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A modified ninhydrin colorimetric analysis for amino acids
ARCHIVES
OF
BIOCBEMISTRY
AND
BIOPBYSICS
67, 10-15 (1957)
A ModSed Ninhydrin Calorimetric Analysis for Amino Acids Hyman Rosen From the Department of Surgical Metabolism, Division of Surgery, Walter Reed Army Institute of Research, Walter Reed Army Medical Center, Washington, D. C. Received July 25, 1956
INTRODUCTION In 1948, Stein and Moore published the first practical procedure for the chromatographic separation of amino acids on starch columns, and a photometric method for the quantitative analysis of the effluent fractions (1, 2). Their procedure was based on the reaction of amino acids with reduced ninhydrin (triketohydrindenhydrate) at pH 5; stannous chloride served as the reducing agent. The method is reliable and reproducible. This procedure is used almost exclusively in column chromatography, since the method is quantitative for individual amino acids. The very large number of analyses required for the completion of a chromatogram (about 600) make it important to use as efficient and automatic a procedure as possible. To this end, a number of modifications have appeared since the introduction of the method. Troll and Cannan (3) were able to increase the sensitivity slightly, but at the expense of introducing pyridine and phenol into the procedure. Moore and Stein (4) themselves dispensed with the stannous chloride by using reduced ninhydrin prepared with ascorbic acid. This procedure obviates precipitation of tin salts by phosphate buffers. A drawback in any method which uses solutions of reduced ninhydrin is the instability of this reagent in air. The solutions must either be stored and manipulated under nitrogen, or prepared fresh every few days. Yemm and Cocking (5) adapted the method of Troll and Cannan for routine use. Their procedure involves the preparation of cyanide-ninhydrin solutions, which are also unstable. The following is a modifica10
COLORIMETRIC
-4NhLYSIS
FOR AMINO
ACIDS
11
tion of the Yemm and Cocking method, adapted for use in column chromatography. It differs from the latter in that it does not require the prior preparation of an unstable ninhydrin reagent. Only two reagents are used, both stable indefinitely at room temperature. With t,he proper equipment, several hundred analyses can be performed by one worker in an hour. REaGENTS 1. Stock N&N: 0.01 M (490 mg./l.). 2. Acetate Bugler: 2700 g. NaOAc.3HsO + 2 1. H20 + 500 ml. glacial HOAc. Make up to 7.5 1. with H?O. This buffer is adapted from t,hat of Stein and Moore (4). Its pH should be 5.3-5.4. It is quite concentrated, and makes preliminary pH adjustment.s of samples, e.g., effluent fractions, usually unnecessary. In new procedures, however, it should be determined whether, in fact., t.he reaction mixture is at pH 5.0-5.2 before proceeding with the analysis. 3. Acetate-Cyanide: 0.0002 Y NaCN in acetate buffer; 20 ml. of Soln. 1 made to 1 1. with Soln. 2. 4. Ninhydrin: 3% in hIethyl Cellosolve (ethylene glycol monomethyl ether). The suitability of the Cellosolve should be checked by t.he procedure of Stein and Moore (2). Ninhydrin suitable for use without recrystallizing can be obt.ained from Dougherty Chemical Co., 87-34 134th St., Richmond Hill 18, N. Y. 5. Diluent: Isopropyl alcohol-water, 1:l. METHOD To a l-ml. sample containing 0.02-0.40 pmole of an amino acid, add aa ml. cyanide-acetate buffer and >s ml. 3yo ninhydrin solution in Methyl Cellosolve. Heat 15 min. in a 100’ water bath. Maximal color is developed after 10-12 min. Immediately after removing from the water bath, add rapidly 5 ml. of isopropyl alcohol-water diluent. Shake vigorously. Allow to cool to room temperature, and read in a calorimeter at 570 rnp (proline and hydroxyproline at 440 mp). If the color density in a given series of tubes is too high to determine accurately, further 5-ml. portions of diluent may be added, until the optical densities are below 0.8. RESULTS
All samples when removed from the water bath are deep red. The color fades rapidly when the mixture is shaken and/or cooled, and the resultant blank is colorless, or faint blue, in the isopropyl alcohol-water diluent. All amino acids, except proline and hydroxyproline, give a purple color with ninhydrin; the latter two give a yellow color. All colors are stable, fading only l-2 % in 24 hr. Figure 1 relates micromoles of leucine per tube (per milliliter) to optical densities at several dilutions. Beer’s law holds at all these dilutions. All
12
HYMAN
ROSEN
other amino acids give very similar results except proline and hydroxyproline. These compounds are imino acids (secondary amines) and give a different reaction product. The ninhydrin color yield of these compounds is much lower than the other amino acids, even when read at 440 rnp, which is their region of maximum absorption. Table I gives color yields of other amino acids based on leucine as 100%. The constancy of the color yields indicates that a mixt#ure of amino acids could be quantitatively analyzed in this way with reasonable accuracy. Many classes of compounds containing amino groups will, to a greater or lesser extent, react with ninhydrin. These include a-amino acids, imino acids, amino alcohols, primary amides, etc. In a biological mixture, such as plasma or tissue extract, only urea generally exists in concentrations high enough to interfere seriously with the analysis for a-amino acids. In urine, ammonium ion might also interfere. Figure 2 shows the effect of heating time on the color development of ninhydrin-urea. The color is so slight that protein-free plasma filtrates can be analyzed without prior removal of urea, provided that it is not unusually high. Analysis of urine requires prior removal of urea and preformed ammonia.
DILUENT
MICROMOLES
FIG.
LEUCINE
PER
TUBE
1. Ninhydrin-leucine color yields obtained on heating for 15 min. at 100°C. (570 mp).
COLORIMETRIc
.4NALPSIS
TABLE Per Cent Color
Yield
of Amino
FOR
AMINO
13
ACIDS
I
Compounds
Based on Leucine
= 100%
Color yield %
103 100 100 100 100 100 100 100 100 100 100 100 97 97 97 96 96 about 70
Lysine Isoleucine Histidine Ornithine Arginine Vsline Alanine Glycine Glutamine Glutamic acid Aspartic acid Taurine Threonine Serine blethionine Phenylalanine Tyrosine Ethanolamine Ammonia Proline Hydroxyproline
about
60
Read at 440 rnp Read at 440 rnp DISCUSSION
This method is intended primarily for the analysis of effluent fractions of individual amino acids from chromatographic columns. It can, however, be applied, with appropriate precautions and somewhat less precision, to amino acid mixtures. For example, protein-free normal plasma filtrates give values of about -I mg. % a-amino nitrogen. In the procedure itself, a number of precautions should be noted: 1. The two solutions, cyanide-buffer and ninhydrin-Cellosolve should not be mixed prior to the analysis. The resultant solution is unstable. 2. The reaction mixture, having been removed from the hot water bath should be immediately diluted wi-ith t.he water-isopropyl alcohol mixt,ure. Cooling at t,his point mill result in high blanks. Furthermore, the stream of diluent should be directed at the center of the test tube so as t,o entrain a maximum of air. 3. The diluted reaction mixtSures are now allowed to cool. Minimum blanks will be obtained only when these solutions reach room temperature.
HYMAN ROSEN
FIG. 2. Effect
of heating
TIME ( MINUTES) time on urea-ninhydrin (1OO”C., 570 mj.8).
color density
4. It is important to maintain the water-Cellosolve ratio as given (1: 1). Excess Cellosolve increases the blank value, while water tends to precipitate the ninhydrin reagent. If this method is applied to column chromatography, color densities of individual amino acids can be plotted. The areas enclosed by the resulting curves are a direct measure of the total amount of the pure amino acids. Integration can be accomplished easily with a planimeter such as Keuffel and Esser B 4236. The area under a curve, expressed in the arbitrary numbers given by the planimeter, is proportional to the amount of amino acid represented by the curve. This proportionality is converted to an equality by a constant, empirically derived from the planimeter reading and the units in which the results are desired to be expressed. ACKNOWLEDGMENTS The work of Dr. Sterling data, is much appreciated.
Chaykin,
who correlated
much
of the experimental
COLORIMETRIC
24NtlLYSIs
FOR AMINO
ACIDS
15
The advice and interest of Dr. Stanley M. Levenson are gratefully acknowledged. SUMMARY
A calorimetric method for the quantitative analysis of pure amino acids is described. It is a modification of one reported by Yemm and Cocking, which employs cyanide and ninhydrin. The present method avoids the necessity for the preparation of solutions of reduced ninhydrin, which is an unstable reagent difficult to prepare and impracticable to store. The method is applicable to amino acid mixtures when allowances are made for slight variability of color yields, and the presence of interfering compounds. REFERENCES STEIN, W. H., AND MOORE, S., J. Biol. Chem. 176, 337 (1948). MOORE, S., AND STEIN, W. H., J. Biol. Chem. 176, 367 (1948). TROLL, W., AND CANNAN, R. K., J. Biol. Chem. 200,803 (1953). MOORE, S., AND STEIN, W. H., J. Biol. Chem. 211,907 (1954). 5. YEI+~M, E. W., AND COCKING, E. C., Analyst 80, 209 (1955).
1. 2. 3. 4.
OF
BIOCBEMISTRY
AND
BIOPBYSICS
67, 10-15 (1957)
A ModSed Ninhydrin Calorimetric Analysis for Amino Acids Hyman Rosen From the Department of Surgical Metabolism, Division of Surgery, Walter Reed Army Institute of Research, Walter Reed Army Medical Center, Washington, D. C. Received July 25, 1956
INTRODUCTION In 1948, Stein and Moore published the first practical procedure for the chromatographic separation of amino acids on starch columns, and a photometric method for the quantitative analysis of the effluent fractions (1, 2). Their procedure was based on the reaction of amino acids with reduced ninhydrin (triketohydrindenhydrate) at pH 5; stannous chloride served as the reducing agent. The method is reliable and reproducible. This procedure is used almost exclusively in column chromatography, since the method is quantitative for individual amino acids. The very large number of analyses required for the completion of a chromatogram (about 600) make it important to use as efficient and automatic a procedure as possible. To this end, a number of modifications have appeared since the introduction of the method. Troll and Cannan (3) were able to increase the sensitivity slightly, but at the expense of introducing pyridine and phenol into the procedure. Moore and Stein (4) themselves dispensed with the stannous chloride by using reduced ninhydrin prepared with ascorbic acid. This procedure obviates precipitation of tin salts by phosphate buffers. A drawback in any method which uses solutions of reduced ninhydrin is the instability of this reagent in air. The solutions must either be stored and manipulated under nitrogen, or prepared fresh every few days. Yemm and Cocking (5) adapted the method of Troll and Cannan for routine use. Their procedure involves the preparation of cyanide-ninhydrin solutions, which are also unstable. The following is a modifica10
COLORIMETRIC
-4NhLYSIS
FOR AMINO
ACIDS
11
tion of the Yemm and Cocking method, adapted for use in column chromatography. It differs from the latter in that it does not require the prior preparation of an unstable ninhydrin reagent. Only two reagents are used, both stable indefinitely at room temperature. With t,he proper equipment, several hundred analyses can be performed by one worker in an hour. REaGENTS 1. Stock N&N: 0.01 M (490 mg./l.). 2. Acetate Bugler: 2700 g. NaOAc.3HsO + 2 1. H20 + 500 ml. glacial HOAc. Make up to 7.5 1. with H?O. This buffer is adapted from t,hat of Stein and Moore (4). Its pH should be 5.3-5.4. It is quite concentrated, and makes preliminary pH adjustment.s of samples, e.g., effluent fractions, usually unnecessary. In new procedures, however, it should be determined whether, in fact., t.he reaction mixture is at pH 5.0-5.2 before proceeding with the analysis. 3. Acetate-Cyanide: 0.0002 Y NaCN in acetate buffer; 20 ml. of Soln. 1 made to 1 1. with Soln. 2. 4. Ninhydrin: 3% in hIethyl Cellosolve (ethylene glycol monomethyl ether). The suitability of the Cellosolve should be checked by t.he procedure of Stein and Moore (2). Ninhydrin suitable for use without recrystallizing can be obt.ained from Dougherty Chemical Co., 87-34 134th St., Richmond Hill 18, N. Y. 5. Diluent: Isopropyl alcohol-water, 1:l. METHOD To a l-ml. sample containing 0.02-0.40 pmole of an amino acid, add aa ml. cyanide-acetate buffer and >s ml. 3yo ninhydrin solution in Methyl Cellosolve. Heat 15 min. in a 100’ water bath. Maximal color is developed after 10-12 min. Immediately after removing from the water bath, add rapidly 5 ml. of isopropyl alcohol-water diluent. Shake vigorously. Allow to cool to room temperature, and read in a calorimeter at 570 rnp (proline and hydroxyproline at 440 mp). If the color density in a given series of tubes is too high to determine accurately, further 5-ml. portions of diluent may be added, until the optical densities are below 0.8. RESULTS
All samples when removed from the water bath are deep red. The color fades rapidly when the mixture is shaken and/or cooled, and the resultant blank is colorless, or faint blue, in the isopropyl alcohol-water diluent. All amino acids, except proline and hydroxyproline, give a purple color with ninhydrin; the latter two give a yellow color. All colors are stable, fading only l-2 % in 24 hr. Figure 1 relates micromoles of leucine per tube (per milliliter) to optical densities at several dilutions. Beer’s law holds at all these dilutions. All
12
HYMAN
ROSEN
other amino acids give very similar results except proline and hydroxyproline. These compounds are imino acids (secondary amines) and give a different reaction product. The ninhydrin color yield of these compounds is much lower than the other amino acids, even when read at 440 rnp, which is their region of maximum absorption. Table I gives color yields of other amino acids based on leucine as 100%. The constancy of the color yields indicates that a mixt#ure of amino acids could be quantitatively analyzed in this way with reasonable accuracy. Many classes of compounds containing amino groups will, to a greater or lesser extent, react with ninhydrin. These include a-amino acids, imino acids, amino alcohols, primary amides, etc. In a biological mixture, such as plasma or tissue extract, only urea generally exists in concentrations high enough to interfere seriously with the analysis for a-amino acids. In urine, ammonium ion might also interfere. Figure 2 shows the effect of heating time on the color development of ninhydrin-urea. The color is so slight that protein-free plasma filtrates can be analyzed without prior removal of urea, provided that it is not unusually high. Analysis of urine requires prior removal of urea and preformed ammonia.
DILUENT
MICROMOLES
FIG.
LEUCINE
PER
TUBE
1. Ninhydrin-leucine color yields obtained on heating for 15 min. at 100°C. (570 mp).
COLORIMETRIc
.4NALPSIS
TABLE Per Cent Color
Yield
of Amino
FOR
AMINO
13
ACIDS
I
Compounds
Based on Leucine
= 100%
Color yield %
103 100 100 100 100 100 100 100 100 100 100 100 97 97 97 96 96 about 70
Lysine Isoleucine Histidine Ornithine Arginine Vsline Alanine Glycine Glutamine Glutamic acid Aspartic acid Taurine Threonine Serine blethionine Phenylalanine Tyrosine Ethanolamine Ammonia Proline Hydroxyproline
about
60
Read at 440 rnp Read at 440 rnp DISCUSSION
This method is intended primarily for the analysis of effluent fractions of individual amino acids from chromatographic columns. It can, however, be applied, with appropriate precautions and somewhat less precision, to amino acid mixtures. For example, protein-free normal plasma filtrates give values of about -I mg. % a-amino nitrogen. In the procedure itself, a number of precautions should be noted: 1. The two solutions, cyanide-buffer and ninhydrin-Cellosolve should not be mixed prior to the analysis. The resultant solution is unstable. 2. The reaction mixture, having been removed from the hot water bath should be immediately diluted wi-ith t.he water-isopropyl alcohol mixt,ure. Cooling at t,his point mill result in high blanks. Furthermore, the stream of diluent should be directed at the center of the test tube so as t,o entrain a maximum of air. 3. The diluted reaction mixtSures are now allowed to cool. Minimum blanks will be obtained only when these solutions reach room temperature.
HYMAN ROSEN
FIG. 2. Effect
of heating
TIME ( MINUTES) time on urea-ninhydrin (1OO”C., 570 mj.8).
color density
4. It is important to maintain the water-Cellosolve ratio as given (1: 1). Excess Cellosolve increases the blank value, while water tends to precipitate the ninhydrin reagent. If this method is applied to column chromatography, color densities of individual amino acids can be plotted. The areas enclosed by the resulting curves are a direct measure of the total amount of the pure amino acids. Integration can be accomplished easily with a planimeter such as Keuffel and Esser B 4236. The area under a curve, expressed in the arbitrary numbers given by the planimeter, is proportional to the amount of amino acid represented by the curve. This proportionality is converted to an equality by a constant, empirically derived from the planimeter reading and the units in which the results are desired to be expressed. ACKNOWLEDGMENTS The work of Dr. Sterling data, is much appreciated.
Chaykin,
who correlated
much
of the experimental
COLORIMETRIC
24NtlLYSIs
FOR AMINO
ACIDS
15
The advice and interest of Dr. Stanley M. Levenson are gratefully acknowledged. SUMMARY
A calorimetric method for the quantitative analysis of pure amino acids is described. It is a modification of one reported by Yemm and Cocking, which employs cyanide and ninhydrin. The present method avoids the necessity for the preparation of solutions of reduced ninhydrin, which is an unstable reagent difficult to prepare and impracticable to store. The method is applicable to amino acid mixtures when allowances are made for slight variability of color yields, and the presence of interfering compounds. REFERENCES STEIN, W. H., AND MOORE, S., J. Biol. Chem. 176, 337 (1948). MOORE, S., AND STEIN, W. H., J. Biol. Chem. 176, 367 (1948). TROLL, W., AND CANNAN, R. K., J. Biol. Chem. 200,803 (1953). MOORE, S., AND STEIN, W. H., J. Biol. Chem. 211,907 (1954). 5. YEI+~M, E. W., AND COCKING, E. C., Analyst 80, 209 (1955).
1. 2. 3. 4.