The separation of isomeric pentose phosphates from each other and the preparation of d -xylulose 5-phosphate and d -ribulose 5-phosphate by column chromatography

ANALYTICAL

BI~HEM~TRY

1x8,4-9

(1981)

The Separation of lsomeric Other and the Preparation o-Ribulose 5Phosphate ARMANDO GASCON,

TERRY

Pentose Phosphates from Each of D-Xyiutose 5Phosphate and by Column Chromatography WOOD,

AND LUCIE CHITEMERERE

Department of Biochemistry, University of Zimbabwe, P. 0. Box MP f67, Mount Pleasant, Salisbury, Zimbabwe Received January 5, 1981 The binding of the S-phosphates of ribose, arabinose, ribulose, and xyluiose to a dihydroxyboryl-substituted cellulose column was investigated under various conditions of pH, salt content, and the presence of ethanol. Ribose S-phosphate was bound to the column at pH 8.1 or above at a high ionic strength when hydrazine was absent but not when it was present. Xylulose 5-phosphate was bound with hydrazine present while ribulose Sphosphate only bound strongly when both hydrazine and ethanol were present. The structural basis for this behavior is discussed. Procedures are described for isolating ribulose Sphosphate from an isomerase equilibrium mixture with ribose 5-phosphate, and for isolating xylulose 5-phosphate from a mixture containing ribulose and ribose 5-phosphates.

D-Ribulose S-phosphate may readily be prepared as an equilibrium mixture with Dribose 5-phosphate by treatment of the latter with D-ribose 5-phosphate ketol isomerase (EC 5.3.1.6). Further addition of D-ribulose 5-phosphate 3-epimerase (EC 5.1.3.1) gives an equilibrium mixture containing some 40% of D-xylulose 5-phosphate (I ). Although ribose 5-phosphate and ribulose 5-phosphate may be separated on a column of Dowex-i formate in the presence of borate (21, or by a salt gradient on a column of DEAE-Sephadex A-25 (T. Wood, unpublished), to our knowledge a separation of the two isomeric ketopentose phosphates by column chromatography has never been reported. Such a separation would provide a simple method for preparing D-xylulose 5-phosphate for kinetic studies of D-ribulose 5-phosphate 3epimerase (EC 5.1.3. I ), transketolase (EC 2.1.2.1), and phosphoketolase (EC 4.1.2.9>, and for other purposes. In 1970, Weith et al. (3) synthesized cellulose derivatives containing the dihydroxyboryl group and showed that they interacted

specifically with the 1,2-cis-diol groups of nucleic acid components, sugars, and other polyols. More recently, Goitein and Parsons (4) have shown that 5-phospho-~-D-ribose I-diphosphate passed through a column of dihydroxyboryl-cellulose while D-ribose .5phosphate was retarded. We report here the use of this material to separate the 5-phosphates of D-ribose, D-arabinose, D-ribulose, and D-xylulose from each other and the isolation of pure samples of the two ketopentose phosphates. EXPERIMENTAL

Materials. Acetytated DBAE-cellulose (N - [N’ - (m - dihydroxyborylphenyl)suc cinamyllaminoethyl-cellulose)’ prepared as described by Weith et al. (3) was obtained from Collaborative Research Inc., Waltham, Massachusetts. D-Ribose 5-phosphate disodium salt, D-arabinose 5-phosphate diso’ Abbreviation used: DBAE-cellulose, N-[N-(m-dihydroxyborylphenyl)succinamyl]aminoethyl - cellulose. 4

QOO3-2697/S l/170004-06$02.00/O Copyright Q 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

SEPARATION OF PENTOSE PHOSPHATES dium salt, DL-glyceraldehyde 3-phosphate diethylacetal monobarium salt, P-hydroxypyruvic acid lithium salt, and D-ribose 5phosphate ketol isomerase, type I from spinach, were all obtained from Sigma Chemical Company. Transketolase (22 units/mg) was prepared from Can&da utilis (5) and D-ribulose 5-phosphate 3-epimerase was purified from calf liver (6). D-Ribose 5-phosphate ketol isomerase was freed from contamination with D-ribulose 5-phosphate 3-epimerase by elution from DEAE-Sephadex A-50 with successive concentrations of 50, 100, 200, and 500 mM KC1 in 50 mM triethanolamine-HCl buffer, pH 7.4, the last fraction to be eluted being free of the contaminating enzyme. This fraction was used to prepare mixtures of D-ribose 5-phosphate and D-ribulose 5-phosphate (1). A mixture containing D-xylulose 5-phosphate as well was prepared by the action of the isomerase and Dribulose 5-phosphate 3-epimerase ( 1). D-Xylulose 5-phosphate free of other pentose phosphates was prepared by condensing hydroxypyruvate and D-glyceraldehyde 3phosphate together in the presence of transketolase; in the absence of further purification it was contaminated with L-glyceraldehyde 3-phosphate and excess hydroxypyruvate (7) but could be used to establish the behavior of D-xylulose 5-phosphate alone on the column as the L-glyceraldehyde 3-phosphate gave a blue color while hydroxypyruvate did not react in the cysteine-carbazole reaction. The Minit molecular building system was supplied by Cochranes of Oxford Ltd., Leafield, Oxford, United Kingdom. Methods. The ketopentose phosphates were determined calorimetrically by the cysteine-carbazole reaction as described by Axelrod and Jang (8) using a heating time of 2 h at 37°C to develop the color. Under these conditions 0.1 pmol of ketopentose 5phosphate gives an absorbance at 540 nm of 0.32 (1). Ribose 5-phosphate and arabinose 5-phosphate were measured without interference from ketopentose phosphates by

5

the phloroglucinol method of Dische and Borenfreund (9). D-Ribulose 5-phosphate and D-xylulose 5-phosphate were measured enzymatically with transketolase and D-ribulose 5-phosphate 3-epimerase as described previously (1). Hydrazine was measured by reaction with para-dimethylaminobenzaldehyde ( 10). The capacity of the DBAE-cellulose was determined using a 10 mM solution of D-ribose 5-phosphate in 50 mM morpholine-HCl buffer, pH 8.1, containing 0.1 M magnesium chloride. This solution was passed through the column until ribose 5phosphate appeared in the effluent. All column runs were carried out in the cold room at 5°C. The behavior of the various pentose phosphates was determined using a short 2 X 0.6-cm-diameter column of DBAE-cellulose (0.4 g; capacity, 100 pmol of D-ribose 5-phosphate) previously washed with starting buffer. The sample containing 2 to 10 pmol of the pentose phosphate was diluted in 2 ml of the starting buffer, a IOfold concentration of hydrazine was added when required, and the pH readjusted to the original value on the meter. The sample was then placed on the column and washed through with 20 ml of starting buffer. The starting buffer was usually followed by a second buffer designed to elute adsorbed ketopentose phosphate and was followed invariably with a third buffer of 0.2 M lithium chloride in 50 mM sodium acetate buffer, pH 5.0, to remove all compounds still remaining adsorbed to the column. Fractions of 2 ml were collected, the pH of alkaline fractions was adjusted to pH 6.0-6.5 and the tubes were stored frozen at - 15°C. Next day the contents were thawed and reacted with cysteine-carbazole and with phloroglucinol and, when necessary, assayed enzymatically. RESULTS

Separation of Ribose 5-Phosphate and Ribulose S-Phosphate

When a mixture of D-ribose 5-phosphate and D-ribulose 5-phosphate was chromato-

6

GASCON,

WOOD, AND CHITEMERERE

graphed on DBAE-cellulose at pH 9.2 in 50 mM morpholine-HCl buffer neither substance was adsorbed although the ribose 5phosphate was retarded. In the presence of 2 M potassium chloride or 0.1 M magnesium chloride at pH 8.1 or above, ribose 5-phosphate was completely adsorbed while the ketopentose phosphate passed through the column (Fig. 1). The unadsorbed material gave a small peak of absorbance at 552 nm after reaction with phloroglucinol (Fig. 1). This was due to the brown color produced when ribulose 5-phosphate is heated with phloroglucinol and not to the presence of ribose 5-phosphate which gives a bright cherry red color with the reagent. Pure ribulose 5-phosphate was precipitated from the unadsorbed material with barium and ethanol as described later for the isolation of xylulose 5-phosphate. Separation of Ribulose 5-Phosphate and Xylulose j-phosphate

When a mixture of ribose 5-phosphate, ribulose 5-phosphate, and xylulose 5-phos-

20 Fraction

No

(2ml 1

FIG. 1. Separation of ribose 5-phosphate and ribulose 5-phosphate on a 2 X 0.6-cm column. The sample containing 7.5 pmol of D-ribose 5-phosphate and 2.5 rmol of D-ribulose 5-phosphate was washed through with 50 mM morpholine-HCI buffer containing 0.1 M magnesium chloride, pH 8.1. At A the column was eluted with 0.2 M LiCl in 50 mM sodium acetate buffer, pH 5.0. -,mM D-ribulose 5-phosphate; - - -, absorbance at 552 nm after reaction of a O.I-ml sample with phloroglucinol. Abbreviations used: R-5-P. ribose 5-phosphate; Ru-5-P, ribulose 5-phosphate.

phate was used as a sample, part of the ketopentose phosphate was adsorbed when 25% ethanol was incorporated into the buffer containing 0.1 M magnesium chloride at pH 8.7. The adsorbed material was recovered by elution with ethanol-free buffer. Although an adsorbed and a nonadsorbed ketopentose phosphate fraction were obtained in this way, these two fractions did not correspond to pure ribulose 5-phosphate and xylulose 5phosphate, respectively, but were mixtures of the two compounds in different proportions. It was concluded that the divalent magnesium ion was crosslinking the two compounds together so the 0.1 M magnesium chloride was replaced by 1 or 2 M potassium chloride with beneficial results. Even in the absence of magnesium ion a tendency of the two ketopentose phosphates to associate together was observed. An attempt to block the keto group and prevent dimer formation was made by adding hydrazine to the sample. This improved the separation of the two ketopentose phosphates from each other and dramatically changed the behavior of ribose 5-phosphate which passed through the column in the presence of hydrazine and was adsorbed in its absence (Table I ). Xylulose 5-phosphate alone, in the absence of hydrazine, passed through the column in buffers containing 1 M KC1 and 25% ethanol at pH 8.7 but was partially retained at pH 9.2 (Table I). Raising the pH from 8.7 to 9.2 similarly improved the separation of the three pentose phosphates when hydrazine was present and ethanol was absent. As ribulose 5-phosphate tended to bind to the column in the presence of ethanol, ethanol was omitted in later experiments. The system finally adopted used hydrazine, high salt, and a raised pH to promote adsorption of xylulose 5-phosphate and a lowering of the salt concentration and pH to bring about its desorption. When the short trial column was replaced by a 5 X 0.6-cm column (1 g DBAE-cellulose; capacity, 250 pmol D-ribose Sphosphate), essentially all the xylulose 5-phos-

SEPARATION

OF PENTOSE TABLE

7

PHOSPHATES

I

OF PENTOSE PHOSPHATES ON A 2 X 0.6Cm-DIAMETER COLUMN OF DBAE-CELLULOSE THROUGH WITH STARTING BUFFER OF 50 mM MORPHOLINE-HCI AND THE ADDITIONAL

BEHAVIOR WASHED

‘Additions

to starting

buffer

Presence of hydrazine

Ribose 5phosphate

WHEN COMPOUNDS

Ribulose Sphosphate

Xylulose 5phosphate

1 M KC1 + 25% EtOH,

pH 8.7

+ ret

ret, + -

1 M KCI

pH 9.2

+ ret

ret, 4 ret, +

+ 25% EtOH,

2 M

KCI. pH 8.7

ret -

ret, -C

2 M

KCI, pH 9.2

ret -

ret, + -

Note. The addition (-) neither adsorbed some retarded.

of a IO-fold nor markedly

excess of hydrazine to the sample is indicated by a (-I-) in the second column. retarded; (+) adsorbed; (ret) markedly retarded; (ret, +) some adsorbed and

phate in an equilibrium mixture was adsorbed while the ribose Sphosphate, hydrazine, and ribulose Sphosphate passed through the column with some slight retardation of the latter. Once the D-xylulose S-phosphate or Dribulose Sphosphate had been separated on the column, the tubes containing the desired material were combined, concentrated, and the sugar phosphate precipitated as the barium salt with ethanol. In order to avoid the concomitant precipitation of large quantities of salt from the buffer potassium chloride was replaced by lithium chloride which gave the same elution pattern but did not precipitate when ethanol was added to precipitate the barium salt. Separation of Ribose S-Phosphate Arabinose 5-Phosphate

and

The affinity of the adsorbent for ribose 5phosphate has been used to separate a mixture of ribose Sphosphate and arabinose 5phosphate. In 50 mM morpholine-HCl buffer, pH 8.1, containing 0.2 M magnesium chloride, D-ribose Sphosphate was adsorbed while D-arabinose Sphosphate passed through the column (I 1). Sample recovery. Although ketopentose

phosphates are alkali labile, over 90% of Dribulose Sphosphate was found intact by enzymatic assay after exposure to a pH of 8.7 for 3 h at room temperature. Xylulose Sphosphate is more stable than ribulose 5phosphate and in the column runs at pH 9.2 no noticeable loss of xylulose 5-phosphate was observed. However, in several runs carried out with a mixture of the three pentose phosphates at pH 9.2 about 5- 10% of strongly adsorbed material that reacted with cysteine-carbazole was eluted by the pH 5 buffer. This material (the third peak in Fig. 2) did not react with transketolase in the assay for D-xylulose 5-phosphate and D-ribulose 5-phosphate, nor did it bind to an anion exchange resin. It appeared to consist of free keto~ntoses formed by hydrolysis of the corresponding phosphates. isolation of o-Xylulose S-Phosphate from an Equilibrium mixture with D-Ribose 5-Phosphate and o-Ribulose SPhosphate An equilibrium mixture containing 15 Fmol of D-xylulose 5-phosphate was chromatographed on a 5 X 0.6cm column at pH 9.2 (Fig. 2). Column fractions were adjusted

8

GASCON,

Froct~on

No

WOOD,

AND

12 ml I

FIG. 2. Separation of ribulose Sphosphate and xylulose 5-phosphate on a 5 X 0.6-cm-diameter column. The sample contained approximately 1.5Kmol of D-xy&lose Sphosphate, 5 pmol D-ribuiose 5-phosphate, 15 amol D-ribose S-phosphate, 100 pmol of hydrazine and 2 M LiCl in 5 ml 50 mM morpholine-HC1 buffer, pH 9.2. It was washed through with 2 M LiCl in 50 mM morpho~ine-HCI buffer, pH 9.2, followed at A by 0.2 M Lit3 in the same buffer, pH 8.7. At B the column was eluted with 0.2 M LiCl in 50 mM sodium acetate buffer, pH 5.0. The position of the ribose S-phosphate peak coincided with that of the hydrazine peak. -, Ketopentose phosphate; - - -, hydrazine. Abbreviations used: Ru-5-P, ribulose 5-phosphate; Xu-5-P, xylulose 5-phosphate. to pH 6.0-6.5 and frozen. After assay, tubes 13 to 17 (Fig. 2) were combined and concentrated to 2 ml by lyophilization. The solution was adjusted to pH 6.5, cooled in ice, 0.2 ml of 25% (w/v) barium acetate, and 8 ml of ice-cold ethanol were added and the suspension of the barium salt was kept for 12 h at 5°C with occasional shaking. The barium salt was centrifuged down and converted to the sodium salt by ion exchange as described previously (12). Enzymatic assay of the clear colorless solution obtained showed the presence of 7.0 pmol D-xylulose Sphosphate, and 0.4 hmol of D-ribulose 5phosphate. These figures indicated a recovery of 80% of the 9.2 pmol of ketopentose phosphate in tubes 13 to 17 and a 5% contamination with D-ribulose Sphosphate. The product contained 3.5% hydrazine. DISCUSSION

It has been suggested that the tetrahedral boronate anion -Ph[B(OH),]is the active

CHITE~ERERE

species that complexes with polyols (3,13). Although the pK vafue of the dihydroxyborylphenyl group attached to cellulose has not been determined it is probably close to that of benzeneboronic acid (Ph[ B(OH),]) itself which is 8.86 ( 14). Ribose Sphosphate and ketopentose phosphates were not bound to the adsorbent in simple buffer at pH values up to 9.2. In view of the alkali lability of sugars in general and the ketopentose phosphates in particular it was not considered advisable to try to achieve binding by raising the pH further so alternative means were sought. The apparent pK value of acids dissociating to give a negatively charged anion may be lowered by increasing the ionic strength (15). Thus, at a pH of 9.2, binding was increased by the addition of salt to the buffer and, in the presence of salt and/or organic solvents at pH 8.7, the degree of binding could be increased by raising the pH to 9.2 (Table 1). Similar results were obtained with nucleic acid derivatives and it has been pointed out (3,13) that the addition of salt both lowers the apparent pK of the dihydroxyborylphenyl group and reduces the mutual repulsion of the sugar phosphate and boronate anions. Lowering the dielectric constant would have the latter effect and could account for the increased binding observed in the presence of ethanol. The ability of borylated celluloses to bind polyols has been attributed to the ability of triols to form particularly stable six-membered ring complexes with phenylboronic acid (16). The binding has also been explained as being due to the presence of cishydroxyl groups with a coplaner oxygencarbon-carbon-oxygen arrangement (3,I3). D-Xylulose 5-phosphate and D-ribulose 5phosphate exist in solution predominantly in the open-chain form ( 17) and the D-xylulose compound having neighboring hydroxyls tram to each other binds more tightly to the adsorbent than D-ribulose Sphosphate in which the hydroxyl groups are cis. After building Minit molecular models it was con-

SEPARATION

OF PENTOSE

eluded that these results were explicable by either theory. Bringing the hydroxyls at carbons 1, 3, and 4 of D-xylulose Sphosphate into a coplaner configuration leaves the carbon chain extended whereas with D-ribulose Sphosphate the chain is folded back on itself. Consequently, on therm~ynamic grounds the binding of D-xylulose Sphosphate appears to be favored. In solution, D-ribose Sphosphate is 98% in the closed ring form (18) and was firmly adsorbed at a relatively low pH (8.1) in the presence of salt and organic solvents. Under all the conditions used, the addition of hydrazine destroyed its affinity for the adsorbent, presumably as a result of its conversion to the straight chain hydrazone. Bringing the hydroxyls on carbons, 2, 3, and 4, or on 3 and 4 only, into a contiguous position for ph~nylboronate complexing would result in a contracted configuration of the carbon chain and would possibly be prevented by steric hindrance of the hydrazine substituent. Whatever the theoretical explanation for the results, we have been able to separate Dxylulose S-phosphate from D-ribulose 5phosphate and prepare each of the ketopentose phosphates free from contamination by the other isomers. The separation of D-ribose 5-phosphate and D-arabinose Sphosphate as a result of the nonbinding of the latter is readily explained by the absence from the ring form of the arabinose compound of cishydroxyl groups able to react with the dihydroxyboryl groups of the absorbent.

9

PHOSPHATES

ACKNOWLEDGMENTS This work was supported by a grant and a research fellowship to A. Gascon from the University of Zimbabwe Research Board.

REFERENCES 1. Wood, T. ( 1975) in Methods in Enzymology (Wood, W. A., ed.), Vol. 41, pp. 37-41, Academic Press, New York. 2. Dickens, F., and Williamson, D. H. ( 1956) Biochem. J. 64, 567-578. 3. Weith, H. L., Wiebers, J. L., and Giiham, P. T. ( 1970)

Biochemistry

9, 4396-440

I.

4. Goitein, R. K., and Parsons, S. M. (1978) Anal. Biochem.

87,637-640.

5. Wood, T. (1981) Biochim. Biophys. Acta 659,233243. 6. Wood, T. ( 1979) Biochim. Biophys. Acta 570,352362. 7. Wood, T. ( 1973) Prep. Biochem. 3, 509-515. 8. Axelrod, B., and Jang, R. (19.54) J. Biol. Chem. 209,84’7-855. 9. Dische, Z., and Borenfreund, E. (19.57) Biochim. Biophys.

Acta

23,639-642.

IO. Snell, F. C., Snefl, C. T., and Snell, C. A. (1959) Methods of Calorimetric Analysis, Vol. HA, p. 707. Van Nostrand, New York. II. Wuod, T. and Gascon, A. (1980) Arch. Biuehem. Biophys.

203, 727-733.

12. Wood, T. ( 1968) J. Chromatogr. 35, 352-361. 13. Rosenberg, M., Wiebers, J. L., and Gilham, P. T, (I 972) Bi~~emistry 11, 3623-3638. 14. Branch, G. E. K., Yabroff, D. L., and Bettman, B. (1934)

J. Amer.

Chem.

Sot.

S&937-941.

15. Long, C. (ed.) (1961) Biochemists’ Handbook, pp. 23-24, Spon, London. 16. Ferrier, R. .I. (1978) Advun. Curbohyd. Chem. Biochem.

35, 3 I-80.

17. Gray. G. R., and Barker, R. ( 1970) Biochemisrry 9, 2454-2462. 18. Serianni, A., Pierce, J., and Barker, R. (1979) Biochemistry

18, 1192- 1 t 99.