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Detection of fluorescamine-labeled amino acids, peptides, and other primary amines on thin-layer chromatograms
Detection and
of Fluorescamine-Labeled
Other
Primary
KAZUHIRO
IRIAI, AND
Roche Zrtslil~de
Amino
Acids,
Amines
on Thin-Layer
PETER SIDNEY
BOHLEN, STANLEY UDENFRIEND
of Molecular Received
Biology, October
Xutley,
Peptides,
Chromatograms STEIN,
Sew Jersey 07110
17, 1973
Amino acids, peptides, catecholamines, and polyamines were reacted with fluorescamine and slibjected to thin-layer chromatography. These fluorescamine derivatives of primary amines were detectable at levels below 100 pmoles. Methods of preparat,ion and rhromatography of these fluorophors are presented.
Fluorrscaminr has been introduced as a fluorogrnic reagent for the assay of primary amines (1, 2). A procedure has recently been drvcloped for detecting primary amines on thin-layer chromatograms by spraying with fluorescamine (3). The method involves prespraying and then postspraying with triethylaminc. hlthough the procedure is simple, sensitive, and generally useful, it dots not take full advantage of the inherent sensitivity of fluorcscaminc. The present report describes a diffcrcnt, approach for dctccting amino acids, peptides, catecholamines, and ot’her primary amincs on thinlayer plat’rs. Solutions containing primary aminrs arc t’reated with fluorescaminc and the resulting mixture of derivatives is analyzed by thin-layer chromat’ography. MATERIALS All the solvents were analytical grade. Amino acids, bradykinin tetraacetate, 3,4-dihydroxy-Lphenylalanine, 3-met,hoxytyramine, uL-normetanephrine, plltrescine, spermidine, and spermine were from Sigma (St. Louis, MO). L-Alanyl-Lalanine, glycyl-r,-proline, L-phenylalanylglycine, glycylglycyl-L-alanine, I,-tyrosyl-L-leucine, Lcarnosine, and dopamine hydrochloride were from Schwarz/Mann (Orangeburg, NY). L-Norepinephrine bitartrate was purchased from Calbiothem (Los Angeles, CA). Fluorescamine was obtained from Hoffmann-La Roche, Inc. (Nutley, NJ). Precoated silica gel plates (without fluorescent indicator) were from Brinkman (Westbury, NY). Siliclad was from Clay Adams (Parsippany, NJ).
Preparation of Fluorophors Method A. A solution containing 4-80 nanomoles of each amine in 0.1 M sodium borate, pH 9.0, was added to a 13 X loo-mm tube. With the tube held on a vortex mixer, 200 ~1 of fluorescamine in acetone (20 mg/lOO ml) was added at room temperature. One microliter containing 10, 20, 30, 50, 100, or 200 picomoles of each amine was spotted. For catecholamines, 100 ~1 of each amine in water was first mixed with 100 ~1 of fluorescamine in acetone (20 mg/lOO ml) and then with 200 11 of 0.1 M sodium bicarbonate, pH 8.3. With catecholamines the reaction was performed at 4°C. Method B. An aliquot of solution containing 20-200 pmoles of the amine was evaporated to dryness under nitrogen at 40°C in a 6 X 50-mm tube. Five microliters of 0.1 M sodium borate or sodium bicarbonate was added to the tube. With the tube held on a vortex mixer, 5 ~1 of fluorescamine in acetone (20 mg/lOO ml) was added from a Hamilton syringe. Five microliters of each solution was transferred with a Hamilton syringe to t,he thin-layer plates. Reaction tubes (6 X 50 mm) were siliconized with lyO siliclad prior to use.
Thin-Layer
Chromatography
Chromatography was performed on 10 X lo-cm or 10 X 20.cm glass plates of silica gel. The following solvent systems were used: A, isopropanol, chloroform, 28y0 ammonia, water (70:30:18:7); B, t/-butanol, acetic acid, water (1OO:l:lOO); C, ?I-butanol, acetic acid, water (4:l:l); D, benzene, dioxane, acetic acid (90:25:5). Chromatograms were developed in the dark for 30-90 min. After development, the plates were air-dried for a few 161
Copyright @ 1974by Academic Press, Inc. All rights of reproduction in any fnrrn reserven
METHODS
162
IMAI
minutes and then sprayed for 10 set with 10% triethanolamine in chloroform. The plates were then dried uuder a stream of cold air for a few minutes and sprayed again with 10% triethanolamine for 20 sec. The plates were observed under a long-wave (366 nm) ultraviolet light. RESULTS
AND
TABLE
FIG. 1. Thin-layer chromatography of amino acids prelabeled with fluorescamine. Aspartic acid, leucine, and arginine were spotted at varying concentrations in 1.0~~1 volumes. The mixture containing 50 pmoles of each of the three amino acids were spotted in a 5-~1 volume. The amounts of amino acid used were 20, 30, 50, 100, and 200 pmoles. The amino acid mixture was labeled by Method B, while each individual amino acid was labeled by Method A. Chromatography was performed in solvent system A.
~10.100~ GlY.PRO
LIO-100--l TYR-LEIJ
FIG. 2. Thin-layer chromatography of peptides prelabeled with fluorescamine. Fluorophors of L-alanyl-L-alanine (left), glycylproline (center), and L-tyrosyl-L-leucine (right) were spotted in 1.0~~1 volumes containing 10, 20, 30, 50, and 100 pmoles (from left to right). Method A was used for fluorophor preparation, and solvent system B was used for chromatography.
I
R, VALUES OF VARIOUS FLUORESCAMINE DERIVATIVES OF PRIMARY AMINES IN DIFFERENT SOLVENT SYSTEMS Solvent system” B
DISCUSSION
The limit of detection for amino acids, as well as peptides, catecholamines, and poly-
ALA-ALA
ET AL.
Alanylalanine Carnosine Glycylproline Phenylalanylglycine Tyrosylleucine Glycylglycylalanine Bradykinin 3,4-Dihydroxyphenylalanine Dopamine Norepinephrine 3-Methoxytyramine Normetanephrine Putrescine” Spermidine” Spermine*
0.22 0.38 0.19 0.09 0.87 0.45 0.07 0.73 0.34 0.07
D
-
0.07 0.28 0.15 0.52 0.35 -
a See Methods section for details. I A second minor spot appeared on the plate with each sample. This may be due to the presence of a small percentage of monosubstituted polyamine in addition to the disubstituted polyamine.
amines, is below 100 pmoles (Figs. 1 and 2). With many compounds as little as 10 pmoles could be detected (Figs. 1 and 2). This sensitivity is far better than the 250 to 500 picomole level achieved by spraying fluoressescamine on chromatograms of unlabeled amines. Two of the possible explanations for the lower sensitivity obtained by spraying plates with fluorescamine may be considered. One is that the reaction of fluorescamine with primary amines does not proceed as far to completion on surfaces as it does in solution under optimal conditions. The second explanation is that the background fluorescence of thin-layer chromatograms is greater when they are sprayed with fluorescamine. This background fluorescence would arise from the formation of fluorescamine derivatives of ammonia or primary amine contaminants in the silica gel, in the solvents used for chromatography or in the triethylamine solution used for prespraying. Several solvent systems have been used for chromatography. No single solvent system was found to be capable of completely re-
AMINE
DETECTION
solving complex fluorescamine-labeled amine mixtures. The data in Table I indicate that system B was suitable for chromatography of small peptides and polyamines and that system D was suitable for catecholamines. System C was useful for resolving larger peptides. Typically, two-dimensional chromatography is used for resolving complex mixtures, such as amino acids. Because of t,he possible light, heat, and acid lability of the fluorophors, it is advisable to use a basic solvent system for the first dimension and to dry the plates in vacua over a desiccant at room temperature overnight in t’he dark. It should be noted that reaction of chiral amines with fluorescamine gives rise to two diastereomeric fluorophors. Two wellresolved spots have been obtained on thinlayer chromatograms with fluorescamine derivatives of n-amino acids (4). However, with the solvent systems presented in this report,, this problem was not encountered. With both methods used for preparing fluorophors, the reaction proceeded rapidly under mild conditions. Method A was used for checking the feasibility of working with fluorescamine-labeled amines at the picomole level, A shortcoming of this procedure is that only a small portion of the sample could be applied to t’he plate. Method B provides for the transfer of most of the sample to the plate and is applicable in most cases. The sodium borate and the small amount of salt present in the sample solution do not interfere. Spraying the chromatograms with triethanolamine made the spots stable for several days when Dhe plates were stored in the dark. Triethanolamine, which has also been useful for stabilization of dansyl fluorophors (5), was found to be superior to triethylamine (3). The use of fluorescamine for prelabeling of primary amines offers distinct advantages over the commonly used dansyl procedure (5). The fluorescence properties and intensities of the fluorescamine and the dansyl fluorophors are comparable. One difficulty with dansylation arises from the presence of the fluorescent reaction byproducts, dansylacid and dansyl-amide. Usually these must be removed by tedious extraction procedures (5, 6). Dansyl chloride not only
ON CHROMATOGRAMS
163
reacts with the amino groups, but also reacts at other sites: such as the hydroxyl group of tyrosine or the imidazole nitrogen of histidine. Because of these side reactions, in many cases more than one fluorophor is produced from an amine. With fluorescamine, each amino acid, peptide, and catecholamine gave one product. A fluorescent spot, which has the same Rf as the ammonia derivative, can be seen in each sample in Fig. 1. This apparently arises from reaction of ammonia in solvent system A with excess fluorcscamine in the sample. Furthermore, dansylation of peptides or amino acids may take several hours as opposed to the fen- seconds required for t’he fluorescamint reaction. The purpose of this report is to demonstrate the feasibility of working with fluorescamine-labeled amines at the picomole level. Although methods of preparation and subsequent thin layer chromatography of these fluorophors have been presented, they are intended to be only preliminary procedures, which may be improved considerably. Current investigations in our laboratory include the separation of fluorescamine-labeled amines by high-efficiency column chromatography. Other separation techniques such as electrophoresis or paper chromatography should also be possible. BCKNOWLEDGMENTS The authors thank Dr. A. M. Felix for his interest and suggestions during the course of the work. Thanks are also due to Mr. R. Welborn for the photographic work. REFERENCES 1. WEIGELE, M., DEBERNARDO, S., TENGI, J. P., AND LEIMORUBER, W. (1972) J. Amer. Chem. Sot. 94, 5927-5928. 2. UDENFRIEND, S., STEIN, S., B~HLEN, P., DAIRMAN, W., LEIMGRUBER, W., AND WEIGELE, M., (1972) Science 178, 871-872. 3. FELIX, A. M., AND JIMENEZ, M. H., (1974) J. Chromatogr., in press. 4. WEIGELE, M., personal communication. 5. SEILER, N. (1970) Methods Biochem. Anal. 18, 259-337. 6. TAMURA, Z., NAKAJIMA, T., NAKAYAMA, T., PIS.4N0, J. J., AND UDENFRIEND, S. (1973) il?laZ. Biochem. 62, 595406.
of Fluorescamine-Labeled
Other
Primary
KAZUHIRO
IRIAI, AND
Roche Zrtslil~de
Amino
Acids,
Amines
on Thin-Layer
PETER SIDNEY
BOHLEN, STANLEY UDENFRIEND
of Molecular Received
Biology, October
Xutley,
Peptides,
Chromatograms STEIN,
Sew Jersey 07110
17, 1973
Amino acids, peptides, catecholamines, and polyamines were reacted with fluorescamine and slibjected to thin-layer chromatography. These fluorescamine derivatives of primary amines were detectable at levels below 100 pmoles. Methods of preparat,ion and rhromatography of these fluorophors are presented.
Fluorrscaminr has been introduced as a fluorogrnic reagent for the assay of primary amines (1, 2). A procedure has recently been drvcloped for detecting primary amines on thin-layer chromatograms by spraying with fluorescamine (3). The method involves prespraying and then postspraying with triethylaminc. hlthough the procedure is simple, sensitive, and generally useful, it dots not take full advantage of the inherent sensitivity of fluorcscaminc. The present report describes a diffcrcnt, approach for dctccting amino acids, peptides, catecholamines, and ot’her primary amincs on thinlayer plat’rs. Solutions containing primary aminrs arc t’reated with fluorescaminc and the resulting mixture of derivatives is analyzed by thin-layer chromat’ography. MATERIALS All the solvents were analytical grade. Amino acids, bradykinin tetraacetate, 3,4-dihydroxy-Lphenylalanine, 3-met,hoxytyramine, uL-normetanephrine, plltrescine, spermidine, and spermine were from Sigma (St. Louis, MO). L-Alanyl-Lalanine, glycyl-r,-proline, L-phenylalanylglycine, glycylglycyl-L-alanine, I,-tyrosyl-L-leucine, Lcarnosine, and dopamine hydrochloride were from Schwarz/Mann (Orangeburg, NY). L-Norepinephrine bitartrate was purchased from Calbiothem (Los Angeles, CA). Fluorescamine was obtained from Hoffmann-La Roche, Inc. (Nutley, NJ). Precoated silica gel plates (without fluorescent indicator) were from Brinkman (Westbury, NY). Siliclad was from Clay Adams (Parsippany, NJ).
Preparation of Fluorophors Method A. A solution containing 4-80 nanomoles of each amine in 0.1 M sodium borate, pH 9.0, was added to a 13 X loo-mm tube. With the tube held on a vortex mixer, 200 ~1 of fluorescamine in acetone (20 mg/lOO ml) was added at room temperature. One microliter containing 10, 20, 30, 50, 100, or 200 picomoles of each amine was spotted. For catecholamines, 100 ~1 of each amine in water was first mixed with 100 ~1 of fluorescamine in acetone (20 mg/lOO ml) and then with 200 11 of 0.1 M sodium bicarbonate, pH 8.3. With catecholamines the reaction was performed at 4°C. Method B. An aliquot of solution containing 20-200 pmoles of the amine was evaporated to dryness under nitrogen at 40°C in a 6 X 50-mm tube. Five microliters of 0.1 M sodium borate or sodium bicarbonate was added to the tube. With the tube held on a vortex mixer, 5 ~1 of fluorescamine in acetone (20 mg/lOO ml) was added from a Hamilton syringe. Five microliters of each solution was transferred with a Hamilton syringe to t,he thin-layer plates. Reaction tubes (6 X 50 mm) were siliconized with lyO siliclad prior to use.
Thin-Layer
Chromatography
Chromatography was performed on 10 X lo-cm or 10 X 20.cm glass plates of silica gel. The following solvent systems were used: A, isopropanol, chloroform, 28y0 ammonia, water (70:30:18:7); B, t/-butanol, acetic acid, water (1OO:l:lOO); C, ?I-butanol, acetic acid, water (4:l:l); D, benzene, dioxane, acetic acid (90:25:5). Chromatograms were developed in the dark for 30-90 min. After development, the plates were air-dried for a few 161
Copyright @ 1974by Academic Press, Inc. All rights of reproduction in any fnrrn reserven
METHODS
162
IMAI
minutes and then sprayed for 10 set with 10% triethanolamine in chloroform. The plates were then dried uuder a stream of cold air for a few minutes and sprayed again with 10% triethanolamine for 20 sec. The plates were observed under a long-wave (366 nm) ultraviolet light. RESULTS
AND
TABLE
FIG. 1. Thin-layer chromatography of amino acids prelabeled with fluorescamine. Aspartic acid, leucine, and arginine were spotted at varying concentrations in 1.0~~1 volumes. The mixture containing 50 pmoles of each of the three amino acids were spotted in a 5-~1 volume. The amounts of amino acid used were 20, 30, 50, 100, and 200 pmoles. The amino acid mixture was labeled by Method B, while each individual amino acid was labeled by Method A. Chromatography was performed in solvent system A.
~10.100~ GlY.PRO
LIO-100--l TYR-LEIJ
FIG. 2. Thin-layer chromatography of peptides prelabeled with fluorescamine. Fluorophors of L-alanyl-L-alanine (left), glycylproline (center), and L-tyrosyl-L-leucine (right) were spotted in 1.0~~1 volumes containing 10, 20, 30, 50, and 100 pmoles (from left to right). Method A was used for fluorophor preparation, and solvent system B was used for chromatography.
I
R, VALUES OF VARIOUS FLUORESCAMINE DERIVATIVES OF PRIMARY AMINES IN DIFFERENT SOLVENT SYSTEMS Solvent system” B
DISCUSSION
The limit of detection for amino acids, as well as peptides, catecholamines, and poly-
ALA-ALA
ET AL.
Alanylalanine Carnosine Glycylproline Phenylalanylglycine Tyrosylleucine Glycylglycylalanine Bradykinin 3,4-Dihydroxyphenylalanine Dopamine Norepinephrine 3-Methoxytyramine Normetanephrine Putrescine” Spermidine” Spermine*
0.22 0.38 0.19 0.09 0.87 0.45 0.07 0.73 0.34 0.07
D
-
0.07 0.28 0.15 0.52 0.35 -
a See Methods section for details. I A second minor spot appeared on the plate with each sample. This may be due to the presence of a small percentage of monosubstituted polyamine in addition to the disubstituted polyamine.
amines, is below 100 pmoles (Figs. 1 and 2). With many compounds as little as 10 pmoles could be detected (Figs. 1 and 2). This sensitivity is far better than the 250 to 500 picomole level achieved by spraying fluoressescamine on chromatograms of unlabeled amines. Two of the possible explanations for the lower sensitivity obtained by spraying plates with fluorescamine may be considered. One is that the reaction of fluorescamine with primary amines does not proceed as far to completion on surfaces as it does in solution under optimal conditions. The second explanation is that the background fluorescence of thin-layer chromatograms is greater when they are sprayed with fluorescamine. This background fluorescence would arise from the formation of fluorescamine derivatives of ammonia or primary amine contaminants in the silica gel, in the solvents used for chromatography or in the triethylamine solution used for prespraying. Several solvent systems have been used for chromatography. No single solvent system was found to be capable of completely re-
AMINE
DETECTION
solving complex fluorescamine-labeled amine mixtures. The data in Table I indicate that system B was suitable for chromatography of small peptides and polyamines and that system D was suitable for catecholamines. System C was useful for resolving larger peptides. Typically, two-dimensional chromatography is used for resolving complex mixtures, such as amino acids. Because of t,he possible light, heat, and acid lability of the fluorophors, it is advisable to use a basic solvent system for the first dimension and to dry the plates in vacua over a desiccant at room temperature overnight in t’he dark. It should be noted that reaction of chiral amines with fluorescamine gives rise to two diastereomeric fluorophors. Two wellresolved spots have been obtained on thinlayer chromatograms with fluorescamine derivatives of n-amino acids (4). However, with the solvent systems presented in this report,, this problem was not encountered. With both methods used for preparing fluorophors, the reaction proceeded rapidly under mild conditions. Method A was used for checking the feasibility of working with fluorescamine-labeled amines at the picomole level, A shortcoming of this procedure is that only a small portion of the sample could be applied to t’he plate. Method B provides for the transfer of most of the sample to the plate and is applicable in most cases. The sodium borate and the small amount of salt present in the sample solution do not interfere. Spraying the chromatograms with triethanolamine made the spots stable for several days when Dhe plates were stored in the dark. Triethanolamine, which has also been useful for stabilization of dansyl fluorophors (5), was found to be superior to triethylamine (3). The use of fluorescamine for prelabeling of primary amines offers distinct advantages over the commonly used dansyl procedure (5). The fluorescence properties and intensities of the fluorescamine and the dansyl fluorophors are comparable. One difficulty with dansylation arises from the presence of the fluorescent reaction byproducts, dansylacid and dansyl-amide. Usually these must be removed by tedious extraction procedures (5, 6). Dansyl chloride not only
ON CHROMATOGRAMS
163
reacts with the amino groups, but also reacts at other sites: such as the hydroxyl group of tyrosine or the imidazole nitrogen of histidine. Because of these side reactions, in many cases more than one fluorophor is produced from an amine. With fluorescamine, each amino acid, peptide, and catecholamine gave one product. A fluorescent spot, which has the same Rf as the ammonia derivative, can be seen in each sample in Fig. 1. This apparently arises from reaction of ammonia in solvent system A with excess fluorcscamine in the sample. Furthermore, dansylation of peptides or amino acids may take several hours as opposed to the fen- seconds required for t’he fluorescamint reaction. The purpose of this report is to demonstrate the feasibility of working with fluorescamine-labeled amines at the picomole level. Although methods of preparation and subsequent thin layer chromatography of these fluorophors have been presented, they are intended to be only preliminary procedures, which may be improved considerably. Current investigations in our laboratory include the separation of fluorescamine-labeled amines by high-efficiency column chromatography. Other separation techniques such as electrophoresis or paper chromatography should also be possible. BCKNOWLEDGMENTS The authors thank Dr. A. M. Felix for his interest and suggestions during the course of the work. Thanks are also due to Mr. R. Welborn for the photographic work. REFERENCES 1. WEIGELE, M., DEBERNARDO, S., TENGI, J. P., AND LEIMORUBER, W. (1972) J. Amer. Chem. Sot. 94, 5927-5928. 2. UDENFRIEND, S., STEIN, S., B~HLEN, P., DAIRMAN, W., LEIMGRUBER, W., AND WEIGELE, M., (1972) Science 178, 871-872. 3. FELIX, A. M., AND JIMENEZ, M. H., (1974) J. Chromatogr., in press. 4. WEIGELE, M., personal communication. 5. SEILER, N. (1970) Methods Biochem. Anal. 18, 259-337. 6. TAMURA, Z., NAKAJIMA, T., NAKAYAMA, T., PIS.4N0, J. J., AND UDENFRIEND, S. (1973) il?laZ. Biochem. 62, 595406.