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[63] Ion-exchange chromatography of citric acid cycle components and related compounds
[63]
ION-EXCHANGE CHROMATOGRAPHY
425
and then ground to ensure against metabolic alteration of acid constituents. Sulfuric acid is used in preference to other acids such as trichloroacetic or perchloric acid where it is necessary to terminate a biochemical reaction. Most of the commonly used protein precipitating reagents appear as peaks on the chromatogram and will swamp a particular area of analysis. The pH of the sample is adjusted to below 2 to keep the acids in a nonionized form. Due to the increased sensitivity of the method, the requirement for preliminary extraction and concentration is unnecessary in most instances. Hence better yield, fewer artifacts, and smaller losses from volatilization or polymerization of certain substances are obtained. Sources of Error. Limitations in reproducibility are the result of a number of factors, of which the calculation of the peak area may be of considerable magnitude. The height-width method for determining area is the most convenient procedure when utilizing a nonlinear absorbance photometer. Errors may also arise from lack of photometric stability or potentiometer reproducibility and deviations in the pump rates of the two or three pumps used. When biological specimens are to be evaluated, it is of utmost importance that the investigator be aware of potential losses during the sample preparation.
[63] I o n - E x c h a n g e C h r o m a t o g r a p h y of C i t r i c A c i d Cycle Components and Related Compounds B y R. W. VON KORFF
Ion-exchange chromatography of citric acid cycle components and related compounds was first applied to biochemical studies by Busch et al., 1 who used Dowex 1 in the formate cycle and a formic acid gradient to elute organic acids. The variants ~,a of this procedure include use of ammonium formate-formic acid 4 and acetate columns with an acetic acid gradient? ,6 These procedures are used widely to separate appreciable amounts of H. Busch, R. B. Hurlbert, and V. R. Potter, J. Biol. Chem. 196, 717 (1952). A. W. Norman and H. F. De Luca, Bioehem. J. 91, 124 (1964). ' G. R. Bartlett, J. Biol. Chem. 234, 459 (1959). ' J. L. Gamble, J. Biol. Chem. 240, 2668 (1965). BIt. Busch, Cancer Res. 13, 789 (1953). • See Vol. III [70].
426
SEPARATION AND ASSAY METtIODS
[53]
organic acids (5-200 micromoles).1 The removal of large amounts of formic or acetic acids, or their ammonium salts, is time-consuming, and it is not well adapted for either the separation of nanomole quantities of citric acid cycle components or the simultaneous monitoring of radioactivity where isotopic acids are employed. Bartlett et al./used Dowex 1 (CI-) with HC1 as the eluting agent to chromatograph carbohydrate intermediates and related cofactors of erythrocytes. They observed that HC1 facilitates recovery of compounds by evaporation, rechromatography after neutralization and dilution, and sampling for 14C radioactivity assay on "infinitely thin" planchets. The use of Dowex 1 (C1-) columns with an HC1 gradient offers several advantages: 1. Washed analytical grade resin in the (Cl:) cycle is readily available. 2. The organic acids, in general, are most stable in acid solution, and the eluates may be stored frozen with little decomposition. 3. The column eluate may be passed through a scintillation flow cell for simultaneous monitoring of radioactivity. 4. Fractions containing volatile acids, e.g., acetate, lactate, or pyruvate, may be neutralized to pH 8--9, without introduction of excessive amounts of salt, and concentrated to dryness. However, quantities of organic acids greatly in excess of 5 micromoles of individual components may alter the pH and lead to peak distortion and displacement or both. For example, succinate and malate, separated readily in amounts up to 5 mieromoles, merge as the amounts increase; the succinate region increases in size, while malate decreases as the acids apparently interact. Complete separation of acids eluting in closely adjacent regions is also a function of the relative amounts of the two compounds. While two acids may elute in separate peaks in a ratio of 5A to 1B (or 1A to 1B) resolution may be poor at a ratio of 1A to 5B. The procedure may be modified for particular situations but is most generally useful in separating trace amounts of radioactive materials. Chromatographic Procedure. Dowex l-X8 (C1-), 200--400 mesh (analytical grade) 8 is washed with water to remove impurities and acid. After washings reach pH 4.5, the resin suspension in water is neutralized to pH 6.8, and stored as a slurry. After about one month the resin should be rewashed and neutralized. G. R. Bartlett, E. Savage, L. Hughes, and A. R. Marlow, J. Applied Physiol. 6, 51 (1953). Dowex I-X10 does not appear to offer any advantages over the X8 resin.
[63]
ION-EXCHANGE CHROMATOGRAPHY
427
A colunm (1 X 17 cm) is formed by pouring water-suspended resin into a tube (1 X 30 cm) having a sintered disk, a 12/2 ball of a (S~) joint at the base and an 18/9 socket at the top. Flow can be controlled by a Teflon needle valve (Manostat Corporation, New York City) having a 12/2 socket (Sj) at one end and a drip tip at the other. About 2 mm of water should remain above the resin. Add 1-2 ml of organic acid sample, 9 pH 6.8-7.2, to the resin bed using a long-tip pipette, and drain it into the resin. Wash the sample container with about 1 ml of water, drain it into the resin. Close the stopcock until the sample is eluted. First, neutralize perchloric acid filtrates 1° to pH 6.8 with 4 N KOH, and keep at 0 ° until the KC10~ has settled; then add the fluid to the column and wash the KC1Q once with cold water. For elution of the acids, fill the column with water and attach a 250ml Erlenmeyer flask with a 35/20 socket (S~) and a 12 cm side arm ending in a right-angle with an 18/9 ball (Sj) ; fill the flask with water, add a stirring bar, and mount above a magnetic stirrer. At the upper end of the flask attach a 35/20 ball (Sj) through which a 5 mm tube extends to within 1 inch of the bottom. If radioactivity is monitored with a scintillation flow cell, connect this tube to a micropump (Buchler Instruments, Inc., Fort Lee, New Jersey) to supply a constant rate of flow. Remove the stopcock from the lower end of the column, attach a 12/2 socket (S~), in turn attached to Teflon tubing passing to a scintillation flow cell assembly. To begin elution, connect the pump to a source of 0.05 N HC1. After 50 fractions of 2 ml have been collected, connect the pump to a source of 0.10 N HC1. The approximate positions of a number of organic and amino acids chromatographed with this system are listed in the table. Oxaloacetic acid cannot be recovered from Dowex 1 as it undergoes decomposition on the basic nitrogenous resin. If the sample is nonradioactive, or if fractions are to be counted individually, gravity flow from a separatory funnel extending to within 1 inch of the bottom of the mixing flask may be used in place of the micropump assembly. Fractions may be collected directly into tubes using the elution schedule described above. Where certain acids elute together (overlap), chemical reduction permits a rapid second separation using the same chromatographic system. For example, citrate and pyruvate overlap, while suecinate and acetoacetate, and fumarate and a-ketoglutarate elute as single peaks. In Samples at high salt concentration should be diluted to reduce the salts to less than 0.15M. At higher initial salt concentrations, acids may be partially eluted in abnormal postions in the ehromatogram. loWhen 14C0, is present, the perchloric acid solution should first be treated with pieces of dry ice to displace the radioactive C01.
428
[63]
SEPARATION AND ASSAY METHODS
PROFILE OF ORGANICACID ELUTIONFROMDOWEX 1-(XS) (C1-) COLUMNS USING AN H C I GRADIENT
Acid Alanine and other monoamino monocarboxylic acids COs, aspartate, glutamate, B-hydroxybutyrate, acetate Lactate Butyrate" ~-Methyl-fl-hydroxyglutarate Suceinate, acetoacetate a-Hydroxyglutarate Glyoxylate Malate Isocitrate Citrate Pyruvate, malonate Fumarate, a-ketoglutarate
Approximate volume at peak (ml) 8 46 52 56 62 66 72 80 86 108 130 150 204
a Unsubstituted acids of greater chain length than C-4 do not elute from the Dowex 1 columns. These may be separated on silicic acid columns. each case the combined fraction containing the compounds to be separated is neutralized to p H 6-7 and treated with 1 to 2 mg of sodium borohydride to reduce the keto acids? 1 After acidification to destroy excess borohydride and neutralization to p H 7, the sample is placed on a Dowex 1 column and rerun as described previously. The reduced samples containing citrate and lactate, succinate and fl-hydroxybutyrate, and fumarate and a-hydroxyglutarate are separated easily by chromatography on Dowex 1 with an HCI gradient. To date, a separation of acetate, fl-hydroxybutyrate, glutamate, and aspartate has not been effected with the chloride system. Lactate follows these compounds and m a y overlap partially. I t m a y be separated completely, however, by using 0.01 N HC1 to form the initial gradient. In this case, subsequent peaks, caused by a gradual acidity gradient, m a y be broader and appear later. After elution of lactate, change the HC1 from 0.01 N to 0.05 N. The Bessman TM Gradient Elution Device (National Instruments Laboratories, Inc., Rockville, Maryland) is useful for further experimentation with different gradients.
Separation o] Acetate plus fl-Hydroxybutyrate and Glutamate plus 11In the case of acetoa~etate, this should be done as soon as possible after chromatography to avoid possible losses. uS. P. Bessman, Anal. Biochem. 18, 256 (1967).
[63]
ION-EXCHANGE CHROMATOGRAPHY
429
Aspartate. The peak containing these acids is neutralized to pH 7 and passed through a 1 X 3 cm column of Dowex 50-X8, 200-400 mesh (analytical grade). Acetate and fl-hydroxybutyrate are not held on the column. Wash the column with 5-6 times the volume of sample, usually not less than 40 ml. The eluate is then neutralized; if radioactivity is to be determined, aliquots are taken for counting to determine the total activity present. Concentrate the remainder of the sample to dryness on a rotating evaporator. The acetate and fl-hydroxybutyrate may be separated on a silicic acid column using a modification of the procedure of Bulen et al.13-15
Wash silicic acid (Mallinckrodt Chemical Works), 100 mesh (labeled "suitable for chromatographic analyses by the method of Ramsey and Peterson"), to remove fines, as described by Bulen, et aI. Dry the washed material thoroughly at 105 ° and prepare the stationary phase as described by Kinnory et al. TM Mix silicic acid, 8.8 g, in a mortar with 5.4 ml of 0.05 N H~SQ until the powder is finely divided. Then wash chloroform in a separatory funnel with a small amount of 0.05 N H~SO, and pass the chloroform layer through dry filter paper to remove water droplets. Bring the silicic acid to a slurry with chloroform in a beaker and pour it into a column (1.4 X 35 cm) that has a small glass-wool plug, a stopcock at the lower end, and a 24/40 joint at the top. Acidify the neutralized sample, not over 0.6 ml in volume, with 0.05 ml of 3 N H~S04. Very small amounts of radioactive acetate and fl-hydroxybutyrate may be determined by adding 5-10 micromoles of nonradioactive carrier acids to aid in locating the peaks. The sample is mixed well with 1.0 g silicic acid and added as a chloroform slurry to the top of the silieic acid column, drained previously until only 1-2 mm of chloroform remains above the silicic acid. After the sample has settled, add to the column a 5-cm layer of chloroform and connect to a reservoir containing 250 ml of acid-washed chloroform and a magnetic mixing bar. A separatory funnel containing 1-butanol-chloroform, 40:60 (v/v), to which 1 ml of 0.05N H2S04 has been added per 100 ml of mixed solvent, is connected to the reservoir, and in turn mounted on a fraction collector and above a magnetic mixer. Fractions (3 ml) are collected into tubes containing 1.0 ml of a neutralized methyl red indicator solution (a 1:20 dilution with water of a 0.04% methyl red solution in ethanol). The diluted methyl red should be neutralized to an orange color and tested for the highest sensitivity to traces of acidity. Acids eluting from the column are detected ~*W. A. Bulen, J. E. Varner, and R. C. Burrell, Anal. Chem. 24, 187 (1952). ~'See Vol. III [64]. ~R. W. Von Korff, J. Biol. Chem. 240, 1351 (1965). ~aD. S. Kinnory, Y. Takeda, and D. M. Greenberg, J. Biol. Chem. 212, 379 (1955).
430
SEPARATION AND ASSAY METHODS
[53]
readily, as 0.1 mieroequivalent of acidity per tube produces a red color. The acids may be determined by-titration_with 2 X 10-8 N NaOH using phenol red indicator (0.04~) and a Vortex mixer to extract all acid into the aqueous layer during titration. To determine radioactive acetate and fl-hydroxybutyrate, combine the tubes for a given peak, and, after extraction of the acid into the aqueous phase (previously made slightly alkaline), count an aliquot to-determine the radioactivity present in the fraction. The total activity in each acid may be calculated from the percentage of activity found in each acid after chromatography multiplied by the total counts in the organic acid fraction prior to chromatography. Elute the glutamate and aspartate retained on the Dowex 50 column during separation of the acetate and fl-hydroxybutyrate with six 2 ml portions of 1 N HC1; concentrate and dissolve the residue in 1 ml of water. When radioactive acids are present, determine total activity on a small aliquot (0.05-0.10 ml). In earlier studies,15 aspartate and glutamate were separated using a procedure of Moore and Stein 17 substituting a 50-cm column for the 100-cm column used by Moore and Stein. The elution volumes (aspartate approximately 40 ml and glutamate 70 ml) are about one-half of those reported here for the 100 cm column. The distribution of radioactivity was determined by passing the eluate through a scintillation flow counter connected to a recorder. The fractional areas under each peak give the percentage of activity in each acid. In more recent work, thin-layer chromatography has been used as recommended by Sch~ifer.TM The solvent system is EtOH-NH,OH, 70:30 (v/v), and slow developing plates TM should be used. Radioactive acids may be counted in a thin-layer chromatogram scanner, or a control strip using nonradioactive acids may be sprayed with ninhydrin to locate the amino acids. In this system glutamate has a higher Rr value than aspartate, whereas in the Moore and Stein system the acids are eluted in the reverse order. The techniques described in this report are useful in studying the dynamic aspects of metabolism in mitochondrial systems. ~5,~°-24
~7S. Moore and W. H. Stein, J. Biol. Chem. 192, 663 (1951) G. Sehiffer, personal communication. Rapid developing plates do not yield separation of the two acids. We have found the Brinkman F2s precoated plates to be satisfactory for this purpose. M. S. Olson and R. W. Yon Korff, J. Biol. Chem. 242, 325 (1967). 21M. S. Olson and R. W. Von Korff, J. Biol. Chem. 242, 333 (1967). "~C. Bauer and R. W. Yon Korff, Biochim. Biophys. Acta 131, 280 (1967). **G. Seh~fer, P. Balde, and W. Lamprecht, Nature 214, 20 (1967). **R. W. Von Korff, Nature 214, 23 (1967).
ION-EXCHANGE CHROMATOGRAPHY
425
and then ground to ensure against metabolic alteration of acid constituents. Sulfuric acid is used in preference to other acids such as trichloroacetic or perchloric acid where it is necessary to terminate a biochemical reaction. Most of the commonly used protein precipitating reagents appear as peaks on the chromatogram and will swamp a particular area of analysis. The pH of the sample is adjusted to below 2 to keep the acids in a nonionized form. Due to the increased sensitivity of the method, the requirement for preliminary extraction and concentration is unnecessary in most instances. Hence better yield, fewer artifacts, and smaller losses from volatilization or polymerization of certain substances are obtained. Sources of Error. Limitations in reproducibility are the result of a number of factors, of which the calculation of the peak area may be of considerable magnitude. The height-width method for determining area is the most convenient procedure when utilizing a nonlinear absorbance photometer. Errors may also arise from lack of photometric stability or potentiometer reproducibility and deviations in the pump rates of the two or three pumps used. When biological specimens are to be evaluated, it is of utmost importance that the investigator be aware of potential losses during the sample preparation.
[63] I o n - E x c h a n g e C h r o m a t o g r a p h y of C i t r i c A c i d Cycle Components and Related Compounds B y R. W. VON KORFF
Ion-exchange chromatography of citric acid cycle components and related compounds was first applied to biochemical studies by Busch et al., 1 who used Dowex 1 in the formate cycle and a formic acid gradient to elute organic acids. The variants ~,a of this procedure include use of ammonium formate-formic acid 4 and acetate columns with an acetic acid gradient? ,6 These procedures are used widely to separate appreciable amounts of H. Busch, R. B. Hurlbert, and V. R. Potter, J. Biol. Chem. 196, 717 (1952). A. W. Norman and H. F. De Luca, Bioehem. J. 91, 124 (1964). ' G. R. Bartlett, J. Biol. Chem. 234, 459 (1959). ' J. L. Gamble, J. Biol. Chem. 240, 2668 (1965). BIt. Busch, Cancer Res. 13, 789 (1953). • See Vol. III [70].
426
SEPARATION AND ASSAY METtIODS
[53]
organic acids (5-200 micromoles).1 The removal of large amounts of formic or acetic acids, or their ammonium salts, is time-consuming, and it is not well adapted for either the separation of nanomole quantities of citric acid cycle components or the simultaneous monitoring of radioactivity where isotopic acids are employed. Bartlett et al./used Dowex 1 (CI-) with HC1 as the eluting agent to chromatograph carbohydrate intermediates and related cofactors of erythrocytes. They observed that HC1 facilitates recovery of compounds by evaporation, rechromatography after neutralization and dilution, and sampling for 14C radioactivity assay on "infinitely thin" planchets. The use of Dowex 1 (C1-) columns with an HC1 gradient offers several advantages: 1. Washed analytical grade resin in the (Cl:) cycle is readily available. 2. The organic acids, in general, are most stable in acid solution, and the eluates may be stored frozen with little decomposition. 3. The column eluate may be passed through a scintillation flow cell for simultaneous monitoring of radioactivity. 4. Fractions containing volatile acids, e.g., acetate, lactate, or pyruvate, may be neutralized to pH 8--9, without introduction of excessive amounts of salt, and concentrated to dryness. However, quantities of organic acids greatly in excess of 5 micromoles of individual components may alter the pH and lead to peak distortion and displacement or both. For example, succinate and malate, separated readily in amounts up to 5 mieromoles, merge as the amounts increase; the succinate region increases in size, while malate decreases as the acids apparently interact. Complete separation of acids eluting in closely adjacent regions is also a function of the relative amounts of the two compounds. While two acids may elute in separate peaks in a ratio of 5A to 1B (or 1A to 1B) resolution may be poor at a ratio of 1A to 5B. The procedure may be modified for particular situations but is most generally useful in separating trace amounts of radioactive materials. Chromatographic Procedure. Dowex l-X8 (C1-), 200--400 mesh (analytical grade) 8 is washed with water to remove impurities and acid. After washings reach pH 4.5, the resin suspension in water is neutralized to pH 6.8, and stored as a slurry. After about one month the resin should be rewashed and neutralized. G. R. Bartlett, E. Savage, L. Hughes, and A. R. Marlow, J. Applied Physiol. 6, 51 (1953). Dowex I-X10 does not appear to offer any advantages over the X8 resin.
[63]
ION-EXCHANGE CHROMATOGRAPHY
427
A colunm (1 X 17 cm) is formed by pouring water-suspended resin into a tube (1 X 30 cm) having a sintered disk, a 12/2 ball of a (S~) joint at the base and an 18/9 socket at the top. Flow can be controlled by a Teflon needle valve (Manostat Corporation, New York City) having a 12/2 socket (Sj) at one end and a drip tip at the other. About 2 mm of water should remain above the resin. Add 1-2 ml of organic acid sample, 9 pH 6.8-7.2, to the resin bed using a long-tip pipette, and drain it into the resin. Wash the sample container with about 1 ml of water, drain it into the resin. Close the stopcock until the sample is eluted. First, neutralize perchloric acid filtrates 1° to pH 6.8 with 4 N KOH, and keep at 0 ° until the KC10~ has settled; then add the fluid to the column and wash the KC1Q once with cold water. For elution of the acids, fill the column with water and attach a 250ml Erlenmeyer flask with a 35/20 socket (S~) and a 12 cm side arm ending in a right-angle with an 18/9 ball (Sj) ; fill the flask with water, add a stirring bar, and mount above a magnetic stirrer. At the upper end of the flask attach a 35/20 ball (Sj) through which a 5 mm tube extends to within 1 inch of the bottom. If radioactivity is monitored with a scintillation flow cell, connect this tube to a micropump (Buchler Instruments, Inc., Fort Lee, New Jersey) to supply a constant rate of flow. Remove the stopcock from the lower end of the column, attach a 12/2 socket (S~), in turn attached to Teflon tubing passing to a scintillation flow cell assembly. To begin elution, connect the pump to a source of 0.05 N HC1. After 50 fractions of 2 ml have been collected, connect the pump to a source of 0.10 N HC1. The approximate positions of a number of organic and amino acids chromatographed with this system are listed in the table. Oxaloacetic acid cannot be recovered from Dowex 1 as it undergoes decomposition on the basic nitrogenous resin. If the sample is nonradioactive, or if fractions are to be counted individually, gravity flow from a separatory funnel extending to within 1 inch of the bottom of the mixing flask may be used in place of the micropump assembly. Fractions may be collected directly into tubes using the elution schedule described above. Where certain acids elute together (overlap), chemical reduction permits a rapid second separation using the same chromatographic system. For example, citrate and pyruvate overlap, while suecinate and acetoacetate, and fumarate and a-ketoglutarate elute as single peaks. In Samples at high salt concentration should be diluted to reduce the salts to less than 0.15M. At higher initial salt concentrations, acids may be partially eluted in abnormal postions in the ehromatogram. loWhen 14C0, is present, the perchloric acid solution should first be treated with pieces of dry ice to displace the radioactive C01.
428
[63]
SEPARATION AND ASSAY METHODS
PROFILE OF ORGANICACID ELUTIONFROMDOWEX 1-(XS) (C1-) COLUMNS USING AN H C I GRADIENT
Acid Alanine and other monoamino monocarboxylic acids COs, aspartate, glutamate, B-hydroxybutyrate, acetate Lactate Butyrate" ~-Methyl-fl-hydroxyglutarate Suceinate, acetoacetate a-Hydroxyglutarate Glyoxylate Malate Isocitrate Citrate Pyruvate, malonate Fumarate, a-ketoglutarate
Approximate volume at peak (ml) 8 46 52 56 62 66 72 80 86 108 130 150 204
a Unsubstituted acids of greater chain length than C-4 do not elute from the Dowex 1 columns. These may be separated on silicic acid columns. each case the combined fraction containing the compounds to be separated is neutralized to p H 6-7 and treated with 1 to 2 mg of sodium borohydride to reduce the keto acids? 1 After acidification to destroy excess borohydride and neutralization to p H 7, the sample is placed on a Dowex 1 column and rerun as described previously. The reduced samples containing citrate and lactate, succinate and fl-hydroxybutyrate, and fumarate and a-hydroxyglutarate are separated easily by chromatography on Dowex 1 with an HCI gradient. To date, a separation of acetate, fl-hydroxybutyrate, glutamate, and aspartate has not been effected with the chloride system. Lactate follows these compounds and m a y overlap partially. I t m a y be separated completely, however, by using 0.01 N HC1 to form the initial gradient. In this case, subsequent peaks, caused by a gradual acidity gradient, m a y be broader and appear later. After elution of lactate, change the HC1 from 0.01 N to 0.05 N. The Bessman TM Gradient Elution Device (National Instruments Laboratories, Inc., Rockville, Maryland) is useful for further experimentation with different gradients.
Separation o] Acetate plus fl-Hydroxybutyrate and Glutamate plus 11In the case of acetoa~etate, this should be done as soon as possible after chromatography to avoid possible losses. uS. P. Bessman, Anal. Biochem. 18, 256 (1967).
[63]
ION-EXCHANGE CHROMATOGRAPHY
429
Aspartate. The peak containing these acids is neutralized to pH 7 and passed through a 1 X 3 cm column of Dowex 50-X8, 200-400 mesh (analytical grade). Acetate and fl-hydroxybutyrate are not held on the column. Wash the column with 5-6 times the volume of sample, usually not less than 40 ml. The eluate is then neutralized; if radioactivity is to be determined, aliquots are taken for counting to determine the total activity present. Concentrate the remainder of the sample to dryness on a rotating evaporator. The acetate and fl-hydroxybutyrate may be separated on a silicic acid column using a modification of the procedure of Bulen et al.13-15
Wash silicic acid (Mallinckrodt Chemical Works), 100 mesh (labeled "suitable for chromatographic analyses by the method of Ramsey and Peterson"), to remove fines, as described by Bulen, et aI. Dry the washed material thoroughly at 105 ° and prepare the stationary phase as described by Kinnory et al. TM Mix silicic acid, 8.8 g, in a mortar with 5.4 ml of 0.05 N H~SQ until the powder is finely divided. Then wash chloroform in a separatory funnel with a small amount of 0.05 N H~SO, and pass the chloroform layer through dry filter paper to remove water droplets. Bring the silicic acid to a slurry with chloroform in a beaker and pour it into a column (1.4 X 35 cm) that has a small glass-wool plug, a stopcock at the lower end, and a 24/40 joint at the top. Acidify the neutralized sample, not over 0.6 ml in volume, with 0.05 ml of 3 N H~S04. Very small amounts of radioactive acetate and fl-hydroxybutyrate may be determined by adding 5-10 micromoles of nonradioactive carrier acids to aid in locating the peaks. The sample is mixed well with 1.0 g silicic acid and added as a chloroform slurry to the top of the silieic acid column, drained previously until only 1-2 mm of chloroform remains above the silicic acid. After the sample has settled, add to the column a 5-cm layer of chloroform and connect to a reservoir containing 250 ml of acid-washed chloroform and a magnetic mixing bar. A separatory funnel containing 1-butanol-chloroform, 40:60 (v/v), to which 1 ml of 0.05N H2S04 has been added per 100 ml of mixed solvent, is connected to the reservoir, and in turn mounted on a fraction collector and above a magnetic mixer. Fractions (3 ml) are collected into tubes containing 1.0 ml of a neutralized methyl red indicator solution (a 1:20 dilution with water of a 0.04% methyl red solution in ethanol). The diluted methyl red should be neutralized to an orange color and tested for the highest sensitivity to traces of acidity. Acids eluting from the column are detected ~*W. A. Bulen, J. E. Varner, and R. C. Burrell, Anal. Chem. 24, 187 (1952). ~'See Vol. III [64]. ~R. W. Von Korff, J. Biol. Chem. 240, 1351 (1965). ~aD. S. Kinnory, Y. Takeda, and D. M. Greenberg, J. Biol. Chem. 212, 379 (1955).
430
SEPARATION AND ASSAY METHODS
[53]
readily, as 0.1 mieroequivalent of acidity per tube produces a red color. The acids may be determined by-titration_with 2 X 10-8 N NaOH using phenol red indicator (0.04~) and a Vortex mixer to extract all acid into the aqueous layer during titration. To determine radioactive acetate and fl-hydroxybutyrate, combine the tubes for a given peak, and, after extraction of the acid into the aqueous phase (previously made slightly alkaline), count an aliquot to-determine the radioactivity present in the fraction. The total activity in each acid may be calculated from the percentage of activity found in each acid after chromatography multiplied by the total counts in the organic acid fraction prior to chromatography. Elute the glutamate and aspartate retained on the Dowex 50 column during separation of the acetate and fl-hydroxybutyrate with six 2 ml portions of 1 N HC1; concentrate and dissolve the residue in 1 ml of water. When radioactive acids are present, determine total activity on a small aliquot (0.05-0.10 ml). In earlier studies,15 aspartate and glutamate were separated using a procedure of Moore and Stein 17 substituting a 50-cm column for the 100-cm column used by Moore and Stein. The elution volumes (aspartate approximately 40 ml and glutamate 70 ml) are about one-half of those reported here for the 100 cm column. The distribution of radioactivity was determined by passing the eluate through a scintillation flow counter connected to a recorder. The fractional areas under each peak give the percentage of activity in each acid. In more recent work, thin-layer chromatography has been used as recommended by Sch~ifer.TM The solvent system is EtOH-NH,OH, 70:30 (v/v), and slow developing plates TM should be used. Radioactive acids may be counted in a thin-layer chromatogram scanner, or a control strip using nonradioactive acids may be sprayed with ninhydrin to locate the amino acids. In this system glutamate has a higher Rr value than aspartate, whereas in the Moore and Stein system the acids are eluted in the reverse order. The techniques described in this report are useful in studying the dynamic aspects of metabolism in mitochondrial systems. ~5,~°-24
~7S. Moore and W. H. Stein, J. Biol. Chem. 192, 663 (1951) G. Sehiffer, personal communication. Rapid developing plates do not yield separation of the two acids. We have found the Brinkman F2s precoated plates to be satisfactory for this purpose. M. S. Olson and R. W. Yon Korff, J. Biol. Chem. 242, 325 (1967). 21M. S. Olson and R. W. Von Korff, J. Biol. Chem. 242, 333 (1967). "~C. Bauer and R. W. Yon Korff, Biochim. Biophys. Acta 131, 280 (1967). **G. Seh~fer, P. Balde, and W. Lamprecht, Nature 214, 20 (1967). **R. W. Von Korff, Nature 214, 23 (1967).