Chapter 8 Nucleotides

191

Chapter 8 NUCLEOTIDES C.K. Lim

1. INTRODUCTION The nucleotides are smaller units which join together to form the oligonucleotides or polynucleotides.The polymer is called a nucleic acid. The basic structure of a mononucleotide consisted of a base linked to a sugar and a phosphate group. The five common nucleotide bases are adenine (A), guanine (G), cytosine (C), uracil (U) and thymine ( Al). and G are derived from purine and C, U and T are from pyrimidine. The sugar is either pentose, ribose or deoxyribose and the phosphate may be mono-, di- or triphosphate. The basic nucleotide components are shown in Fig. 1 and the structure of a typical nucleotide, adenosine triphosphate (ATP), is shown in Fig. 2. 2. HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

Nucleotide analysis is essential in many biomedical and clinical fields because of their importance in cellular metabolism. HPLC has greatly improved the speed, resolution and sensitivity of analysis compared with classical ion-exchange chromatography'. It is the method of.choice particularly when the simultaneous analysis of a range of compounds is required. 2.1. lon-exchangechromatography The nucleotides are predominantly negatively charged because of the low PKal (in the range 1.5 - 4.5 depending on the base group) for the primary phosphate293.The secondary phosphate has pKa2 of between 6.0 and 6.5 in all instances. The charge increases with increasing number of phosphate groups present, i.e., nucleotide mono- < di- < triphosphate. The nucleotides also possess cationic nitrogen atoms at pH below 2-4, depending on the nature of the base. These highly charged molecules are therefore ideal for separation by ion-exchange chrdmatography on which many of the earlier separations were b a ~ e d ~The - ~ later . development of chemically bonded microparticulate (3 - 10 pm) ion-exchange packings substantially improved the resolution and speed of a n a l y ~ i s ~ - ' ~ and the technique is still widely used. The column is usually 5 - 25 cm long with an I.D. of

192 (a)

Purine

Pyrimidine

(b) NH2 I

Guanine

Adenine

I

HN

/

B c\

0

II

CH

o=c I

I

\N/CH H

Cytosine

Thymine

Uracil

,, H

O

3 OH

D

H

O

U

2

3 ,

OH

Ribose

OH

o

H

2 I

-

2 Deoxyribose

Fi . 1. Structures of basic nucleotide components. (a) Purine and pyrimidine ring system; (by nucleotide bases; (c) nucleotide sugars.

- 5 mm, packed with a strong anion exchanger (SAX) such as trimethylaminopropylsilane or a weak anion exchanger such as aminopropylsilane (APS). lon-exchange chromatography of nucleotides involves the displacement of charged species bound to the ion exchanger with counter ions in the eluent. In isocratic separation, this is achieved by using a buffer of controlled ion concentration and pH. lsocratic 4

193

0

0

0

OH

OH

OH

I

II

-

1

OH OH

*+BASE

SUGAR

PHOSPHATES

NUCLEOSIDE

NUCLEOTIDE mono-di-tri-

Fig. 2. Structure of adenosine triphosphate (ATP). elution ion-exchange chromatography generally lacks selectivity but can be useful for the rapid separation of a small number of nucleotides, e.g., those containing the same number of phosphate groups or an identical base group.

!

0

I

4

I

I

8

12

I

16

1

20

I

24

Time ( min

Fig. 3. Separation of nucleotides by strong anion-exchange chromatography. Column, 25 cm x 5 m m Spherisorb-SAX (5 pm ; eluents, 0.04 M KH2P04 (pH 2.9) (solvent A) and 0.5 M KHzP04 (pH 2.9) containing 0. M KCI (solvent B); elution, linear gradient from 0% to 30% B in 30 min.

d

194

12

8

MIN

4

, lo

0

Fig. 4. Separation of nucleotides by weak anion- exchange chromatography. Column, 10 cm x 4.6 mm APS-Hypersil (5 m); eluents, 0.04 M KH2P04 (pH 2.8) (solvent A) and 0.5 M KH2P04 + 0.8 M KCI ( p i 2.7) (solvent B); elution, linear gradient from 0% to 100% B in 15 min. Reproducedfrom ref. 11 with permission. In gradient ion-exchangechromatography, which is necessary when a large number of nucleotides, as in a complex biological extract, are to be analysed, the bound species are eluted from the column with a buffer gradient of increasing ionic strength. The nucleotides are essentially displaced from the column by the counter ions when an appropriate ionic concentration is reached. The length of the column is therefore less important here than in other modes of chromatographic separation. The resolution is influenced by the pH of the eluent, which affects the selectivity of the column. Nucleotides are retained longer when the pH is lowered and eluted faster when the molarity of the buffer is increased. Increasing the column'temperature also leads to a decrease in retention times. The most commonly used buffer for gradient ion-exchange chromatography of nucleotides is potassium dihydrogenphosphate although other buffers, such as ammonium phosphate, have also been employed. The final buffer concentration is usually about 50 times the initial concentration. Figs. 3 and 4 show the separation of nucleotides on a SAX and an APS column respectively. The elution order AMP c ADP cATP reflects the increasing molecular charge in that order. As the overall charge of a nucleotide is also affected by the substituents on the purine or pyrimidine ring, the following elution order is observed on Partisil 10-SAX at pH 4.0 - 4.5: CMP c AMP < UMP < IMP c GMP. The diand triphosphate nucleotides behave similarly. The elution order, however, is pH dependent. One of the major problems with gradient ion-exchange chromatography, in common with other forms of gradient elution chromatography of nucleotides, is the gradual rise of the detector baseline during separation. This is caused by UV-absorbing impurities ad-

195 sorbed on the column at low and then eluted off at high buffer concentrations. The impurities come mainly from the buffer salts” but sample extracts, if not properly purified, and water, if not freshly distilled and deionized, may also contribute to the .baseline rise. Only salts of the highest purity should be used and replacing half the concentration of phosphate in the final buffer with potassium chloride has been shown to reduce the baseline rise”. 2.2. Reversed-phase ion-pair chromatography

The development of chemically bonded reversed-phase (RP) packings such as octadecylsilane (ODS) has naturally led to the investigation of the ion-pairing (IP) technique as an alternative to ion-exchange chromatography. In the presence of a cationic IP agent, e.g., tetrabutylammonium sulphate or hydroxide, nucleotides are well retained by these c ~ I u m n s ~In~RP-IP - ~ ~ .chromatography the concentration of the IP agent on the hydrocarbonaceous stationary phase surface is a direct function of the chain length of the bonded phase, the percentage carbon loading and the relative hydrophobicity of the IP agent2’. Maximum retention is thus provided by a CIS column with tetrabutyl- rather than tetraethyl- or tetramethylammonium salts as IP agents. The optimum concentration in the mobile phase is 25 - 30 mmol/l for most IP agents at pH 5.7 - 6.0. The concentrationof the buffer salts in the mobile phase has a profound effect on the retention of nucleotides, as salt will compete with the solutes for the IP agent; an increase in salt concentration decreases the capacity factors (k’) of nucleotides. Salts such as NaCl can therefore be added to the mobile phase to provide extra control on the retention and resolution of nucleotides. Fig. 5 shows the separation of nucleotides by RP-IP chromatography. The elution order mono- < di- < triphosphate nucleotide is similar to that in anion-exchange chromatography.

I l

a

I ‘

0

1

12

L

24

1

36

48

Minulea

Fig. 5. Reversed-phaseion-pair chromatography of nucleotides. Column, 25 cm x 4.6 mm Spherisorb-ODS (5 pm); eluents and elution, concave gradient (Waters curve 8) from 25 mM tetrabu lammonium hydrogensulphate BAHS) in 0.05 M KH2P04-NH4CIbuffer (pH 3.9) to 0.05 TBAHS in 0.1 M KH2P04-NH4 I buffer (pH 3.4) + 30% (v/v) methanol in 40 min. Reproduced from ref. 16 with permission.

z

&

196 Zwitterion-pairing agents, e.g., 11-aminoundecanoic acid, have also been employed for the separation of nucleotide~~~. The IP agent forms a quadrupolar ion pair with the nucleotides, which are then separated on a reversed-phase column. The optimum conditions were 1 - 2 mmol/l of IF agent in phosphate buffer (pH 5.2 - 5.7),with methanol as the organic modifier. 2.3. Reversed-phasechromatography Nucleotides have been separated on reversed-phase columns with ammonium phosphate, ammonium dihydrogenphosphate or potassium dihydrogenphosphate buffer of controlled pH and methanol as the e l ~ e n ? These ~ ~ . systems are based on suppression of the ionization of nucleotides at low pH. The elution order is that expected for reversedphase chromatography with the more polar compounds eluted before the less polar ones. Thus nucleotide triphosphates elute before the di-‘ and monophosphates. Anomalous behaviour, with reversal of the expected order, however, was observed when..a relatively high concentration (0.6 M) of buffer at pH 3 was used on a C8 column. A probable explanation is that under such conditions ion pairing occurred, resulting in an elution order similar to that in RP-IP chromatography. Reversed-phase chromatography has not been widely used for the separation of nucleotides because most systems do not provide adequate resolution for complex mixtures of nucleotides. However, it has potential for further improvement, particularly by exploiting the solute-solvent-stationary phase interactions, as has been shown by the separation of 38 ribonucleotides, deoxynucleotides, cyclic nucleotides and deoxycyclic nucleotides in 33 min (Fig. 6). The separation was carried out on an ODs-Hypersil column with 83.3 mM triethylammonium phosphate, (pH 6.0) and methanol as the gradient mixture3’, and the method was successfully used

0

b

Fig. 6. Reversed-phase

I’o

20

15

50

25

Tima ( mln)

35

raphy of nucleotides. Column, 25 cm x 5 mm ODSin 83.3 mM trieth lamrnonium phosphate methanol in the same bu er (solvent B). Elution, 0 - 20 min, 0% - 45% B; 20 - 28 min, 34% - 100% B; 28 - 33 min, 100% B.

x

197

I

I

0

I

I

6

12

Time

(

I

10

1

24

min I

Fig. 7. Separation of nucleotides in human red blood cells. HPLC conditions as in Fig. 6. Reproduced from ref. 37 with permission.

to analyse nucleotides in human red blood cells (Fig. 7). The improvement in speed and resolution was attributed to the use as the eluting buffer of triethylammonium phosphate, which possesses excellent chromatographic properties such as masking of residual silano1 groups and acceleration of proton equilibrium, which are particularly favourable for reversed-phase chromat~graphy~~. The system is also applicable to isocratic elution3g and the simultaneous separation of more than 20 nucleotides has been achieved (Fig. 8). lsocratic elution has the advantage of being more reproducible and also significantly eliminates the problem of baseline drift associated with gradient elution. The separation of nucleotides in human lymphocytes (Fig. 9a) and in rat brown adipose tissues (Fig. 9b) are examples of applications. With the triethylammonium phosphate system, the elution orders cytidine c uridine c guanosine c inosine c thymidine c adenosine and ribo- c deoxyribo- c deoxycyclic nucleotides are largely consistent with the solvophobic theory using buffered eluents on reversed-phase columns4. Thus the more hydrophobic nucleotides have larger k’ values than the more polar nucleotides. The elution order nucleotide mono- c di- c triphosphates, however, is the opposite of that expected for reversed-phase chromatography. This clearly indicates a mixture of retention mechanism and is not surprising as triethylamine is an IP agent. The k’ values of the nucleotides separated on an ODS- Hypersil column with 4% (v/v) methanol in 83.3 mM triethylammonium phosphate buffer (pH 6.0) as mobile phase are given in Table I.

0

5

10

15

20

25

30

35

40

--- 120

126

TIME (MIN I

Fig. 8. lsocratic reversed-phase chromatography of nucleotides. Column, ODS-Hypersil (25 cm x 5 mm); mobile phase, methanol-83.3 mM triethylammonium phosphate (pH 6.0) (4% v/v).

199

1

IGTP ‘AD,

(a)

- c -

I

0

I

5

,

AMP

10 Time ( min I

,

-

I

15

I

20

Fig. 9. Separation of nucleotides in (a) rat brown adipose tissue and (b) human lymphocytes. Column, ODs-Hypersil; eluent, methanol - 83.3 mM triethylammonium phosphate (pH 6.0) (6:94, vlv). Rapid separation of the more hydrophobic nucleotides can be achieved by increasing the methanol content in the eluent. The k’ of CAMP, for example, was reduced from 38.2 to 8.2 when the methanol content was increased from 4 to 10% (Wv). The effect of methanol on the k’ values of nucleotides is shown in Fig. 10. Increasing the methanol content can actually lead to better resolution of certain nucleotide pairs, e.g., GTP and AMP, because the effect of methanol on each nucleotide is different, with the triphosphates being more significantly affected than the di- and monophosphates. The k’ values of nucleotides may also be controlled by adjusting the pH of the buffer. The optimum pH for separating purine nucleotides is between 4.5 and 6.0 (Fig. 11). The ionic strength of triethylammonium phosphate also affects the k’ values (Fig. 12) , but to a

200 TABLE I CAPACITY FACTORS (k’) OF RIBONUCLEOTIDES, DEOXYNUCLEOTIDES,CYCLIC NUCLEOTIDES AND DEOXYCYCLIC NUCLEOTIDES ON 5-pm HYPERSIL-ODS WITH METHANOL-83.3 mM TRIETHYIAMMONIUM PHOSPIIATE BUFFER (pH 6.0) (4:96, v/v) AS MOBILE PHASE Compound Cytidine 5’-monophosphate Cvtidine 5’-diphosphate Uiidine 5’-monophosphate 2’-Deoxycytidine 5’-monophosphate Uridine 5’-phosphoglucose Xanthosine 5’-monophosphate Uridine 5’-diphosphate 2’-Deoxycytidine 5’-diphosphate Guanosine 5’-monophosphate Cytidine 5’4riphosphate lnosine 5’-monophosphate 2’-Deoxyuridine 5’-monophosphate Xanthosine 5’-diphosphate Guanosine 5’-diphosphate 2’-Deoxyuridine 5’-diphosphate 2’-Deoxycytidine 5’-triphosphate lnosine 5’-diphosphate Nicotinamide adenine dinucleotide Xanthosine 5’-triphosphate Thymidine 5’-monophosphate 2’-Deoxyinosine 5’-monophosphate 2’-Deoxyguanosine 5’-monophosphate Guanosine 5’-triphosphate Adenosine 5’-monophosphate 2’-Deoxyuridine 5’4riphosphate lnosine 5’-triphosphate Thymidine 5’-diphosphate 2’-Deoxyinosine 5’-diphosphate 2’-Deoxyguanosine 5’-diphosphate Adenosine 5’-diphosphate Thymidine 5’-triphosphate 2’-Deoxyinosine 5’-triphosphate 2’-Deoxyguanosine 5’4riphosphate 2’-Deoxyguanosine 3’:5’-cyclic monophcisphate 2’-Deoxyadenosine 5’-monophosphate Adenosine triphosphate Guanosine 3’5’-cyclic monophosphate 2’-Deoxyadenosine 5’-diphosphate 2’-Deoxyadenosine 5’-triphosphate . . P’-DeoGadenosine3’:5’-cyclic monophosphate Adenosine 3’:5’-cycIic monophosphate

:‘

Abbreviation CMP CDP UMP dCMP UDPG XMP UDP dCDP GMP CTP IMP dUMP XDP GDP dUDP dCTP IDP NAD XTP TM P dlMP dGMP GTP AMP dUTP ITP TDP dlDP dGDP ADP lTP dlTP dGTP dcGMP dAMP ATP cGMP dADP dATP dcAMP CAMP

(k3 0.4 0.7 0.7 0.8 0.9 1.o 1.2 1.2 1.3 1.3 1.4

1.5 1.5

1.9 2.2 2.2 2.3 2.9 2.9 3.2 3.2 3.4 3.5 3.6 4.1 4.3 4.4

4.8 5.2 5.6 8.0 8.1 8.3 8.3 8.5

10.1

11.6 12.3 20.7 22.6 38.2

201 10.0

8.0

--

-.x

o

e

c

6.0

dATP

4.0

dAD P

c )r

P

m n V

d AMP AT P dGTP

#iFP

2.0.

WP G DP GMP 0. 1

5

6

7

Methanol (56, vh 1

Fig. 10. Effect of methanol content on the capacity factors (k') of nucleotides. Column, ODS-Hypersil; mobile phase, methanol - 83.3mM triethylammonium phosphate (pH 5.0).

much smaller extent than the pH. Buffer concentrations between 80 and 85 mM are recommended. 2.4. Metal chelate chromatography

This method, which is rarely used, is based on the interaction between nucleotides and metal ions on a silica column chemically bonded with bidentate ligands such as dithiocarbamate-cobalt(ll1) complexes4'. A small amount of magnesium(l1) is added to the mobile phase (0.1 M potassium dihydrogensulphate, pH 6.0) to provide for retention control. Gradient elution with an increasing concentration of magnesium(l1) in 30 mmol/l triethylamine has also been described fo$ke separation of nucleotides on an ODS The system probably involves both metal chelates and ion pairing.

202 lo.(

81

-

-.Y

6.C

dADP ADP

0

5

P

" m CT

G

dAMP

4.c

AMP dGTP GT P dGDP GD P dGMP GMP

2s

2

3

4

5

6

PH

-

Fig. 11. Effect of pH on capaci factors of nucleotides. Mobile phase, methanol 83.3 mM triethylammonium phosphate $:94, vlv).

3. EXTRACTION OF NUCLEOTIDES FROM CELLS AND TISSUES

Methods for the extraction of nucleotides have been described in detail by Perrett43. Nucleotides are usually extracted from cells and tissues with perchloric acid (PCA) or trichloroacetic acid (TCA). The precipitated proteins are removed by centrifugation. As nucleotides are unstable in acidic solutions, the supernatant should be neutralized or the acid removed by extraction immediately following centrifugation. PCA is neutralized with potassium hydroxide and the precipitated potassium perchlorate is removed by centrifugation. TCA is removed by extraction with water-saturated diethyl ether. Neutralization of acid extracts with tri-n-octylamine in Freon has also been described44. Shryock et al.45 developed a method for the extraction of adenine nucleotides from cultcred endothelial cells using 89% (vlv) methanol containing 0.5 mM EDTA or EGTA at 70°C as the extractant. The extraction efficiency was shown to be better than the PCA or TCA procedures. The method may possibly be applled to the extraction of nucleotides in dispersed cells and frozen, powdered tissues. Whichever method is chosen for the extraction of nucleotides, it is essential to minimize changes in nucleotide composition due to enzyme reactions. This is especially important when whole tissues are analysed. Intercellular

203 enzymes such as 5'-nucleotidases rapidly convert nucleotides with larger numbers of phosphate groups to lower ones and subsequently to nucleosides and bases. Phosphorylating enzymes may in turn transform nucleosides into nucleotides. These enzyme actions can be stopped by free~e-clamping~~ the tissue in situ using metal tongs, with the live animal under light ether or Nembrital anesthesia. 3.1. Extraction of heart tissue The following procedure may be used to extract nucleotides from whole heart tissue: (1) pre-freeze a pair of metal tongs to -196°C with liquid nitrogen; (2) rapidly freeze-clamp the heart by squeezing the tissue between the flat jaws of the tongs; (3) free the frozen

1o.c

9.0

8.0

ZC

--Y

6.0

._ .-0u 54 b

0 c

m

n m

u

40

31:

2.0

1.0

0

-

. 50

100

150

GDP GMP

200

Triethylarnrnonium phosphate ( rnM 1 Fig. 12. Effect of triethylammonium phosphate buffer concentration on capacity factors of nucleotides. Mobile phase, methanol - triethylammonium phosphate (pH 5.0) (6:94, v/v).

204

tissue with a scalpel and cut away any material not held between the jaws; (4) remove a small, accurately weighed sample (10 - 20 mg) for dry weight determination by drying to constant weight; (4) rapidly weigh the remaining tissue in a cold weighing boat; (5) pulverise the tissue to a fine, lumpless powder in a ,percussion mortar at liquid nitrogen temperature; (6) transfer the powder to a cold homogenizer tube containing ice-cold 20% perchloric acid (about 1 ml per 100 mg of tissue) and mix thoroughly; (7) dilute the mixture with an equal volume of ice-cold distilled water and homogenize immediately for 60 - 90 s or until the homogenate appears uniform in colour and texture; (8) centrifuge to remove the protein precipitate at 4°C; the precipitate may be used, after solubilization, for protein determination if required; (9) neutralize the supernatant with potassium hydroxide and remove the potassium perchlorate famed by centrifugation.

3.2.Extraction of liver tissue Liver tissue may be homogenized directly after freeze-clamping. Add the weighed tissue to a homogenizer containing ice-cold 10% TCA (about 1 ml per 100 mg of tissue) and homogenize as described for heart tissue. Excess of TCA is removed by extracting the supernatant with 3 x 4 volumes of water-saturated diethyl ether or by neutralization with tri-n-octylamine in Freon.

3.3.Extraction of red blood cells Heparinized blood is centrifuged at 2000 g for 10 min at 4°C. The plasma and buffy coat are discarded. The red blood cells are washed with a volume of ice-cold 0.9% NaCl and again centrifuged. The packed cells (1 volume) are slowly added to ice-cold 10% TCA (2 volumes) and vortex-mixed. After centrifugation, the supernatant is transferred to a clean tube and the excess of TCA is removed as described above. 3.4. Extraction of lymphocytes

Whole blood (20 ml) is defibrinated and mixed with an equal volume of saline and 40 ml of phosphate-buffered saline (PBS). The mixture is divided into four 20-ml portions and each portion is carefully layered onto 10 ml of Lymphoprep in a glass universal container and centrifuged at 630 g for 10 min. The cloudy interface of lymphocytes is collected and pooled in a glass centrifuge tube. An equal volume of PBS is added to the lymphocyte suspension and the mixture is centrifuged at 1800 g for 10 min. The pellet is resuspended in 1 ml of PBS. To minimize red cell contamination, 3 ml of distilled water are added to the suspension and left to stand on ice for 45 s to lyse the red cells. PBS (20 ml) is then added and the suspension is centrifuged at 1800 g for 10 min. The supernatant is discarded and the lymphocyte pellet is resuspended in 500 pl of PBS. A small volume (50 pl) is removed for cell counting. To the remainder, 10% TCA (100 pI per lo6 cells) is added and the mixture is vortex-mixed. Excess of TCA is removed as described above. It may also be necessary to sonicate or homogenize the extraction mixture to achieve complete disintegration of the cells.

205

The cell preparation and extraction procedure is relatively slow and it is therefore difficult to avoid some degradation of nucleotides as lymphocytes and other white cells are metabolically more active than red cells. 3.5. Extraction of tissue culture cells These cells are collected by centrifugation or by rapid filtration and then extracted with 10% TCA. Shryock et however, found that in the extraction of adenine nucleotides from cultured vascular endothelial cell monolayers, aqueous methanol yielded extracts with a higher ATP content than with PCA or TCA. The extraction solution was 80% methanol containing 0.5 mM EGTA or EDTA at 70°C. The recovery of exogenous ATP added during the extraction process was reported to be generally greater than 90%. EGTA or EDTA was added to the extraction solution to ensure complete inhibition of enzymes involved in nucleotide degradation. Tissue culture cells are significantly more metabolically active than blood cells. 4. HPLC DETECTORS FOR NUCLEOTIDES 4.1. Ultraviolet

Nucleotides have strong UV absorption at in 240-280 nm region and 254 nm is most commonly used for detection. The detection limit, depending on the sensitivity of the detector, is about 10-50 pmol injected. This is adequate for the analysis of most cell and tissue extracts but is insufficiently sensitive for the detection of cyclic nucleotides, e.g., CAMP,in physiological fluids. 4.2. Fluorescence

Adenine nucleotides react with bromo- or chloroacetaldehyde to form highly fluorescent 1,N6-etheno derivatives4’- 52.Thederivatives can be separated by HPLC48-51and the reaction (Fig. 13) can therefore be applied to the sensitive determination of CAMP in plasma, urine and other biological materials.

ri

R

Fig. 13. Reaction of adenine compounds with haloacetaldehydes to form 1,N6-etheno derivatives.

206 4.3. Electrochemical Some nucleotides, particularly those containing the guanine and xanthine ring, are electr~active~~. Guanosine nucleotides, including cGMP, have been separated by RP-IP chromatography and detected electrochemically at 0.95 V vs. Ag/AgCI over the range 1-1000 pmol injected. The method had been applied to the determination of guanonine nucleotides in brain extracts54.

+

4.4. Radioactivity On-line radioactivity measurements with a heterogeneous flow cell has been described for the analysis of lymphoid cell ribonu~leotides~~. The cells were incubated with t4C]uridine and the nucleotides formed were extracted and separated by RP-HPLC. The method is both sensitive and specific. 5. QUANTIFICATION OF NUCLEOTIDES

Nucleotides are quantified by peak-height or peak-area measurement with an electronic integrator. Standards for the construction of calibration graphs are available commercially. An internal standard is added together with the extractant to correct for dilution and sampling errors during sample preparation. The internal standard should ideally be a nucleotide monophosphate which is not naturally occurring. Nucleotide monophosphates are preferred as they are chemically more stable than di- and triphosphates. CMP, XMP, c X M P and ~ ~ 3’-AMP3’ have been used as internal standards. Before quantitative analysis of nucleotides is performed, it is important to ensure that the quality of the extraction is acceptable. This can usually be assessed by measuring the ATP/ADP ratio, which is a sensitive indicator of metabolic changes. The true ATP/ADP ratio for red cells, for example, is 14 and any lowering of this ratio is indicative of enzymic or chemical degradation. The changes in the in vivo nucleotide concentrations during extraction can also be measured by calculating the energy charge (EC), which is an estimate of the energy status of the cells, from the equation ATP + fi ADP ATP + ADP + AMP In cells with maximum energy status, the EC value approaches unity. A good extract should therefore have an EC value between 0.85 and 0.95. EC =

6.PEAK IDENTIFICATION The simplest way of identifying a p a k is by its retention time or by co-chromatography with an authentic standard. This may be adequate when well defined samples are analysed. In situations where the homogeneity of the peak is in doubt, more rigorous peak identification methods are required.

207 6.1. Peak identification by changing the chromatographic conditions The retention times of nucleotides are affected by buffer concentration, pH and organic modifier content in the mobile phase. Alteration d.one or more of these conditions followed by retention time measurement allows reasonable positive identification of the compounds. Changing the chromatographic mode, for example, from ion-exchange to RP-IP or RP chromatography, may also be similarly applied. 6.2. Spectral characteristics Peaks can be identified by their characteristic UV spectra. This is achieved either by trapping the peak in the flow cell (stop-flow technique) for scanning or with a photodiode array detector. Peak homogeneity can also be determined by obtaining the first- and second-order derivatives of the UV spectra. A more commonly used technique is to measure the absorbance ratio at 280 and 254 nm. This ratio is significantly affected if the peak is contaminated. 6.3. Enzymatic shift This method makes use of a specific enzyme to convert a tentatively identified compound in the sample to a known product with an entirely different retention time. Thus, after incubating the sample with an enzyme, the original peak is eliminated and a new peak appears in the chromatogram. For example, nucleotide monophosphates can be identified by treating the sample with 5’-nucleotidase, which converts the nucleotides into nucleosides, and CAMP can be identified by converting it into AMP with phosphodiesterase.

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