Ion-exchange TLC - Separation of Nucleotide Sugars and Nucleoside Monophosphates on PEI-cellulose

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SHORT COMMUNICATIONS

575

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. KLAP PER , M. H., AND HACKETT, D. P., Biochim. Biophys. Acta 96, 272 (1965)

6. LATHE , G. H., AND RUTHVE X,

C. R. J., Biochem. J. 62, 665 (1956).

J.

W. LEE

Wheat

Research

Unit

C. S. I. R. 0.

RHONDA ~UCIVER

North Ryde, N. S. W., Aus tralia

Received August 26, 1965

Ion-Exchange Thin-Layer Chromatography

XIV. Separation of Nucleotide Sugars and Nucleoside

Monophosphates on PEI-Cellulose

Nucleoside diphosphate sugars and nucleoside monophosphates differ-

ing 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 col-

umns 3), paper

(1,

2) or thin layers 4, 5). Our own work 6, 7) had



576

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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 PEI-

cellulose layers (6, 7)) we have developed a fast and sensitive chromato-

graphic 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 solu-

tion4 (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 22-

25°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).



SHORT COMMUNICATIONS 577

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 = UDP-

glucose, 9 = UDP-N-acetylglucosamine,

10 = TDP-glucose, 11 = ADP-ribosr, 12 =

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-



578 SHORT COMMUNICATIONS

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 triphos-

phates 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. Com parison between PEI -cellu lose thin-layer chromatography (PEI-TLC)

and PEI-paper chromatography (PEI-PC). 10, 5, and 1 ~1 of an aqueou s solutio n

containing 6-12 mpm oles/J each of UMP, UDP-N-acetylglucosamine, UDP-glucose,

and

UDP

were app lied to starting spo ts 1, 2, and 3, respectively. Both chromatogram s

were developed usin g System 1 up to 15 cm from the origin. Development time s

121 min (PEI-TLC) and 58 min (PEI-PC). a = UMP, b = UDP -N-acetylglucos-

amine , c = UDP -glucose, d = UDP. A very sm all amount of an unknown impurity

(i) in the mixture show s up only on thin layer, not on paper. Photographed by

short-wave ultraviolet light.



SHORT COMMUNICATIONS 579

PEI-paper chromatography (Fig. 2) shows that, under identical condi-

tions, substance zones on ion-exchange plates are more distinct than on

ion-exchange paper. Mobilities are generally slightly greater on PEI-

paper 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 trans-

ferred 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

Th is work has been supported by grants-in-aid from the U. S. Atom ic Energy

Com miss ion (AT(30-I)-2643), the U. S. Pu blic Health Service (CA 5018-081, the

National Scienc e Foundation (22138), and the Wellcom e Trust. T his is publication

No. 1233 of the Cancer Com miss ion of Harvard University.

REFERENCES

1. KLENOW, H., AND LICHTLER, E., Biochim . Biophys. Acta 23, 6 (1957).

2. CARM INATTI, H., PASSE RON, S., DAN KER T, M., AND RECONDO, E., J. Chromatog.

18, 342 (1965).

3. COHN, W. E., AND BOLLUM, F. J., Bioc him . Biophys . Acta 48, 588 (1961).

4. DIETRICH , C. P., DIETRIC H, S. M. C., AND PON TIS, 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. RAND ERAT H, K., Angew. Chem. 74, 780 (19622); Intern. Ed. 1, 553 (1962).

9. VERAC HTERT, 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. Kapla n, eds.-in-chief). Aca dem ic Pres s, New York, in preparation.

12. RAND ERAT H, K., J. Chromutog. 10, 235 (1963).

K.

RANDERATH

E.

RANDERATH

Bioche mical Research Laboratory and

John Collins W arren Laboratories of the

Huntington Memorial Hos pital of Harvard University

at the Massachusetts General Hospital

Boston, Massachusetts

Received August 31,1966

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Ion-exchange TLC - Separation of Nucleotide Sugars and Nucleoside Monophosphates on PEI-cellulose