Technical Notes Anal. Chem. 1995, 67,1627-1631
Simultaneous Separation of Nucleotides and Nucleotide Sugars Using an lon=Pair ReversedlPhase HPLC: Application for Assaying Glycosyltransferase Activity Isabelle Meynial, Veronique Paquet, and Didiew Combs* Departement de Genie Biochimique et Alimentaire, INSA UR.4 CNRS 544,Centre de Bioingenierie Gilbert Durand, Complexe Scientifique de Rangueil, 31077 Toulouse Cedex, France
Nucleotides and nucleotide sugars were simultaneously separated by high-performance liquid chromatography using an ion-pairreversed-phasemethod. Tetrabutylammonium hydrogen sulfate (IBAHS), which is very hydrophobic, was selected as the counterion. Using a linear elution gradient, the influence of the counterion concentration, the pH in the mobile phase, and the temperature on the solute retention times has been studied. A concentration of 2.5 mM TBAHS in a potassium phosphate buffer, pH 6.9, and ambient temperature were the optimal conditions to separate a mixture of nucleotide sugars and nucleotides. Moreover, this method has allowed the direct separation of the 4-epimeric-uridine5'diphosphate sugars (UDP-galactose and UDP-glucose) without prior formation of a UDP sugar-borate complex. This simple and rapid (20 min) method enabled nucleotides and nucleotide sugars to be detected down to 40 pmol. This technique was particularly atkractive for assaying glycosyltransferase activity. As an example, the quantitative determination of UDP-galactose, NADH, NAD+, and UTP during the lactose synthesis by a galactosyltransferase (EC 2.1.4.22) was successfully investigated. Nucleotides and nucleotide sugars are fundamental compounds. They are involved in many biochemical reactions in prokaryotic and eukaryotic cells. They are implicated in nucleic acid metabolism and are also important as energy storage molecules and as cofactors of numerous biochemical reactions.' Moreover, nucleotide sugars are the glycosyl donors in the synthesis of bacterial peptidoglycan,ZJ fungal cell walls? and (1) Ram,J. D. In Biochemistty; Patterson, N., Ed.; Carolina Biological Supply Co.: Burlington, NC, 1989. (2) Archibald, A R; Hancock, I. C.; Harwood, C. R In Cell wall structure, synthesis and turnover; Sonenshein, A L., Hoch, J. A, Losick, R, Eds.; ASM: Washington, DC, 1993; pp 381-410. (3) Neidhardt, F. C. In Escherichia coli and Salmonella typhimurium; Neidhardt, J. L., lngraham, K. B., Low, B., Magasanik, M., Schaechter, M., Umbarger, H. E., Eds.; ASM: Washington, DC, 1987; Vol. 1.
0003-2700/95/0367-1627$9.00/0 0 1995 American Chemical Society
storage carbohydrates such as gly~ogen.~ Nucleotide sugars are also involved as a general rule in protein glycosylation reactions catalyzed by glycosyltransferases6-* with release of the free nucleotide. In addition, glycosyltransferases are now used to synthesize oligosaccharidesin vitrogJosince they are highly stereoand regiospecitlc enzymes with respect to both the nucleotide sugar donor and acceptor, and consequently no reaction byproducts are formed. They constitute, therefore, an attractive tool for the synthesis of glycans in vitro and for the remodeling of the glycan part of natural or recombinant glycoproteins." Thus, the reconstitution in vitro of the oligosaccharide chain of a glycoprotein should lead to better understanding of the role of these chains. As can be seen from these examples, the qualitative and quantitative analysis of the nucleotides and nucleotide sugars is of great importance in a wide number of fields. High-performance liquid chromatography (HPLC) methods have been reported for the separation of these two types of compounds using anionexchange ~hromatography'~J3 or ion-pair chromat~graphy.'~-~~ Ion-pair reversed-phase chromatography has appeared to be a better alternative to separate nucleotides or nucleotide sugars. The stationary phase (a4218 bound phase) is more stable. The addition into the mobile phase of a counterion of charge opposite (4) Herrera, J. R In Fungal cell wall: structure, synthesis and assembk CRC Press: Boca Raton, FL, 1991; Chapter 5, pp 59-88. (5) Dickinson, J. R In Saccharomyces. Biotechnology handbooks; Tuite, M. F., Oliver, S. G., Eds.; Plenum Press: New York, 1991;Vol. 4, pp 59-100. (6) Abeijon, C.; Hirschberg, C. B. Trends Biochem. Sci. 1992,17,33-36. (7) Korf, U.; Thiem, J. Kontakte 1992,1,3-10. (8) Leloir, L F. Science 1971,172,1299-1303. 215(9) Ichikawa, Y.; Look, G. C.; Wong, C. H. Anal. Biochem. 1992,202, 238. (10) Wong, C. H.; Ichikawa, Y.; Krach, T.; Gautheron-Le-Narvor,C.; Dumas, D. P.; Look, G. C.]. Am. Chem. SOC.1991,113, 8137-8145. (11) Van den Eijnden, D. H.; Nemansky, M.; Wietske, E. C. M. In Protein glycosylation;Conradt, H. S., Ed.; VCH Verlagsgesellschaft mbH Weinheim, 1991;pp 195-205. (12) Hartwick, R A; Brown, P. R ]. Chromatogr. 1975,112,651-662. (13) Olempska-Beer, 2.; Bautz Frees, E. Anal. Biochem. 1984,140,236-245. (14) Hoffman, N. E.; Liao J. C. Anal. Chem. 1977,49, 2231-2234. (15) Perrone, P. A; Brown, P. R]. Chromatogr. 1984,317, 301-310. (16) Perret, D. In Hplc ofsmall molecules a practical approach; Lim, C. IC, Ed.; IRL Press: Oxford, UK, 1986; Chapter 9. (17) Werner, A Chromatographia 1991,31,401-410. (18) Lagunas, R; Diez-Maas, J. C. Anal. Biochem. 1994,216,188-194.
Analytical Chemistry, Vol. 67,No. 9,May 1, 1995 1627
to that of the molecule has permitted these compounds to be analyzed separately and rapidly with a satisfactory ~ i e l d . ' ~ - l ~ In this paper, a simple and sensitive HPLC method was developed to detect simultaneously nucleotides and nucleotide sugars using ion-pair chromatography. The influence of the pH, the counterion concentration in the mobile phase, and the temperature on the retention time of different molecules is reported. Finally, the detection of galactosyltransferase activity is given as an example of the applicability of the method. EXPERIMENTAL SECTION
Materials. Nucleotide sugars, nucleotides, galactosyltransferase (EC 2.4.1.22), lactalbumin, phosphoenolpyruvate, and pyruvate kinase/lactate dehydrogenase mixture were purchased from Sigma Chemical Co. (St. Louis, MO); TBAHS (HPLC grade) was from Aldrich (Germany) ; potassium dihydrogen phosphate, dipotassium hydrogen phosphate, glucose, manganese chloride, ethylenediaminetetraaceticacid (EDTA); and potassium chloride were from prolabo (France); glycylglycin was from Boehringer Mannheim (Germany); and acetonitrile (analytical grade) was from Carlo Erba (Italy). ChromatographicSystem. Chromatography was performed on an Amino Quant HP 1090 Series II/M liquid chromatograph (Hewlett Packard, France) with a DR5 ternary solvent delivery system, an autoinjector, and a temperaturecontrolled column compartment. A built-in diode array detector monitored the elution of the solutes at 264 and 254 nm. The data were collected and analyzed using an HPLC ChemStation (Hewlett Packard, France). The separationswere accomplished on a Spherisorb ODS I1 5 pm (250 x 4.6 mm) column (ICs, France), connected to a 5 pm guard column (20 x 4.6 nun) with the same properties. Mobile Phase and Sample Preparation. Nucleotide sugars and free nucleotides were dissolved in Milli Q quality water (Millipore, France) as 1g/L stock solutions. These solutions were then diluted to achieve working concentrations and maintained at 4 "C. The mobile phase A was prepared with a 50 mM dipotassium hydrogen phosphate solution. The pH was adjusted to 7 with a 50 mM potassium dihydrogen phosphate solution. The counterion (TJ3AHS) was then added at the desired final concentration. The pH was again measured (6.9) and taken throughout as working PH. The mobile phase B was prepared in the same manner as the mobile phase A. The pH of a 100 mM dipotassium hydrogen phosphate solution was adjusted to 7 with a 100 mM potassium dihydrogen phosphate solution. The counterion was added, and the final pH was measured and not readjusted. This buffer was diluted with 50%acetonitrile. Both mobile phases were filtered through a Teflon filter (Hewlett Packard, France) and degassed prior to use. Chromatographic Analysis. The void volume (VO)of the column was determined by injecting 5 pL of acetone using 100% acetonitrile as mobile phase. The defined retention time (TO) was 1.99 min. The capacity factor k' was calculated from k' = (Tr - To)/To, where T, is the retention time of the solute as defined by Rosset et aL20 (19) Snyder, L. R; Kirchland, J. J. In Introduction to the modern liquid chromatography; Wiley: New York, 1979 Chapter 11.
1628 Analytical Chemistry, Vol. 67,No. 9,May 1, 1995
Prior to analysis, the column was allowed to equilibrate for 90 min with a mixture of 97.5%eluent A and 2.5%eluent B, at a flow rate of 1 mL/min. After a set of experiments, the column was washed with Milli Q quality water (Millipore, France), at 1 mL/min flow rate, and the residual molecules were then eliminated with a linear gradient from 0%to 15%acetonitrile in potassium phosphate buffer. At ambient temperature, 20 pL portions of the samples were injected into the column. All the separations were carried out with a 15 min linear gradient from 2.5% to 30%of eluent B, at a flow rate of 1 mL/min. The absorbance of the column effluent was monitored at 264 nm for the uridine derivatives and at 254 nm for the guanosine derivatives. Quantitative Studies. Calibration curves were established using a gradient elution chromatography. For each point, the injection volume (20 pL) was the same, but the concentration of solute was varied over a range of 1.25 x W3to 5 x g/L. In all cases, four injections were performed. Assay of Galactosyltransferase Activity. The assay of galactosyltransferase activity performed was according to the procedure suggested by Sigma Chemical Co. This method was derived from that of Fitzgerald et al.2l Galactosyltransferase enzyme solution was dissolved just before use at 0.2 units/mL, in a 20 mM Tris-HC1 buffer with 2 mM EDTA and 2 mM 2-mercaptoethanol, pH 7.5. One unit of galactosyltransferase activity is defined as the amount of enzyme that catalyzes the transfer of 1 pmol of galactose from UDP-galactose to glucose per minute and milliliter of reaction mixture, at pH 8.4, 30 "C, in the presence of 0.2 mg of lactalbumin. The reaction mixture contained 51 mM glycylglycine,0.14 mM NADH, 1.3 mM phosphoenolpyruvate, 4.9 mM manganese chloride, 49 mM potassium chloride, 0.36 mM UDP-galactose, 0.02% lactalbumin, 18 mM glucose, 0.64 mM Tris-HC1,0.064mM EDTA, 0.064 mM 2-mercaptoethanol, 17.5 units of pyruvate kinase, 25 units of lactic dehydrogenase, and 0.02 units of galactosyltransf e m e in a total 3.125 mL volume. The reaction was performed in a water bath at 30 "C for 10 min and was stopped by rapid cooling in ice. A control assay was prepared under the same conditions but without galactosyltransferase. The control and assay were diluted Cfold in Milli Q water quality (Millipore, France), and 20 pL of each sample was injected into the column. The quantitative determination of each substrate and product was accomplished using calibration curves. RESULTS AND DISCUSSION
Numerous reports have been published on the ion-pair chromatography of the nucleotide^.^^-^^ This technique is the method of choice for the analysis of a mixture of these compounds using a gradient e1uti0n.I~Indeed, the nucleotides are ionic molecules in the pH range 2-8, and the addition to the mobile phase of a counterion of charge opposite to that of the nucleotides has enabled satisfactory results to be obtained. In this study, the chosen ion-pair reagent was a quaternary ammonium ion, TBAHS. This ion has previously been shown to be the most effective in retarding the elution of the majority of the nucleotide^.'^ The presence of TBAKS,which is more hydrophobic than other ionpair reagents such as ammonium acetate, increased the bond (20) Rosset. R; Caude, M.; Sardy, A. Chromatographie en phase liquide et
supercn'tique; Masson: Paris, 1991. (21) Fitzgerald, D. F.; Brodbeck, U.; Kiyosawa, I.; Mawal, R.; Colvin, B.; Ebner. K E. J. Biol. Chem. 1970,245, 2103-2108.
Table 1. Capacity Factors of Nucleotide Sugars and Nucleotides'
solute
capacity factor, k'
UDP-glucose
2.33 2.65 2.92 2.96 6.75 3.08 5.37 3.35 1.81 5.77 8.57 2.35 5.89 8.21
UDP-galactose UDP-GalNac
UDP-GlcNac UDP-GkUA GDP-mannose AD P-glucose
GDP-glucose UMP UDP UTP GMP
GDP GTP
Experimental conditions: 15 min linear gradient from 2.5%to 30% of eluent B, pH 6.9, 2.5 mM TBAHS.
strength of the nucleotides to the stationary Thus, Tl3AHS enables solute elution to be sufficiently delayed to facilitate a separation of nucleotide sugars. This is an important factor in view of the hydrophilic nature of these compounds. Table 1 shows the retention times and the capacity factors for the majority of nucleotide sugars and free nucleotides involved in the glycosylation pathway. Results were in agreement with those of Perrone et al.15 and Wemer:17 the elution order of nucleotides was monophosphate, diphosphate, and triphosphate, as predicted. The more hydrophilic nucleotide sugars were eluted first. The presence of a carboxylic acid group on the UDP-gluUA (k' = 3.08) increased the retention time in comparison to that of the UDP-glucose (k' = 2.33). Indeed, at pH 6.9, the carboxylic acid group is in the form RCOO-, and the negative charge of the molecule was amplified. The capacity factors of all tested molecules were different, so the chosen chromatographic conditions enabled the efficient separation of a mixture of nucleotide sugars and free nucleotides. Additionally,this system allows the analysis of both type of nucleotides: guanoside derivatives (purine bases) as well as uridine derivatives (pyrimidine bases) (Table 1) * Influence of the pH of the Mobile Phase, the Counterion Concentration, and the Temperature on the Separation. The majority of the separation methods for nucleotides presented in the literature were at the pH range 3-6.15J6J8 However, nucleotides are ionic molecules over the pH range 2-8. In order to study the influence of the mobile phase pH on the separation, three pH values have been tested: 5.7, 6.3, and 6.9. This study was performed with each molecule specified in Table 1. Results for UDP-galactose, UDP, and UTP are plotted on Figure 1. Increasing the pH value from 5.7 to 6.9 led to a slightly decreased retention time for all molecules. This effect was more prononced for UDP-galactose than for the other free nucleotides. Despite this, the elution order of all molecules tested was not modified by varying the pH value (results not shown) and was always in agreement with the results of Perrone et al.15 and Hoffman et d.14 At this pH range, the negative charge of the molecules is directly proportionalto the number of phosphate groups, and consequently (22) Pingoud, A; Flies, A; Pingoud, V. In HPLC of macromolecules a practical approach; Oliver, R W. A, Ed.; IRL Press: Oxford, U q 1989 Chapter 7, pp 188-189.
UDP-ealactose 5.6
5.8
6.0
6.2
6.4
6.6
6.8
7.0
PH Figure 1. Effect of the mobile phase pH on the capacity factors of UDP-galactose, UDP, and UTP. Experimental conditions: eluent A, K2HPOdKH2POd 50 mM, indicated pH, 2.5 mM TBAHS; eluent 6, K2HP0dKH2PO450 mM, indicated pH, 2.5 mM TBAHS/5O% acetonitrile; 15 min linear gradient from 2.5% to 30% of eluent 9;flow rate 1 mumin.
the elution order was m o m , di-, and triphosphate. It can be concluded that only the phosphate moiety is involved in the mechanism of the separation. In the case of nucleotide sugars, the same phenomenon has been observed. Moreover, the presence of a substituent on the glycosyl residue, such as an N-acetyl group, decreased the hydrophilic character of the molecule and consequently increased the retention time of the nucleotide-sugar relative to that of the non-acetylated compound: as an example, at pH 5.7, k ' u ~ p ~ = a l3.93 ~ ~ and ~ k ' u ~ p .= ~ ~3.53. A similar result has been observed at pH 6.9. Thus, ion-pair chromatographyof a mixture of nucleotide sugars and nucleotides could be performed at pH = 7 with significant gain in analysis time (19 min) without modification of the elution order. This result was of interest for the application of this method to determine glycosyltransferase activity. Since the majority of reactions catalyzed by glycosyltransferases are achieved at the pH range 7-8, the disappearance of the substrates and the appearance of the products could be quantiiled at a similar pH. The hydrolysis of the glycosyltransferase substrates (nucleotide sugars), which are particularly unstable in solution,23 was diminished, and consequently more satisfactory results were obtained. A study of the influence of the counterion concentration and the temperature on the capacity factor was performed. Three concentrations of "l3AHS have been tested: 1.5,2.5, and 4 mM (results not shown). Normally, increasing the concentration of the counterion in the mobile phase causes an increase in capacity factor The results obtained here were in agreement with the literature. Increasing the temperature to 40 "C did not enhance the analysis; therefore, the further separations were undertaken at ambient temperature. This present study suggests that a concentration of 2.5 mM Tl3AI-Ewas the optimal counterion concentration with the chromatographic system used for the analysis of mixtures of nucleotide sugars and free nucleotides. Separation of a Nucleotide Sugars and Free Nucleotides Mixture. The optimized chromatographic conditions for the separation were used to analyze a mixture of nucleotide sugars and free nucleotides (see Figure 2). Resolution and selectivity were satisfactory. A comparison between Table 1 and Figure 2 confirms that the elution order of the mixture of the molecules follows the predicted order for uridine derivatives as well as for k'.19820
(23) Keppler, D.; Decker, K In Methods of Enzymatic Analysis; Bergmeyer, H. U., Ed.; Verlag Chemie: Weinheim, New York, 1974; Vol. 4, pp 22212224.
Analytical Chemistry, Vol. 67,No. 9,May 1, 1995
1629
3 v
B
3
350
,
300
UDP-glucose
250
-
200
g
150
f
a
100
9
50
UDP-galactose
5
hl
0
700
600
3
500
9
400
h
v
5.g
0
3
i'1
6
t
9
12
1s
18
0.01
0.00
0.02
0.03
0.05
0.04
Concentration (g/l)
I\
300 200
; 3 100 I 0
0 0
3
6
9
12
15
2
18
Retention time (min)
Figure 2. Gradient elution separation of a mixture of nucleotide sugars and nucleotides. In each case, the separations were performed at pH 6.9 and 2.5 mM TBAHS. The injected amount of each product was over a range of 5-10 nmol.
6 8 Retention time (min) 4
10
I 12
Figure 3. Quantitative studies. (a) Calibration curves of UDPglucose, UDP-N-acetylglucosamine, UDP-galactose, and UTP over a range of 40 pmol to 2 nmol. (b) Response obtained to an injection of 40 pmol of UDP-galactose.
following reaction:24 guanosine derivatives. Additionally, the chromatographic system even allowed us to separate the epimeric nucleotide sugars: UDPgal and UDP-glc (Figure 2a). A similar result has already been achieved by Lagunas et a1.,18but this procedure necessitated the formation of a complex between the UDP sugar and borate prior to analysis. Consequently, the method described here is simpler and elution more rapid than those of Lagunas et a1.P epimeric sugars eluted in less than 10 min as compared to elution times of between 10 and 16 min. Moreover, this rapid technique allows the simultaneous quantification of UDP, UTP, and nucleotide sugars, and hence is particularly adapted for assaying glycosyltransferase activity. Quantitative Studies. In order to obtain quantitative studies using the chromatographic system developed here, calibration curves have been established. In all cases, the relation between peak area response and concentration of injected molecule gave a straight line over a range of 40 pmol to 2 nmol, with zero ordinate-abscissa intercepts (Figure 3a). The method investigated is particularly sensitive and enabled detection of nucleotide and nucleotide-sugar down to 40 pmol (Figure 3b). The chromatographic procedure presented here is more sensitive and simpler than those reported earlier.I4J8Furthemore, the range of reaction products analyzed has been extented to include both free nucleotides and nucleotide sugars. Assay of Galactosyltransferase Activity. The galactosyltransferase @C 2.4.1.22) or ~-D-N-acetylglucosaminide-~-1-4galactosyltransferase is a glycosyltransferase which catalyzes the transfer of galactose from UDP-galactose to N-acetylglucosamine or its glycosides, forming a p-1-4 linkage according to the 1630 Analytical Chemistry, Vol. 67, No. 9, May 1, 1995
UDP-galactose
+ acceptor + NADH galactose ,L?-1-4 acceptor
+ UDP + NAD'
Glucose and its glycosides are also acceptors in the presence of lactalbumin. This galactosyltransferase has been extensively studied, because it is involved in the biosynthesis of the glycan moieties of glycopr~teins.~J~ Current assays for galactosyltransferase activity are based on a spectrophotometricdetermination. During the reaction, the disappearance of NADH is recorded at 340 nm, indicating the formation of NAD+, which is coupled to the synthesis of UDP.21,24This technique is simple, but the quantitative determination of the substrates and products is not possible. Therefore, the chromatographic system described here has been tested to detect the appearance of the galactosyltransferase reaction products. The chosen acceptor was glucose. To avoid the feedback inhibition of the UDP released, phosphoenolpyruvate in the presence of a mixture of pyruvate kinase and lactate dehydrogenase has been added to the reaction mixture in order to transform UDP to In the chromatograms of control and assay samples (Figure 4), it can be seen that the disappearance of UDP-galactose and NADH is correlated to the appearance of NAD+ and UTP, and the results are quantitative: 0.7 pmol of galactose was transferred to glucose over the 10 min reaction period. (24) Ebner, K E. In n t e Enzymes; Boyer, P. D., Ed.; Academic Press: New York, 1973; Vol. 9, pp 363-377. (25) Wong, C. H.Tibtech 1992, 10,337-341.
Control
8o
...,.......
70
3
60
Assay
ACKNOWLEWMENT The authors are very grateful to Monique Suderie for her technical assistance and to Dr. N. D. Lindley for improving the English of the manuscript,
-
v
B
50-
3; 40 8
{
30-
3 4
20 10
ferase activity. This result is particularly attractive considering the growing interest in oligosaccharide synthesis in vitro using glycosyltransferases,
................I
...
..,........
Thus the galactosyltransferaseactivity can be determined using chromatographic procedures. Additionally,this procedure can be used to determine rapidly the substrate specilicity of an enzyme and consequently to discover novel substrates? The ion-pair reversed-phase HPLC method described permitted the simultaneous quantitative and qualitative determination of free nucleotides and nucleotide sugars. Satisfactory separationswere carried out at pH 6.9, at ambient temperature, and at 2.5 mM concentration of TBAHS. The successful application of this chromatographic system for assaying galactosyltransferaseactivity indicated that it can be used generally to evaluate glycosyltrans-
LIST OF ABBREVIATIONS GMP guanosine 5’-monophosphate GDP guanosine 5’-diphosphate UMP uridine 5’-monophosphate UDP uridine 5’diphosphate GTP guanosine 5’-triphosphate UTP uridine 5’-triphosphate GDP-man guanosine 5’diphosphomannose UDP-glc uridine 5’-diphosphoglucose UDP-gal uridine 5’diphosphogalactose UDP-GalNac uridine 5’-diphospho-N-acetylgalactosamine UDP-GlcNac uridine 5’-diphospho-N-acetylglucosamine UDP-glcUA uridine 5’-diphosphoglucuronic acid ADP-glucose adenosine-5’-diphosphoglucose GDP-glucose guanosine 5’-diphosphoglucose NAD+ nicotinamide adenine dinucleotide (oxidized form) NADH nicotinamide adenine dinucleotide (reduced form) TBAHS tetrabutylammonium hydrogen sulfate Received for review September 28, 1994. February 9, 1995.@
Accepted
AC940963E @
Abstract published in Advance ACS Abstracts, March 15, 1995.
Analytical Chemistry, Vol. 67, No. 9, May 1, 1995
1631
