Separation of bases, ribonucleosides and deoxyribonucleosides by anion-exclusion and partition chromatography on cation-exchange resin: Application to the assay of ribonucleotide reductase, deaminase and nucleosidase

ANALYTICAL

BIOCHEMISTRY

Separation

67, 625-633 ( 1975)

of Bases,

Deoxyribonucleosides Partition

Ribonucleosides by Anion-Exclusion

Chromatography Resin: Application

Ribonucleotide

and

on Cation-Exchange to the Assay of

Reductase, and

and

Deaminase

Nucleosidasel

BIMAL C. PAL,' JAMES D. REGAN, AND FRANKLIN D. HAMILTON Biology Division, of Tennessee-Oak

Oak Ridge Natiorlal Laboratory and the University Ridge Graduate School of Biomedical Sciences, Oak Ridge’. Tennessee 37830

Received January 28, 1975; accepted March 18, 1975

[5-"H]CDP and CTP are used as substrates in the assay of ribonucleotide reductase. deaminase and nucleosidase activity in crude enzyme preparations. After incubation. the nucleotides are hydrolyzed to nucleosides by sequential treatment with potato apyrase and alkaline phosphatase. An aliquot is then chromatographed on a cation-exchange column at 50°C with 0.1 M boric acid, adjusted to pH 7.4 with ammonia, used as eluant. The pyrimidines Ura, Urd, dUrd, Cyt, Cyd and dCyd are separated and eluted in about 50 min in small volumes. Assays by this procedure of CTP reductase activity in crude fractions of ribonucleotide reductase from Euglena gracilis gave results comparable to those obtained by the standard method. The new procedure is also applicable when adenine or guanine nucleotides are used as substrates. The adenine derivatives Ade, Ado. dAdo, Hyp, Ino. dlno as well as the guanine derivatives Cua, Guo. dGuo, Xan, Xao are separated from each other in this chromatographic system in about an hour.

The key role played by the ribonucleotide reductase in the control of the intracellular synthesis of DNA has prompted many investigators to devise suitable assays for this enzymatic activity (l-8). The original procedure of Reichard (I), based on the separation of CMP and dCMP on Dowex 50, is the most reliable method to date. It is possible to use 14C-, 3H- or 32P-labeled substrates, but the analysis is limited to the cytidine nucleotides and requires more than 24 hr. Samples are also eluted in large volumes. The pioneering investigations of Cohn (9) (see particularly his reference to work by Khym) into the use of borate complexes to 1 Research sponsored by the U.S. Atomic Energy Commission Union Carbide Corporation. ” To whom reprint requests should be directed. 625 Copyright Q 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

under contract with

626

PAL,

REGAN

AND

HAMILTON

separate ribonucleosides from deoxyribonucleosides led successive workers in this area to exploit this principle in developing different assay methods. Chromatographic analyses on columns, paper, thin layer plates, and columns of borate covalently linked to cellulose have been used, but none of these has proved entirely satisfactory for one reason or another. Often paper and thin layer chromatography require multiple development in several solvent systems (3,5,7). Methods based on column chromatography were plagued by troubles in the regeneration of the column (2) and, in one case, by the instability of the covalently linked borate-cellulose column (7). The time required in all these assays is also considerable. Samples are often eluted in large volumes that must be reduced before the samples can be counted for radioactivity. The spectrophotometric assay of Holmgren et al. (8) is limited to highly purified enzymes. In the assay described in this paper, the nucleotides in the incubation mixture are hydrolyzed enzymatically to nucleosides and then separated on a column of cation-exchange resin using the principle of anionexclusion (10) and partition chromatography. This method minimizes most of the problems encountered in other methods of assay. MATERIALS

[3H]CDP and [3H]CTP were obtained from Schwarz/Mann and used as such. The cation exchanger, Aminex A-6 (17 ? 2.5 pm), was purchased from Bio-Rad Laboratories, Richmond, CA, and was cleaned up as described previously ( 10). Alkaline phosphatase (Worthington BAPF) was dialyzed against water. Potato apyrase (Sigma) was used without further purification. Other nucleosides and chemicals were obtained from a variety of commercial sources. A jacketed column (Chromatronix, Catalog No. LC-6M-13), 33 X 0.63 cm, was used for the A-6 resin. These columns do not have any dead volume at the ends and are connected with Teflon tubing (i.d., 0.3 mm) so as to minimize zone spreading. An off-column septum injecting device (similar to Chromatronix, Catalog No. 164A 11) was used for injecting samples of 5-25 ~1 into the column with a Hamilton syringe fitted with a hypodermic needle. The column effluent was continuously monitored and recorded by essentially the same procedure as described in detail by Uziel et al. (11). METHODS

The feasibility of separating related nucleosides and bases was studied by using three different standard mixtures: (i) Ura, Urd, dUrd, Cyt, Cyd, dCyd; (ii) Ade, Ado, dAdo, Hyp, Ino, dIno; and (iii) Gua, Guo, dGuo, Xan, Xao. These types of mixtures were selected because crude preparations of ribonucleotide reductase are often contaminated with

REDUCTASE.

DEAMINASE

AND

NUCLEOSIDASE

627

deaminase and nucleosidase. This is especially true for crude cell extracts. Five- to ten-microliter samples of these three standard mixtures were injected into the column (20 x 0.63 cm) of A-6 maintained at 50°C and eluted with 0.1 M boric acid, adjusted with 1 M ammonia to pH 7.4 in the case of(i) and pH 7.3 in the cases of (ii) and (iii). The effluent was monitored at 260 nm. Ribonucleotide reductase from Euglena gracilis was incubated with [“H]CTP as described before (12) and assayed by the present method and the method of Reichard (1) for comparison. A 50% suspension of HeLa cells in 0.02 M phosphate buffer, pH 7, and 0.01 M P-mercaptoethanol was sonicated and centrifuged at 100,OOOg for 3 hr. The clear supernatant was used as such for assay of the ribonucleotide reductase activity. Protein concentration was measured by the procedure of Lowry et al. (13) and found to be 13 mg/ml. The incubation was carried out as described by Moore (4). The complete reaction mixture contained (in a final volume of 0.12 ml): Potassium phosphate buffer, pH 7, I pmole; NaF, 1 pmole; Mg(OAc),, 0.3 pmole: FeCl,, 0.007 pmole; dithiothreitol, 0.74 pmole; ATP, 0.5 pmole; HeLa cell supernatant fraction, 50 ~1; and [3H]CDP (sp act, 20 X lo6 cpm/pmole), 100 nmoles. After incubation, the reaction mixture was heated in a boiling-water bath for 2 min to denature the enzyme, 0.05 pmole of cold carrier dCyd * HCl was added, and the nucleotides were converted into nucleosides by the sequential action of potato apyrase and alkaline phosphatase, essentially as described by Moore (4). The incubation mixture, using ribonucleotide reductase from Euglena gracilis, was treated in the same manner. A 5-20-~1 aliquot was then injected into the column of A-6 for analysis; the column, maintained at 50°C was eluted at a rate of 0.3 1 ml/min with 0.1 M boric acid, adjusted to pH 7.4 with 1 M ammonia. At the same time the column was continuously monitored at 260 nm. One-minute fractions were collected in scintillation vials, and each fraction was counted in a 5-ml mixture of 5 g of PPO (2,5-diphenyloxazole) and 100 g of naphthalene dissolved in 1 liter of dioxane with a Beckman LS-233 liquid scintillation counter. RESULTS

AND

DISCUSSION

We have spent considerable time and effort determining the column temperature and the pH and buffer concentration of the eluant that effect the best separation of nucleosides and bases. The optimum separation of mixture i (Ura, Urd, dUrd, Cyt, Cyd, dCyd (Fig. 1)) was achieved at 50°C with 0.1 M boric acid, adjusted to pH 7.4 with 1 M ammonia. As expected, Urd and Cyd were eluted first from the column since these are the two components capable of forming anions by com-

628

PAL, REGAN

AND HAMILTON ELUTION

10 0.8

r

1.55

3.10

4.65

6.20

7.75

VOLUME 9.30

(ml) 10.85

12.40

13.95

(5.50

35

40

45

50

2 1

0.6

0.4

2 4” 0.2

0.1

0 ’ 0

5

10

15

20

25 TIME

30

55

(mid

FIG. 1. Separation of standard mixture i of pyrimidine bases and nucleosides (Ura. Urd, dUrd, Cyt, Cyd, dCyd) on an Aminex A-6 column (20 x 0.63 cm). A 5+1 sample was injected into the column, maintained at 50°C and eluted with 0. I M boric acid adjusted to pH 7.4 with 1 M ammonia at 0.3 1 mi/min (50 psi pressure).

plexing with borate. All the components are completely separated and eluted in small volumes suitable for direct counting without prior concentration. The analysis is complete in about 50 min and the length of time can be further reduced to 30 min if one is not looking for cytosine. The formation of Urd and dUrd as a result of deaminase activity can easily be detected and quantitated. The nucleosidase activity can also be monitored since the system also separates Ura and Cyt. The completeness of the enzymatic dephosphorylation can also be easily checked since CDP and CMP are eluted before Urd by virtue of their high anionic charges, and considerable radioactivity in this region of the chromatogram can be taken as evidence of incomplete hydrolysis. We found that hydrolysis was essentially complete under the conditions of our experiments. In the case of mixture ii (Ade, Ado, dAdo, Hyp, Ino, dIno) all the components are separated, but there is a slight overlap between the Ino and Ado peaks and between the dIno and Hyp peaks. Here also, the Ino and Ado come out first by virtue of the formation of anionic borate complexes (Fig. 2). The analysis is complete in about 65 min and, if one is not looking for Ade, the time can be cut down further to about 45 min. All the components of mixture iii (Gus, Guo, dGuo, Xan, Xao) are also separated in this system (Fig. 3). Gua was found to be very insolu-

REDUCTASE,

DEAMINASE

AND

ELUTION 1.55

3.to

4.65

6.20

7.75

9.30

5

10

15

20

25

30

0.6 ,

VOLUME

629

NUCLEOSIDASE (ml1

10.85

12.40

13.95

(5.50

1705

35

40

45

50

55

16.04

20.15

60

65

0.4

4

Cl g 0.2

0.1

0

TIME

70

hnl

FIG. 2. Separation of standard mixture ii of bases and nucleosides (Ade, Ado, dAdo. Hyp, Ino, dlno) on an Aminex A-6 column (30 X 0.63 cm). A lo-~1 sample was injected into the column, maintained at 50°C and eluted with 0.1 M boric acid adjusted to pH 7.3 with 1 M ammonia at 0.31 ml/min (50 psi pressure).

ble in the eluting solvent; the position of this elution in the chromatogram was determined by running a separate sample of Gua through the column. Xao and Guo are eluted first because of the formation of anionic borate complexes. The Xan peak is interposed between the Xao and Guo peaks because of its partial anionic nature at pH 7.3 (pK = 7.44).

FIG. 3. Separation of standard mixture iii of bases and nucleosides (Gus, Guo, dGuo. Xan, Xao) on an Aminex A-6 column (20 X 0.63 cm). A IO-PI sample was injected into the column, maintained at 50°C and eluted with 0.1 M boric acid adjusted to pH 7.3 with 1 M ammonia at 0.3 I ml/min (7 1 psi pressure).

630

PAL,

REGAN

AND

HAMILTON

FIG. 4. Chromatographic analysis of the incubation mixture containing HeLa cell extract and [“H]CDP which was subsequently hydrolyzed with potato apyrase and alkaline phosphatase. A 20-~1 aliquot was injected into the column (Aminex A-6, 20 x 0.63 cm), maintained at 50°C and eluted with 0. I M boric acid adjusted to pH 7.4 with 1 M ammonia at 0.31 ml/min (81 psi pressure). One-milliliter fractions were collected and counted for radioactivity.

Next we tested the system under actual assay conditions using a HeLa cell supernatant fraction and [3H]CDP as substrate and ribonucleotide reductase from Euglena gracilis with [3H]CTP as substrate. The results in the former case are shown in Figs. 4 and 5. There are small amounts of radioactive impurities in the sample of [3H]CDP as shown in the profile of the radioactivity of the column effluent in the blank experiment (Fig. 5). The radioactivity indicated in the sample chromatogram can be corrected by this amount. Since the recovery from the column is essentially quantitative, there is no correction factor involved. Materials eluting in the positions of Urd, Ura, and dCyd were radioactive. Presumably Urd and dCyd are formed as a result of the action of deaminase and ribonucleotide reductase, respectively. Ura can be formed as a result of the combined action of deaminase and nucleosidase. It is noteworthy that no dUrd or Cyt was detected. The deaminase, nucleosidase, and ribonucleotide reductase activities in the HeLa cell supernatant fluid were calculated to be 20.3, 2.0 and 0.1 units per mg of protein after correcting for the background obtained from the blank experiment (without the enzyme). A unit of activity is defined as

REDUCTASE,

DEAMINASE

AND

NUCLEOSIDASE

631

r

FIG. 5. Chromatographic analysis of the incubation mixture without the HeLa cell extract but containing everything else, including [SH]CDP, and treated in a manner similar to that described in the legend for Fig. 4.

the amount of enzyme that catalyzes the conversion of 1 nmole of substrate in 30 min at 37°C. Lack of pronounced ribonucleotide reductase activity in the HeLa cell supernatant fluid may be due to the presence of inhibitors since no attempt was made to remove them by dialysis. A sample of ribonucleotide reductase from Euglena gracilis was assayed by the present method and the method of Reichard (1) in parallel and found to yield comparable results (Table 1). The elution times for the peaks shown in Figs. l-5 are highly reproducTABLE ASSAY

OF THE REDUCTION REDUCTASE

Method Reichard (1) Present

1

OF [VH]CTP BY THE RIBONUCLEOTIDE FROM Euglena gracilis

nMoles/incubation

tube”

9.54* 9.83”

o Number of nmoles of [VH]CTP reduced was calculated by multiplying the number of nmoles of [VH]CTP in the incubation tube by the factor (cpm in the fractions containing [VH]dCMP/totaL cpm in the eluate) in Reichard’s procedure; or by the factor (cpm in the fractions containing [S-W]dCyd/total cpm in the eluate) in the present method. b An average of two determinations.

632

PAL,

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ible, and there is no interference from purine nucleosides in a pyrimidine assay or vice versa. We have not attempted to identify all the peaks in Fig. 4. The enzymes, DTT (dithiothreitol), oxidized DTT, Cyd, dCyd and Ado in the incubation mixture show absorbance at 260 nm. Adenosine and cytidine peaks overlap. There is a lag of about I5 set between the time the sample is monitored by uv absorption in the flow cell and collection of the sample in the counting vial. This lag is negligible and has not been taken into account in plotting the radioactivity in Figs. 4 and 5. The Urd, Cyd, and dCyd peaks appear at 10, 13 and 27.75 min, respectively in Fig. 1. The radioactive peaks due to Urd, Cyd, and dCyd appear at 10, I3 and 29 min, respectively in Fig. 4. The position of the uv peak due to dCyd in Fig. 4 is 27.75. Thus the elution times are prac‘tically constant when one compares Fig. 1 with Fig. 4. If one wishes to adapt this method for assay of a large number of samples for cytidine ribonucleotide reductase, the uv monitor and collection of I-min fractions can be eliminated after the column is standardized by running a standard mixture such as shown in Fig. I; then only one fraction containing dCyd needs to be collected and counted, and dCyd is eluted in 30 min. If one sets up ten columns and ten pumps, one should be able to do 160 assays per day. In conclusion, we may state that the ribonucleotide reductase assay described in this paper has several advantages over those previously published (l-8). (a) It can be used to determine UDP, UTP, CDP, CTP, ADP, ATP, GDP and GTP reductase activity. (b) It is rapid, the chromatographic step requiring only 30-65 min. (c) It is reliable, accurate, and highly reproducible. Since the recovery from the column is essentially quantitative, no correction needs to be made on this account. (d) The column is very stable and can be used almost indefinitely. (e) Although this method does involve the enzymatic hydrolysis of the nucleotides to nucleosides, the completion of this step can easily be checked by the elution of radioactive fractions in front of the uridine peak position. (f) The method is suitable for use with crude enzyme preparations and cell extracts. It can be used for the assay of deaminase and nucleosidase activities individually or at the same time. (g) Errors due to the presence of radioactive impurities in the substrates can be corrected very easily. (h) One can use both “H- and 14C-labeled substrates; ““P-labeled substrates cannot be used, however, since the assay is made at the nucleoside level. (i) The components are eluted from the column in small volumes suitable for direct counting without prior concentration. ACKNOWLEDGMENT Thanks

are due to Dr.

Waldo

E. Cohn

for his interest

and encouragement.

REDUCTASE,

DEAMINASE

AND

NUCLEOSIDASE

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REFERENCES I. 2. 3. 4. 5. 6. 7. 8.

Reichard, P. (1958) Acfa Chem. Stand. 12, 3048. Steeper, J. R., and Steuart, C. D. (1970) Anal. Biochem. 34, 123-130. Fujioka, S., and Silber, R. ( 1970) J. Biol. Chem. 245. 1688- 1693. Moore, E. C. (1967) in Methods in Enzymology (Grossman, L., and Moldave, K., eds.), Vol. 12, Part A, pp. 15.5-164, Academic Press, New York. Yeh, Y-C., and Tessman. 1. (1972) J. Biol. Chem. 247, 3752-3154. Cory. J. G., Russell, F. A.. and Mansell. M. M. (1973) Anal. Biochem. 55, 449-456. Moore. E. C.. Peterson, D., Yang, L. Y.. Yeung. C. Y.. and Neff, N. F. (1974) Biochemistry 13, 1904-2907. Holmgren. A.. Reichard. P., and Thelander. L. ( 1965) Proc. Nat. Acad. Sri. C/SA 54, 830-836.

9.

10. 11. 12. 13.

Cohn, W. E. (1955) in The Nucleic Acids (Chargti. E., and Davidson. J. N., eds.). Vol. 1, pp. 21 I-241, Academic Press, New York: and references cited therein. Singhal. R. P. (1972) Arch. Biochem. Biophys. 152, 800-810. Uziel, M., Koh. C. K., and Cohn, W. E. ( 1968) Anal. Biochem. 25, 77-98. Hamilton. F. D. (1974) J. Biol. Chem. 249, 4438-4434. Lowry. 0. H., Rosebrough, A. L.. Farr. A. L.. and Randall, R. J. (1951) J. Bio[. Chem. 193, 265-275.