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Simultaneous multiple-column chromatography: Its application to the separation of the adenine nucleotides of human erythrocytes
ASALYTICAL
4, 39-45
BIOCHEMISTRY
Simultaneous
(1962)
Multiple-Column
Its Application to Adenine Nucleotides CHESTER
DE
Chromatography:
the Separation of the of Human Erythrocytesl
LUCA, JOSEPH H. STEVENSON, EUGENE KAPLAN
JR.,
AND From The
the Departments Johns Hopkins
of Pediatrics, University School Received
Sinai Hospital of Baltimore, and of Medicine, Balfimore, Maryland
October
18, 1961
INTRODUCTION
In recent years the technique of column chromatography has become increasingly popular as an analytical tool for the experimentalist. When used for this purpose, it is the custom, usually, to process a single column many times employing a given set of conditions. By analogy to general laboratory practice, it would be desirable to employ a minimum of two columns simultaneously, one as a control for the other; a multiplicity of columns developed simultaneously would be more ideal. This is possible to a limited extent with column chromatography. Directions for building an apparatus for developing at least six columns at once have been reported by Vestergaard (1). Described here is a method for easily handling three columns concurrently employing only readily available commercial equipment. To illustrate the feasibility and facility of using such an apparatus, results are presented from an application to separating the adenine nucleotides and quantitating levels of ATP? in human erythrocytes. MATERIALS
AND
Chromatography
METHODS
Apparatus
The chromatographic apparatus, shown in Fig. 1, is a model V 15’ fraction collector manufactured by Gilson Medical Electronic Co. This model has a three-section, square collecting stage which executes boustro‘This work Service. ‘Abbreviations tri-phosphate,
was
supported
used: respectively
AMP, ; rbc,
by
Grant
No.
28-5271
ADP, and ATP; red blood cell(s). 39
of the
U.
S. Public
adenosined’-mono-,
Health di-,
and
40
DE
LUCA,
FIG. 1. The Gilson fraction simultaneous multiple-column
STEVENSON,
AND
collector equipped chromatography.
KAPLAN
as
described
in
the
text
for
phedon movement. During the chromatography process a column is placed over the first tube position of each section of the stage. The fraction collector may be equipped with any of the various devices available for activating the mechanism which changes the position of the collecting tubes. When either t,he drop-counting or fixed-volume type of tripping device is used, only one column need be aligned with it. The other two columns merely communicate with their respective collecting tubes through appropriately placed thistle tubes. A common elution system is positioned above t.he columns. This communicates with all columns below through a radially symmetrical manifold. In the present study a nonlinear gradient elution is employed (2).
MULTIPLE-COLCMN
CHROMATOGRAPHY
41
This is achieved by connecting, in series, t,wo separatory funnels as shown in the figure. The uppermost funnel serves as a reservoir and the lower funnel acts as a mixing chamber. The latter is equipped with a Teflon-covered stirring bar and a magnetic stirrer of the type manufactured by Tri-R Instrument Co. The eluting mixture, adapted from the work of Hurlbert (2), was modified to give a one-stage, continuous gradient system capable of an uninterrupted elution of the t,hree common nucleotides of adeninc. The chromat,ographic medium used is Dowex l-X2 ion-exchange resin, 2OWlOO mesh, purchased from J. T. Baker Chemical Co. The resin, obtained in the chloride form, is first cycled through NaOH and HCI, then washed with acetone as recommended by Cohn (3). Finally, it is converted to the formate form and washed exhaustively with distilled water until no ultraviolet light, absorbing material is eluted. Preparation
of Erythrocyte
Extract
One to three milliliters of heparinized, fresh blood is centrifuged at 3000 rpm for 10 min in a refrigerated Lourdes centrifuge, using the high-speed angle rotor SRA-V. The plasma and buffy coat are removed by suction. The red blood cells are then washed twice by resuspension in about two volumes of cold 0.9% NaCl and centrifuged as before. The cells are transferred to a graduated vessel with repeated washings of isotonic saline, and the suspension is finally adjusted to an exact volume equal to three to four times that originally occupied by the cells alone. A sample is removed at this point with a capillary tube, and a microltcmatocrit is determined using the Adams Micro-Hematocrit Centrifuge (Clay-Adams, Inc., New York). The remaining cell suspension is hemolyzed with cold distilled water during quantit,ative transfer to a 40-ml Lusteroid centrifuge tube and adjustment of the final volume to at least five times that of the saline suspension. Deproteinization is effect.ed by adding 1.1 ml of cold 3 M HClO, for every 10 ml of the hemolyzate and blending for 1 min with a Lourdes Multi-Mixer at a rheostat setting of 50. The blade assembly is then washed down with 0.3 nl HClO, from a polyethylene wash bottle. The washings are collected in the homogenate, and the entire coagulum is centrifuged at lO,OQO-12,000 rpm for 10 min. The supernatant fluid is decanted as quantitatively as possible and neutralized immediately with cold 2M KOH. The resulting precipitate of potassium perchlorate is removed by a final centrifugation. The neutral ext.ract is stored at -15°C. During the extraction procedure, all operations are carried out as rapidly as possible, and all materials are kept on ice.
42
DE
LUCA,
STEVENSON,
Chromatographic
AND
KAPLAN
Procedure3
Neutralized extracts of red blood cells estimated to contain 0.5-1.0 pmoles of ATP are placed on Dowex columns, measuring 1 X 13 cm, with the aid of slight positive pressure. The extracts are washed into the resin with three 3 ml water rinses, and the columns are closed at their bottoms. Next 1 M formic acid is layered over the resin bed t.o a height of 7 cm. For the adapted gradient elution system, 1 M formic acid is adjusted in the mixing chamber to a precalibrated 400-ml mark after the flow lines to the manifold and columns are filled and all air is excluded. One liter of 0.5 M ammonium formate in 4 M formic acid is then placed in the reservoir, which is positioned above the mixing chamber. The connections from the manifold to each column are now secured. To begin the elution all stopcocks are opened sequentially from the uppermost on the reservoir to those at t,he bottoms of the columns. A variety of steps may be designed for this procedure so that no air is trapped in the system. A flow rate of 1 ml/min from each column is used routinely. Eluate volumes to be collected may be set at 1.5 or 2.0 ml per tube. Pressure systems may help to maintain a constant flow rate and to insure the collection of constant volumes when the tube changer operates on a time basis. Calibrated tubes may be placed at regular intervals on the collecting stage to serve to spot check the actual volumes obtained. Using a volumetric device rout,inely, we have found a gravity feed system to be adequate. The slight change in rate of flow observed is readily adjusted by the use of Kimble stopcocks equipped with Teflon plugs with metering valves. The total eluting time is 34 hr. The eluates are read in the Beckman Model DU spectrophotometer at 260 mp against water. For general chromatography the use of a multiple column system provides a ready opportunity for obtaining a true blank when the eluting agent contributes to the values obtained by the analytical method employed. We find it unnecessary to include a blank column each time as its contribution to the absorbance at 260 rnp equals the baseline values seen in the nucleotide profile. Correction for this background due to the formate ion is made only in the area where the ATP levels are determined. These levels are obtained by a summation of values of individual tubes comprising the nucleotide peak. For this quantitization, a molar absorbancy of 14.2 X 1O-3 for ATP was used (5). ‘For greater detail on the technique of chromatography (2), (3), and (4) are recommended to the reader.
of nucleotides,
references
MULTIPLE-COLUMN
43
CHROMATOGRAPHY
RESULTS E’igure 2 shows a typical with the system described
OJ i lb : b
:
elution above.
IO : A
profile obtained during a single run It can be seen that the one-stage
: 20 : i FRACTION
: k
: b
: 9, : ,;,
NUMBER
FIG. 2. Typical elution patterns observed during the simultaneous of three different extracts of human erythrocytes. This separation 1 x 13 cm columns of Dowex I-12 ion exchange resin using ammonium formate gradient as described in the text. The arrow marks the initiation of the clution process. Two-milliliter fractions n rate of 1 ml/min.
chromatography is obtained on a formic acidat the abscissa are collected at
eluting system adopted is capable of distinct separation of the majol nucleotides cont,ained by the human red blood cell. Also illustrated here
44
DE
LUCA,
STEVENSON,
AND
KAPLAK
is the typical perfect correspondence of peak position that is repeatedly possible only with simultaneous multiple column development.. Experiments with known mixtures of nucleotides average 95% recovery. Table 1 gives a summary of mean values for ATP obtained in this study on red cells of normal human adults and newborn infants up to 36 hr of life. St.atistical analysis of the data shows a significantly higher ATP content in the erythrocytes of the newborn infant. The p value for the difference in the ATP levels of t.hese two groups is ca. 0.001 (6). TABLE 1 COMPARATIVE LEVELS OF ATP IN HUMAN ERYTHROCYTES ATP
(pmole/lOO
ml rbc)
69.2 f 14.3 86.4 f 10.4
Adult (14)a Newborn (14)
a The figures in parentheses denote the number of samples.
While this report was in preparation, the work of Stave and Cara on whole blood appeared (7). Their results indicate a difference exists in the ATP levels of whole blood of infants and adults. Many others have published ATP values for adults only; for examples see references (8)) (9), and (10). DISCUSSION
Sequential chromatography of single columns, although remarkably reproducible when performed with care, leaves something to be desired as a strict analytical procedure. The packing of columns using a standard method and the same batch of prepared resin can be expected to yield relatively uniform columns and, therefore, need not be a fact.or limiting replicate development. More difficult to at,tain is the identity of elution which is t,he factor most important for precise reproducibility. The rigorous comparability needed for certain analyses can be claimed only for a set, of columns processed as a unit,. That the manner and rate of change of composit,ion of the eluant be the same for every column can be insured only with a single eluting system common to all columns. Described here is an approach to a minimum analytical unit of the type described above. The biological system for which it was adopted is relatively simple in that the nucleotides det,ectable, with t,he methods and amounts of blood employed, are limited. The technique of multiple chromatography is applied here not only to conserve time, but also to insure exact replication of analysis. The use of this method would be most important where the qualit,ative examination of peak position is of primary concern. Such would be the case, for example, wit.h the current
YULTIPLE-COLUMS
45
CHROMATOGRAPHY
work on the multiple molecular chromatography (11).
forms
of enzymes
separable
by column
SUMMARY
A readily available apparatus is described for the simult.aneoun development of three chromatographic columns. Also described is a single-stage, nonlinear gradient elution system capable of the uninterrupted separation of the adenine nucleot’ides of human erythrocytes. The practicality of these methods is exemplified in their application to the determination of the ATP content of red cells of normal adults and full-term newborn infants. The results show a significantly higher level of t,he triphosphate in the cells of the infant as compared to those of t,he adult. REFERENCES 1. VESTERGAARD, P., J. Chlomatog. 3, 554, 560 (1960). 2. HURLBERT, R. B., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. III, p. 785. Academic Press, New York, 1957. 3. COHN, W. E., see reference (2)) p. 724. 4. HURLBERT, R. B., SCHMITZ, H., BRUMM, A. F., .~ND POTTER, V. R., J. Biol. Chem. 209, 23 (1954). 5. Pabst Laboratories Circular OR-lo, p. 2 (1956). 6. FISHER, R. A., AND YATES, F., “Statistical Tables for Biological, Agricultural and Medical Research,” 4th ed., p. 40. Hafner Publishing Co., New York, 1953. 7. STAVE, U., AND CAM, J., Viol. Neonatorum 3, 160 (1961). 8. BARTLETT, G. R., J. Viol. Chem. 234, 449 (1959). 9. BISHOP, C., RASKINE, D. M., AND TALBOT, J. H., J. Biol. Chem. 234, 1233 (1959). 10. OVERGARD-HANSEN, K., AND J@RGENSEX, S., &and. J. Clin. Lab. Znvestig. 12, 10 (1960). 11. KAJI, A., TK~~s~GR, K. -4., AND COLOR-ICK, S. P., irs “Multiple Molecular Forms of Enzymes” (F. Wroblewski, ed.), An,l. N. E’. Acntl. Sci.. 94, Art. 3, 798 (1961)
4, 39-45
BIOCHEMISTRY
Simultaneous
(1962)
Multiple-Column
Its Application to Adenine Nucleotides CHESTER
DE
Chromatography:
the Separation of the of Human Erythrocytesl
LUCA, JOSEPH H. STEVENSON, EUGENE KAPLAN
JR.,
AND From The
the Departments Johns Hopkins
of Pediatrics, University School Received
Sinai Hospital of Baltimore, and of Medicine, Balfimore, Maryland
October
18, 1961
INTRODUCTION
In recent years the technique of column chromatography has become increasingly popular as an analytical tool for the experimentalist. When used for this purpose, it is the custom, usually, to process a single column many times employing a given set of conditions. By analogy to general laboratory practice, it would be desirable to employ a minimum of two columns simultaneously, one as a control for the other; a multiplicity of columns developed simultaneously would be more ideal. This is possible to a limited extent with column chromatography. Directions for building an apparatus for developing at least six columns at once have been reported by Vestergaard (1). Described here is a method for easily handling three columns concurrently employing only readily available commercial equipment. To illustrate the feasibility and facility of using such an apparatus, results are presented from an application to separating the adenine nucleotides and quantitating levels of ATP? in human erythrocytes. MATERIALS
AND
Chromatography
METHODS
Apparatus
The chromatographic apparatus, shown in Fig. 1, is a model V 15’ fraction collector manufactured by Gilson Medical Electronic Co. This model has a three-section, square collecting stage which executes boustro‘This work Service. ‘Abbreviations tri-phosphate,
was
supported
used: respectively
AMP, ; rbc,
by
Grant
No.
28-5271
ADP, and ATP; red blood cell(s). 39
of the
U.
S. Public
adenosined’-mono-,
Health di-,
and
40
DE
LUCA,
FIG. 1. The Gilson fraction simultaneous multiple-column
STEVENSON,
AND
collector equipped chromatography.
KAPLAN
as
described
in
the
text
for
phedon movement. During the chromatography process a column is placed over the first tube position of each section of the stage. The fraction collector may be equipped with any of the various devices available for activating the mechanism which changes the position of the collecting tubes. When either t,he drop-counting or fixed-volume type of tripping device is used, only one column need be aligned with it. The other two columns merely communicate with their respective collecting tubes through appropriately placed thistle tubes. A common elution system is positioned above t.he columns. This communicates with all columns below through a radially symmetrical manifold. In the present study a nonlinear gradient elution is employed (2).
MULTIPLE-COLCMN
CHROMATOGRAPHY
41
This is achieved by connecting, in series, t,wo separatory funnels as shown in the figure. The uppermost funnel serves as a reservoir and the lower funnel acts as a mixing chamber. The latter is equipped with a Teflon-covered stirring bar and a magnetic stirrer of the type manufactured by Tri-R Instrument Co. The eluting mixture, adapted from the work of Hurlbert (2), was modified to give a one-stage, continuous gradient system capable of an uninterrupted elution of the t,hree common nucleotides of adeninc. The chromat,ographic medium used is Dowex l-X2 ion-exchange resin, 2OWlOO mesh, purchased from J. T. Baker Chemical Co. The resin, obtained in the chloride form, is first cycled through NaOH and HCI, then washed with acetone as recommended by Cohn (3). Finally, it is converted to the formate form and washed exhaustively with distilled water until no ultraviolet light, absorbing material is eluted. Preparation
of Erythrocyte
Extract
One to three milliliters of heparinized, fresh blood is centrifuged at 3000 rpm for 10 min in a refrigerated Lourdes centrifuge, using the high-speed angle rotor SRA-V. The plasma and buffy coat are removed by suction. The red blood cells are then washed twice by resuspension in about two volumes of cold 0.9% NaCl and centrifuged as before. The cells are transferred to a graduated vessel with repeated washings of isotonic saline, and the suspension is finally adjusted to an exact volume equal to three to four times that originally occupied by the cells alone. A sample is removed at this point with a capillary tube, and a microltcmatocrit is determined using the Adams Micro-Hematocrit Centrifuge (Clay-Adams, Inc., New York). The remaining cell suspension is hemolyzed with cold distilled water during quantit,ative transfer to a 40-ml Lusteroid centrifuge tube and adjustment of the final volume to at least five times that of the saline suspension. Deproteinization is effect.ed by adding 1.1 ml of cold 3 M HClO, for every 10 ml of the hemolyzate and blending for 1 min with a Lourdes Multi-Mixer at a rheostat setting of 50. The blade assembly is then washed down with 0.3 nl HClO, from a polyethylene wash bottle. The washings are collected in the homogenate, and the entire coagulum is centrifuged at lO,OQO-12,000 rpm for 10 min. The supernatant fluid is decanted as quantitatively as possible and neutralized immediately with cold 2M KOH. The resulting precipitate of potassium perchlorate is removed by a final centrifugation. The neutral ext.ract is stored at -15°C. During the extraction procedure, all operations are carried out as rapidly as possible, and all materials are kept on ice.
42
DE
LUCA,
STEVENSON,
Chromatographic
AND
KAPLAN
Procedure3
Neutralized extracts of red blood cells estimated to contain 0.5-1.0 pmoles of ATP are placed on Dowex columns, measuring 1 X 13 cm, with the aid of slight positive pressure. The extracts are washed into the resin with three 3 ml water rinses, and the columns are closed at their bottoms. Next 1 M formic acid is layered over the resin bed t.o a height of 7 cm. For the adapted gradient elution system, 1 M formic acid is adjusted in the mixing chamber to a precalibrated 400-ml mark after the flow lines to the manifold and columns are filled and all air is excluded. One liter of 0.5 M ammonium formate in 4 M formic acid is then placed in the reservoir, which is positioned above the mixing chamber. The connections from the manifold to each column are now secured. To begin the elution all stopcocks are opened sequentially from the uppermost on the reservoir to those at t,he bottoms of the columns. A variety of steps may be designed for this procedure so that no air is trapped in the system. A flow rate of 1 ml/min from each column is used routinely. Eluate volumes to be collected may be set at 1.5 or 2.0 ml per tube. Pressure systems may help to maintain a constant flow rate and to insure the collection of constant volumes when the tube changer operates on a time basis. Calibrated tubes may be placed at regular intervals on the collecting stage to serve to spot check the actual volumes obtained. Using a volumetric device rout,inely, we have found a gravity feed system to be adequate. The slight change in rate of flow observed is readily adjusted by the use of Kimble stopcocks equipped with Teflon plugs with metering valves. The total eluting time is 34 hr. The eluates are read in the Beckman Model DU spectrophotometer at 260 mp against water. For general chromatography the use of a multiple column system provides a ready opportunity for obtaining a true blank when the eluting agent contributes to the values obtained by the analytical method employed. We find it unnecessary to include a blank column each time as its contribution to the absorbance at 260 rnp equals the baseline values seen in the nucleotide profile. Correction for this background due to the formate ion is made only in the area where the ATP levels are determined. These levels are obtained by a summation of values of individual tubes comprising the nucleotide peak. For this quantitization, a molar absorbancy of 14.2 X 1O-3 for ATP was used (5). ‘For greater detail on the technique of chromatography (2), (3), and (4) are recommended to the reader.
of nucleotides,
references
MULTIPLE-COLUMN
43
CHROMATOGRAPHY
RESULTS E’igure 2 shows a typical with the system described
OJ i lb : b
:
elution above.
IO : A
profile obtained during a single run It can be seen that the one-stage
: 20 : i FRACTION
: k
: b
: 9, : ,;,
NUMBER
FIG. 2. Typical elution patterns observed during the simultaneous of three different extracts of human erythrocytes. This separation 1 x 13 cm columns of Dowex I-12 ion exchange resin using ammonium formate gradient as described in the text. The arrow marks the initiation of the clution process. Two-milliliter fractions n rate of 1 ml/min.
chromatography is obtained on a formic acidat the abscissa are collected at
eluting system adopted is capable of distinct separation of the majol nucleotides cont,ained by the human red blood cell. Also illustrated here
44
DE
LUCA,
STEVENSON,
AND
KAPLAK
is the typical perfect correspondence of peak position that is repeatedly possible only with simultaneous multiple column development.. Experiments with known mixtures of nucleotides average 95% recovery. Table 1 gives a summary of mean values for ATP obtained in this study on red cells of normal human adults and newborn infants up to 36 hr of life. St.atistical analysis of the data shows a significantly higher ATP content in the erythrocytes of the newborn infant. The p value for the difference in the ATP levels of t.hese two groups is ca. 0.001 (6). TABLE 1 COMPARATIVE LEVELS OF ATP IN HUMAN ERYTHROCYTES ATP
(pmole/lOO
ml rbc)
69.2 f 14.3 86.4 f 10.4
Adult (14)a Newborn (14)
a The figures in parentheses denote the number of samples.
While this report was in preparation, the work of Stave and Cara on whole blood appeared (7). Their results indicate a difference exists in the ATP levels of whole blood of infants and adults. Many others have published ATP values for adults only; for examples see references (8)) (9), and (10). DISCUSSION
Sequential chromatography of single columns, although remarkably reproducible when performed with care, leaves something to be desired as a strict analytical procedure. The packing of columns using a standard method and the same batch of prepared resin can be expected to yield relatively uniform columns and, therefore, need not be a fact.or limiting replicate development. More difficult to at,tain is the identity of elution which is t,he factor most important for precise reproducibility. The rigorous comparability needed for certain analyses can be claimed only for a set, of columns processed as a unit,. That the manner and rate of change of composit,ion of the eluant be the same for every column can be insured only with a single eluting system common to all columns. Described here is an approach to a minimum analytical unit of the type described above. The biological system for which it was adopted is relatively simple in that the nucleotides det,ectable, with t,he methods and amounts of blood employed, are limited. The technique of multiple chromatography is applied here not only to conserve time, but also to insure exact replication of analysis. The use of this method would be most important where the qualit,ative examination of peak position is of primary concern. Such would be the case, for example, wit.h the current
YULTIPLE-COLUMS
45
CHROMATOGRAPHY
work on the multiple molecular chromatography (11).
forms
of enzymes
separable
by column
SUMMARY
A readily available apparatus is described for the simult.aneoun development of three chromatographic columns. Also described is a single-stage, nonlinear gradient elution system capable of the uninterrupted separation of the adenine nucleot’ides of human erythrocytes. The practicality of these methods is exemplified in their application to the determination of the ATP content of red cells of normal adults and full-term newborn infants. The results show a significantly higher level of t,he triphosphate in the cells of the infant as compared to those of t,he adult. REFERENCES 1. VESTERGAARD, P., J. Chlomatog. 3, 554, 560 (1960). 2. HURLBERT, R. B., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. III, p. 785. Academic Press, New York, 1957. 3. COHN, W. E., see reference (2)) p. 724. 4. HURLBERT, R. B., SCHMITZ, H., BRUMM, A. F., .~ND POTTER, V. R., J. Biol. Chem. 209, 23 (1954). 5. Pabst Laboratories Circular OR-lo, p. 2 (1956). 6. FISHER, R. A., AND YATES, F., “Statistical Tables for Biological, Agricultural and Medical Research,” 4th ed., p. 40. Hafner Publishing Co., New York, 1953. 7. STAVE, U., AND CAM, J., Viol. Neonatorum 3, 160 (1961). 8. BARTLETT, G. R., J. Viol. Chem. 234, 449 (1959). 9. BISHOP, C., RASKINE, D. M., AND TALBOT, J. H., J. Biol. Chem. 234, 1233 (1959). 10. OVERGARD-HANSEN, K., AND J@RGENSEX, S., &and. J. Clin. Lab. Znvestig. 12, 10 (1960). 11. KAJI, A., TK~~s~GR, K. -4., AND COLOR-ICK, S. P., irs “Multiple Molecular Forms of Enzymes” (F. Wroblewski, ed.), An,l. N. E’. Acntl. Sci.. 94, Art. 3, 798 (1961)