A N A L Y T I C A L BIOCHEMISTRY 7 0 , 4 0 3 - 4 1 2
(1976)
A Rapid Radiometric Assay for Adenosine Deaminase RAYMOND J. JACKSON AND RICHARD G. KEIGHTLEY
Departments of Microbiology and Pediatrics, University of Alabama in Birmingham, University Station, Birmingham, Alabama, 35294 Received June 30, 1975; accepted September 11, 1975 A rapid radiochemical procedure for the measurement of adenosine deaminase is described. The method employs phospho-Sephadex, a weak cation exchanger, which permits the enzymic product inosine to pass unretarded through the gel while the radioactive substrate adenosine is retained. Use of a Millipore filter manifold permits rapid processing of samples and eliminates time-consuming column chromatographic, electrophoretic, or paper chromatographic techniques required for separation of product and substrate. The activity of adenosine deaminase was examined in spleen cell preparations prepared from normal CBA mice. Excellent agreement of results was obtained when the radioactive method was compared with two other independent assay techniques.
Adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) catalyzes the conversion of adenosine to inosine and ammonia. The enzyme is present in a wide variety of organisms and tissues (1-5). Methods currently employed for the assay of ADA 1 include: (a) a spectrophotometric assay based on the decrease in absorbance at 265 nm (6,7), (b) the determination of the ammonia liberated from adenosine by formation of colored complexes that can be quantitated spectrophotometrically (8), and (c) radioactive assays employing either high voltage electrophoresis for separation of adenosine and inosine (9) or elution of inosine from a Dowex-50 H + column with a solution of 0.5 M LiCL in 0.01 y H C L (10). The new method is based on the selective adsorption of unreacted [8-14C]adenosine to a phospho-Sephadex gel in the hydrogen ion form while the enzymic product, inosine, passes through the gel and is quantitatively recovered in the eluate. Using a Millipore filter manifold, up to 30 reaction mixtures may be analyzed at one time.
MATERIALS [8-14ClAdenosine 59 mCi/mmol and [8-14C]hypoxanthine > 50 mCi/ mmol were purchased from Amersham/Searle Corporation (Arlington Heights, Illinois). [6-14C]Uric acid was a gift of Dr. J. A. Nelson of t The abbreviations used are: ADA, adenosine deaminase; PNPase, purine nucleoside phosphorylase.
403 Copyright © 1976 by Academic Press. Inc. All rights of reproduction in any form reserved.
404
JACKSON AND K E I G H T L E Y
Southern Research Institute, Birmingham, Alabama. Adenosine, inosine, dithiothreitol, Trizma base, and adenosine deaminase Type I were obtained from Sigma Chemical Co., St. Louis, Missouri. Uric acid was a product of Fisher Scientific, Fairlawn, N.J. Xanthine oxidase was purchased from Boehringer Mannheim Corporation, New York, N.Y. Sephadex G-25 was obtained from Pharmacia Fine Chemicals, Piscataway, N.J. All other chemicals were of reagent grade. Phospho-Sephadex (11) was prepared from Sephadex G-25 and phosphorus oxychloride according to the method of Peterson and Sober (12) for the corresponding cellulose derivative and was prepared for use by washing in 0.1 N H C L followed by washing with deionized water. The synthesis of phosphoSephadex can be completed in 4 to 5 hr. For use in the assay, 2 vol of settled gel were suspended in 1 vol of deionized water.
METHODS
Preparation of murine spleen extracts. Spleens were aseptically removed from CBA mice and lymphocytes obtained by gently teasing the spleens in serum-free Waymouth's medium MB 752/1. The cell suspensions were centrifuged at 300g for 10 min and washed once in 0.85% N a C L in 10 mM T r i s - H C l buffer, pH 7.5. The cell pellet was resuspended in 10 mM T r i s - H CI buffer, pH 7.5, containing I mM dithiothreitol. Cell suspensions were then disrupted in a model W185 D Heat Systems-Ultrasonics cell disruptor for 30 sec in an ice bath. The sonicate was centrifuged at 27,000 g for 1 hr and the resultant supernatant employed as the source of enzyme. The enzyme preparations were stored at 0°C. No detectable loss in activity was detected after 4 weeks of storage. Enzyme assays. (a) Radioactive Assay. The reaction mixtures employed for the radioactive assay contained 0.2/zmol [8-14C]adenosine (466,000 dpm//zmol) in 10 mM T r i s - H C l buffer, p H 7.5, containing 1 mM dithiothreitol and enzyme in a total volume of 400/zl. When crude spleen sonicates were employed as the enzyme source, 8 × 10-3 units of xanthine oxidase were also incorporated into the reaction mixture. The enzymatic activity was quantitated in the manner described below. Two milliliters of the phospho-Sephadex gel suspension was pipetted into the well of a Millipore filter manifold (Model 3025), and the excess water was removed by suction leaving a " p a d " of moist gel. Following incubation of the reaction mixtures at 25°C for the desired period of time, the entire reaction mixture was taken up in a pasteur pipet and applied directly to the gel. A slight vacuum was left in the manifold and thus the reaction mixture was immediately absorbed into the gel pad upon application. This procedure stops the reaction. The 10 × 75 mm reaction tube was rinsed with 1 ml of 1 mM Tris-HC1 buffer, pH 7.5, and also applied to the gel followed by a 4-ml wash with the same buffer. One milliliter of the gel eluate was counted for radioactivity in 8 ml of a modified Bray's solution (13). The
ASSAY OF ADENOSINE DEAMINASE
405
counting efficiency was approximately 74% in eluates derived from reaction mixtures containing crude lymphocyte supernatants. Counts per minute were converted to dpm by the external standardization procedure. (b) Spectrophotometric Assay. The spectrophotometric assay employed for assay of ADA activity was a modification of Kalchar's method (6) as described by Battistuzzi et al. (14). Following incubation, 1.6 ml of 8.7% perchloric acid was added to the reaction mixture and the solution placed in a boiling water bath for 10 min in order to stop the reaction. The solution was subsequently neutralized with 0.32 ml of 5 M K2COa, centrifuged, and read at 268 nm in a Gilford spectrophotometer. The amount of adenosine consumed was estimated employing a AE268m°l of 6.77 × 103 (14). (c) Ammonia Liberation Assay. The ammonia liberated as a result of the catalysis of adenosine to inosine was quantitated employing the phenol-hypochlorite reagent described by Muftic (15). A standard curve consisting of 0-100 nmol of ammonia (as anhydrous ammonium sulfate) was prepared each time reaction mixtures were assayed by this method. Following color development, the samples were read at 655 nm in a Gilford spectrophotometer. Paper chromatography. Paper chromatography was performed on Whatmann # 1 paper at room temperature. The solvents employed were: Solvent A, saturated ammonium sulfate:H~O:propanol, 80:18:2; Solvent B, isobutyric acid:H20:ammonia, 66:33:1; Solvent C 1.8 M ammonium formate in 2% boric acid, pH 7.0. The compounds were visualized with an ultraviolet lamp. Miscellaneous methods. Protein was estimated by the method of Lowry et al. (16). Radioactivity was quantitated either in a Packard Model 3375 scintillation spectrometer or in the case of chromatograms with a Packard model 7201 radiochromatogram scanner. RESULTS Initially commercial calf intestine ADA was employed to test the efficacy of the radioactive method. Approximately 6 × 10-a units of ADA were added to the standard assay mixture (see Methods), and the reaction mixtures incubated at 25°C for various periods of time. Figure I depicts the results obtained with the assay method. As shown in the figure, the assay was linear with time for approximately 10 min under the conditions employed. Eluates of reaction mixtures in which enzyme was omitted had counts of approximately 170 cpm, i.e., less than 0.04% of the total adenosine counts applied to the gel. Background counts ranged from 50-200 cpm depending on the particular batch of gel employed in the assay. The total filtrate counts were obtained by multiplying the counts per minute per milliliter by the total volume of the filtrate. However, the constancy of filtrate volume was such that the results could be expressed in counts per
406
JACKSON AND K E I G H T L E Y r
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FI6. 1. Time-dependent, linear accumulation of radioactivity in gel eluates following incubation of [8-14C]adenosine with calf intestinal ADA.
minute per milliliter (see insert Fig. 1) and the total counts calculated by multiplying by a factor of 5.4. This factor varies slightly depending upon the batch of gel used. Linearity of the assay was also shown to be a function of the protein concentration added to the reaction mixtures (data not shown). The validity of the assay was further investigated by incubating [8-14C]adenosine (3.75 /zmol, 475,000 dpm//.~mol) with approximately 8 × 10-3 units of calf intestine ADA. Incubation was allowed to proceed for 3 hr at 25°C. Following incubation, the reaction mixture was applied to a column of phospho-Sephadex. Elution of the column was accomplished with water. Figure 2 depicts the elution profile obtained under these conditions. As shown in the figure, a single sharp radioactive peak was obtained. The radioactive fractions were pooled and concentrated by rotoevaporation to approximately 1 ml. An absorption spectrum of the unknown radioactive product was performed at pH 3.0 (see insert Fig. 2) and at pH 11 with an Aminco DW-2 spectrophotometer, and were indistinguishable from authentic inosine. Chromatography of the concentrated radioactive material was performed in three solvent systems (Table 1). The chromatograms were examined for ultraviolet absorbing material and subsequently scanned for radioactivity. Figure 3 illustrates the results obtained with Solvent A. In Fig. 3A, a single radioactive peak was detected when approximately 32,000 cpm of the unknown was cochromatographed with 2/zmol of authentic inosine. Moreover, the radioactive peak (Rs0.46) was coincident with the uv absorbing inosine. Figure 3B is a chromatogram of [8-14C]adenosine establishing its purity. Adenosine exhibited an Rs of 0.21 in this solvent system. These data coupled with the spectral data provide evidence that the radioactive material eluted from the phospho-
ASSAY OF ADENOSINE DEAMINASE
407
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FIG. 2. P h o s p h o - S e p h a d e x elution profile of a reaction mixture following incubation of [8-14C]adenosine with calfADA. The reaction mixture was applied to a 2.5 × 7.5 cm column of p h o s p h o - S e p h a d e x and elution accomplished with water. Fractions of 3 ml were collected and counted for radioactivity. The radioactive fractions were pooled, concentrated, and an absorption spectrum taken (see Fig. 2 inset).
Sephadex gel after incubation of [8-'4C]adenosine with ADA is [814C]inosine. Following identification of the radioactive material in the phosphoSephadex eluate as inosine, an experiment was designed in order to determine the percentage recovery of inosine from the gel under the assay conditions. Five separate mixtures were applied to the gel and washed as TABLE 1 PAPER CHROMATOGRAPHY OF STANDARD COMPOUNDS AND UNKNOWN PRODUCT
Solvents," R I values Compound
A
B
C
Adenosine Hypoxan:hine Inosine Unknown lnosine + u n k n o w n
0.21 0.60 0.46 0.46 ~ 0.4d'
0.84 0.63 0.54 0.53 b 0.53 b
0.53 0.60 0.75 0.75 ° 0.75 °
" The composition of solvents A, B, and C are given in Methods. b Indicates radioactive spot coincident with uv absorbance.
408
JACKSON A N D K E I G H T L E Y I
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FIG. 3. Paper chromatography (Solvent A) of the radioactive product. (A) Approximately 32,000 cpm of the radioactive fraction was cochromatographed with 2/zmol of authentic inosine. The chromatogram was scanned for radioactivity and the carrier inosine located by ultraviolet absorption. (B) Chromatogram of the substrate [8-14C]adenosine establishing its purity.
described previously. As indicated in Table 2, an average recovery of 97% of the inosine applied to the gel was obtained with this procedure. In crude extracts it is likely that other enzymes of the purine salvage pathway may be present. Since inosine may be converted to hypoxanthine by purine nucleoside phosphorylase (EC 2.4.2.1), extracts contaminated with this enzyme may give deceptively low values if hypoxanthine is not TABLE 2 INOSINE RECOVERY
Sample number 1a 2 3 4 5 All Samples
FROM PHOSPHO-SEPHADEX
Total DPM recovered
Recovery (%)
93,873 95,466 91,839 95,931 95,919
96.2 97.8 94.2 98.4 98.4 97.0 _+ 3.6 b
" Samples consisted of 97,520 dpm [8-14C]inosine, 1 mM Cleland's reagent in 10 mM Tris-HCI buffer, pH 7.5. Total volume was 400/zl. b Standard deviation.
409
ASSAY OF ADENOSINE DEAMINASE TABLE 3
URIC ACID RECOVERY FROM PHOSPHO-SEPHADEX Sample number
Total DPM recovered
Recovery (%)
141,276 137,180 138,342 142,077 143,938
99.2 96.4 97.1 98.8 101.1 98.5 ± 2.9 b
l" 2 3 4 5 All s a m p l e s
(1976)
A Rapid Radiometric Assay for Adenosine Deaminase RAYMOND J. JACKSON AND RICHARD G. KEIGHTLEY
Departments of Microbiology and Pediatrics, University of Alabama in Birmingham, University Station, Birmingham, Alabama, 35294 Received June 30, 1975; accepted September 11, 1975 A rapid radiochemical procedure for the measurement of adenosine deaminase is described. The method employs phospho-Sephadex, a weak cation exchanger, which permits the enzymic product inosine to pass unretarded through the gel while the radioactive substrate adenosine is retained. Use of a Millipore filter manifold permits rapid processing of samples and eliminates time-consuming column chromatographic, electrophoretic, or paper chromatographic techniques required for separation of product and substrate. The activity of adenosine deaminase was examined in spleen cell preparations prepared from normal CBA mice. Excellent agreement of results was obtained when the radioactive method was compared with two other independent assay techniques.
Adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) catalyzes the conversion of adenosine to inosine and ammonia. The enzyme is present in a wide variety of organisms and tissues (1-5). Methods currently employed for the assay of ADA 1 include: (a) a spectrophotometric assay based on the decrease in absorbance at 265 nm (6,7), (b) the determination of the ammonia liberated from adenosine by formation of colored complexes that can be quantitated spectrophotometrically (8), and (c) radioactive assays employing either high voltage electrophoresis for separation of adenosine and inosine (9) or elution of inosine from a Dowex-50 H + column with a solution of 0.5 M LiCL in 0.01 y H C L (10). The new method is based on the selective adsorption of unreacted [8-14C]adenosine to a phospho-Sephadex gel in the hydrogen ion form while the enzymic product, inosine, passes through the gel and is quantitatively recovered in the eluate. Using a Millipore filter manifold, up to 30 reaction mixtures may be analyzed at one time.
MATERIALS [8-14ClAdenosine 59 mCi/mmol and [8-14C]hypoxanthine > 50 mCi/ mmol were purchased from Amersham/Searle Corporation (Arlington Heights, Illinois). [6-14C]Uric acid was a gift of Dr. J. A. Nelson of t The abbreviations used are: ADA, adenosine deaminase; PNPase, purine nucleoside phosphorylase.
403 Copyright © 1976 by Academic Press. Inc. All rights of reproduction in any form reserved.
404
JACKSON AND K E I G H T L E Y
Southern Research Institute, Birmingham, Alabama. Adenosine, inosine, dithiothreitol, Trizma base, and adenosine deaminase Type I were obtained from Sigma Chemical Co., St. Louis, Missouri. Uric acid was a product of Fisher Scientific, Fairlawn, N.J. Xanthine oxidase was purchased from Boehringer Mannheim Corporation, New York, N.Y. Sephadex G-25 was obtained from Pharmacia Fine Chemicals, Piscataway, N.J. All other chemicals were of reagent grade. Phospho-Sephadex (11) was prepared from Sephadex G-25 and phosphorus oxychloride according to the method of Peterson and Sober (12) for the corresponding cellulose derivative and was prepared for use by washing in 0.1 N H C L followed by washing with deionized water. The synthesis of phosphoSephadex can be completed in 4 to 5 hr. For use in the assay, 2 vol of settled gel were suspended in 1 vol of deionized water.
METHODS
Preparation of murine spleen extracts. Spleens were aseptically removed from CBA mice and lymphocytes obtained by gently teasing the spleens in serum-free Waymouth's medium MB 752/1. The cell suspensions were centrifuged at 300g for 10 min and washed once in 0.85% N a C L in 10 mM T r i s - H C l buffer, pH 7.5. The cell pellet was resuspended in 10 mM T r i s - H CI buffer, pH 7.5, containing I mM dithiothreitol. Cell suspensions were then disrupted in a model W185 D Heat Systems-Ultrasonics cell disruptor for 30 sec in an ice bath. The sonicate was centrifuged at 27,000 g for 1 hr and the resultant supernatant employed as the source of enzyme. The enzyme preparations were stored at 0°C. No detectable loss in activity was detected after 4 weeks of storage. Enzyme assays. (a) Radioactive Assay. The reaction mixtures employed for the radioactive assay contained 0.2/zmol [8-14C]adenosine (466,000 dpm//zmol) in 10 mM T r i s - H C l buffer, p H 7.5, containing 1 mM dithiothreitol and enzyme in a total volume of 400/zl. When crude spleen sonicates were employed as the enzyme source, 8 × 10-3 units of xanthine oxidase were also incorporated into the reaction mixture. The enzymatic activity was quantitated in the manner described below. Two milliliters of the phospho-Sephadex gel suspension was pipetted into the well of a Millipore filter manifold (Model 3025), and the excess water was removed by suction leaving a " p a d " of moist gel. Following incubation of the reaction mixtures at 25°C for the desired period of time, the entire reaction mixture was taken up in a pasteur pipet and applied directly to the gel. A slight vacuum was left in the manifold and thus the reaction mixture was immediately absorbed into the gel pad upon application. This procedure stops the reaction. The 10 × 75 mm reaction tube was rinsed with 1 ml of 1 mM Tris-HC1 buffer, pH 7.5, and also applied to the gel followed by a 4-ml wash with the same buffer. One milliliter of the gel eluate was counted for radioactivity in 8 ml of a modified Bray's solution (13). The
ASSAY OF ADENOSINE DEAMINASE
405
counting efficiency was approximately 74% in eluates derived from reaction mixtures containing crude lymphocyte supernatants. Counts per minute were converted to dpm by the external standardization procedure. (b) Spectrophotometric Assay. The spectrophotometric assay employed for assay of ADA activity was a modification of Kalchar's method (6) as described by Battistuzzi et al. (14). Following incubation, 1.6 ml of 8.7% perchloric acid was added to the reaction mixture and the solution placed in a boiling water bath for 10 min in order to stop the reaction. The solution was subsequently neutralized with 0.32 ml of 5 M K2COa, centrifuged, and read at 268 nm in a Gilford spectrophotometer. The amount of adenosine consumed was estimated employing a AE268m°l of 6.77 × 103 (14). (c) Ammonia Liberation Assay. The ammonia liberated as a result of the catalysis of adenosine to inosine was quantitated employing the phenol-hypochlorite reagent described by Muftic (15). A standard curve consisting of 0-100 nmol of ammonia (as anhydrous ammonium sulfate) was prepared each time reaction mixtures were assayed by this method. Following color development, the samples were read at 655 nm in a Gilford spectrophotometer. Paper chromatography. Paper chromatography was performed on Whatmann # 1 paper at room temperature. The solvents employed were: Solvent A, saturated ammonium sulfate:H~O:propanol, 80:18:2; Solvent B, isobutyric acid:H20:ammonia, 66:33:1; Solvent C 1.8 M ammonium formate in 2% boric acid, pH 7.0. The compounds were visualized with an ultraviolet lamp. Miscellaneous methods. Protein was estimated by the method of Lowry et al. (16). Radioactivity was quantitated either in a Packard Model 3375 scintillation spectrometer or in the case of chromatograms with a Packard model 7201 radiochromatogram scanner. RESULTS Initially commercial calf intestine ADA was employed to test the efficacy of the radioactive method. Approximately 6 × 10-a units of ADA were added to the standard assay mixture (see Methods), and the reaction mixtures incubated at 25°C for various periods of time. Figure I depicts the results obtained with the assay method. As shown in the figure, the assay was linear with time for approximately 10 min under the conditions employed. Eluates of reaction mixtures in which enzyme was omitted had counts of approximately 170 cpm, i.e., less than 0.04% of the total adenosine counts applied to the gel. Background counts ranged from 50-200 cpm depending on the particular batch of gel employed in the assay. The total filtrate counts were obtained by multiplying the counts per minute per milliliter by the total volume of the filtrate. However, the constancy of filtrate volume was such that the results could be expressed in counts per
406
JACKSON AND K E I G H T L E Y r
28
'~0
24
× E o_
ff
r
ff
r
r
/
20
o o LJ_
12
2
4
6
8
I0
M i n u t e s at 2 5 %
FI6. 1. Time-dependent, linear accumulation of radioactivity in gel eluates following incubation of [8-14C]adenosine with calf intestinal ADA.
minute per milliliter (see insert Fig. 1) and the total counts calculated by multiplying by a factor of 5.4. This factor varies slightly depending upon the batch of gel used. Linearity of the assay was also shown to be a function of the protein concentration added to the reaction mixtures (data not shown). The validity of the assay was further investigated by incubating [8-14C]adenosine (3.75 /zmol, 475,000 dpm//.~mol) with approximately 8 × 10-3 units of calf intestine ADA. Incubation was allowed to proceed for 3 hr at 25°C. Following incubation, the reaction mixture was applied to a column of phospho-Sephadex. Elution of the column was accomplished with water. Figure 2 depicts the elution profile obtained under these conditions. As shown in the figure, a single sharp radioactive peak was obtained. The radioactive fractions were pooled and concentrated by rotoevaporation to approximately 1 ml. An absorption spectrum of the unknown radioactive product was performed at pH 3.0 (see insert Fig. 2) and at pH 11 with an Aminco DW-2 spectrophotometer, and were indistinguishable from authentic inosine. Chromatography of the concentrated radioactive material was performed in three solvent systems (Table 1). The chromatograms were examined for ultraviolet absorbing material and subsequently scanned for radioactivity. Figure 3 illustrates the results obtained with Solvent A. In Fig. 3A, a single radioactive peak was detected when approximately 32,000 cpm of the unknown was cochromatographed with 2/zmol of authentic inosine. Moreover, the radioactive peak (Rs0.46) was coincident with the uv absorbing inosine. Figure 3B is a chromatogram of [8-14C]adenosine establishing its purity. Adenosine exhibited an Rs of 0.21 in this solvent system. These data coupled with the spectral data provide evidence that the radioactive material eluted from the phospho-
ASSAY OF ADENOSINE DEAMINASE
407
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20
30
40
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FIG. 2. P h o s p h o - S e p h a d e x elution profile of a reaction mixture following incubation of [8-14C]adenosine with calfADA. The reaction mixture was applied to a 2.5 × 7.5 cm column of p h o s p h o - S e p h a d e x and elution accomplished with water. Fractions of 3 ml were collected and counted for radioactivity. The radioactive fractions were pooled, concentrated, and an absorption spectrum taken (see Fig. 2 inset).
Sephadex gel after incubation of [8-'4C]adenosine with ADA is [814C]inosine. Following identification of the radioactive material in the phosphoSephadex eluate as inosine, an experiment was designed in order to determine the percentage recovery of inosine from the gel under the assay conditions. Five separate mixtures were applied to the gel and washed as TABLE 1 PAPER CHROMATOGRAPHY OF STANDARD COMPOUNDS AND UNKNOWN PRODUCT
Solvents," R I values Compound
A
B
C
Adenosine Hypoxan:hine Inosine Unknown lnosine + u n k n o w n
0.21 0.60 0.46 0.46 ~ 0.4d'
0.84 0.63 0.54 0.53 b 0.53 b
0.53 0.60 0.75 0.75 ° 0.75 °
" The composition of solvents A, B, and C are given in Methods. b Indicates radioactive spot coincident with uv absorbance.
408
JACKSON A N D K E I G H T L E Y I
t
I
I
A
5
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~
8
8
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6
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0.2 0.3 0.4 0.5 0 6 Rf I
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0,3 0 4 Rf
0.5 0.6
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FIG. 3. Paper chromatography (Solvent A) of the radioactive product. (A) Approximately 32,000 cpm of the radioactive fraction was cochromatographed with 2/zmol of authentic inosine. The chromatogram was scanned for radioactivity and the carrier inosine located by ultraviolet absorption. (B) Chromatogram of the substrate [8-14C]adenosine establishing its purity.
described previously. As indicated in Table 2, an average recovery of 97% of the inosine applied to the gel was obtained with this procedure. In crude extracts it is likely that other enzymes of the purine salvage pathway may be present. Since inosine may be converted to hypoxanthine by purine nucleoside phosphorylase (EC 2.4.2.1), extracts contaminated with this enzyme may give deceptively low values if hypoxanthine is not TABLE 2 INOSINE RECOVERY
Sample number 1a 2 3 4 5 All Samples
FROM PHOSPHO-SEPHADEX
Total DPM recovered
Recovery (%)
93,873 95,466 91,839 95,931 95,919
96.2 97.8 94.2 98.4 98.4 97.0 _+ 3.6 b
" Samples consisted of 97,520 dpm [8-14C]inosine, 1 mM Cleland's reagent in 10 mM Tris-HCI buffer, pH 7.5. Total volume was 400/zl. b Standard deviation.
409
ASSAY OF ADENOSINE DEAMINASE TABLE 3
URIC ACID RECOVERY FROM PHOSPHO-SEPHADEX Sample number
Total DPM recovered
Recovery (%)
141,276 137,180 138,342 142,077 143,938
99.2 96.4 97.1 98.8 101.1 98.5 ± 2.9 b
l" 2 3 4 5 All s a m p l e s
