ANALYTICAL
BIOCHEMISTRY
72.473-479
(1976)
Fluorometric Detection of Peptides after Column Chromatography or on Paper: o-Phthalaldehyde and Fluorescamine E. Roche
Institute
Received
AND J. G. GAVILANES'
MENDEZ yf‘Molecrdrrr
December
Biology,
Nutlry.
1, 1975; accepted
New
January
Jersey
071 IO
8. 1976
The fluorogenic reagents, o-phthalaldehyde and fluorescamine. have been used for procedures to detect peptides on fractions collected during column chromatographies. Maximal fluorescence was obtained with both reagents after alkaline hydrolysis of the peptides. Comparisons of the fluorescence obtained with ophthalaldehyde and fluorescamine in aqueous solutions and on paper chromatography have been studied.
INTRODUCTION
Until several years ago, ninhydrin was essentially the only reagent used for the detection of amino acids and peptides with a sensitivity in the nanomol level. Fluorescamine was recently introduced as a reagent which reacts with primary amines in the picomol range (l-3), and a number of applications of this reagent has been described (4- 16). Another reagent, o-phthalaldehyde (OPA) has been used for many years in various fluorescent analytical reactions (17- 19). More recently it has been shown to react with primary amines, in the presence of 2-mercaptoethanol to yield highly fluorescent products (20-2 1). Application of OPA to the automated assay of amino acids at the picomol level has also been reported (22). We report procedures to detect peptides with OPA after column chromatography, either directly or after alkaline hydrolysis of the peptides, and for the detection of amino acids and peptides on paper chromatography. Comparisons are also made with fluorescamine. MATERIALS
AND METHODS
Fluorescamine was provided by the Chemical Research Division of Hoffman-La Roche, Inc.; o-phthalaldehyde was obtained from Sigma Chemical Company; boric acid from Mallinckrodt; 2-mercaptoethanol and triethylamine from Eastman Organic Chemicals. The water purifica1 Departamento
de Bioquimica.
Universidad
de Madrid, 473
Copyright 0 1976 by Academic Press. Inc. All rights of reproduction in any form reserved.
Spain.
474
MENDEZ
tion systems were from Hydro Carolina.
AND
GAVILANES
Service and Supplies,
Durham,
North
Direct Assay with Fluorescamine or o-Phthalaldehyde. Aliquots from the columns were placed in borosilicate culture tubes (13 x 100 mm) Corning Glass Works and dried at 110°C. For assay with fluorescamine, 1.6 ml of 0.5 M sodium borate pH 8.5 was added to each tube. While the tube was vigorously shaken on a vortex type mixer, two 0.2 ml aliquots of fluorescamine (0.3 mg/ml in acetone) were added and mixed for IO- 15 sec. For assay with o-phthalaldehyde, 1.6 ml of 0.5111or 0.25 M sodium borate pH 10, containing 0.05% mercaptoethanol was added to each tube and then 0.4 ml of o-phthalaldehyde, (0.3 mg/ml in water) was added and mixed for 2-3 sec. Measurements were carried out in the same tubes in an Aminco-Bowman Spectrofluorometer (American Instrument Company) with excitation at 390 nm and emission at 475 nm for fluorescamine and excitation at 340 and emission at 455 nm for o-phthalaldehyde. Typically, 30 min or more elapsed before fluorescence was measured. Alkaline
Hydrolysis
After drying the aliquots from the column, 0.2 ml of 0.5 M NaOH (11) was added to each tube and hydrolysis was carried out at 120°C in an autoclave for 30 min. After cooling, 0.2 ml of 0.5 M HCl and 1.2 ml of 0.5 M sodium borate, pH 8.5 or 0.25 M sodium borate, pH 10.0 containing 0.05% mercaptoethanol was added. followed by the additions of fluorescamine or o-phthalaldehyde, as described above. Detection
of Amino Acids and Peptides on Paper
The detection of amino acids and peptides on paper chromatograms with fluorescamine has been described (12). The procedure for detection with o-phthalaldehyde on paper was as follows. After electrophoresis or chromatography the paper was dried at 50°C for 1 hr. The paper was washed with acetone and it was then dipped in a tray containing a solution of 1% triethylamine and 0.05% 2-mercaptoethanol in acetone. After 5 min at room temperature, the paper was dipped in a tray containing a solution of OPA, 0.3 mg/ml in acetone and allowed to dry for 5 min at room temperature. Finally the paper was washed with acetone and dried. The fluorescent spots were detected under a long wave (366 nm) ultraviolet lamp. Photographs were taken as described elsewhere (12). RESULTS
AND DISCUSSION
In order to establish the optimal conditions for a simple manual procedure of OPA assay, we reinvestigated the conditions previously
FLUOROMETRIC
DETECTION
475
OF PEPTIDES
p loo ! 50+G--uy; F s
-
2
RUIAMACEl OCA-ACE1 I
0
50
FLUINbEl-OH / _ ‘
1 I I!50
12 loo
TIME
x I
200’
. I
24hr
(minutor)
FIG. 1. Stability of the amino acids labeled with OPA or fluorescamine at room temperature. Reaction with fluorescamine or OPA was under the conditions described in Methods, except that the fluorogenic reagents were dissolved in the solvents indicated on the graph. A protein hydrolysate calibration mixture, giving a final concentration of 2.5 nmol/ml of each amino acid, was used.
described (2 1). Although individual amino acids have different pH maxima for the reaction between pH 8.0 and 11 .O, except for Lys, which has its maximum at pH 6.0 to 7.0 (21). We chose pH 10.0 as the best compromise for OPA assay. The OPA reaction was not significantly influenced when the mercaptoethanol concentration was varied from 0.05 to l%, nor when the borate concentration was varied from 0.1 to 0.5 M. Maximal fluorescence was found 5 min after mixing the reagent (21) and the fluorophors were stable for hours (Fig. 1) at room temperature. The fluorescamine reaction was carried out at pH 8.5 according to the conditions previously described (4). As shown in Fig. 1, maximal fluorescence was obtained for OPA when the reagent was dissolved in ethanol or in water. Acetone was found to be a very poor solvent for OPA, while the reverse was found for fluorescamine. For the manual procedure, OPA was dissolved in water rather than ethanol because the reagent is relatively stable in the former (at least for several months) and also to avoid salt precipitation by the organic solvent. Additionally, with the typically large number of fractions collected in the column chromatographies, mixing two aqueous solutions is less time-consuming than mixing organic solvent with water. Although OPA, in the presence of 2-mercaptoethanol, reacts poorly with peptides (23), the reverse was found with amino acids (Fig. 1); OPA with amino acids yields higher fluorescence than with fluorescamine. It has previously been. demonstrated with ninhydrin (24) and with fluorescamine (11) that peptides collected after column chromatography should be hydrolyzed with alkali prior to the fluorogenic reaction. This is because peptide bond hydrolysis increases several-fold the number
476
MENDEZ
AND GAVILANES
of primary amino groups available for reaction. Furthermore, some peptides such as those having proline, hydroxyproline or blocked amino acid at the N-terminal, yield no fluorescent products unless they are hydrolyzed. Although the reaction with fluorescamine yielded more fluorescence with the intact peptides, after alkaline hydrolysis of the peptides OPA yielded approximately twice as much fluorescence than fluorescamine (Fig. 2). However, from the experiments shown in Figs. 1 and 2, it is incorrect to assume which reagent has greater sensitivity. The sensitivity of an analytical procedure is, by definition, the lowest quantity of sample that can be measured and this can only be determined as one approaches the limits of detection. Sensitivity is best represented by the ratio of sample fluorescence to blank fluorescence. In one typical experiment, individual tubes containing 5 nmol of leucine, as well as blanks, were carried through the alkaline hydrolysis procedure. The leucine samples yielded four times higher fluorescence on reaction with OPA than with fluorescamine. However, the blanks were also four times higher with OPA than with fluorescamine. Since standard deviations as high as 6.9% are commonly obtained at these low levels, fluorescence values several times higher than blank are required for accuracy. With leucine, for example, 2.5 nmol/ml produced a fivefold increase over blank when assayed by either reagent. A higher sample to blank fluorescence ratio was achieved
o-CHTHALALDEWVDE
FlUOlESCAMlW
-After
*--
hydmlysh Bdm hydmtysb
TUBE
NUMBER
FIG. 2. Chromatography of a tryptic-peptic digest of human (K) Bence Jones protein. The hydrolyzate (10 mg) was prepared as previously described (12) and applied to a Sephadex G-25-F column (2 x 90 cm), equilibrated with 0.1 M NH,HCO,, pH 8.5. The column was eluted with 0.1 M NH,HCO,, pH 8.5, at a flow rate of 19 ml/hr and fractions were collected every 7 min. Aliquots ofO.1 ml were used for reaction with fluorescamine (A) and o-phthalaldehyde (B) either by direct fluorometric assay (a-++ ) or after alkaline hydrolysis ( ) (see Methods).
FLUOROMETRIC
DETECTION
WA
F
A
B
OF PEPTIDES
F
OFA
c
D
477
FIG. 3. Ultraviolet-photography showing the fluorescent spots from amino acids and peptides detected on paper with fluorescamine or o-phthalaldehyde. Amino acids (500 picomol each) were spotted on paper and stained with (A) o-phthalaldehyde or (B) fluorescamine as described in Methods. A tryptic digest of cholera toxin (5 mol of each peptide) detected with fluorescamine (C) or OPA (D) after electrophoresis at pH 3.5.
for aspartic acid with OPA. On the other hand, a higher sample to blank fluorescence ratio was achieved for arginine with fluorescamine. Thus, under the conditions used in these experiments, OPA is a more sensitive reagent for aspartic, fluorescamine is a more sensitive reagent for arginine, and equivalent sensitivities are obtained for leucine with both reagents. Identical sample and blank fluorescence values were obtained with both Sigma and Durrum OPA. Since the fluorescence of the blank is the prime factor limiting sensitivity, suitable precautions must be taken to avoid solvent contamination by amines and other reactive compounds. For these studies, a water purification system, producing water with a resistance greater than 4 x lo5 ohms, was utilized. The distilled water was filtered to eliminate microorganisms,
478
MENDEZ
AND GAVILANES
twice deionized and filtered to eliminate organic compounds. However, distilled or deionized water from several sources has, at times, introduced high blanks with OPA, but not with fluorescamine. Borate buffers, prepared from boric acid obtained from several commercial sources, also introduced high fluorescence blanks with OPA, but not with fluorescamine (the lowest background with OPA was obtained with boric acid from Mallinckrodt). Ammonium ions, which yield poor fluorescence with fluorescamine, might be the major contaminating species. However, the ammonium bicarbonate used for the column chromatography presented in Fig. 2 is removed during the drying step (see Methods). Pyridine acetate, another volatile buffer, is also satisfactory. For detection of amino acids and peptides on paper chromatograms, conditions similar to those employed for fluorescamine (12) were found to be optimal. As Fig. 3 illustrates, amino acids are more readily visualized at the 500 pmole level with OPA than with fluorescamine, while peptides are more easily discernible with fluorescamine. Whereas the fluorescent spots produced by fluorescamine remained visible for several weeks, the spots produced by OPA disappeared after several hours. Varying the concentration of mercaptoethanol did not enhance the fluorescence intensity nor the stability of the spots produced with OPA. Additionally, a greater sensitivity for detection of peptides on thin layer chromatograms was found with fluorescamine. In conclusion, it may be stated that OPA is a useful reagent for amino acid and peptide detection, provided that the proper precautions are taken. It is hoped that this report will dispel any confusion about the inherent sensitivity of these two reagents. Proper comparisons between these two reagents in flow systems remain to be performed. The most important point is that the introduction of fluorescent techniques, with these as well as other fluorogenic reagents, has advanced the field of peptide biochemistry. ACKNOWLEDGMENTS We wish to thank Drs. S. Udenfriend, S. Stein, and 0. Tsolas for their valuable discussions, Mr. R. Welbom for his excellent technical assistance in the photographic recordings, and Mrs. A. Dugan for secretarial work.
REFERENCES 1. Weigeie, M., Blount, J. F., Tengi, J. P., Czaijkowki, (1972)
J. Amer.
Chem.
2. Weigele, M., DeBernardo. Chem.
Sot.
Sot.
R. C., and Leimgruber,
W.
94, 4052.
S. L., Tengi, J. P., and Leimgruber,
W. (1972) J. Amer.
94, 5927.
3. Udenfriend, S., Stein, S., Bohlen, P., and Dairman, W. (1972) in Third American Peptide Symposium, Boston (Meinhofer, J.. ed.), pp. 655-663, Ann ArborHumphery Science, Ann Arbor, Michigan.
FLUOROMETRlC 4. Udenfriend, (1972)
DETECTION
OF PEPTIDES
S., Stein, S., Bbhlen, P., Dairman, W.. Leimgruber,
Science
479 W., and Weigele, M.
178, 871-872.
5. Biihlen, P., Stein, S., Dairman, W., and Udenfriend, S. (1973) Arch. Biochem. Biophys. 155, 213-220. 6. Felix, A., and Jimenez, M. H. (1974) J. Chromutogr. 89, 361. 7. Imai, K., Bohlen, P., Stein, S.. and Udenfriend, S. (1974) Arch. Biochem. Biophys. 161,
161-
170.
8. Pace, J. L.. Kemper, D. L., and Ragland, W. L. (1974) Biochem. Biophys. Res. Common. 57, 482-487. 9. Vandekerckhove, J., and Van Montagu, M. (1974) Eltr. J. Biochem. 44, 279-288. IO. Stein, S., Chang, C. H., Bohlen, P.. Imai, K., and Udenfriend. S. (1974) Anctl. Biochem. 60, 272-277. Il. Nakai, N.. Lai, C. Y., and Horecker, B. L. (1974)J. Anal. Biochem. 58, 563-575. 12. Mendez. E., and Lai, C. Y. (1975)Anal. Biochem. 65, 281-292. 13. Felix, A., Jimenez, H., Vergona, R., and Cohen, M. (1974) Int. J. Peptide Protein Res. 7, 11-22. 14. Bbhlen, P., Stein, S., Stone, J. and Udenfriend, S. (1975)Am7/. Biochem. 67,438-445. 15. Stein, S., Bohlen, P., Stone, J., Dairman, W. and Udenfriend, S. (1973)Arch. Biochem. Biophys. 155, 202-212. 16. Stein, S., Bohlen, P. and Udenfriend, S. (1975)Arch. Bioclrem. Biophys. 163,400-403. 17. Cohn, V. H. and Lyle (1966) Anal. Biochem. 14, 434. 18. Shore, P. A., Burkhalter, A.. and Cohn, V. H., Jr. (1959) /. Ph~rmaco/. Expr. Therap. 127, 182. 19. Hakanson, T., Ronnberg, A. L., and Sjolund, K. (1974)Anal. Biochem. 59, 98-109. 20. Roth, M. (1971) Anal. Biochem. 43, 880-882. 21. Roth, M.. and Hampai, A. (1973)./. Chromatogr. 83, 353-356. 22. Benson, J. and Hare, P. (1974) Proc. Nut. Acud. Sci. 72, 619-622. 23. Taylor, S. and Tappel. A. (1973) Anal. Biochem. 56, 140-148. 24. Hits, C. H. W. (1%7) in Methods in Enzymology (Hirs, C. H. W., ed.), Vol. I I. pp. 325-329. Academic Press, New York.
BIOCHEMISTRY
72.473-479
(1976)
Fluorometric Detection of Peptides after Column Chromatography or on Paper: o-Phthalaldehyde and Fluorescamine E. Roche
Institute
Received
AND J. G. GAVILANES'
MENDEZ yf‘Molecrdrrr
December
Biology,
Nutlry.
1, 1975; accepted
New
January
Jersey
071 IO
8. 1976
The fluorogenic reagents, o-phthalaldehyde and fluorescamine. have been used for procedures to detect peptides on fractions collected during column chromatographies. Maximal fluorescence was obtained with both reagents after alkaline hydrolysis of the peptides. Comparisons of the fluorescence obtained with ophthalaldehyde and fluorescamine in aqueous solutions and on paper chromatography have been studied.
INTRODUCTION
Until several years ago, ninhydrin was essentially the only reagent used for the detection of amino acids and peptides with a sensitivity in the nanomol level. Fluorescamine was recently introduced as a reagent which reacts with primary amines in the picomol range (l-3), and a number of applications of this reagent has been described (4- 16). Another reagent, o-phthalaldehyde (OPA) has been used for many years in various fluorescent analytical reactions (17- 19). More recently it has been shown to react with primary amines, in the presence of 2-mercaptoethanol to yield highly fluorescent products (20-2 1). Application of OPA to the automated assay of amino acids at the picomol level has also been reported (22). We report procedures to detect peptides with OPA after column chromatography, either directly or after alkaline hydrolysis of the peptides, and for the detection of amino acids and peptides on paper chromatography. Comparisons are also made with fluorescamine. MATERIALS
AND METHODS
Fluorescamine was provided by the Chemical Research Division of Hoffman-La Roche, Inc.; o-phthalaldehyde was obtained from Sigma Chemical Company; boric acid from Mallinckrodt; 2-mercaptoethanol and triethylamine from Eastman Organic Chemicals. The water purifica1 Departamento
de Bioquimica.
Universidad
de Madrid, 473
Copyright 0 1976 by Academic Press. Inc. All rights of reproduction in any form reserved.
Spain.
474
MENDEZ
tion systems were from Hydro Carolina.
AND
GAVILANES
Service and Supplies,
Durham,
North
Direct Assay with Fluorescamine or o-Phthalaldehyde. Aliquots from the columns were placed in borosilicate culture tubes (13 x 100 mm) Corning Glass Works and dried at 110°C. For assay with fluorescamine, 1.6 ml of 0.5 M sodium borate pH 8.5 was added to each tube. While the tube was vigorously shaken on a vortex type mixer, two 0.2 ml aliquots of fluorescamine (0.3 mg/ml in acetone) were added and mixed for IO- 15 sec. For assay with o-phthalaldehyde, 1.6 ml of 0.5111or 0.25 M sodium borate pH 10, containing 0.05% mercaptoethanol was added to each tube and then 0.4 ml of o-phthalaldehyde, (0.3 mg/ml in water) was added and mixed for 2-3 sec. Measurements were carried out in the same tubes in an Aminco-Bowman Spectrofluorometer (American Instrument Company) with excitation at 390 nm and emission at 475 nm for fluorescamine and excitation at 340 and emission at 455 nm for o-phthalaldehyde. Typically, 30 min or more elapsed before fluorescence was measured. Alkaline
Hydrolysis
After drying the aliquots from the column, 0.2 ml of 0.5 M NaOH (11) was added to each tube and hydrolysis was carried out at 120°C in an autoclave for 30 min. After cooling, 0.2 ml of 0.5 M HCl and 1.2 ml of 0.5 M sodium borate, pH 8.5 or 0.25 M sodium borate, pH 10.0 containing 0.05% mercaptoethanol was added. followed by the additions of fluorescamine or o-phthalaldehyde, as described above. Detection
of Amino Acids and Peptides on Paper
The detection of amino acids and peptides on paper chromatograms with fluorescamine has been described (12). The procedure for detection with o-phthalaldehyde on paper was as follows. After electrophoresis or chromatography the paper was dried at 50°C for 1 hr. The paper was washed with acetone and it was then dipped in a tray containing a solution of 1% triethylamine and 0.05% 2-mercaptoethanol in acetone. After 5 min at room temperature, the paper was dipped in a tray containing a solution of OPA, 0.3 mg/ml in acetone and allowed to dry for 5 min at room temperature. Finally the paper was washed with acetone and dried. The fluorescent spots were detected under a long wave (366 nm) ultraviolet lamp. Photographs were taken as described elsewhere (12). RESULTS
AND DISCUSSION
In order to establish the optimal conditions for a simple manual procedure of OPA assay, we reinvestigated the conditions previously
FLUOROMETRIC
DETECTION
475
OF PEPTIDES
p loo ! 50+G--uy; F s
-
2
RUIAMACEl OCA-ACE1 I
0
50
FLUINbEl-OH / _ ‘
1 I I!50
12 loo
TIME
x I
200’
. I
24hr
(minutor)
FIG. 1. Stability of the amino acids labeled with OPA or fluorescamine at room temperature. Reaction with fluorescamine or OPA was under the conditions described in Methods, except that the fluorogenic reagents were dissolved in the solvents indicated on the graph. A protein hydrolysate calibration mixture, giving a final concentration of 2.5 nmol/ml of each amino acid, was used.
described (2 1). Although individual amino acids have different pH maxima for the reaction between pH 8.0 and 11 .O, except for Lys, which has its maximum at pH 6.0 to 7.0 (21). We chose pH 10.0 as the best compromise for OPA assay. The OPA reaction was not significantly influenced when the mercaptoethanol concentration was varied from 0.05 to l%, nor when the borate concentration was varied from 0.1 to 0.5 M. Maximal fluorescence was found 5 min after mixing the reagent (21) and the fluorophors were stable for hours (Fig. 1) at room temperature. The fluorescamine reaction was carried out at pH 8.5 according to the conditions previously described (4). As shown in Fig. 1, maximal fluorescence was obtained for OPA when the reagent was dissolved in ethanol or in water. Acetone was found to be a very poor solvent for OPA, while the reverse was found for fluorescamine. For the manual procedure, OPA was dissolved in water rather than ethanol because the reagent is relatively stable in the former (at least for several months) and also to avoid salt precipitation by the organic solvent. Additionally, with the typically large number of fractions collected in the column chromatographies, mixing two aqueous solutions is less time-consuming than mixing organic solvent with water. Although OPA, in the presence of 2-mercaptoethanol, reacts poorly with peptides (23), the reverse was found with amino acids (Fig. 1); OPA with amino acids yields higher fluorescence than with fluorescamine. It has previously been. demonstrated with ninhydrin (24) and with fluorescamine (11) that peptides collected after column chromatography should be hydrolyzed with alkali prior to the fluorogenic reaction. This is because peptide bond hydrolysis increases several-fold the number
476
MENDEZ
AND GAVILANES
of primary amino groups available for reaction. Furthermore, some peptides such as those having proline, hydroxyproline or blocked amino acid at the N-terminal, yield no fluorescent products unless they are hydrolyzed. Although the reaction with fluorescamine yielded more fluorescence with the intact peptides, after alkaline hydrolysis of the peptides OPA yielded approximately twice as much fluorescence than fluorescamine (Fig. 2). However, from the experiments shown in Figs. 1 and 2, it is incorrect to assume which reagent has greater sensitivity. The sensitivity of an analytical procedure is, by definition, the lowest quantity of sample that can be measured and this can only be determined as one approaches the limits of detection. Sensitivity is best represented by the ratio of sample fluorescence to blank fluorescence. In one typical experiment, individual tubes containing 5 nmol of leucine, as well as blanks, were carried through the alkaline hydrolysis procedure. The leucine samples yielded four times higher fluorescence on reaction with OPA than with fluorescamine. However, the blanks were also four times higher with OPA than with fluorescamine. Since standard deviations as high as 6.9% are commonly obtained at these low levels, fluorescence values several times higher than blank are required for accuracy. With leucine, for example, 2.5 nmol/ml produced a fivefold increase over blank when assayed by either reagent. A higher sample to blank fluorescence ratio was achieved
o-CHTHALALDEWVDE
FlUOlESCAMlW
-After
*--
hydmlysh Bdm hydmtysb
TUBE
NUMBER
FIG. 2. Chromatography of a tryptic-peptic digest of human (K) Bence Jones protein. The hydrolyzate (10 mg) was prepared as previously described (12) and applied to a Sephadex G-25-F column (2 x 90 cm), equilibrated with 0.1 M NH,HCO,, pH 8.5. The column was eluted with 0.1 M NH,HCO,, pH 8.5, at a flow rate of 19 ml/hr and fractions were collected every 7 min. Aliquots ofO.1 ml were used for reaction with fluorescamine (A) and o-phthalaldehyde (B) either by direct fluorometric assay (a-++ ) or after alkaline hydrolysis ( ) (see Methods).
FLUOROMETRIC
DETECTION
WA
F
A
B
OF PEPTIDES
F
OFA
c
D
477
FIG. 3. Ultraviolet-photography showing the fluorescent spots from amino acids and peptides detected on paper with fluorescamine or o-phthalaldehyde. Amino acids (500 picomol each) were spotted on paper and stained with (A) o-phthalaldehyde or (B) fluorescamine as described in Methods. A tryptic digest of cholera toxin (5 mol of each peptide) detected with fluorescamine (C) or OPA (D) after electrophoresis at pH 3.5.
for aspartic acid with OPA. On the other hand, a higher sample to blank fluorescence ratio was achieved for arginine with fluorescamine. Thus, under the conditions used in these experiments, OPA is a more sensitive reagent for aspartic, fluorescamine is a more sensitive reagent for arginine, and equivalent sensitivities are obtained for leucine with both reagents. Identical sample and blank fluorescence values were obtained with both Sigma and Durrum OPA. Since the fluorescence of the blank is the prime factor limiting sensitivity, suitable precautions must be taken to avoid solvent contamination by amines and other reactive compounds. For these studies, a water purification system, producing water with a resistance greater than 4 x lo5 ohms, was utilized. The distilled water was filtered to eliminate microorganisms,
478
MENDEZ
AND GAVILANES
twice deionized and filtered to eliminate organic compounds. However, distilled or deionized water from several sources has, at times, introduced high blanks with OPA, but not with fluorescamine. Borate buffers, prepared from boric acid obtained from several commercial sources, also introduced high fluorescence blanks with OPA, but not with fluorescamine (the lowest background with OPA was obtained with boric acid from Mallinckrodt). Ammonium ions, which yield poor fluorescence with fluorescamine, might be the major contaminating species. However, the ammonium bicarbonate used for the column chromatography presented in Fig. 2 is removed during the drying step (see Methods). Pyridine acetate, another volatile buffer, is also satisfactory. For detection of amino acids and peptides on paper chromatograms, conditions similar to those employed for fluorescamine (12) were found to be optimal. As Fig. 3 illustrates, amino acids are more readily visualized at the 500 pmole level with OPA than with fluorescamine, while peptides are more easily discernible with fluorescamine. Whereas the fluorescent spots produced by fluorescamine remained visible for several weeks, the spots produced by OPA disappeared after several hours. Varying the concentration of mercaptoethanol did not enhance the fluorescence intensity nor the stability of the spots produced with OPA. Additionally, a greater sensitivity for detection of peptides on thin layer chromatograms was found with fluorescamine. In conclusion, it may be stated that OPA is a useful reagent for amino acid and peptide detection, provided that the proper precautions are taken. It is hoped that this report will dispel any confusion about the inherent sensitivity of these two reagents. Proper comparisons between these two reagents in flow systems remain to be performed. The most important point is that the introduction of fluorescent techniques, with these as well as other fluorogenic reagents, has advanced the field of peptide biochemistry. ACKNOWLEDGMENTS We wish to thank Drs. S. Udenfriend, S. Stein, and 0. Tsolas for their valuable discussions, Mr. R. Welbom for his excellent technical assistance in the photographic recordings, and Mrs. A. Dugan for secretarial work.
REFERENCES 1. Weigeie, M., Blount, J. F., Tengi, J. P., Czaijkowki, (1972)
J. Amer.
Chem.
2. Weigele, M., DeBernardo. Chem.
Sot.
Sot.
R. C., and Leimgruber,
W.
94, 4052.
S. L., Tengi, J. P., and Leimgruber,
W. (1972) J. Amer.
94, 5927.
3. Udenfriend, S., Stein, S., Bohlen, P., and Dairman, W. (1972) in Third American Peptide Symposium, Boston (Meinhofer, J.. ed.), pp. 655-663, Ann ArborHumphery Science, Ann Arbor, Michigan.
FLUOROMETRlC 4. Udenfriend, (1972)
DETECTION
OF PEPTIDES
S., Stein, S., Bbhlen, P., Dairman, W.. Leimgruber,
Science
479 W., and Weigele, M.
178, 871-872.
5. Biihlen, P., Stein, S., Dairman, W., and Udenfriend, S. (1973) Arch. Biochem. Biophys. 155, 213-220. 6. Felix, A., and Jimenez, M. H. (1974) J. Chromutogr. 89, 361. 7. Imai, K., Bohlen, P., Stein, S.. and Udenfriend, S. (1974) Arch. Biochem. Biophys. 161,
161-
170.
8. Pace, J. L.. Kemper, D. L., and Ragland, W. L. (1974) Biochem. Biophys. Res. Common. 57, 482-487. 9. Vandekerckhove, J., and Van Montagu, M. (1974) Eltr. J. Biochem. 44, 279-288. IO. Stein, S., Chang, C. H., Bohlen, P.. Imai, K., and Udenfriend. S. (1974) Anctl. Biochem. 60, 272-277. Il. Nakai, N.. Lai, C. Y., and Horecker, B. L. (1974)J. Anal. Biochem. 58, 563-575. 12. Mendez. E., and Lai, C. Y. (1975)Anal. Biochem. 65, 281-292. 13. Felix, A., Jimenez, H., Vergona, R., and Cohen, M. (1974) Int. J. Peptide Protein Res. 7, 11-22. 14. Bbhlen, P., Stein, S., Stone, J. and Udenfriend, S. (1975)Am7/. Biochem. 67,438-445. 15. Stein, S., Bohlen, P., Stone, J., Dairman, W. and Udenfriend, S. (1973)Arch. Biochem. Biophys. 155, 202-212. 16. Stein, S., Bohlen, P. and Udenfriend, S. (1975)Arch. Bioclrem. Biophys. 163,400-403. 17. Cohn, V. H. and Lyle (1966) Anal. Biochem. 14, 434. 18. Shore, P. A., Burkhalter, A.. and Cohn, V. H., Jr. (1959) /. Ph~rmaco/. Expr. Therap. 127, 182. 19. Hakanson, T., Ronnberg, A. L., and Sjolund, K. (1974)Anal. Biochem. 59, 98-109. 20. Roth, M. (1971) Anal. Biochem. 43, 880-882. 21. Roth, M.. and Hampai, A. (1973)./. Chromatogr. 83, 353-356. 22. Benson, J. and Hare, P. (1974) Proc. Nut. Acud. Sci. 72, 619-622. 23. Taylor, S. and Tappel. A. (1973) Anal. Biochem. 56, 140-148. 24. Hits, C. H. W. (1%7) in Methods in Enzymology (Hirs, C. H. W., ed.), Vol. I I. pp. 325-329. Academic Press, New York.
