Spectrophotometric assay for d -ribose-5-phosphate ketol-isomerase and for d -ribulose-5-phosphate 3-epimerase

ANALYTICAL

BIOCHEMISTRY

33,

297-306

Spectrophotometric

(1970)

Assay

Ketol-isomerase

for

and

phosphate

D-Ribose&phosphate

for

D-Ribulose&

3-Epimerase T. WOOD

Department

of Biochemistry,

McGill

University,

Montreal, Quebec, Canada

Received July 11, 1969 A number of methods for the assay of n-ribosed-phosphate ketolisomerase (ribosephosphate isomerase EC 5.3.1.6) have been reviewed by Knowles et al. (1). A procedure employed in this laboratory has been described (2) in which, using n-ribose 5-phosphate as a substrate, the oxidation of NADH was followed continuously at 340 rnp after the addition of n-ribulose-5-phosphate 3-epimerase (EC 5.1.3.1)) transketolase (EC 2.2.1.1) and a mixture of glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) and triosephosphate isomerase (EC 5.3.1.1). Details of a related assay for n-ribulose-5-phosphate 3-epimerase were also published (2). The above assays have a number of disadvantages. The ribosephosphate isomerase assay at 340 rnp involves three consecutive auxiliary reactions to couple the isomerase reaction to NADH oxidation and a considerable excess of two enzymes that are not commonly available. The assay for the epimerase requires n-ribulose ‘5-phosphate, a substrate that is expensive and difficult to isolate. The commonly used calorimetric procedure of Dische and Borenfreund (3) suffers from drawbacks due to variations in the depth of color given by a standard quantity of ribulose 5-phosphate and interference by the presence of n-ribulose-5phosphate 3-epimerase. These disadvantages are overcome in the method of Bruns et al. (4) in which the disappearance of n-ribose 5-phosphate is followed using the phloroglucinol reaction (5), but, even with careful technique, considerable variations in the depth of color given by a standard amount of ribose 5-phosphate were found. The method for n-ribose-5-phosphate ketol-isomerase described here is based upon the observation of Knowles and Pon (6) that an absorption band with a peak at 280 rnp appears when the enzyme is mixed with n-ribose 5-phosphate. It is similar to the assay at 286 mp described recently by Knowles et al. (1) except that a wavelength of 290 rnp is 297

298

T. WOOD

used to reduce interference from proteins and nucleic acids. The same principle is applied for the assay of n-ribulose-&phosphate 3-epimerase by employing as substrate an equilibrium mixture of D-ribose &phosphate and D-ribulose &phosphate in the presence of excess ribosephosphate isomerase and following the increase in absorption at 290 w as further ketopentose phosphate is formed. The millimolar extinction coefficient for ketopentose phosphate was determined by measuring the change in absorption at 290 mp occurring when ketopentose phosphate was produced or removed in three different enzyme reactions. EXPERIMENTAL

Chemicals and Enzymes. n-Ribose 5-phosphate was obtained from Sigma Chemical Company; in solution it showed negligible absorption over the range 240-360 ~J.L. 6Phosphogluconate trimonocyclohexylammonium salt was from the same source and when assayed with the dehydrogenase (7) was found to contain 77% of the n-form. Potassium pyruvate and thiamine pyrophosphate were from Nutritional Biochemicals Corp. Rabbit muscle lactate dehydrogenase (EC 1.1.1.27)) glyceroll-phosphate dehydrogenase, and triosephosphate isomerase were obtained from Boehringer Mannheim Corp., and yeast 6-phosphogluconate dehydrogenase (EC 1.1.1.44), type VI, 46 units/mg, free from ribosephosphate isomerase, was from Sigma. n-Ribulose-5-phosphate 3epimerase was prepared from rabbit muscle by the method of Tabachnik et al. (8) and was also prepared from ox liver. Ribosephosphate isomerase was from Sigma and had a specific activity of 55 units/mg when assayed at pH 7.1, 37”, and 3.1 mM n-ribose 5-phosphate (9, 10). It contained about 2% of n-ribulose-5-phosphate 3-epimerase when assayed at pH 7.4, 37”, and 2.5 mM n-ribulose 5-phosphate by the method described later in this paper. The proportion of epimerase was reduced to ca. 0.3% with only a 20% loss in isomerase activity by heating a solution containing 4 mg/ml of the isomerase at 70°C for 1 min; this enzyme preparation will be referred to as “heat-treated isomerase.” Spectrophotomstric measurements. All measurements in the ultraviolet were made in a 1 cm cuvet at 37” in a Beckman DB double-beam spectrophotometer connected to a Varicord 43 linear-log recorder (Photovolt Corp., 115 Broadway, New York 10010). For measurements at 290 rnp a slit width of 0.25 mm was used, corresponding to a spectral slit width of 2.8 ~Q.L. All solutions placed in the spectrophotometer were allowed to come to thermal equilibrium before the enzyme reaction was started. Cobrimetric Measurements. Ketopentose phosphate was estimated

ISOMERASE

AND

EPIMERASE

299

ASSAY

by the cysteine-carbazole reaction (3) using a heating time of 15 min at 37”. Ribose 5-phosphate was estimated by the phloroglucinol reaction (5). Ultrafiltration. Protein was removed by ultrafiltration in the cold room through a UM-10 membrane using a 10 ml capacity ultrafiltration cell obtained from Scientific Systems Division, Amicon Corporation, Lexington, Mass. 02173. Assay of D-Ribose-5-phosphate Ketol-isomerase. A solution containing 100 miW triethanolamine buffer, pH 7.4, and 0.20 ml of 100 mM n-ribose 5phosphate was placed in a 1 cm cuvet and allowed to come to 37” in the spectrophotometer. The volume of buffer was chosen to give a final volume of 2.09 ml after the addition of the sample. A suitable dilution of the enzyme sample was added and the change in absorption at 290 rnp recorded for 10 to 15 min. The activity in pmoles/min/ml sample was calculated from the slope of the initial linear portion of the progress curve using the factor 0.072 (see Table 1). Assay of D-Ribulose-5-phosphate S-,Epimerase. The assay mixture was the same as for the isomerase assay except that 2.2 units of heattreated phosphoriboisomerase was added. The isomerase reaction was complete after 10 min and the absorption of the mixture thereafter Absorption

TABLE 1 Changes and Millimolar Extinction Coefficients at 290 mp for Format.ion and Disappearance of Ketopentose Phosphate in a 1 cm Cell at 37”

Conversion

procedure’

Absorption

10 mM R-5-P to 7.5 mM R-5-P + 2.5 mM Ru-5-P 7.5 mM R-5-P + 2.5 mM Ru-5-P to 1.4 mM Ru-5-P + 4.3 m&I Xu-5-P + 4.3 mM R-5-P 1.92 mM D-6-PG to Ru-5-P

2.94 mM

D-6-PG

to Ru-5-P

3.85 mM D-6-PG to Ru-5-P 2.94 m&I Ru-5-P to 0.73 mM + 2.21 mil4 R-5-P Value adopted

Ru-5-P

0.180

change f

0.007b

Millimolar extinction coefficient 0.072

0.305 0.310

0.095

0.103” 0.103” 0.117” 0. 224c 0.235” 0. 254c 0.106 0.090

0.054 0.054 0.061 0.076 0.080 0.066 0.048 0.041 0.072

f 0.003”

0.097 P

a Abbreviations: R-5-P, n-ribose 5-phosphate; Ru-5-P, Xu-5-P, n-xylulose 5-phosphate; D-bPG, n+phosphogluconate. h Mean f st,andard deviation of six determinations. c Corrected for fall in absorpt,ion of pyruvat,e consumed.

n-ribttlose

5-phosphate;

300

T. WOOD

increased at a slow steady rate due to the traces of epimerase in the isomerase. A suitable dilution of the epimerase was added to give a linear rate and the activity of the enzyme was calculated, using the factor 0.072 from the rate of reaction after correction for the slow initial rate before adding the epimerase sample. RESULTS Conversion of D-Ribosed-p,hosphate to D-Rib&se-5-phosphate. 2 ml of a solution of 10 mM D-ribose &phosphate in 50 mM triethanolamine buffer, pH 7.4, was placed in the spectrophotometer and allowed to come to thermal equilibrium, and the spectrum between 240 m,u and 360 rnp recorded. Insignificant absorption was found over this range. Heat-treated ribosephosphate isomerase (2.2 units) was added and the change in absorption at 290 rn@ recorded. After 10 min the reaction had come to equilibrium and the absorption was constant except for a very slow linear increase due to the traces of ribulose-5-phosphate epimerase present. The spectrum of the mixture was recorded again. After correction for the contribution of the enzyme protein, an absorption curve for the product was obtained showing a broad peak with X,,, = 278 rnp and a minimum at 240 n-+ as reported previously (6). The ratio of the height of the peak at 280 rnp to the height at 290 ~IJ.Lwas 1.18 compared to a ratio of 1.14 measured from the curve published by Knowles et al. (1). At 37” and pH 7.0 to 7.5, ribulose 5-phosphate forms 25% of the total pentose phosphate at equilibrium (8, 11). Using this fact, the millimolar extinction coefficient at 290 rnp for the formation of Dribulose 5-phosphate was calculated (Table 1). That the change in absorption at 290 rnp was actually measuring the ribosephosphate isomerase reaction was checked by withdrawing 0.02 ml samples from the cuvet at intervals and freezing them in dry iceacetone. The samples, together with ribose 5-phosphate standards, were later subjected to the phloroglucinol reaction and the number of micromoles of ribose 5-phosphate converted at each time was calculated. The values followed the spectrophotometer plot closely. The chromophore generated by the action of ribosephosphate isomerase on ribose 5-phosphate did not change markedly in absorption at 290 ~,LL after freezing and thawing the solution, after lyophilization and resolution, or after removal of the enzyme by ultrafiltration. Conversion of D-Ribose 5-Phosphate to D-Rib&se 5-Phosphate and D-Xylulose 5-Phosphate. A solution containing 20 pmoles of n-ribose

5-phosphate was placed in the spectrophotomcter and heat-treated ribosephosphate isomerase was added as described above. After 9 min,

ISOMERASE

AND

EPIMERASE

301

ASSAY

when the reaction had come to equilibrium, n-ribulose-5-phosphate 3epimerase was added and the reaction followed until a new equilibrium had been reached and the absorption at 290 ~JL was constant, 35 min later. The product had the same absorption spectrum over the range of 240 to 360 ~JU as the product obtained with isomerase alone. The assumption was made that ribulose 5-phosphate and xylulose 5-phosphate had the same absorption coefficient at 290 ~.IJL and this coefficient was calculated (Table 1) from the observed change, after correction for added protein, from the equilibrium ratios of n-ribose 5-phosphate:Dribulose 5-phosphate : n-xylulose 5-phosphate = 75 : 25 : 75 reported at 37” and pH 7.5 by Tabachnik et al. (8). Absorption

Change

in Ribose-5-phosphate

Ketol-isomeruse

Reaction

at Different pH Values. The isomerase reaction was repeated in 0.1 M imidazole buffers, pH 6.5, 7.0, 7.5, and 8.0, and followed to equilibrium. The over-all changes in absorption at pH 6.5, 7.0, and 7.5 were the same as in triethanolamine buffer, pH 7.5, but at pH 8.0 the change was greater and variable. This result was not unexpected since ketopentose phosphates are unstable at pH values much above 7.0. Assay Procedures Ribulose-5-phosphate

for D-Ribose-5-phosphate S-Epimerase. The

Ketol-isomerase

and

D-

assay procedures finally adopted are described in the “Experimental” section. In both assays the initial rate of change of absorption at 290 mp was linear with enzyme concentration up to 0.2 unit/ml. Conversion of DL-GPhosphogluconate to D-Ribulose 5-Phosphate. A solution containing 3.80 mM 6-phosphogluconate trimonocyclohexylammonium salt (2.94 mM n-6-phosphogluconate), 0.2 mM NADP, 10 mM triethanolamine buffer, 10 mM magnesium chloride, and 6 mM potassium pyruvate was adjusted to pH 7.5 and 2.0 ml was placed in a 1 cm cuvet in the spectrophotometer. After equilibration at 37”C, lactate dehydrogenase (26 units) was added followed by 6-phosphogluconate dehydrogenase (0.84 unit). The reaction was followed at 290 mu; 0.05 ml samples were withdrawn at intervals and the ketopentose phosphate produced was estimated by the cysteine-carbazole reaction. A plot of the optical densities at 540 rnp from the cysteine-carbazole reaction followed the spectrophotometric plot very closely. The experiment was repeated, without the calorimetric measurement, using concentrations of 1.92 mM and 3.85 mM n-6-phosphogluconate. The millimolar extinction coefficient for the formation of n-ribulose 5phosphate was calculated from the measured increase in absorption after correcting for the changes due to the addition of enzyme protein and the disappearance of absorption at 290 IQL of the pyruvate consumed in the reaction (Table 1). A 1 mM solution of lithium lactate

302

T.

WOOD

had negligible absorption at 290 ~JU and 1 mM potassium pyruvate had an absorption of 0.025. At the end of the reaction the absorption remained constant. At this stage, a mixture of ribosephosphate isomerase and n-ribulose-5-phosphate 3-epimerase was added and the fall in absorption at 290 w due to the conversion of n-ribulose 5-phosphate into an equilibrium mixture of D-ribulose 5-phosphate, n-xylulose 5-phosphate, and n-ribose 5phosphate was followed. An extinction coefficient was again calculated (Table 1) from the fall in absorption and the known composition of the equilibrium mixture (8). Comparison of Assay for D-ribulose-5-p,hosphate S-Epimerase at 290 rnp with Assay at 340 rnp. A mixture was prepared in a 1 cm cuvet

containing the following substances in a volume of 2.0 ml: 50 mM glycylglycine, pH 7.4, 0.1 mM thiamine pyrophosphate, 0.13 mM NADH, 0.26 unit glycerol-l-phosphate dehydrogenase, 1.6 units triosephosphate isomerase, and 2 mM D-ribose 5-phosphate brought to equilibrium with 2.2 units of ribosephosphate isomerase. The absorption was recorded at 340 ~JU and 37”; 0.2 unit of transketolase was then added and, when the absorption was almost constant with time, various amounts of n-ribulose-5-phosphate Q-epimerase from ox liver were added. The activity of the epimerase was calculated from the rate of fall of absorption at 346 ~J.L The same volumes of enzyme solution were assayed at 290 rnp, as described earlier, only using 50 mM glycylglycine buffer, pH 7.4, and 10 mM n-ribose !&phosphate brought to equilibrium with 2.2 units of ribosephosphate isomerase in a total volume of 1.0 ml. The results for the two assays were plotted as micromoles per minute against microliters of enzyme solution added to the cuvet (Fig. 1). Because of the different substrate concentrations required in the two assays and the 85-fold difference in extinction coefficients of the chromophores a direct comparison was difficult. However, in one experiment using a 2 mM concentration of n-ribose 5-phosphate and 20 ,~l of epimerase the rate at 290 ~-IJJ was found to be the same, within the limits of experimental error, as that measured in the 340 rnp assay (Fig. 1). DISCUSSION

Some uncertainty was expressed by Knowles and Pon (6) as to the structure of the molecule absorbing in the region around 280 rnp and they suggested from their experimental evidence that it was a fi-diketone phosphate which was convertible to ribulose 5-phosphate by addition of a molecule of water. As a consequence it was considered of prime importance in the present work to measure the changes in absorption

ISOMERASE

AND

EPIMERASD

303

ASSAY

GO-

0 -

50-

-0

x .i 40

-

E : i5

E30-

/

-0

5 microliters

15

10

of

20

enzyme

FIG. 1. Relation between initial rate of reaction (gmoles/min X 10”) of Dribulose&phosphate 3-epimeraae and amount of enzyme (microliters enzyme solution) in assay at 290 ma and in assay at 340 rnp. See text for details. Rate at 290 rnh and 2.5 mM n-ribnlose 5-phosphate (O), at 340 mp and 0.5 mM n-ribulose 5phosphate CO), and at 290 rnp and 0.5 mM n-ribulose 5-phosphate CM).

when ribulose 5-phosphate was formed from both ribose 5-phosphate and 6-phosphogluconate and to show that the absorption change was independent of the route by which it was brought about. Furthermore, the changes resulting from the conversion of ribulose 5-phosphate to ribose 5-phosphate and to xylulose 5-phosphate were also measured. Throughout these enzymic interconversions the changes in absorption at 290 q followed closely the progress of the reactions, measured calorimetrically, and were in accordance with the known equilibria of the reactions involved and an extinction coefficient of a 1 mM solution of ribulose 5-phosphate of 0.072 (Table 1). No interference was noticed in the assay from other ultraviolet-absorbing species of the type described by Knowles and Pon (6)) and it was concluded that the absorption

was

principally

due to ketopentose

phosphate.

The

chromo-

phore was stable to freezing and thawing, lyophilization, and ultrafiltrations, so the presence of a transient isomeric form of the ketopentose phosphate seemed to be excluded. Since D-ribulose 5-phosphate and D-xylulose 5-phosphate differ only in their configuration about carbon-3 we would expect them to have the same absorption due to their keto group. This was confirmed when

304

T. WOOD

n-ribulose-5-phosphate 3-epimerase was added to a sample of n-ribulose 5-phosphate and no change in absorption resulted. The millimolar extinction coefficient for the formation or disappearance of ketopentose phosphate using a variety of reactions was calculated (Table 1). The figures obtained with 6-phosphogluconate dehydrogenase were considered the least reliable on account of the several corrections involved and because of a possible contribution from NADPH remaining at the end of the reaction. By contrast, the conversion of D-ribose 5-phosphate to an equilibrium mixture of n-ribose 5-phosphate and D-ribulose 5-phosphate could be reproduced readily and easily and it is the value obtained in this reaction that was adopted for routine use in assays of n-ribose-5-phosphate ketol-isomerase and n-ribulose-5-phosphate 3-epimerase. The value adopted of 0.072 is greater than the corresponding figure of 0.0586 reported by Knowles et al. (1) at 280 rnp. Possible reasons for this discrepancy may lie in differences in purity of the n-ribose 5-phosphate used. Since some batches of ribose 5-phosphate may contain considerable amounts of ketopentose phosphate, which is formed on storage, a smaller absorption increase will occur with such a batch compared to that obtained with a sample free of ketopentose phosphate. Furthermore, Knowles et al. (1) give no indication of the number of experiments on which their figure is based. The Michaelis constant of erythrocyte ribosephosphate isomerase has been reported as 2.1 to 2.2 mM (4,12) while lower values of 0.46 mM have been obtained for the enzyme from yeast (13) and 0.74 mhI for the spinach enzyme (14). With a K, value of 2.2 mM and the 10 mM substrate concentration chosen for the assay, the enzyme would be nearly saturated and operating at 82% of its maximal velocity. The only value traceable in the literature of the Michaelis constant of n-ribulose 5-phosphate for n-ribulose-5-phosphate 3-epimerase was a value of 1 m&I determined by Hurwitz and Horecker (15) using the enzyme from Lactobacillus pentosus. If we assume that epimerases from other sources have a similar Michaelis constant and that at the start of the reaction the ribulose 5-phosphate concentration is the equilibrium value of 2.5 mM (8)) then, in the epimerase assay, the enzyme would be operating at 72% of maximal velocity. Although a higher concentration of ribose 5-phosphate could be used to produce a higher equilibrium concentration of ribulose 5-phosphate to saturate the enzyme, it has been observed that at these higher concentrations of ribulose B-phosphate a nonenzymic reaction may be initiated resulting in a rapid linear increase in absorption at 290 mp. This reaction occurs with ribulose 5-phosphate formed from both 6-phosphogluconate and ribose 5-phos-

ISOMERASE

AND

EPIMERASE

305

ASSAY

phate and is believed to be either a condensation or a form of the “Maillard” or “browning” reaction. In concentrated solutions of ribulose &phosphate formed from &phosphogluconate the sudden increase in absorption described above was accompanied by the development of a yellow color. The assay procedures described here have been applied to the purified enzymes and to extracts of rat muscle, rat kidney, rat liver, ox muscle, ox liver, rabbit muscle, and pigeon liver, with satisfactory results. Since both ribulose 5-phosphate and xylulose 5-phosphate have the same absorption at 290 rnp, the presence of n-ribulose-5-phosphate 3epimerase in tissue extracts would not interfere with the isomerase assay. Transketolase could interfere by removing xylulose 5-phosphate as soon as it is formed, but in many of the tissues so far investigated the transketolase activity is much lower than the isomerase and epimerase activities (16-18). SUMMARY

Spectrophotometric assays for n-ribose-&phosphate ketol-isomerase and n-ribulose-5-phosphate 3-epimerase are reported. These assays are based upon the increase in absorption at 290 w due to the keto group of the products formed from ribose 5-phosphate. The millimolar extinction coefficient for the formation or disappearance of ketopentose phosphate was determined by a number of methods and a value of 0.072 was adopted. The applications of the assays and possible sources of interference are discussed. ACKNOWLEDGMENT This Canada.

work

was

supported

by

a grant

from

the

Medical

Research

Council

of

REFERENCES F. C., PON, M. K., AND PON, N. G., And. Biochem. 29, 40 (1969). KIELY. M. E., TAN, E. L., AND WOOD, T., Can. J. Biochem. 47, 455 (1969). DISCHE, Z., AND BORENFREUND, E.. J. Biol. Chem. 192, 583 (1951). BRUNS, F. H., NOLTMANN, E.. AND VALHAUS, El, Biochem. 2. 330, 483 (1958). DISCHE, Z., AND BORENFREUND, E.. Biochim. Biophys. Acta 23, 639 (1957). KNOWLES, F. C., AND PON. N. G,. 1. Am. Chem. Sot. 90, 6536 (1968). HOHORST. H.-J., in “Methods of Enzymatic Analysis” (Bergmeyer, H.-U., ed.), p. 143. Academic Press, New York, 1965. ‘I’ABACHNIK, M., SRERE, P. A.. COOPER, J., AND RACKER, E., Arch. Biochem. Biophys. ‘74, 315 (1958). HORICCK~, B. L., Huawrrz, J., AND WEISSBACH, A., in “Biochemical Preparations” (C. S. Vestling, ed.). Vol. 6, p. 83. Wiley, New York, 1958. Data sheet on phosphoriboisomerase (ribosephosphate isomerase) published by Sigma Chemical Co.

1. KNOWLES,

2. 3. 4. 5. 6.

7. 8.

9. 10.

306 11. 12. 13. 14. 15.

16. 17. 18.

T. WOOD

B., AND JANG, R., J. Biol. Chem. 209, 847 (1954). M., AND TSTJBOI, K. K., Arch. Biochem. Biophys. 103, 1 (1963). H., AND TOMOEDA, M., Agr. Biol. Chem. (Tokyo) 30, 61 (1966). A. C., AND LANE, M. D., 156th National Meeting, American Chemical Society, Abstract No. 147 (1968). H~~WITZ, J., AND HORECKER, B. L., J. Biol. Chem. 223, 993 (1956). $RIYASTAVA, L. M., AND HUESCHER, G., B&hem. J. 101, 48 (1968). NOVELLO, F., AND MCLEAN, P., Biochelm. J. 107, 775 (1968). TAN, E. L., AND WOOD, T., Camp. Biochem. Physiol., 31, 635 (196%.

AXIDLF~OD, URNETZKY, HORITSU, RUTNER,