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A coupled optical assay for determination of adenine in mixtures
ANALYTICAL
BIOCHEMISTRY
A Coupled
98,
Optical
UMBERTO
273-277
(1979)
Assay for Determination
MURA,
of Adenine
MAURIZIO ROMANO, FRANCESCO AND PIER LUIGI IPATA
Received
September
in Mixtures
SGARRELLA,
26. 1978
A rapid and specific spectrophotometric assay for the determination of adenine is described. The method is based on the absorbance change at 265 nm which accompanies the ribose l-phosphate-dependent conversion of adenine into inosine. catalyzed by the successive action of adenosine phosphorylase and adenosine deaminase. Common purine and pyrirnidine bases. nucleosides. and nucleotides do not interfere. The assay was tested in various biochemical situations. in which there was both adenine formation and utilization.
Almost all common purine bases. nucleosides, and mononucleotides can be measured spectrophotometrically by coupled optical enzyme tests: adenosine. hypoxanthine, and guanine by using adenosine deaminase, xanthine oxidase, and guanase plus xanthine oxidase, respectively (1,2): guanosine and inosine after conversion to bases by the action of purine nucleoside phosphorylase (2); and 5’-AMP and 5’-IMP may be determined after conversion to nucleosides catalyzed by 5’-mononucleotidase (3). The specificity of these auxiliary enzymes allows the measurement of each of the compounds listed above present in a mixture (3). Adenine. however. is generally isolated by chromatographic (4,5) or electrophoretic (6,7) procedures, before its quantitative determination; other methods are based on adenine adsorption on cellulose acetate filters (8), or on fluorometric measurement of adenine and its derivatives either directly (9) or after interaction with chloroacetaldehyde (10) or glyoxal hydrate trimer (I 1). In 1952, Klenow described a spectrophotometric assay for adenine (12), based on its conversion to 2,8-dihydroxyadenine by milk xanthine oxidase followed at 305 nm: however, this method is not suitable 273
to determine adenine in the presence of hypoxanthine andlor xanthine, even though it might be possible to distinguish the 2,8dihydroxyadenine from uric acid by adding uricase to the mixture after completion of the reaction, to determine the contribution of hypoxanthine and xanthine to the change in absorbance at 305 nm. Adenase has also been used to measure adenine (13). However, this enzyme has a limited distribution and has not been obtained in a form suitable for routine assay. Recently, we have identified in several species of Bacillus a specific adenosine phosphorylase, distinct from purine nucleoside phosphorylase (14,15). This enzyme has been conveniently utilized for the spectrophotometric determination of ribose lphosphate (16). In this paper we show that the following coupled reactions: adenine + ribose l-phosphate E adenosine + Pi, [I] adenosine + H,O + inosine + NH3,
[21
allowing the spectrophotometric determination of the sugar-phosphate, can be conveniently employed also for the determination of adenine. 0003.2697/79/140273-05$02.00/O CopyrIght All right,
‘r 1979 by Academic Prerr. Inc 01 repmduct~~n in any tbrm rrrrned.
214
MURA
MATERIALS
AND METHODS
Bases, nucleosides, and nucleotides were obtained from Sigma Chemical Company. Adenosine phosphorylase was prepared from Bacillus cereus as described previously (16). Adenosine deaminase (EC 3.5.4.4) was obtained from Boehringer und Soehne and was diluted with water to give a concentration of 20 pg of protein/ml before use. 5’-Nucleotidase (EC 3.1.3.5) was obtained from Sigma Chemical Company and diluted with water to 0.3 mg of protein/ml before use. The experiments were carried out at 37°C in I-cm-pathlength cuvettes and the change in optical density at 265 nm was determined with a recording Beckman ACTA C-III spectrophotometer. The difference in molar extinction coefficients at 265 nm between adenine and inosine was taken as 6390 for our experimental conditions (see Ref. (16)). The detailed standard procedure for the determination of adenine was as follows: 1.0 ml of 0.2 M Tris-Cl buffer, pH 7.4, was pipetted into one cuvette, followed by different amounts of adenine in a maximal volume of 0.2 ml, 100 ~1 of adenosine deaminase, 50 ~1 of adenosine phosphorylase, and water to bring the volume to 1.850 ml. The absorbance at 265 nm was first recorded for several minutes against a reference cuvette in which adenine was substituted by water: finally, 0.15 ml of 1.5 mM ribose l-phosphate was added to both cuvettes, and the total change in optical density was recorded. Modifications of the standard conditions are described in the presentation of the experimental data. B. cereus extracts were prepared as previously described (16) and contained 0.3 mg of protein/ml. The alfalfa (Medicugo sativa) juice, prepared by squeezing the leaves at 110 atm, contained 15.8 mg of protein/ml, and was a kind gift of Dr. R. Fiorentini, of the Institute of Agricultural Industry of the University of Pisa. Determinations of inosine and hypoxanthine were carried out as previously described (3). The molarities of all
ET AL
purine and pyrimidine standard solutions were determined spectrophotometrically from the extinction coefficients at suitable wavelengths (18). RESULTS
AND DISCUSSION
Figure 1 shows the results of an experiment in which four different amounts of adenine were determined. In the absence of ribose l-phosphate the absorbance at 265 nm remained constant, indicating the absence of adenase activity in the auxiliary enzymes. Adenine at concentrations as low as 5 PM can easily be measured by the method proposed, as shown from the calibration curves presented in Fig. 2, where adenine was measured in the presence and absence of a series of purine and pyrimidine bases, nucleosides, and nucleotides as possible interfering compounds. It is evident that there was practically no interference, even at the lowest level of adenine used. The method was used to measure adenine in various biochemical situations. First, we
r-
.----adenasine --adenosine
OIJ
-
IO
deaminase
1
uhosphorylase
20
30 TIME [min)
FIG. 1. Spectrophotometric determination of adenine: 10.6 (a), 29.0 (b), 49.0 (c), and 76.0 (d) nmol of adenine were determined spectrophotometrically as described in the text. The change in absorbance accounted for 11.3 (a), 29.4 (b), 50.7 (c), and 77.9 (d) nmol of adenine.
COUPLED
FIG. 2. Calibration bases (A), nucleosides final concentration.
OPTICAL
ASSAY
FOR
ADENINE
DETERMINATION
curve of adenine. Adenine was measured alone (B), and nucleotides (C and D). Each compound
(0). or in the presence of was added at the indicated
deaminase
FIG. 3. Formation of adenine from adenosine catalyzed by alfalfa juice at 37°C. The reaction mixture contained, in a final volume of 30 ml, 2.0 pmol of adenosine, 97.3 mM Tris-Cl buffer, pH 7.4. and 75 ~1 of alfalfa juice; 2.5.ml portions of the reaction mixture were withdrawn and heated 2 min at 100°C. After cooling at 37°C. 100 ~1 of adenosine deaminase was added to 1.7 ml of the heated reaction mixture to measure the residual adenosine. When the reaction was completed, 50 ~1 of the adenosine phosphorylase preparation was added in the same cuvette, followed by 0.15 ml of 1.5 mM ribose l-phosphate, to measure adenine. Adenine formed (0) and residual adenosine (0) are shown as a function of time. The inset shows the spectrophotometric determination of adenosine and adenine at the 5th (a) and the 30th (b) min of incubation. The difference in molar extinction coefficients. between adenosine and inosine was taken as 8110 (see Ref. (3)).
275
276
MURA
1 ;?1
radenosine phosphorylase ribase -1~ phosphate r
I T
IO
20
30 TIME(min)
FIG. 4. Spectrophotometric determination of adenosine. AMP, and adenine in a mixture. The assay mixture contained, in a final volume of 1.680 ml, 60 mM Tris-Cl buffer, pH 7.4, 26.4 nmol of adenosine, 21.3 nmol of AMP. and 41.6 nmol of adenine. At the times indicated by arrows 100 ~1 of adenosine deaminase. 20 ~1 of 5’-nucleotidase. and 50 ~1 of adenosine phosphorylase plus 0.15 ml of 1.5 mM ribose l-phosphate were added. The change in absorbance accounted for 26.6 nmol of adenosine. 19.3 nmol of AMP, and 40.1 nmol of adenine. The difference in molar extinction coefficients at 265 nm between AMP and inosine was taken as 8540 (see Ref. (3)).
ET AL
phosphorylase, and purine nucleoside phosphorylase activities (17) was analyzed for its ability to produce and to utilize adenine. As expected, in the presence of inorganic phosphate adenosine was transformed into inosine, hypoxanthine, and adenine. accounting for all the nucleoside consumed (Fig. 5). In the absence of inorganic phosphate, inosine was the only reaction product formed during the entire incubation period. Utilization of adenine was investigated by incubating crude extracts of B. cereus in the presence of adenine and ribose lphosphate. As shown in Fig. 6, adenine was converted into adenosine and inosine, most likely through the successive action of adenosine phosphorylase and adenosine deaminase.
selected an enzymatic reaction, catalyzed by adenosine nucleosidase (EC 3.2.2.7) present in alfalfa juice (Ipata et crl., unpublished results): adenosine + H,O + adenine + ribose. It is evident from Fig. 3 that the amount of adenine formed accounted for all the adenosine consumed. No other possible reaction products were detectable. It is noteworthy, as shown in the inset of Fig. 3, that adenosine and adenine can be measured in the same cuvette. The versatility of the procedure is further illustrated in Fig. 4, showing that it is possible to measure in the same cuvette a mixture of AMP, adenosine, and adenine. Second, a crude extract of B. cererrs possessing adenosine deaminase, adenosine
Fw. 5. Formation of adenine, inosine. and hypoxanthine from adenosine catalyzed by crude extract of B. wwus at 37°C. The reaction mixture contained. in a final volume of 40 ml, 1.9 Fmol of adenosine, 82.5 mM Tris-Cl buffer, pH 7.4, 41.3 mM KCI to prevent adenosine deaminase inactivation (Muraet (I/. , unpublished data), 10.0 mM KH,PO,, and 2.0 ml of B. ccwus extract; 4.5-ml portions of the reaction mixture were withdrawn and heated 2 min at 100°C. After cooling at 37°C. 1.2 ml of the heated reaction mixture was diluted to 1.7 ml with 0.2 M Tris-Cl buffer, pH 7.4, for the measurement of adenosine and adenine as described in Fig. 3. Hypoxanthine and inosine were determined on a 1.2.ml portion. Residual adenosine (0). adenine (O), inosine (A). and hypoxanthine (A) formed are shown as a function of time.
COUPLED
OPTICAL
ASSAY
FOR
ADENINE
277
DETERMlNATION
ACKNOWLEDGMENT This work was supported C.N.R. (Progetti Finalizzati).
by a grant
from
the Italian
REFERENCES
FIG. 6. Formation of adenosine and inosine from adenine catalyzed by crude extract of B. ~CTL’IIJ at 37°C. The reaction mixture contained, in a final volume of 50 ml, 2.4 wmol of adenine. 3.2 pmol of ribose l-phosphate, 83.6 mM Tris-Cl buffer. pH 7.4. 41.8 mM KCI. and 2.5 ml of B. cercas extract. Adenosine and adenine were measured as described in Fig 3. Inoslne was determined on a 1.2-ml portion. Residual adenine (O), adenosine (0). and inosine (A) formed are shown as a function of time. There was no formation of hypoxanthine.
In our hands, the method described in this paper htas proved to be both practicable and useful for the rapid determination of adenine in various experimental situations. Taken together with the other spectrophotometric methods for the determination of AMP, IMP, adenosine, inosine, and hypoxanthine (3). and of ribose l-phosphate (16) present in mixtures, it allows the complete spectrophotometric analysis of all the products of AMP and IMP degradation.
I. Kalckar, H. M. (1947) J. Biol. Chenl. 167, 445459. 2. Kalckar, H. M. (1947)5. Bid/. Chrrn. 167,429-443. 3. Mura. U., Sgarrella. F., Felicioli. R. A., Senesi, S., and Ipata. P. L. (1976) Bull. M
BIOCHEMISTRY
A Coupled
98,
Optical
UMBERTO
273-277
(1979)
Assay for Determination
MURA,
of Adenine
MAURIZIO ROMANO, FRANCESCO AND PIER LUIGI IPATA
Received
September
in Mixtures
SGARRELLA,
26. 1978
A rapid and specific spectrophotometric assay for the determination of adenine is described. The method is based on the absorbance change at 265 nm which accompanies the ribose l-phosphate-dependent conversion of adenine into inosine. catalyzed by the successive action of adenosine phosphorylase and adenosine deaminase. Common purine and pyrirnidine bases. nucleosides. and nucleotides do not interfere. The assay was tested in various biochemical situations. in which there was both adenine formation and utilization.
Almost all common purine bases. nucleosides, and mononucleotides can be measured spectrophotometrically by coupled optical enzyme tests: adenosine. hypoxanthine, and guanine by using adenosine deaminase, xanthine oxidase, and guanase plus xanthine oxidase, respectively (1,2): guanosine and inosine after conversion to bases by the action of purine nucleoside phosphorylase (2); and 5’-AMP and 5’-IMP may be determined after conversion to nucleosides catalyzed by 5’-mononucleotidase (3). The specificity of these auxiliary enzymes allows the measurement of each of the compounds listed above present in a mixture (3). Adenine. however. is generally isolated by chromatographic (4,5) or electrophoretic (6,7) procedures, before its quantitative determination; other methods are based on adenine adsorption on cellulose acetate filters (8), or on fluorometric measurement of adenine and its derivatives either directly (9) or after interaction with chloroacetaldehyde (10) or glyoxal hydrate trimer (I 1). In 1952, Klenow described a spectrophotometric assay for adenine (12), based on its conversion to 2,8-dihydroxyadenine by milk xanthine oxidase followed at 305 nm: however, this method is not suitable 273
to determine adenine in the presence of hypoxanthine andlor xanthine, even though it might be possible to distinguish the 2,8dihydroxyadenine from uric acid by adding uricase to the mixture after completion of the reaction, to determine the contribution of hypoxanthine and xanthine to the change in absorbance at 305 nm. Adenase has also been used to measure adenine (13). However, this enzyme has a limited distribution and has not been obtained in a form suitable for routine assay. Recently, we have identified in several species of Bacillus a specific adenosine phosphorylase, distinct from purine nucleoside phosphorylase (14,15). This enzyme has been conveniently utilized for the spectrophotometric determination of ribose lphosphate (16). In this paper we show that the following coupled reactions: adenine + ribose l-phosphate E adenosine + Pi, [I] adenosine + H,O + inosine + NH3,
[21
allowing the spectrophotometric determination of the sugar-phosphate, can be conveniently employed also for the determination of adenine. 0003.2697/79/140273-05$02.00/O CopyrIght All right,
‘r 1979 by Academic Prerr. Inc 01 repmduct~~n in any tbrm rrrrned.
214
MURA
MATERIALS
AND METHODS
Bases, nucleosides, and nucleotides were obtained from Sigma Chemical Company. Adenosine phosphorylase was prepared from Bacillus cereus as described previously (16). Adenosine deaminase (EC 3.5.4.4) was obtained from Boehringer und Soehne and was diluted with water to give a concentration of 20 pg of protein/ml before use. 5’-Nucleotidase (EC 3.1.3.5) was obtained from Sigma Chemical Company and diluted with water to 0.3 mg of protein/ml before use. The experiments were carried out at 37°C in I-cm-pathlength cuvettes and the change in optical density at 265 nm was determined with a recording Beckman ACTA C-III spectrophotometer. The difference in molar extinction coefficients at 265 nm between adenine and inosine was taken as 6390 for our experimental conditions (see Ref. (16)). The detailed standard procedure for the determination of adenine was as follows: 1.0 ml of 0.2 M Tris-Cl buffer, pH 7.4, was pipetted into one cuvette, followed by different amounts of adenine in a maximal volume of 0.2 ml, 100 ~1 of adenosine deaminase, 50 ~1 of adenosine phosphorylase, and water to bring the volume to 1.850 ml. The absorbance at 265 nm was first recorded for several minutes against a reference cuvette in which adenine was substituted by water: finally, 0.15 ml of 1.5 mM ribose l-phosphate was added to both cuvettes, and the total change in optical density was recorded. Modifications of the standard conditions are described in the presentation of the experimental data. B. cereus extracts were prepared as previously described (16) and contained 0.3 mg of protein/ml. The alfalfa (Medicugo sativa) juice, prepared by squeezing the leaves at 110 atm, contained 15.8 mg of protein/ml, and was a kind gift of Dr. R. Fiorentini, of the Institute of Agricultural Industry of the University of Pisa. Determinations of inosine and hypoxanthine were carried out as previously described (3). The molarities of all
ET AL
purine and pyrimidine standard solutions were determined spectrophotometrically from the extinction coefficients at suitable wavelengths (18). RESULTS
AND DISCUSSION
Figure 1 shows the results of an experiment in which four different amounts of adenine were determined. In the absence of ribose l-phosphate the absorbance at 265 nm remained constant, indicating the absence of adenase activity in the auxiliary enzymes. Adenine at concentrations as low as 5 PM can easily be measured by the method proposed, as shown from the calibration curves presented in Fig. 2, where adenine was measured in the presence and absence of a series of purine and pyrimidine bases, nucleosides, and nucleotides as possible interfering compounds. It is evident that there was practically no interference, even at the lowest level of adenine used. The method was used to measure adenine in various biochemical situations. First, we
r-
.----adenasine --adenosine
OIJ
-
IO
deaminase
1
uhosphorylase
20
30 TIME [min)
FIG. 1. Spectrophotometric determination of adenine: 10.6 (a), 29.0 (b), 49.0 (c), and 76.0 (d) nmol of adenine were determined spectrophotometrically as described in the text. The change in absorbance accounted for 11.3 (a), 29.4 (b), 50.7 (c), and 77.9 (d) nmol of adenine.
COUPLED
FIG. 2. Calibration bases (A), nucleosides final concentration.
OPTICAL
ASSAY
FOR
ADENINE
DETERMINATION
curve of adenine. Adenine was measured alone (B), and nucleotides (C and D). Each compound
(0). or in the presence of was added at the indicated
deaminase
FIG. 3. Formation of adenine from adenosine catalyzed by alfalfa juice at 37°C. The reaction mixture contained, in a final volume of 30 ml, 2.0 pmol of adenosine, 97.3 mM Tris-Cl buffer, pH 7.4. and 75 ~1 of alfalfa juice; 2.5.ml portions of the reaction mixture were withdrawn and heated 2 min at 100°C. After cooling at 37°C. 100 ~1 of adenosine deaminase was added to 1.7 ml of the heated reaction mixture to measure the residual adenosine. When the reaction was completed, 50 ~1 of the adenosine phosphorylase preparation was added in the same cuvette, followed by 0.15 ml of 1.5 mM ribose l-phosphate, to measure adenine. Adenine formed (0) and residual adenosine (0) are shown as a function of time. The inset shows the spectrophotometric determination of adenosine and adenine at the 5th (a) and the 30th (b) min of incubation. The difference in molar extinction coefficients. between adenosine and inosine was taken as 8110 (see Ref. (3)).
275
276
MURA
1 ;?1
radenosine phosphorylase ribase -1~ phosphate r
I T
IO
20
30 TIME(min)
FIG. 4. Spectrophotometric determination of adenosine. AMP, and adenine in a mixture. The assay mixture contained, in a final volume of 1.680 ml, 60 mM Tris-Cl buffer, pH 7.4, 26.4 nmol of adenosine, 21.3 nmol of AMP. and 41.6 nmol of adenine. At the times indicated by arrows 100 ~1 of adenosine deaminase. 20 ~1 of 5’-nucleotidase. and 50 ~1 of adenosine phosphorylase plus 0.15 ml of 1.5 mM ribose l-phosphate were added. The change in absorbance accounted for 26.6 nmol of adenosine. 19.3 nmol of AMP, and 40.1 nmol of adenine. The difference in molar extinction coefficients at 265 nm between AMP and inosine was taken as 8540 (see Ref. (3)).
ET AL
phosphorylase, and purine nucleoside phosphorylase activities (17) was analyzed for its ability to produce and to utilize adenine. As expected, in the presence of inorganic phosphate adenosine was transformed into inosine, hypoxanthine, and adenine. accounting for all the nucleoside consumed (Fig. 5). In the absence of inorganic phosphate, inosine was the only reaction product formed during the entire incubation period. Utilization of adenine was investigated by incubating crude extracts of B. cereus in the presence of adenine and ribose lphosphate. As shown in Fig. 6, adenine was converted into adenosine and inosine, most likely through the successive action of adenosine phosphorylase and adenosine deaminase.
selected an enzymatic reaction, catalyzed by adenosine nucleosidase (EC 3.2.2.7) present in alfalfa juice (Ipata et crl., unpublished results): adenosine + H,O + adenine + ribose. It is evident from Fig. 3 that the amount of adenine formed accounted for all the adenosine consumed. No other possible reaction products were detectable. It is noteworthy, as shown in the inset of Fig. 3, that adenosine and adenine can be measured in the same cuvette. The versatility of the procedure is further illustrated in Fig. 4, showing that it is possible to measure in the same cuvette a mixture of AMP, adenosine, and adenine. Second, a crude extract of B. cererrs possessing adenosine deaminase, adenosine
Fw. 5. Formation of adenine, inosine. and hypoxanthine from adenosine catalyzed by crude extract of B. wwus at 37°C. The reaction mixture contained. in a final volume of 40 ml, 1.9 Fmol of adenosine, 82.5 mM Tris-Cl buffer, pH 7.4, 41.3 mM KCI to prevent adenosine deaminase inactivation (Muraet (I/. , unpublished data), 10.0 mM KH,PO,, and 2.0 ml of B. ccwus extract; 4.5-ml portions of the reaction mixture were withdrawn and heated 2 min at 100°C. After cooling at 37°C. 1.2 ml of the heated reaction mixture was diluted to 1.7 ml with 0.2 M Tris-Cl buffer, pH 7.4, for the measurement of adenosine and adenine as described in Fig. 3. Hypoxanthine and inosine were determined on a 1.2.ml portion. Residual adenosine (0). adenine (O), inosine (A). and hypoxanthine (A) formed are shown as a function of time.
COUPLED
OPTICAL
ASSAY
FOR
ADENINE
277
DETERMlNATION
ACKNOWLEDGMENT This work was supported C.N.R. (Progetti Finalizzati).
by a grant
from
the Italian
REFERENCES
FIG. 6. Formation of adenosine and inosine from adenine catalyzed by crude extract of B. ~CTL’IIJ at 37°C. The reaction mixture contained, in a final volume of 50 ml, 2.4 wmol of adenine. 3.2 pmol of ribose l-phosphate, 83.6 mM Tris-Cl buffer. pH 7.4. 41.8 mM KCI. and 2.5 ml of B. cercas extract. Adenosine and adenine were measured as described in Fig 3. Inoslne was determined on a 1.2-ml portion. Residual adenine (O), adenosine (0). and inosine (A) formed are shown as a function of time. There was no formation of hypoxanthine.
In our hands, the method described in this paper htas proved to be both practicable and useful for the rapid determination of adenine in various experimental situations. Taken together with the other spectrophotometric methods for the determination of AMP, IMP, adenosine, inosine, and hypoxanthine (3). and of ribose l-phosphate (16) present in mixtures, it allows the complete spectrophotometric analysis of all the products of AMP and IMP degradation.
I. Kalckar, H. M. (1947) J. Biol. Chenl. 167, 445459. 2. Kalckar, H. M. (1947)5. Bid/. Chrrn. 167,429-443. 3. Mura. U., Sgarrella. F., Felicioli. R. A., Senesi, S., and Ipata. P. L. (1976) Bull. M