Journal of Virological Methods, Elsevier
180 (1987) 281-290
Improved method for the measurement ribonucleotide reductase activity Allan J. Darling, Bernadette
M. Dutia’ and Howard S. Marsden
MRC Virology Unit, Institute of Virology, Church Street, Glasgow, U.K. (Accepted
An improved method for the measurement of herpes simplex virus type 1 encoded ribonucleotide reductase has been developed. The enzyme which catalyses the conversion of ribonucleoside diphosphates to deoxyribonucleoside diphosphates was determined by first converting the ribonucleotide substrate and deoxyribonucleotide product to the corresponding nucleosides by treatment with snake venom phosphodiesterase. Then nucleosides were separated by HPLC and measured by flow through scintillation counting and by monitoring their absorbance at 254 nm. Under the conditions used in the experiment cytidine and deoxcytidine, the derivitised substrate and product respectively, eluted from the column at approximately 4 min 33 s and 6 min 24 s. Peak heights and areas were automatically calculated by computer to ascertain the amount of product formed and thus quantitate the assay. Automation of the assay from sample injection to analysis provides a significant saving in time and an improvement in the efficiency of measurement of ribonucleotide reductase activity over other published methods. HSV-1; Ribonucleotide
HPLC; Flow through scintillation counting
Ribonucleotide reductase (E.C. 126.96.36.199) catalyses the first unique step in DNA synthesis by converting the 4 ribonucleotides to the corresponding deoxyribonuCorrespondence Gil 5JR, U.K.
‘Present address: Department Edinburgh, EH9 lQH, U.K. 0166-0934/87/$03.50
cleotides (Thelander and Reichard, 1979; Reichard and Ehrenberg, 1983). Several in vitro assay methods have been developed for the measurement of ribonucleotide reductase activity. A colourimetric method using diphenylamine to measure the conversion of ATP to dATP by the adenosylcobalamin-dependant ribonucleotide reductase of Lactobacillus leichmannii has been described (Blakely, 1978). Adenosylcobalamin-dependant ribonucleotide reductase activity can also be determined by measuring 3H exchange between .5’-3H, adenosylcobalamin and water (Abeles and Beck, 1967; Hogenkamp et al., 1968). Ribonucleotide reductase activity can be measured spectrophotometrically by following the oxidation of NADPH, the ultimate hydrogen donor for ribonucleotide reduction, in an assay containing the ribonucleoside di- or triphosphate, thioredoxin and thioredoxin reductase (Thelander et al., 1978). This method requires the purification of the ribonucleotide reductase, thioredoxin and thioredoxin reductase and is thus unsuitable for assaying activity in crude or partially purified extracts. Commonly for impure extracts, ribonucleotide reductase is measured directly by following the conversion of a 3H- or 32P-labelled ribonucleotide into the deoxyribonucleotide (Thelander et al., 1978; Holmgren, 1981). Determination of the amount of product formed is usually accomplished after conversion of both the substrate and product either into their corresponding nucleoside monophosphates by acid hydrolysis (Thelander et al., 1978) or into the corresponding nucleosides by treatment with snake venom phosphodiesterase (Dutia et al., 1986). The nucleosides or nucleoside monophosphates can then be separated by thin layer chromatography, the spots visualised by UV and scraped into scintillation vials for counting (Hopper, 1978; Turk et al., 1986). Alternatively, separation can be achieved by ion-exchange chromatography on a Dowex column and eluted fractions can be collected and analysed by liquid scintillation counting (Thelander et al., 1978; Averett et al., 1983). These methods, for impure extracts, are inherently labour intensive and are thus not suitable for screening large numbers of samples. There is also a large potential for error in both the recovery and measurement of the enzymes substrate and product. In this paper we describe an automated method for the determination of herpes simplex virus type l-encoded ribonucleotide reductase activity based on the separation of the ribonucleoside from the deoxyribonucleoside by HPLC and subsequent measurement by flow through scintillation counting although the methodology can be equally applied to the measurement of ribonucleotide reductase from any source.
Cells and virus BHK-21 clone 13 cells (Macpherson and Stoker, 1962) were grown in Eagle’s medium supplemented with 10% tryptose phosphate broth and 10% calf serum. Herpes simplex virus type 1 (HSV-1) strain 17 (Brown et al., 1973) was used in all experiments at a multiplicity of infection of 10 pfu/cell.
Preparation of extracts for ribonucleotide
Partially purified extracts containing HSV-1 ribonucleotide reductase were prepared as described previously (Dutia et al., 1986). Extracts were made 15 h post infection at 31°C. Ribonucleotide
HSV-1 ribonucleotide reductase activity was measured by following the conversion of [3H]CDP into 13H]dCDP. The assay mixture contained in addition to enzyme in a final reaction volume of 90 ~1: 200 mM Hepes, pH 8.2; 10 mM DTT; 100 PM CDP and 1 &i [3H]CDP (Amersham International plc, specific activity 37.7 mCi/mg). After incubation at 37°C for 30 min the reaction was stopped by heating at 100°C for 2 min. After cooling, nucleotides were converted to their corresponding nucleosides by the addition of 2 mg of Crotalux adamentus venom (10 ~1 of a 200 mg ml-’ solution) and MgC12 to a final concentration of 15 mM followed by incubation at 37°C for 2 h. Samples were then heated at 100°C for 2 min and the precipitate formed was removed by centrifugation at 2000 x g for 5 min. If required, 10 ~1 of a 1mM solution of ‘cold’ deoxycytidine was added to each sample as a marker. The supernatants were then removed and aliquoted into vials for analysis by HPLC. HPLC
and flow through scintillation counting
Samples were analysed on a Waters HPLC system incorporating a Wisp 710B automatic injection system all under the direction of a Waters system controller model 720. Reverse phase chromatography was performed on a p Bondapack Cl8 cartridge column fitted inside a Waters Z-module (radial compression separation system). The eluent from the column was monitored continuously at 254 nm using a Waters UV monitor model 441 and also 3H was measured continuously using a Ramona Raytest flow through scintillation counter. Data from the UV monitor and from the flow through scintillation counter were collected and stored on an IBM PC running under the ‘Ramona Radio-Chromatography System’ programme version 5.4e (Raytest Instruments). Samples were loaded into the Wisp automatic injection system and the run time and equilibration period were programmed into this machine. The first sample was then injected onto the column and eluted as described later, gradient formation and sample injection being controlled by the system controller. The column was then equilibrated automatically with the starting buffer before subsequent injections. After all the samples have been run the data was analysed automatically by the computer and the results stored and/or printed out.
A schematic diagram showing the HPLC/flow through scintillation counter used to measure HSV-1 ribonucleotide reductase activity is shown in Fig. 1. Currently up to 77 separate runs can be analysed although the automatic injector has the facility to hold 100 samples.
Flow To Waste
Fig. 1. Schematic of ribonucleotide
diagram of the HPLCiflow through scintillation counter system for the measurement reductase activity. Arrows (b) represent the direction of solvent and scintillant flow and dashed lines (---) represent communication cables.
Gradient for the separation of cytidine from deoxycytidine by reverse phase HPLC apack column. Buffer A was 0.1 M KH?PO,, pH 5.4, and Buffer B methanol:water Time (min)
Flow rate (ml min
0 3 8 11 19 24
3 3 3 3 3 0.1
100% A IOW A 45% A 55% B 100%A 100% A 100% A
on a Cl8 )L Bond(80:20). through
In all experiments the column was equilibrated with 0.1 M KH,PO,, pH 5.4 (buffer A) and elution was accomplished with a linear gradient of methanol:water (80:20, buffer B). The optimal gradient for the separation of cytidine from deoxycytidine is shown in Table 1. After an initial 3 min elution with buffer A, a linear gradient increasing to 55% B between 3 and 8 min is applied. The column is then re-equilibrated for the next run by linearly reducing the concentration of buffer B to 0% between 8 and 11 min followed by an 8 min wash with buffer A. If no more samples are detected by the system controller then the flow rate is reduced to 0.1 ml min-’ at 24 min. Fig. 2 shows the separation of a mixture of cytidine and deoxycytidine standards using this separation gradient. Under these conditions cytidine eluted as a broad peak at 4 min 33 s whereas deoxycytidine eluted as a sharp peak at 6 min 25 s. These elution times remain constant from experiment to experiment and from sample to sample within about 3 s. It is therefore possible to completely separate the derivatised substrate and product of the ribonucleotide reductase reaction by almost 2 min thus facilitating an accurate determination of enzyme activity. The results of an actual experiment to determine ribonucleotide reductase activity in a partially purified extract are shown in Fig. 3. The top trace corresponds to the output from the UV detector as in Fig. 2. The positions of cytidine (at 4 min 25 s) and deoxycytidine (at 6 min 25 s) are clearly distinguished from other UV absorbing components in the assay mixture which elute throughout the run. The position of the deoxycytidine peak is easily distinguished as unlabelled deoxycytidine was added to the sample as a marker after snake venom phosphodiesterase treatment. The bottom trace shows the corresponding output from the flow through scintillation counter, the bottom trace being
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of cytidine and deoxycytidine by reverse phase HPLC on a Cl8 )L Bondapack col(50 ~1 of a 50 PM cytidine, 100 PM deoxycytidine solution in buffer A) was eluted as described in the text and the absorbance at 254 nm monitored.
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Fig. 3. Separation of cytidine and deoxycytidine from an HSV-1 ribonucleotide reductase assay. The enzyme (50 )11 of a partially purified extract. protein concentration 30 mg ml-‘) was assayed as described in Materials and Methods and the nucleotides were converted to nucleosides by snake venom phosphodiesterase. A sample (50 ~1) was then analysed by HPLCiflow through scintillation counting. The top trace shows the absorbance at 254 nm of the eluant from the column and the bottom trace shows the ‘H trace as measured by the flow through scintillation counter.
time shifted to compensate approximately for the delay between sample detection by the UV monitor and the scintillation counter. The main radioactive peak eluted from the column at 4 min 26 s and this corresponds to [‘Hlcytidine and represents unused substrate. The product of the reaction, deoxycytidine, gives a small peak in this experiment which eluted from the column at 6 min 25 s and is completely separated from the cytidine peak. A small peak of “H label eluted at 1 min 12 s and this probably occurs due to the incomplete hydrolysis of all the nucleotides by
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Fig. 4. Automatic evaluation of the Ribonucleotide Reductase assay shown in Fig. 3. Abbreviations used in the Table headings: CH = channel; R/T = retention time; Integr. = integral of peak (cps for 3H and arbitary units for UV); ROI = regions of interest (identified by the computer programme as a peak); c/c ROI = peak integrated value expressed as a percentage of the sum of the values of all the ROIs; % Total = peak integrated value expressed as a percentage of all the total 3H or UV.
the snake venom phosphodiesterase as it also occurs in the absence of ribonucleotide reductase activity but is not present in the [3H]CDP supplied by the manufacturer. The identity of the compounds in the other very minor peaks is not known. The data from both traces were integrated by the computer and printed out to give the numer of peaks, their retention times, widths and sizes (Fig. 4). In this sample the total number of counts detected was 236520. The cytidine peak accounted for 198470 of these counts or 83.91% of the total. The deoxycytidine peak, the measure of ribonucleotide reductase activity, contained 27 108 counts or 11.46% of the total. The UV trace is calculated similarly.
This communication describes the use of reverse phase HPLC and flow through scintillation counting to introduce a high level of automation into an assay for ribonucleotide reductase. The main advantages of this technique over other methods are the accuracy and reproducibility of the measurements, personal errors being kept to a minimum, and the ability to perform many assays quickly and with ease. This is particularly important in the measurement of HSV encoded ribonucleotide reductase activity as the enzyme is regarded as a potential target for anti-viral therapy (Frame et al., 1985; Spector and Jones, 1985; Spector et al., 1985; Cohen et al., 1986; Dutia et al., 1986) and the screening of potential inhibitors of the enzyme involves performing large numbers of assays. Measurement of HSV-1 ribonucleotide reductase activity using other ribonucleoside diphosphates as substrates can also be achieved using this system although the solvent gradient for the separation of the ribonucleoside and deoxyribonucleoside must be modified according to the substrate used. If required, the sentivity of the assay can also be increased by decreasing the amount of unlabelled ribonucleoside diphosphate substrate in the assay. The measurement of ribonucleotide reductase activity using this technique is not restricted to HSV-1. Ribonucleotide reductase activity from any source can be measured. As an alternative to hydrolysis of nucleoside diphosphates to nucleosides by snake reductase activity can be venom phosphodiesterase treatment, ribonucleotide measured by acid hydrolysis of the nucleoside diphosphates to nucleoside monophosphates (Thelander et al., 1978). However the relative separation between the ribonucleoside monophosphates and deoxyribonucleoside monophosphates on reverse phase HPLC is not as great as between the ribonucleosides and deoxyribonucleosides, the maximum separation of cytidine monophosphate from deoxycytidine monophosphate achieved is 48 s compared with approximately 2 min for cytidine and deoxycytidine. We conclude that the automation of the assay using HPLC and flow through scintillation counting represents a significant improvement in both the ease and accuracy in the measurement of ribonucleotide reductase activity.
We wish to thank Professor J.H. Subak-Sharpe and Dr. M. Frame for their interest and advice and for critical reading of the manuscript. We also thank Miss F. Conway for typing the manuscript.
References Abeles, R.H. and Beck, W.S. (1967) J. Biol. Chem. 242, 3589-3593. Averett, D.R., Lubber, C., Elion, G.B. and Spector, T. (1983) J. Biol. Chem. 258, 9831-9838. Blakely, R.L. (1978) Methods Enzymol. 51, 246259. Brown, S.M., Ritchie, D.A. and Subak-Sharpe, J.H. (1973) J. Gen. Virol. 18, 329-346. Cohen, E.A., Gaudreau, P., Brazeau, P. and Langelier, Y. (1986) Nature 321, 441-443. Dutia, B.M., Frame, M.C., Subak-Sharpe, J.H., Clark, W.N. and Marsden, H.S. (1986) Nature 321, 439-441. Frame, M.C., Marsden, H.S. and Dutia, B.M. (1985) J. Gen. Virol. 66, 1581-1587. Hogenkamp, H.P.C., Ghambeer, R.K., Brownson, C., Blakely, R.L. and Vitols, E. (1968) J. Biol. Chem. 243, 799808. Holmgren, A. (1981) Curr. Top. Cell. Regul. 19, 47-76. Hopper, S. (1978) Methods Enzymol. 51, 237-246. Macpherson, I. and Stoker, M. (1962) Virology 16, 147-151. Reichard, A. and Ehrenberg, A. (1983) Science 221, 514-519. Spector, T., Averett, D.R., Nelson, D.J., Lambe, C.V., Morrison, R.W., St. Clair, M.H. and Furman, P.A. (1985) Proc. Natl. Acad. Sci. USA 82, 4254-4257. Spector, T. and Jones, T.E. (1985) J. Biol. Chem. 260, 8694-8697. Thelander, L. and Reichard, P. (1979) Ann. Rev. Biochem. 48, 133-158. Thelander, L., Sjoberg, B.-M. and Eriksson, S. (1978) Methods Enzymol. 51, 227-237. Turk, S.R., Shipman, C. and Drach, J.C. (1986) J. Gen. Virol. 67, 1625-1632.