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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 2225C. 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 (60C) 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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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 = 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