- Email: [email protected]
[4] High-voltage paper electrophoresis
32
AMINO ACID ANALYSIS AND RELATED PROCEDURE8
[4]
[4] High-Voltage Paper Electrophoresis By W. J. DREYER and E. BYNUM
Introduction The column chromatographic methods of amino acid analysis, discussed elsewhere (see this volume [1, 2]), are indispensable tools whenever quantitative data of high precision are required. There are several procedures, however, which necessitate accurate qualitative analysis, but wherein quantitation of the order of _ 1 0 % is satisfactory. Under such circumstances more rapid methods may be used. The technique of one-dimensional high-voltage electrophoresis of amino acids, described below, permits the separation of 10-20 samples in a period of 115 minutes. The procedure provides qualitative identification of amino acids found in peptide hydrolyzates containing as little as 1 X 10-9 mole of each amino acid, and quantitative results on samples containing 550 X 10-9 mole of each. The technique has found application in a number of procedures. It has been used very extensively for the determination of amino acid composition of peptides. 1 Very simple techniques have been developed to identify the carboxyl terminal amino acid in peptides or proteins following hydrazinolysis. 2 The method has also been an invaluable aid in connection with the phenylisothiocyanate (PTH) sequential degradation procedures. The P T H amino acids released during sequential degradation are hydrolyzed prior to analysis in this case. Alternatively, peptides remaining after removal of terminal residues may be hydrolyzed and analyzed. In enzymatic methods of sequential degradation, kinetic studies have been carried out, on the rate of release of amino acids from the amino terminal end of a polypeptide by aminopeptidase. The volatile buffer triethylamine is utilized to avoid desalting and to permit direct application of the aliquots t~ the paper. Alternatively, carboxypeptidase is dissolved in 2 M ammonium bicarbonate and kinetic studies are utilized to determine carboxyl terminal sequences of polypeptide chains. Finally, the procedure has found application in miscellaneous procedures that involve the routine examination of large numbers of clinical samples. One example of this is in the screening of urine of newborn children for abnormalities in the content of amino acids, peptides, and a variety of 1R. E. Canfield and C. B. Anfinsen, in "The Proteins" (H. Neurath, ed.), 2rid Ed., Vol. 1. Academic Press, New York, 1963. J. C. Bennett and W. J. Dreyer, in preparation.
[4]
HIGH-VOLTAGE PAPER ELECTROPHORESIS
33
other compounds, many of which have not yet been identified. Early diagnosis of a number of types of hereditary disease is thus simplified. Many aspects of these methods have been discussed elsewhere in this volume and for this reason they have been mentioned here only briefly, witch special reference to their use in conjunction with the rapid high-voltage analytical system. The general and historical aspects of electrophoresis have been covered in earlier volumes of this ~ries as well as in a number of review and reference works2 -7 These sources should also be consulted for application of the general methodology to be discussed here to a variety of charged molecules other than amino acids. General Pririciples of the Method Ideally, the mobility # of an ion of net charge q, and diffusion coefficient D, is given by the expression
qD
= ~
(1)
where T is the absolute temperature, and k is the Boltzmann constant. Equation (1) is independent of the size and shape of the molecule. In practice the mobility of amino acids on paper only approximates that predicted by this expression, since interaction of the amino acids with other molecules or with the supporting medium is not taken into account. The utilization of additional variables has permitted separations that might have been theoretically impossible. Thus in the procedure described below, careful attention must be paid to details such as pH, temperature, voltage, type and length of paper, etc. If these conditions are followed, separations such as those illustrated can be accomplished easily and routinely. Apparatus
The analyses are carried out in a large bifurcated Fiberglas tank. The tank contains 2 liters of buffer in the bottom of each of the two chambers, which are divided by a center partition. The remainder of the electrophoretic chamber is filled with an inert petroleum distillate (such as Varsol). A tubular stainless steel heat exchanger is submerged in the Varsol along the top walls of the chamber. The analytical paper 3A. Tiselius, Vol. IV [1]. 4A. H. F. Laurell, Vol. IV [la]. M. Bier, Vol. V [3]. eC. J. 0. R. Morris and P. Morris, "Separation Methods in Biochemistry." Wiley (Interscience), New York, 1963. E. Heftmann (ed.), "Chromatography." Reinhold, New York, 1961.
34
A M I N O ACID A N A L Y S I S AND R E L A T E D
PROCEDURES
[4]
is placed in the chamber on a suitable rack such that the two ends of the paper are submerged in the buffer in the bottom of the respective limbs of the apparatus. The paper is completely covered by the organic phase~ and heat generated during electrophoresis is dissipated by conduction and convection. The Varsol is in turn cooled by circulating an appropriate coolant through the heat exchanger. Chilled water, brine, and Freon all serve equally well. The apparatus contains a D.C. power supply rated for 10,000 volts and 500 milliamps, which supplies a potential to the electrode vessels via platinum wires. Suitable apparatus is commercially available and is illustrated in Fig. 1.
Fro. 1. High voltage (10,000 volt) electrophoretic apparatus used for peptide and amino acid separation. (Courtesy of Gilson Medical Electronics, Middleton, Wisconsin.)
[4]
HIGH-VOLTAGE PAPER ELECTROPHORESIS
35
Acid Hydrolysis of Polypeptides A typical application of this high-voltage technique involves the amino acid analysis of many dozens of peptide samples obtained by the usual techniques from proteolytic hydrolyzates of proteins. Final purification is often accomplished by the preparative use of the same highvoltage electrophoretic equipment used for amino acid analysis. Aliquots of the peptide samples are placed in 10 X 75 mm test tubes labeled with a diamond pencil. After the samples have been taken to dryness in vacuo, 400 #1 constant boiling (b.p. 108 °) hydrochloric acid is added to each tube. The tubes can then be evacuated, flushed with nitrogen, and sealed in the normal fashion if desired. However, when large numbers of peptide samples are to be studied, it is more convenient to place the unsealed tubes in an appropriate noncorrosive rack within a small desiccator. Approximately 10 ml constant boiling HC1 is placed in the bottom of the desiccator. The tubes may be covered with glass beads or teardrops if desired. A specially constructed clamp is used to hold the lid of the desiccator in place. Silicone high-vacuum grease is applied, but a Viton or Teflon seal should also suffice. The clamp is constructed from two aluminum rings that fit around the desiccator base. One ring can be slipped up to the flange; the second is placed over the lid in such a way that bolts with wing nuts can be used to tighten the ground-glass seal. The desiccator is then evacuated. A three-way stopcock is used to flush the desiccator with a good grade of pure nitrogen. The container is then alternately evacuated and flushed with nitrogen at least 3 times in order to eliminate the last trace of oxygen. The evacuated desiccator is then placed in an oven at 105 ° for 20 hours. A well-insulated oven is required to provide uniform heating of the hydrolysis vessel. Air must be admitted before removing the desiccator from the oven to minimize the chances of bumping. The top is most conveniently removed after cooling by use of a special leverage device designed for the purpose. Alternatively, the edge of a razor blade may be slipped beneath the lid and tapped lightly a few times in order to loosen the lid. The tubes are cooled and the hydrolyzates are taken to dryness in vacuo over sodium hydroxide pellets. Application of Samples to Paper Of the many types of paper tested for this purpose, Whatman No. 3MM chromatography grade paper has been found to be the most satisfactory for general applications. It is available in rolls 18" wide X 300' in length. A suitable length of paper is cut from the roll and an origin line drawn across the paper at a position that will be 6" above the end of the rack after proper mounting of the paper. The samples are taken
36
AMINO ACID ANALYSIS AND RELATED PROCEDURES
[4]
up in a volume of water such that 10 ~I will contain 10-60 X 10-9 moles of each of the amino acids to be analyzed. A small amount of phenol red is normally added at the same time to provide a visual aid during sample application and moistening of the papers. A Beckman Instrument Co. sample striper (_~320005) provides a convenient means to apply a 10-~1 sample. A glass rod is placed under the origin line to aid in the positioning of the sample striper. The samples are so spaced as to permit the sheet of paper to be cut into strips for scanning in a densitometer or for elution of the ninhydrin color. The application of the samples as small spots is also satisfactory and permits as many as 20 samples to be applied to one paper. The latter approach is particularly well suited for qualitative screening purposes. The paper is fastened onto the supporting rack with the origin line if' above the end. (The buffer level within the tank should be just sufficient to cover the paper clamps on the ends of the rack.) The rack and paper are then unfolded and placed on an inexpensive grade of absorbent filter paper. The paper is creased along the origin line and supported a few inches above the table top by placing a small glass rod underneath the origin. A good grade of 98 + % formic acid is diluted 1 : 16 in a large carboy to give 1.64 M formic acid. The pH of this solution should be 1.58 when read at 25 °. A siphon tube fitted with a Teflon stopcock and a blunt glass tip is used to moisten the paper carefully and slowly at positions about F' on each side of the origin line. The solution is allowed to spread in such a way as to meet in a straight line at the origin. Care must be taken in this operation, and in subsequent additions the solution should always be applied well behind the advancing front as it approaches the origin. The remainder of the paper is rapidly moistened in the meantime. The paper should be very wet but any excess formic acid is blotted from the paper to prevent running. As soon as the solvent has moistened the origin, the rack is folded and the rollers are carefully tightened. The paper is placed in the electrophoretie chamber with the origin at the positive electrode. A potential of 7800 volts is applied for 115 minutes. The current should be approximately 420 milliamps. A lower current probably indicates that the paper is too dry and in danger of drying out during the run. A temperature of 430-44 ° is maintained by the use of a built-in thermistor sensing device and a controller unit which operates a solenoid valve that admits the flow of coolant into the lower of the two heat-exchanger coils. The top coil is provided with a simple thermostat set to cool continuously at temperatures over 42 ° . This arrangement provides continuous convective circulation of the organic solvent. The formic acid concentration and the temperature must both be controlled with care t~ achieve the desired resolution of the amino acids. For this reason the electrophoretic chamber is
[4]
HIGH-VOLTAGE PAPER ELECTROPHORESIS
37
prewarmed to 44 ° prior to the first run of each day. This aids in providing a constant temperature and also presaturates the organic solvent with the aqueous phase so as to eliminate any tendency of the paper to dry out during a run. A blank paper moistened with the solvent is utilized for this purpose. Electrophoresis for 5-10 minutes with the blank paper is sufficient to raise the temperature to the control level. Immediately after electrophoresis the paper is dried in a ventilated chromatographic drying oven. Color Development with Cadmium-Ninhydrin Reagent A stock solution is made up in the following proportions: water, 200 ml; acetic acid, 40 ml; cadmium acetate, 2.0 g. A 24-mi aliquot of this stock solution is added to 200 ml acetone followed by 2.0 g ninhydrin (Pierce Chemical Co.). The papers are dipped in this reagent and placed in a glass or other suitable sealed enclosure containing a small vessel of concentrated sulfuric acid. Color development is allowed to proceed for 18 hours at 25 °, or for appropriately shorter periods of time at h~gher temperatures. (A test should be made to determine optimum time o~ development at higher temperatures.) The resulting cadmium complex of the ninhydrin color is much more stable than the usual pigment and the background paper blanks are exceptionally low. Following color development, papers are routinely photographed with back-lighting such as is provided by an X-ray view box. A 500-mt~ interference filter (Bausch and Lomb) greatly enhances the contrast of the photographs so that amino acid levels below 1 X 10-9 moles are recorded. In laboratories where large numbers of amino acid analyses are carried out such photographic records provide an invaluable means of recording data. A Polaroid MP-3XL camera with type 55PN film is particularly useful. Figure 2 illustrates the results of this procedure as applied to peptide hydrolyzates.
Quantitative Analysis The chromatography paper is appropriately marked with a straightedge and cut into strips. When rapid semiquantitative information is required these strips are scanned in a recording densitometer with a 500-mtk interference filter. The height of each peak is measured. Most amino acids reveal an approximately linear relationship between concentration and the absorbance at the top of the peak on the recording tracing. Standard curves are used for this correlation. Much more satisfactory results are in general obtained with a modification of the elution procedure described by Atfield and Morris. s In this +G. N. Atfield and C. J. 0. R. Morris, Biochem. d. 81, 606 (1961).
38
AMINO ACID ANALYSIS AND RELATED PROCEDURES
[4]
® LYS
ARG HIS
GLY
ALA
VAL SER LEU ILU (PRO) THR MET GLU PHE ASP TYR TRY
CYSTEIC
Fro. 2. Typical separation of amino acids found in peptide hydrolyzates. This separation was accomplished in 120 minutes using 7800 volts. The origin and approximately one third of the paper has been removed from the bottom portion in the photograph. The most common difficulty encountered in first attempts at using this method is illustrated in the center columns of this photograph. The irregularity in the zones observed was caused by nonuniform convergence of buffer at the origin
[5]
THI]~-LAYER CHROMATOGRAPHY
39
procedure the band containing a given amino acid is carefully excised from the strip and cut into several longitudinal strips to fit into an 8 X 75 mm test tube. Absolute methanol (3.0 ml) is added to each tube with an accurate automatic syringe or pipetting device. The tubes are sealed with No. 000 gum rubber stoppers (Emil Greiner, Inc., New York). The racks of tubes are placed horizontally on a platform rocking device for 1 hour, and then permitted to stand for 15 minutes so that any paper fibers present may settle to the bottom. The optical density of these solutions is determined at 500 m~ in a spectrophotometer fitted with a 1ml cell. A linear relationship is found for the measured absorbance (after subtraction of the small blank value) as related to concentration of a given amino acid. Thus for this type of analysis the quantity of each amino acid is determined by simply multiplying the absorbance by a factor derived from the slope of standard curves. Both of these methods are found to be satisfactory for all amino acids found in a hydrolyzate of an oxidized (cysteic acid) or a reduced and alkylated (S-carboxymethylcysteine) polypeptide chain, with the exception of proline. This amino acid gives a pale yellow color with the cadmium reagent. When analysis of proline is required, the method recommended by Atfield and Morris (with a cadmium-isatin reagent) is used. s
[5] T h i n - L a y e r C h r o m a t o g r a p h y ( T L C ) of A m i n o A c i d s
By M. BRENNER and A. NIEDERWIESER I. Introduction TLC is an experimentally simple and inexpensive method that permits very rapid and efficient qualitative and even semiquantitative analysis of amino acids and amino acid mixtures.1 It lends itself especially to the comparison of protein hydrolyzates (mapping), analysis of the course of hydrolysis experiments, characterization of peptides and their hydrolyzates, and purity control. An occasional ambiguity in the interpretation of a result is more than compensated for by the versatility of the method and by the minimal requirements with regard to sample size. 1M. Brenner, A. Niederwieser, and G. Pataki, in "Thin-Layer Chromatography, A Laboratory Handbook" (E. Stahl, ed.), p. 391. Published jointly with Springer, Berlin and Academic Press, New York, 1965 (German Ed., "Diinnschicht-Chromatographie, Ein Laboratoriumshandbuch."Springer, Berlin, 1962). line. S-Carboxymethylcysteineruns slower than tryptophan, and cysteic acid moves to a point several inches below the origin. In the case of analysis of urine a large number of unidentified compounds move slower than tryptophan.
AMINO ACID ANALYSIS AND RELATED PROCEDURE8
[4]
[4] High-Voltage Paper Electrophoresis By W. J. DREYER and E. BYNUM
Introduction The column chromatographic methods of amino acid analysis, discussed elsewhere (see this volume [1, 2]), are indispensable tools whenever quantitative data of high precision are required. There are several procedures, however, which necessitate accurate qualitative analysis, but wherein quantitation of the order of _ 1 0 % is satisfactory. Under such circumstances more rapid methods may be used. The technique of one-dimensional high-voltage electrophoresis of amino acids, described below, permits the separation of 10-20 samples in a period of 115 minutes. The procedure provides qualitative identification of amino acids found in peptide hydrolyzates containing as little as 1 X 10-9 mole of each amino acid, and quantitative results on samples containing 550 X 10-9 mole of each. The technique has found application in a number of procedures. It has been used very extensively for the determination of amino acid composition of peptides. 1 Very simple techniques have been developed to identify the carboxyl terminal amino acid in peptides or proteins following hydrazinolysis. 2 The method has also been an invaluable aid in connection with the phenylisothiocyanate (PTH) sequential degradation procedures. The P T H amino acids released during sequential degradation are hydrolyzed prior to analysis in this case. Alternatively, peptides remaining after removal of terminal residues may be hydrolyzed and analyzed. In enzymatic methods of sequential degradation, kinetic studies have been carried out, on the rate of release of amino acids from the amino terminal end of a polypeptide by aminopeptidase. The volatile buffer triethylamine is utilized to avoid desalting and to permit direct application of the aliquots t~ the paper. Alternatively, carboxypeptidase is dissolved in 2 M ammonium bicarbonate and kinetic studies are utilized to determine carboxyl terminal sequences of polypeptide chains. Finally, the procedure has found application in miscellaneous procedures that involve the routine examination of large numbers of clinical samples. One example of this is in the screening of urine of newborn children for abnormalities in the content of amino acids, peptides, and a variety of 1R. E. Canfield and C. B. Anfinsen, in "The Proteins" (H. Neurath, ed.), 2rid Ed., Vol. 1. Academic Press, New York, 1963. J. C. Bennett and W. J. Dreyer, in preparation.
[4]
HIGH-VOLTAGE PAPER ELECTROPHORESIS
33
other compounds, many of which have not yet been identified. Early diagnosis of a number of types of hereditary disease is thus simplified. Many aspects of these methods have been discussed elsewhere in this volume and for this reason they have been mentioned here only briefly, witch special reference to their use in conjunction with the rapid high-voltage analytical system. The general and historical aspects of electrophoresis have been covered in earlier volumes of this ~ries as well as in a number of review and reference works2 -7 These sources should also be consulted for application of the general methodology to be discussed here to a variety of charged molecules other than amino acids. General Pririciples of the Method Ideally, the mobility # of an ion of net charge q, and diffusion coefficient D, is given by the expression
qD
= ~
(1)
where T is the absolute temperature, and k is the Boltzmann constant. Equation (1) is independent of the size and shape of the molecule. In practice the mobility of amino acids on paper only approximates that predicted by this expression, since interaction of the amino acids with other molecules or with the supporting medium is not taken into account. The utilization of additional variables has permitted separations that might have been theoretically impossible. Thus in the procedure described below, careful attention must be paid to details such as pH, temperature, voltage, type and length of paper, etc. If these conditions are followed, separations such as those illustrated can be accomplished easily and routinely. Apparatus
The analyses are carried out in a large bifurcated Fiberglas tank. The tank contains 2 liters of buffer in the bottom of each of the two chambers, which are divided by a center partition. The remainder of the electrophoretic chamber is filled with an inert petroleum distillate (such as Varsol). A tubular stainless steel heat exchanger is submerged in the Varsol along the top walls of the chamber. The analytical paper 3A. Tiselius, Vol. IV [1]. 4A. H. F. Laurell, Vol. IV [la]. M. Bier, Vol. V [3]. eC. J. 0. R. Morris and P. Morris, "Separation Methods in Biochemistry." Wiley (Interscience), New York, 1963. E. Heftmann (ed.), "Chromatography." Reinhold, New York, 1961.
34
A M I N O ACID A N A L Y S I S AND R E L A T E D
PROCEDURES
[4]
is placed in the chamber on a suitable rack such that the two ends of the paper are submerged in the buffer in the bottom of the respective limbs of the apparatus. The paper is completely covered by the organic phase~ and heat generated during electrophoresis is dissipated by conduction and convection. The Varsol is in turn cooled by circulating an appropriate coolant through the heat exchanger. Chilled water, brine, and Freon all serve equally well. The apparatus contains a D.C. power supply rated for 10,000 volts and 500 milliamps, which supplies a potential to the electrode vessels via platinum wires. Suitable apparatus is commercially available and is illustrated in Fig. 1.
Fro. 1. High voltage (10,000 volt) electrophoretic apparatus used for peptide and amino acid separation. (Courtesy of Gilson Medical Electronics, Middleton, Wisconsin.)
[4]
HIGH-VOLTAGE PAPER ELECTROPHORESIS
35
Acid Hydrolysis of Polypeptides A typical application of this high-voltage technique involves the amino acid analysis of many dozens of peptide samples obtained by the usual techniques from proteolytic hydrolyzates of proteins. Final purification is often accomplished by the preparative use of the same highvoltage electrophoretic equipment used for amino acid analysis. Aliquots of the peptide samples are placed in 10 X 75 mm test tubes labeled with a diamond pencil. After the samples have been taken to dryness in vacuo, 400 #1 constant boiling (b.p. 108 °) hydrochloric acid is added to each tube. The tubes can then be evacuated, flushed with nitrogen, and sealed in the normal fashion if desired. However, when large numbers of peptide samples are to be studied, it is more convenient to place the unsealed tubes in an appropriate noncorrosive rack within a small desiccator. Approximately 10 ml constant boiling HC1 is placed in the bottom of the desiccator. The tubes may be covered with glass beads or teardrops if desired. A specially constructed clamp is used to hold the lid of the desiccator in place. Silicone high-vacuum grease is applied, but a Viton or Teflon seal should also suffice. The clamp is constructed from two aluminum rings that fit around the desiccator base. One ring can be slipped up to the flange; the second is placed over the lid in such a way that bolts with wing nuts can be used to tighten the ground-glass seal. The desiccator is then evacuated. A three-way stopcock is used to flush the desiccator with a good grade of pure nitrogen. The container is then alternately evacuated and flushed with nitrogen at least 3 times in order to eliminate the last trace of oxygen. The evacuated desiccator is then placed in an oven at 105 ° for 20 hours. A well-insulated oven is required to provide uniform heating of the hydrolysis vessel. Air must be admitted before removing the desiccator from the oven to minimize the chances of bumping. The top is most conveniently removed after cooling by use of a special leverage device designed for the purpose. Alternatively, the edge of a razor blade may be slipped beneath the lid and tapped lightly a few times in order to loosen the lid. The tubes are cooled and the hydrolyzates are taken to dryness in vacuo over sodium hydroxide pellets. Application of Samples to Paper Of the many types of paper tested for this purpose, Whatman No. 3MM chromatography grade paper has been found to be the most satisfactory for general applications. It is available in rolls 18" wide X 300' in length. A suitable length of paper is cut from the roll and an origin line drawn across the paper at a position that will be 6" above the end of the rack after proper mounting of the paper. The samples are taken
36
AMINO ACID ANALYSIS AND RELATED PROCEDURES
[4]
up in a volume of water such that 10 ~I will contain 10-60 X 10-9 moles of each of the amino acids to be analyzed. A small amount of phenol red is normally added at the same time to provide a visual aid during sample application and moistening of the papers. A Beckman Instrument Co. sample striper (_~320005) provides a convenient means to apply a 10-~1 sample. A glass rod is placed under the origin line to aid in the positioning of the sample striper. The samples are so spaced as to permit the sheet of paper to be cut into strips for scanning in a densitometer or for elution of the ninhydrin color. The application of the samples as small spots is also satisfactory and permits as many as 20 samples to be applied to one paper. The latter approach is particularly well suited for qualitative screening purposes. The paper is fastened onto the supporting rack with the origin line if' above the end. (The buffer level within the tank should be just sufficient to cover the paper clamps on the ends of the rack.) The rack and paper are then unfolded and placed on an inexpensive grade of absorbent filter paper. The paper is creased along the origin line and supported a few inches above the table top by placing a small glass rod underneath the origin. A good grade of 98 + % formic acid is diluted 1 : 16 in a large carboy to give 1.64 M formic acid. The pH of this solution should be 1.58 when read at 25 °. A siphon tube fitted with a Teflon stopcock and a blunt glass tip is used to moisten the paper carefully and slowly at positions about F' on each side of the origin line. The solution is allowed to spread in such a way as to meet in a straight line at the origin. Care must be taken in this operation, and in subsequent additions the solution should always be applied well behind the advancing front as it approaches the origin. The remainder of the paper is rapidly moistened in the meantime. The paper should be very wet but any excess formic acid is blotted from the paper to prevent running. As soon as the solvent has moistened the origin, the rack is folded and the rollers are carefully tightened. The paper is placed in the electrophoretie chamber with the origin at the positive electrode. A potential of 7800 volts is applied for 115 minutes. The current should be approximately 420 milliamps. A lower current probably indicates that the paper is too dry and in danger of drying out during the run. A temperature of 430-44 ° is maintained by the use of a built-in thermistor sensing device and a controller unit which operates a solenoid valve that admits the flow of coolant into the lower of the two heat-exchanger coils. The top coil is provided with a simple thermostat set to cool continuously at temperatures over 42 ° . This arrangement provides continuous convective circulation of the organic solvent. The formic acid concentration and the temperature must both be controlled with care t~ achieve the desired resolution of the amino acids. For this reason the electrophoretic chamber is
[4]
HIGH-VOLTAGE PAPER ELECTROPHORESIS
37
prewarmed to 44 ° prior to the first run of each day. This aids in providing a constant temperature and also presaturates the organic solvent with the aqueous phase so as to eliminate any tendency of the paper to dry out during a run. A blank paper moistened with the solvent is utilized for this purpose. Electrophoresis for 5-10 minutes with the blank paper is sufficient to raise the temperature to the control level. Immediately after electrophoresis the paper is dried in a ventilated chromatographic drying oven. Color Development with Cadmium-Ninhydrin Reagent A stock solution is made up in the following proportions: water, 200 ml; acetic acid, 40 ml; cadmium acetate, 2.0 g. A 24-mi aliquot of this stock solution is added to 200 ml acetone followed by 2.0 g ninhydrin (Pierce Chemical Co.). The papers are dipped in this reagent and placed in a glass or other suitable sealed enclosure containing a small vessel of concentrated sulfuric acid. Color development is allowed to proceed for 18 hours at 25 °, or for appropriately shorter periods of time at h~gher temperatures. (A test should be made to determine optimum time o~ development at higher temperatures.) The resulting cadmium complex of the ninhydrin color is much more stable than the usual pigment and the background paper blanks are exceptionally low. Following color development, papers are routinely photographed with back-lighting such as is provided by an X-ray view box. A 500-mt~ interference filter (Bausch and Lomb) greatly enhances the contrast of the photographs so that amino acid levels below 1 X 10-9 moles are recorded. In laboratories where large numbers of amino acid analyses are carried out such photographic records provide an invaluable means of recording data. A Polaroid MP-3XL camera with type 55PN film is particularly useful. Figure 2 illustrates the results of this procedure as applied to peptide hydrolyzates.
Quantitative Analysis The chromatography paper is appropriately marked with a straightedge and cut into strips. When rapid semiquantitative information is required these strips are scanned in a recording densitometer with a 500-mtk interference filter. The height of each peak is measured. Most amino acids reveal an approximately linear relationship between concentration and the absorbance at the top of the peak on the recording tracing. Standard curves are used for this correlation. Much more satisfactory results are in general obtained with a modification of the elution procedure described by Atfield and Morris. s In this +G. N. Atfield and C. J. 0. R. Morris, Biochem. d. 81, 606 (1961).
38
AMINO ACID ANALYSIS AND RELATED PROCEDURES
[4]
® LYS
ARG HIS
GLY
ALA
VAL SER LEU ILU (PRO) THR MET GLU PHE ASP TYR TRY
CYSTEIC
Fro. 2. Typical separation of amino acids found in peptide hydrolyzates. This separation was accomplished in 120 minutes using 7800 volts. The origin and approximately one third of the paper has been removed from the bottom portion in the photograph. The most common difficulty encountered in first attempts at using this method is illustrated in the center columns of this photograph. The irregularity in the zones observed was caused by nonuniform convergence of buffer at the origin
[5]
THI]~-LAYER CHROMATOGRAPHY
39
procedure the band containing a given amino acid is carefully excised from the strip and cut into several longitudinal strips to fit into an 8 X 75 mm test tube. Absolute methanol (3.0 ml) is added to each tube with an accurate automatic syringe or pipetting device. The tubes are sealed with No. 000 gum rubber stoppers (Emil Greiner, Inc., New York). The racks of tubes are placed horizontally on a platform rocking device for 1 hour, and then permitted to stand for 15 minutes so that any paper fibers present may settle to the bottom. The optical density of these solutions is determined at 500 m~ in a spectrophotometer fitted with a 1ml cell. A linear relationship is found for the measured absorbance (after subtraction of the small blank value) as related to concentration of a given amino acid. Thus for this type of analysis the quantity of each amino acid is determined by simply multiplying the absorbance by a factor derived from the slope of standard curves. Both of these methods are found to be satisfactory for all amino acids found in a hydrolyzate of an oxidized (cysteic acid) or a reduced and alkylated (S-carboxymethylcysteine) polypeptide chain, with the exception of proline. This amino acid gives a pale yellow color with the cadmium reagent. When analysis of proline is required, the method recommended by Atfield and Morris (with a cadmium-isatin reagent) is used. s
[5] T h i n - L a y e r C h r o m a t o g r a p h y ( T L C ) of A m i n o A c i d s
By M. BRENNER and A. NIEDERWIESER I. Introduction TLC is an experimentally simple and inexpensive method that permits very rapid and efficient qualitative and even semiquantitative analysis of amino acids and amino acid mixtures.1 It lends itself especially to the comparison of protein hydrolyzates (mapping), analysis of the course of hydrolysis experiments, characterization of peptides and their hydrolyzates, and purity control. An occasional ambiguity in the interpretation of a result is more than compensated for by the versatility of the method and by the minimal requirements with regard to sample size. 1M. Brenner, A. Niederwieser, and G. Pataki, in "Thin-Layer Chromatography, A Laboratory Handbook" (E. Stahl, ed.), p. 391. Published jointly with Springer, Berlin and Academic Press, New York, 1965 (German Ed., "Diinnschicht-Chromatographie, Ein Laboratoriumshandbuch."Springer, Berlin, 1962). line. S-Carboxymethylcysteineruns slower than tryptophan, and cysteic acid moves to a point several inches below the origin. In the case of analysis of urine a large number of unidentified compounds move slower than tryptophan.