eds.), a volume of “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.-in-chief). Academic Press, New York, in preparation. 12. RANDERATH, K., J. Chromutog. 10, 235 (1963). K. RANDERATH

E. Biochemical Research Laboratory and John Collins Warren Laboratories of the Huntington Memorial Hospital of Harvard at the Massachusetts General Hospital Boston, Massachusetts Received August 31,1966

University

RANDERATH