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Comparison of barium and zinc precipitation procedures in the isolation of adenosine phosphates from mung bean seedlings
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
67, go-94 (1957)
Comparison of Barium and Zinc Precipitation Procedures in the Isolation of Adenosine Phosphates from Mung Bean Seedlings Eleanor Goldstein and Harry G. Albaum From the Biology
Research
Laboratory,
Brooklyn
College,
Brooklyn,
New
York
Received June 25, 1956
A zinc precipitation procedure devised by Owens (5) for the preparat,ion of nucleotides from the mold Neurospora sitophila is reported to give a yield two to nine times greater than that obtained with the classic barium method. After anion-exchange column fractionation of both preparations, a greater proportion of adenosine diphosphate (ADP) to adenosine triphospha te (ATP) resulted from hhat of the zinc. We decided t,o test the applicability of this zinc precipitation procedure in the isolation of adenosine phosphate from higher plant tissue, and to compare the results with t.hose obtained using the barium method. bh3~H0D
The adenosine phosphates were ext,racted from mung bean seedlings (Phaseolus aureus) using the method of Albaum (2). The mung beans were obtained in the sprouted condition at a Chinese grocery, thus eliminating the necessity of germinating them in the laboratory. The precipitation procedures employed were those described by Albaum for barium (2) and by Owens for zinc (5). The compounds ATP and ADP were separated on a Dower 1 anion-exchange column by the differential elution method of Cohn and Carter (3), and identified by the adenine t,o labile phosphate ratio (1). The total purine determinations were made in the Beckman spectrophotometer using quartz cells with a light path of 1.0 cm. All 26OO-A.-absorbing material was assumed to be adenine.
Preparation
of Column
Enough Dowex 1 anion-exchange resin for several columns was prepared according to Kornberg and Pricer (4). Some 2OCMOO-meshresin was washed repeatedly by centrifugation in bottles with about 4 vol. of 3 N HCl until it no longer displayed absorption at 2600 A. This could not be determined with t.he super90
PRECIPITATION
BY
BARITJM
AND
ZINC
91
natant which contained filterable particles in suspension. To overcome the difficulty, a 3-cm. column was set up and the clear effluent was tested. The bulk resin was then charged by washing with 2 M sodium formate until the supernatant was free of chloride ions, and stored under the solution at 2’C. The bottom of a lOO-ml. buret was packed with a wad of glass wool. Over this was poured resin in 2 M sodium formate in an amount to make a packed column of the desired height. The resin was sedimented and washed by having about 200 ml. of distilled water pass through under pressure.
Extractirm. Approximately 1 lb. of mung bean sprouts of a 40-50-mm. hypocotyl length was homogenized in 200 ml. of 10% trichloroacetic acid in a Waring blendor. This and the subsequent steps in the extraction and precipitation procedures were carried out at 3°C. The homogenate was allowed to stand for 1 hr. and then squeezed in several layers of cheesecloth. The turbid fluid was filtered under pressure through paper pulp. An adjustment in pH from 1.5 to 8.0 was made, and the extract was again subjectedtofiltrat,ion. One volume of 95% ethanol was added to the filtrate (approximately 400 ml.) and permitted to st,and overnight. The next day, the extract was freed of precipitated polysaccharides by slow filtration (filtration under pressure proved inadequate here), and the filtrate was divided into equal volumes for barium and zinc precipitation.
Precipitation To 400 ml. of the extract was added 4.0 ml. of 25% barium acetate or 20 ml. of 25% zinc acetate. Both precipitants are in slight excess. After 30 min., the precipitates were collected by centrifugation, washed once with 2 vol. of 95% ethanol, twice with 1 vol. of peroxide-free ether, and spread thinly to facilitate drying. The material was kept in vacua in the cold for at least 4 days.
Column h?xtionation
of Barium Preparation
The barium precipitate was dissolved in cold 2 N HCl and brought up to a volume of 11 ml. with distilled water. To this was added 2.0 ml. of 1 lli sodium sulfate. The highly insoluble barium sulfate formed was removed by centrifugation and washed once with 2 ml. of distilled water, and the adenine concentration of the combined supernatants was determined. The pH of the supernatant was adjusted to 10.5 with cold 1 N NH,OH. The alkaline solution was added to the previously chilled column and allowed to run through six times at a rate up to 3 ml./min., the effluent being collected in an ice bath. The column was then washed with distilled water to pH 4.5, and eluted with Soln. No. 3 (0.003 Al HCl, pH 2.67), Soln. No. 4 (0.02 M NaCl in 0.01 M HCl, pH 2.21), Soln.No. 4A (0.11 llf NaCl in0.01 M HCl,pH 2.18), Soln.No. 5 (0.2 M NaCl in 0.01 III HCl, pH 2.15), and Soln. No. 6 (0.02 211NaCl in 0.1 M HCl, pH 1.20). Fifteen milliliter fractions were collected and the pH of each was determined.
92
ELElNOIt
GOLDSTEIN
AND
HARRP
TABLE Purine
Content
G. ALBAuM
I
of Bariu.m
and Zinc Precipitates Run I
Barium
Run II Zinc
(a) Tissue, wet wt., g.
225
225
(b) Precipitate, dry wt., mg. (c) Purine, mg. (al) Purine, 100 (c)/(b), To
689
299
3.61 0.524
Column Fractionation
Barium
Zinc
225 677 3.21 0.474
3.15 1.05
225 391 3.72 0.951
of Zinc Preparation
The zinc precipitate was dissolved in 15 ml. of cold 1 N NHIOH, and the adenine concentration of the solution was determined. The material was then added to a chilled column and allowed to run through four times. Two 15-ml. port,ions of 1 N NH,OH were passed through to remove zinc ions. This was followed by a washing with distilled water until a constant pH of 4.5 was attained. The column was eluted as for the barium preparation. RESULTS
AND
DISCUSSION
The quant.ities of barium and zinc precipitate obt,ained in two experimental runs are shown in Table I. It will be noted that in both casesa much greaber amount of precipit,ate was obbained by the barium procedure. In spit,e of the fact, however, that more precipitate was obtained, the amount of 2600-A.-absorbing material was approximat,ely the same. This suggests that the barium precipitation carries down a larger quant,ity of nonabsorbing impurit,ies. TABLE Adsorption
of Barium
II
and Zinc Preparations on and Elution Anion-Exchange Resin Colun1n.s Run I Zinc Barium
Purine’
(a) added to column, mg. (b) Rejected by column,
Run II Barium Zinc
from Dowex
1
Run III Barium Zinc
3.57 1.53
3.11 0
2.35 0.560
3.65 0
3.66 0.460
2.40 0
2.04
3.11
1.79
3.65
3.20
2.40
mg.
(c) Adsorbed
on column,
mg. (d) Adsorbed,
% (e) Eluted
100(c)/(b),
from
column,
57.1 1.08
100 1.71
76.2 1.20
100 1.73
87.4 2.45
100 1.97
mg.
(f) Eluted, 100(e)/(c), % 52.9 3 Column length, cm. * Material absorbing at 2600 A.
55.0 3
67.0 3
47.1 3
80.3 7
82.1 3
PRECIPITATION
BY
BARIUM
AND
93
ZINC I
Effluent, ml.
FIG. 1. Fractionation on a Dowex 1 anion-exchange column of a barium preparation, run II. Solution changes are indicated by arrow8 and effluent pH changes by the broken line.
When the barium and zinc preparations are poured through the column (run I, Table II) and when the columns are of equal size, it is clear t.hat all of the purine is adsorbed by the column in the case of the zinc preparation, but little more than half is taken up in the case of the barium preparation. On elution, however, both preparations behave in t.he same way. It appeared probable that less material was adsorbed in t.he case of the barium procedure because more impurities were present and these “saturated” the column so that not all of the purine could be adsorbed. This could be tested in two ways: by cutting down on the
1.0
0 360
3i5
‘$0
825
600
900
1050
1140
Effluent. ml.
Fractionation on a Dowex 1 anion-exchange column of a zinc preparation, run II. Solution changes are indicated by arrows and effluent pH changes by the broken line. FIG.
2.
94
ELEANOR GOLDSTEIN AND HARRY G. ALBAUM
amount of the barium preparation added to the column while keeping the column the same length, or by increasing the column. This was done in runs II and III. It will be noted that where the column length remains the same but the amount of t,he barium preparation is cut down, the quantity of purine adsorbed increases from 57 to 76 %; and, finally, where the amount of barium preparation is kept the same but the column length is increased, the amount of material adsorbed increases to 87%. The quantities eluted in ea.ch case again remain approximately t,he same. The claim by Owens (5) that a higher ratio of ADP ho ATP is obtained with the zinc procedure does not appear to be valid here. The elution diagrams, Figs. 1 and 2, show that the proportion of ADP to ATP by either procedure is the same. The difference in t,he absolut,e amount of ADP and ATP eluted is related to t,he difference in the quantity of t,he two preparations present on the columns. SUMMARY
1. Adenosine phosphates were extracted from mung bean sprouts (Phaseolus uureus) with trichloroacetic acid, precipitated as both barium and zinc salts for comparison of yield, separated into ADP and ATP fractions by anion-exchange chromatography, and identified on the basis of t,he adenine to labile phosphate ratio. 2. With the zinc precipit’ation procedure, approximately 1.5 times more purine was obtained after column fractionation. This was attributed to t,he incomplebe adsorption of the barium sample on the resin. 3. Both with the barium and zinc precipitation met,hods, 29% of the total purine was identified as ADP and 71% as ATP. REFERENCES Methods of Plant Analysis,” Vol. 4, p. 20. Springer1. ALBAUM, H. G., “Modern Verlag, Berlin, 1955. 2. ALBAUM, H. G., OGUR, M., AND HIRSHFELD, A., Bach. Biochem. 27, 130 (1950). 3. COHN, W. E., AND CURTER, C. E., J. Am. Chem. Sot. 72,4273 (1950). 4. KORNBERG, A., AND PRICER, W. E., JR., J. Biol. Ch.em. 188, 557 (1950). 5. OWENS, R. G., Science 122, 415 (1955).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
67, go-94 (1957)
Comparison of Barium and Zinc Precipitation Procedures in the Isolation of Adenosine Phosphates from Mung Bean Seedlings Eleanor Goldstein and Harry G. Albaum From the Biology
Research
Laboratory,
Brooklyn
College,
Brooklyn,
New
York
Received June 25, 1956
A zinc precipitation procedure devised by Owens (5) for the preparat,ion of nucleotides from the mold Neurospora sitophila is reported to give a yield two to nine times greater than that obtained with the classic barium method. After anion-exchange column fractionation of both preparations, a greater proportion of adenosine diphosphate (ADP) to adenosine triphospha te (ATP) resulted from hhat of the zinc. We decided t,o test the applicability of this zinc precipitation procedure in the isolation of adenosine phosphate from higher plant tissue, and to compare the results with t.hose obtained using the barium method. bh3~H0D
The adenosine phosphates were ext,racted from mung bean seedlings (Phaseolus aureus) using the method of Albaum (2). The mung beans were obtained in the sprouted condition at a Chinese grocery, thus eliminating the necessity of germinating them in the laboratory. The precipitation procedures employed were those described by Albaum for barium (2) and by Owens for zinc (5). The compounds ATP and ADP were separated on a Dower 1 anion-exchange column by the differential elution method of Cohn and Carter (3), and identified by the adenine t,o labile phosphate ratio (1). The total purine determinations were made in the Beckman spectrophotometer using quartz cells with a light path of 1.0 cm. All 26OO-A.-absorbing material was assumed to be adenine.
Preparation
of Column
Enough Dowex 1 anion-exchange resin for several columns was prepared according to Kornberg and Pricer (4). Some 2OCMOO-meshresin was washed repeatedly by centrifugation in bottles with about 4 vol. of 3 N HCl until it no longer displayed absorption at 2600 A. This could not be determined with t.he super90
PRECIPITATION
BY
BARITJM
AND
ZINC
91
natant which contained filterable particles in suspension. To overcome the difficulty, a 3-cm. column was set up and the clear effluent was tested. The bulk resin was then charged by washing with 2 M sodium formate until the supernatant was free of chloride ions, and stored under the solution at 2’C. The bottom of a lOO-ml. buret was packed with a wad of glass wool. Over this was poured resin in 2 M sodium formate in an amount to make a packed column of the desired height. The resin was sedimented and washed by having about 200 ml. of distilled water pass through under pressure.
Extractirm. Approximately 1 lb. of mung bean sprouts of a 40-50-mm. hypocotyl length was homogenized in 200 ml. of 10% trichloroacetic acid in a Waring blendor. This and the subsequent steps in the extraction and precipitation procedures were carried out at 3°C. The homogenate was allowed to stand for 1 hr. and then squeezed in several layers of cheesecloth. The turbid fluid was filtered under pressure through paper pulp. An adjustment in pH from 1.5 to 8.0 was made, and the extract was again subjectedtofiltrat,ion. One volume of 95% ethanol was added to the filtrate (approximately 400 ml.) and permitted to st,and overnight. The next day, the extract was freed of precipitated polysaccharides by slow filtration (filtration under pressure proved inadequate here), and the filtrate was divided into equal volumes for barium and zinc precipitation.
Precipitation To 400 ml. of the extract was added 4.0 ml. of 25% barium acetate or 20 ml. of 25% zinc acetate. Both precipitants are in slight excess. After 30 min., the precipitates were collected by centrifugation, washed once with 2 vol. of 95% ethanol, twice with 1 vol. of peroxide-free ether, and spread thinly to facilitate drying. The material was kept in vacua in the cold for at least 4 days.
Column h?xtionation
of Barium Preparation
The barium precipitate was dissolved in cold 2 N HCl and brought up to a volume of 11 ml. with distilled water. To this was added 2.0 ml. of 1 lli sodium sulfate. The highly insoluble barium sulfate formed was removed by centrifugation and washed once with 2 ml. of distilled water, and the adenine concentration of the combined supernatants was determined. The pH of the supernatant was adjusted to 10.5 with cold 1 N NH,OH. The alkaline solution was added to the previously chilled column and allowed to run through six times at a rate up to 3 ml./min., the effluent being collected in an ice bath. The column was then washed with distilled water to pH 4.5, and eluted with Soln. No. 3 (0.003 Al HCl, pH 2.67), Soln. No. 4 (0.02 M NaCl in 0.01 M HCl, pH 2.21), Soln.No. 4A (0.11 llf NaCl in0.01 M HCl,pH 2.18), Soln.No. 5 (0.2 M NaCl in 0.01 III HCl, pH 2.15), and Soln. No. 6 (0.02 211NaCl in 0.1 M HCl, pH 1.20). Fifteen milliliter fractions were collected and the pH of each was determined.
92
ELElNOIt
GOLDSTEIN
AND
HARRP
TABLE Purine
Content
G. ALBAuM
I
of Bariu.m
and Zinc Precipitates Run I
Barium
Run II Zinc
(a) Tissue, wet wt., g.
225
225
(b) Precipitate, dry wt., mg. (c) Purine, mg. (al) Purine, 100 (c)/(b), To
689
299
3.61 0.524
Column Fractionation
Barium
Zinc
225 677 3.21 0.474
3.15 1.05
225 391 3.72 0.951
of Zinc Preparation
The zinc precipitate was dissolved in 15 ml. of cold 1 N NHIOH, and the adenine concentration of the solution was determined. The material was then added to a chilled column and allowed to run through four times. Two 15-ml. port,ions of 1 N NH,OH were passed through to remove zinc ions. This was followed by a washing with distilled water until a constant pH of 4.5 was attained. The column was eluted as for the barium preparation. RESULTS
AND
DISCUSSION
The quant.ities of barium and zinc precipitate obt,ained in two experimental runs are shown in Table I. It will be noted that in both casesa much greaber amount of precipit,ate was obbained by the barium procedure. In spit,e of the fact, however, that more precipitate was obtained, the amount of 2600-A.-absorbing material was approximat,ely the same. This suggests that the barium precipitation carries down a larger quant,ity of nonabsorbing impurit,ies. TABLE Adsorption
of Barium
II
and Zinc Preparations on and Elution Anion-Exchange Resin Colun1n.s Run I Zinc Barium
Purine’
(a) added to column, mg. (b) Rejected by column,
Run II Barium Zinc
from Dowex
1
Run III Barium Zinc
3.57 1.53
3.11 0
2.35 0.560
3.65 0
3.66 0.460
2.40 0
2.04
3.11
1.79
3.65
3.20
2.40
mg.
(c) Adsorbed
on column,
mg. (d) Adsorbed,
% (e) Eluted
100(c)/(b),
from
column,
57.1 1.08
100 1.71
76.2 1.20
100 1.73
87.4 2.45
100 1.97
mg.
(f) Eluted, 100(e)/(c), % 52.9 3 Column length, cm. * Material absorbing at 2600 A.
55.0 3
67.0 3
47.1 3
80.3 7
82.1 3
PRECIPITATION
BY
BARIUM
AND
93
ZINC I
Effluent, ml.
FIG. 1. Fractionation on a Dowex 1 anion-exchange column of a barium preparation, run II. Solution changes are indicated by arrow8 and effluent pH changes by the broken line.
When the barium and zinc preparations are poured through the column (run I, Table II) and when the columns are of equal size, it is clear t.hat all of the purine is adsorbed by the column in the case of the zinc preparation, but little more than half is taken up in the case of the barium preparation. On elution, however, both preparations behave in t.he same way. It appeared probable that less material was adsorbed in t.he case of the barium procedure because more impurities were present and these “saturated” the column so that not all of the purine could be adsorbed. This could be tested in two ways: by cutting down on the
1.0
0 360
3i5
‘$0
825
600
900
1050
1140
Effluent. ml.
Fractionation on a Dowex 1 anion-exchange column of a zinc preparation, run II. Solution changes are indicated by arrows and effluent pH changes by the broken line. FIG.
2.
94
ELEANOR GOLDSTEIN AND HARRY G. ALBAUM
amount of the barium preparation added to the column while keeping the column the same length, or by increasing the column. This was done in runs II and III. It will be noted that where the column length remains the same but the amount of t,he barium preparation is cut down, the quantity of purine adsorbed increases from 57 to 76 %; and, finally, where the amount of barium preparation is kept the same but the column length is increased, the amount of material adsorbed increases to 87%. The quantities eluted in ea.ch case again remain approximately t,he same. The claim by Owens (5) that a higher ratio of ADP ho ATP is obtained with the zinc procedure does not appear to be valid here. The elution diagrams, Figs. 1 and 2, show that the proportion of ADP to ATP by either procedure is the same. The difference in t,he absolut,e amount of ADP and ATP eluted is related to t,he difference in the quantity of t,he two preparations present on the columns. SUMMARY
1. Adenosine phosphates were extracted from mung bean sprouts (Phaseolus uureus) with trichloroacetic acid, precipitated as both barium and zinc salts for comparison of yield, separated into ADP and ATP fractions by anion-exchange chromatography, and identified on the basis of t,he adenine to labile phosphate ratio. 2. With the zinc precipit’ation procedure, approximately 1.5 times more purine was obtained after column fractionation. This was attributed to t,he incomplebe adsorption of the barium sample on the resin. 3. Both with the barium and zinc precipitation met,hods, 29% of the total purine was identified as ADP and 71% as ATP. REFERENCES Methods of Plant Analysis,” Vol. 4, p. 20. Springer1. ALBAUM, H. G., “Modern Verlag, Berlin, 1955. 2. ALBAUM, H. G., OGUR, M., AND HIRSHFELD, A., Bach. Biochem. 27, 130 (1950). 3. COHN, W. E., AND CURTER, C. E., J. Am. Chem. Sot. 72,4273 (1950). 4. KORNBERG, A., AND PRICER, W. E., JR., J. Biol. Ch.em. 188, 557 (1950). 5. OWENS, R. G., Science 122, 415 (1955).