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Crystallization of the sodium salt of adenosine triphosphate
VOL. 20 (I956)
BIOCHIMICA ET BIOPHYSICA ACTA
23
CRYSTALLIZATION OF THE SODIUM SALT OF ADENOSINE TRIPHOSPHATE by LOUIS B E R G E R
Sigma Chemical Co., St. Louis, Mo. (U.S.A .)
A method is described for the crystallization of the disodium salt of adenosine triphosphate (ATP)* by precipitation from aqueous solution with ethanol. The effect of variations of temperature, pH, and purity of the ATP are briefly discussed. EXPERIMENTAL
Seven g of high purity amorphous sodium salt of ATP, isolated from muscle**, was dissolved in 40 ml water; the p H was 3.5. Upon addition of an equal volume of 95 % ethanol at room temperature an oily precipitate formed which changed to crystals in about IO minutes (Fig. I). After standing at room temperature for one hour, the solution was clear and the crystals coated the bottom and sides of the glass vessel. The crystals were collected by filtration and washed several times with absolute ethanol. The yield was 5.6 g after air-drying at room temperature overnight. An additional 0.7 g of crystals was obtained in a similar manner b y adding 40 m195 % ethanol to the filtrate. The main crop of crystals was subjected to elemental analysis. As shown in Table I, the observed values agree favorably with Fig. I . Photomicrograph Of Crys-~alsof oiltheory for the disodium salt of ATP con- sodium salt of ATP. Magnification = 400 d!_~meters. taining 3H~O; molecular weight = 605. Inorganic P was determined b y the method of FISKE AND SUBBAROWx on a freshly prepared solution of the crystals. Acid-labile P was estimated by hydrolysing for 7 minutes in N HC1 at lOO% Total P was estimated after wet ashing with H2SO i and * The following abbreviations are used: ATP, adenosine triphosphate; ADPI adenosine diphosphate; AMP, adenosine monophosphate; AtetraP, adenosine tetraphosphate. All temperatures a r e in degrees centigrade. ** Offered by Sigma Chemical Company as, "Chromatographed Grade" of ATP prior to the development of their crystalline preparation.
Re#fences p. 26.
24
L. BERGER
VOL. 2 0 ( I 9 5 6 )
H , O 2. Adenine content of the solution was calculated from the optical d e n s i t y a t 260 m ~ at p H 2 b a s e d on E~60 z 14,2oo 2. E x c e p t for the small a m o u n t of inorganic P, the observed values shown in Table I I correspond to pure ATP. E x c e p t for a higher inorganic P content, the values r e p o r t e d in Table I I also a p p l y to the a m o r p h o u s sodium salt of A T P from which the crystals were prepared. TABLE I ELEMENTAL
Constituent
A N A L Y S I S OF C R Y S T A L L I N E
Found* %
C H N •p Na H20 (loss on drying at Io5°C)
ATP
Theory/or Na2A T P . 3HmO (mot. ~t. = 605) %
19.64 3.39 12.04 15.61 7.56 8.59
i9.85 3.3 I 11.58 I5.38 7.61 8.92
" All determinations except Na were made by Elek Micro Analytical Laboratories of Los Angeles, California• Na was determined by flame photometry. TABLE II PHOSPHORUS CONTENT OF CRYSTALLINE
Inorganic P as % of total P Ratio of acid-labile to total P Atoms of P per mole of adenine
ATP
Found
Theory b! A T P
0.05 0.666 3.00
o.oo 0.666 3.00
While the elemental a n d phosphorus d e t e r m i n a t i o n s r e p o r t e d in Tables I a n d I I i n d i c a t e d t h a t the crystals were essentially pure ATP, BOCK AND ALBERTY3 have p o i n t e d out t h a t these procedures are generally not sensitive enough to rule out the absence of AMP, A D P , or A t e t r a P . However, p a p e r c h r o m a t o g r a p h y did establish the homogeneous n a t u r e of the crystals. Two solvent systems were employed, b o t h of which are c a p a b l e of detecting I to 2 O//o c o n t a m i n a t i o n b y r e l a t e d adenosine compounds in ATP. One was isobutyric acid - conc. N H 4 O H - H 2 0 (66:1:33) p H 3.84. The other was the t w o - l a y e r isoamyl a l c o h o l - 2 o % N a , H P O 4 s y s t e m 5. The ascending b o u n d a r y a n d u l t r a v i o l e t detection m e t h o d s of CARTER6 were used. O n l y one spot was d e t e c t a b l e on the p a p e r after d e v e l o p m e n t for 18 hours in either s y s t e m when crystalline A T P was applied. The a m o r p h o u s sodium salt of A T P which was used to p r e p a r e the crystals was also free of c o n t a m i n a t i o n . A f t e r storage of the crystals at room t e m p e r a t u r e for 3 m o n t h s during the summer, A M P a n d A D P w e r e d e t e c t a b l e b y p a p e r c h r o m a t o g r a p h y . The inorganic P increased to 5 % of t h e , t o t a l P with a corresponding decrease in the acid-labile P. W h e n stored at - - 2 0 ° for the same period of time, no significant a m o u n t of decomposition of the c r y s t a l l i n e A T P was noted. This indicates t h a t A T P decomposes at a r a t e of 2 to 5% per m o n t h at 28 to 33 ° with little or no decomposition at - - 2 0 °. A single melting
Re/erences p. 26.
VOL. 20 (1956)
CRYSTALLIZATION OF ATP SODIUM SALT
25
p o i n t determination was made on the crystals after storage at - - 2 0 ° for 8 days. The sample was placed in an oil bath which had been prewarmed to I IO°, and the temperature was raised 3 ° per minute. The crystals melted with decomposition at 188 ° (corrected).
E//ect o/temperature Addition of 2 volumes of 95% ethanol at 5 ° a n d J i 5 ° to a cold aqueous solution of the crystalline ATP at p H 3.5 resulted in the formation of oily precipitates which remained oily when stored overnight in an ice chest or deep freeze, respectively. However, when these same preparations were placed at room temperature, crystallizatiofi did occur after several hours. Crystallizations or recrystallizations were uniformly successful in about 3o minutes or less when the alcohol temperature was between 18 ° and 32 °. Under otherwise similar conditions potassium ions could not replace sodium ions in the formation of crystalline ATP.
E~ect o/the purity o/the A TP Attempts to crystallize ATP from solutions known to contain significant amounts of other nucleotides were often successful but usually required a longer period of time; up to 2 hours. In other instances, seeding was required to induce crystallization. Generally, the crystalline ATP obtained from solution containing other nucleotides, was not free of these contaminants as evidenced by paper or ion exchange chromatographyL This demonstrates the need of a high purity ATP as the starting material if crystalline ATP of maximum purity is to be obtained; crystallization in itself does not necessarily produce maximum purity ATP.
EBect o/pH A solution of crystalline ATP (52 mg/ml) was adjusted from p H 3.5 to 7.5 with 7N NaOH. The p H was then readjusted to pH o.5 by the dropwise addition of 6N HC1. A I ml sample was removed at each pH indicated in Table I I I as the readjustment of p H was being made. Two volumes of 95% ethanol was added to each sample at room temperature and then observed grossly and microscopically for crystallization. Crystallization took place in 4 ° minutes or less in all samples of p H 4 or lower. No crystals formed at p H 5 or 6.5 even after standing overnight at room temperature. TABLE III EFFECT OF P H ON FORMATION O F CRYSTALLINE A T P
(SEE TEXT)
pH
Yield in rag
Nature o] product
Atoms o] Na/mole of adenine
o. 5 i.o 1.5 2.5 4.0 .5.0 6.5
38 42 45 46 29 -34
Crystalline Crystalline Crystalline Crystalline Crystalline Amorphous Amorphous
i. 73 1.7i i .82 1.9o i .87 -3.32
All precipitates were collected b y centrifugation and washed twice with 5 ml portions of 67 % ethanol, twice with 95 % ethanol, ~and ~hen dried in a vacuum desicRe]erences p. 26.
26
~. BERGER
VOL. 20 (1956)
cator for 30 minutes over alumina. The pH 5 sample was lost on centrifugation. Sodium and adenine were estimated on solutions of these products as previously described. It was assumed that these solutions were free of extraneous Na as NaCI because no chloride ion was detected when tested with AgNO3 in dilute HNOv As shown in Table III, crystalline ATP containing approximately 2 atoms of Na per mole was formed at all pHs of 4 or lower. ACKNOWLEDGEMENT
The author is grateful to Mr work was carried out.
D A N BROIDA
under whose guidance and supervision this
SUMMARY A d d i t i o n of I or 2 v o l u m e s of 95 % e t h a n o l a t room t e m p e r a t u r e to a q u e o u s s o l u t i o n s of t h e s o d i u m s a l t of A T P a t p H 4 or lower, r e s u l t e d in t h e f o r m a t i o n of t h e c r y s t a l l i n e d i s o d i u m s a l t of ATP.
REFERENCES I C. H. FISKE AND Y. SUBBAROW,f. Biol. Chem., 66 (1925) 375. 2 W. E. COHN AND C. E. CARTER, J . Am. Chem. Soc., 72 (195 o) 4273 . 3 R. M. BocK AND R. A. ALBERTY, f . Biol. Chem., 193 (195 I) 435. 4 P e r s o n a l c o m m u n i c a t i o n from Dr. R n o SANADI. 5 P e r s o n a l c o m m u n i c a t i o n from Dr. DAVID LIPKIN. s C. E. CARTER, J. Am. Chem. Soc., 72 (195 o) 1466.
Received December 8th, 1955
BIOCHIMICA ET BIOPHYSICA ACTA
23
CRYSTALLIZATION OF THE SODIUM SALT OF ADENOSINE TRIPHOSPHATE by LOUIS B E R G E R
Sigma Chemical Co., St. Louis, Mo. (U.S.A .)
A method is described for the crystallization of the disodium salt of adenosine triphosphate (ATP)* by precipitation from aqueous solution with ethanol. The effect of variations of temperature, pH, and purity of the ATP are briefly discussed. EXPERIMENTAL
Seven g of high purity amorphous sodium salt of ATP, isolated from muscle**, was dissolved in 40 ml water; the p H was 3.5. Upon addition of an equal volume of 95 % ethanol at room temperature an oily precipitate formed which changed to crystals in about IO minutes (Fig. I). After standing at room temperature for one hour, the solution was clear and the crystals coated the bottom and sides of the glass vessel. The crystals were collected by filtration and washed several times with absolute ethanol. The yield was 5.6 g after air-drying at room temperature overnight. An additional 0.7 g of crystals was obtained in a similar manner b y adding 40 m195 % ethanol to the filtrate. The main crop of crystals was subjected to elemental analysis. As shown in Table I, the observed values agree favorably with Fig. I . Photomicrograph Of Crys-~alsof oiltheory for the disodium salt of ATP con- sodium salt of ATP. Magnification = 400 d!_~meters. taining 3H~O; molecular weight = 605. Inorganic P was determined b y the method of FISKE AND SUBBAROWx on a freshly prepared solution of the crystals. Acid-labile P was estimated by hydrolysing for 7 minutes in N HC1 at lOO% Total P was estimated after wet ashing with H2SO i and * The following abbreviations are used: ATP, adenosine triphosphate; ADPI adenosine diphosphate; AMP, adenosine monophosphate; AtetraP, adenosine tetraphosphate. All temperatures a r e in degrees centigrade. ** Offered by Sigma Chemical Company as, "Chromatographed Grade" of ATP prior to the development of their crystalline preparation.
Re#fences p. 26.
24
L. BERGER
VOL. 2 0 ( I 9 5 6 )
H , O 2. Adenine content of the solution was calculated from the optical d e n s i t y a t 260 m ~ at p H 2 b a s e d on E~60 z 14,2oo 2. E x c e p t for the small a m o u n t of inorganic P, the observed values shown in Table I I correspond to pure ATP. E x c e p t for a higher inorganic P content, the values r e p o r t e d in Table I I also a p p l y to the a m o r p h o u s sodium salt of A T P from which the crystals were prepared. TABLE I ELEMENTAL
Constituent
A N A L Y S I S OF C R Y S T A L L I N E
Found* %
C H N •p Na H20 (loss on drying at Io5°C)
ATP
Theory/or Na2A T P . 3HmO (mot. ~t. = 605) %
19.64 3.39 12.04 15.61 7.56 8.59
i9.85 3.3 I 11.58 I5.38 7.61 8.92
" All determinations except Na were made by Elek Micro Analytical Laboratories of Los Angeles, California• Na was determined by flame photometry. TABLE II PHOSPHORUS CONTENT OF CRYSTALLINE
Inorganic P as % of total P Ratio of acid-labile to total P Atoms of P per mole of adenine
ATP
Found
Theory b! A T P
0.05 0.666 3.00
o.oo 0.666 3.00
While the elemental a n d phosphorus d e t e r m i n a t i o n s r e p o r t e d in Tables I a n d I I i n d i c a t e d t h a t the crystals were essentially pure ATP, BOCK AND ALBERTY3 have p o i n t e d out t h a t these procedures are generally not sensitive enough to rule out the absence of AMP, A D P , or A t e t r a P . However, p a p e r c h r o m a t o g r a p h y did establish the homogeneous n a t u r e of the crystals. Two solvent systems were employed, b o t h of which are c a p a b l e of detecting I to 2 O//o c o n t a m i n a t i o n b y r e l a t e d adenosine compounds in ATP. One was isobutyric acid - conc. N H 4 O H - H 2 0 (66:1:33) p H 3.84. The other was the t w o - l a y e r isoamyl a l c o h o l - 2 o % N a , H P O 4 s y s t e m 5. The ascending b o u n d a r y a n d u l t r a v i o l e t detection m e t h o d s of CARTER6 were used. O n l y one spot was d e t e c t a b l e on the p a p e r after d e v e l o p m e n t for 18 hours in either s y s t e m when crystalline A T P was applied. The a m o r p h o u s sodium salt of A T P which was used to p r e p a r e the crystals was also free of c o n t a m i n a t i o n . A f t e r storage of the crystals at room t e m p e r a t u r e for 3 m o n t h s during the summer, A M P a n d A D P w e r e d e t e c t a b l e b y p a p e r c h r o m a t o g r a p h y . The inorganic P increased to 5 % of t h e , t o t a l P with a corresponding decrease in the acid-labile P. W h e n stored at - - 2 0 ° for the same period of time, no significant a m o u n t of decomposition of the c r y s t a l l i n e A T P was noted. This indicates t h a t A T P decomposes at a r a t e of 2 to 5% per m o n t h at 28 to 33 ° with little or no decomposition at - - 2 0 °. A single melting
Re/erences p. 26.
VOL. 20 (1956)
CRYSTALLIZATION OF ATP SODIUM SALT
25
p o i n t determination was made on the crystals after storage at - - 2 0 ° for 8 days. The sample was placed in an oil bath which had been prewarmed to I IO°, and the temperature was raised 3 ° per minute. The crystals melted with decomposition at 188 ° (corrected).
E//ect o/temperature Addition of 2 volumes of 95% ethanol at 5 ° a n d J i 5 ° to a cold aqueous solution of the crystalline ATP at p H 3.5 resulted in the formation of oily precipitates which remained oily when stored overnight in an ice chest or deep freeze, respectively. However, when these same preparations were placed at room temperature, crystallizatiofi did occur after several hours. Crystallizations or recrystallizations were uniformly successful in about 3o minutes or less when the alcohol temperature was between 18 ° and 32 °. Under otherwise similar conditions potassium ions could not replace sodium ions in the formation of crystalline ATP.
E~ect o/the purity o/the A TP Attempts to crystallize ATP from solutions known to contain significant amounts of other nucleotides were often successful but usually required a longer period of time; up to 2 hours. In other instances, seeding was required to induce crystallization. Generally, the crystalline ATP obtained from solution containing other nucleotides, was not free of these contaminants as evidenced by paper or ion exchange chromatographyL This demonstrates the need of a high purity ATP as the starting material if crystalline ATP of maximum purity is to be obtained; crystallization in itself does not necessarily produce maximum purity ATP.
EBect o/pH A solution of crystalline ATP (52 mg/ml) was adjusted from p H 3.5 to 7.5 with 7N NaOH. The p H was then readjusted to pH o.5 by the dropwise addition of 6N HC1. A I ml sample was removed at each pH indicated in Table I I I as the readjustment of p H was being made. Two volumes of 95% ethanol was added to each sample at room temperature and then observed grossly and microscopically for crystallization. Crystallization took place in 4 ° minutes or less in all samples of p H 4 or lower. No crystals formed at p H 5 or 6.5 even after standing overnight at room temperature. TABLE III EFFECT OF P H ON FORMATION O F CRYSTALLINE A T P
(SEE TEXT)
pH
Yield in rag
Nature o] product
Atoms o] Na/mole of adenine
o. 5 i.o 1.5 2.5 4.0 .5.0 6.5
38 42 45 46 29 -34
Crystalline Crystalline Crystalline Crystalline Crystalline Amorphous Amorphous
i. 73 1.7i i .82 1.9o i .87 -3.32
All precipitates were collected b y centrifugation and washed twice with 5 ml portions of 67 % ethanol, twice with 95 % ethanol, ~and ~hen dried in a vacuum desicRe]erences p. 26.
26
~. BERGER
VOL. 20 (1956)
cator for 30 minutes over alumina. The pH 5 sample was lost on centrifugation. Sodium and adenine were estimated on solutions of these products as previously described. It was assumed that these solutions were free of extraneous Na as NaCI because no chloride ion was detected when tested with AgNO3 in dilute HNOv As shown in Table III, crystalline ATP containing approximately 2 atoms of Na per mole was formed at all pHs of 4 or lower. ACKNOWLEDGEMENT
The author is grateful to Mr work was carried out.
D A N BROIDA
under whose guidance and supervision this
SUMMARY A d d i t i o n of I or 2 v o l u m e s of 95 % e t h a n o l a t room t e m p e r a t u r e to a q u e o u s s o l u t i o n s of t h e s o d i u m s a l t of A T P a t p H 4 or lower, r e s u l t e d in t h e f o r m a t i o n of t h e c r y s t a l l i n e d i s o d i u m s a l t of ATP.
REFERENCES I C. H. FISKE AND Y. SUBBAROW,f. Biol. Chem., 66 (1925) 375. 2 W. E. COHN AND C. E. CARTER, J . Am. Chem. Soc., 72 (195 o) 4273 . 3 R. M. BocK AND R. A. ALBERTY, f . Biol. Chem., 193 (195 I) 435. 4 P e r s o n a l c o m m u n i c a t i o n from Dr. R n o SANADI. 5 P e r s o n a l c o m m u n i c a t i o n from Dr. DAVID LIPKIN. s C. E. CARTER, J. Am. Chem. Soc., 72 (195 o) 1466.
Received December 8th, 1955