ion-exchange tlc - separation of nucleotide sugars and nucleoside monophosphates on pei-cellulose
TRANSCRIPT
7/23/2019 Ion-exchange TLC - Separation of Nucleotide Sugars and Nucleoside Monophosphates on PEI-cellulose
http://slidepdf.com/reader/full/ion-exchange-tlc-separation-of-nucleotide-sugars-and-nucleoside-monophosphates 1/5
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
7/23/2019 Ion-exchange TLC - Separation of Nucleotide Sugars and Nucleoside Monophosphates on PEI-cellulose
http://slidepdf.com/reader/full/ion-exchange-tlc-separation-of-nucleotide-sugars-and-nucleoside-monophosphates 2/5
576
SHORT COMMUNICATIONS
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).
7/23/2019 Ion-exchange TLC - Separation of Nucleotide Sugars and Nucleoside Monophosphates on PEI-cellulose
http://slidepdf.com/reader/full/ion-exchange-tlc-separation-of-nucleotide-sugars-and-nucleoside-monophosphates 3/5
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-
7/23/2019 Ion-exchange TLC - Separation of Nucleotide Sugars and Nucleoside Monophosphates on PEI-cellulose
http://slidepdf.com/reader/full/ion-exchange-tlc-separation-of-nucleotide-sugars-and-nucleoside-monophosphates 4/5
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.
7/23/2019 Ion-exchange TLC - Separation of Nucleotide Sugars and Nucleoside Monophosphates on PEI-cellulose
http://slidepdf.com/reader/full/ion-exchange-tlc-separation-of-nucleotide-sugars-and-nucleoside-monophosphates 5/5
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