AETALYTICAL
65, 396-404
BIOCHEMISTRY
A Fluorometric
(1975)
Method
of the Tryptophan
for the Determination Content
of Proteins
T. SASAKI,' B. ABRAMS, AND B. L. HORECKER Roche institute of Molecular Biology, Nutley, New Jersey 07110 Received November
1, 1974; accepted December 13, 1974
Digestion of proteins with a mixture of chymotrypsin and pronase followed by dilution in 6 M urea eliminates the quenching effects usually observed when tryptophan fluorescence is measured in native or denatured proteins. Following proteolysis, the tryptophan content can be estimated from the fluorescence emission, using free tryptophan as an internal standard. The values obtained for a number of proteins are in agreement with the literature values. The method can be applied to as little as 40 pg of protein sample.
The determination of the tryptophan content of proteins remains difficult and uncertain, despite the existence of a number of published procedures (for a review, see Friedman (1)). Theoretically, the intrinsic fluorescence of tryptophan should provide the basis for a sensitive and rapid analytical method, but the fluorescence is strongly influenced by its microenvironment, even in denatured proteins, and may differ substantially from that of free tryptophan(2). Several attempts have been made to overcome this difficulty. The method of Duggan and Udenfriend (3) employed alkaline hydrolysis prior to the fluorescence measurements, but this treatment can lead to loss of tryptophan (1) or the formation of other fluorescent products. More recently, Shelton and Rogers (4) measured the fluorescence of proteins dissolved in sodium dodecyl sulfate and mercaptoethanol and found it necessary to introduce statistical factors to correct for the low quantum yield. In the present work, partial proteolysis of protein samples at pH 8.0 was found to eliminate the problem of quenching by the microenvironment for all but a few disulfide proteins. Coupling proteolysis with the use of an internal tryptophan standard provides a simple and sensitive method for the determination of the tryptophan content.
’ Present address: National Food Research Institute. Tokyo 13.5,Japan. 396 Copyr&ht @ 1975 by Academic Press. Inc. All rights of reproduction in any form reserved.
FLUOROMETRIC
DETERMINATION
OF
TRYPTOPHAN
397
MATERIALS Pepsin from swine stomach mucosa (twice crystallized), alcohol dehydrogenase from horse liver (twice crystallized), lysozyme from chicken egg white (twice crystallized), DNase from bovine pancreas, chymotrypsin from bovine pancreas, lactate dehydrogenase from beef heart, pyruvate kinase from rabbit muscle and papain (twice crystallized) were purchased from the Worthington Biochemical Corporation, Freehold, NJ. Triose phosphate isomerase from rabbit muscle (Type III), phosphoglucomutase from rabbit muscle, trypsin from bovine pancreas (twice crystallized), cytochrome c from horse heart (Type VI), and glucagon from bovine and porcine pancreas were purchased from the Sigma Chemical Corporation, St. Louis, MO. Glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle was obtained from the Boehringer Mannheim Corporation, New York, NY: bovine serum albumin and ribonuclease from beef pancreas (crystallized five times) were purchased from the Nutritional Biochemical Corporation, Cleveland, OH and Pentex Incorporated, Kankakee, IL, respectively. Bovine hemoglobin (twice crystallized) and horse heart myoglobin (twice crystallized) were purchased from Schwarz/Mann Research Laboratories, Orangeburg, NY. Pronase from Streptomyces griveus (B grade) and L-tryptophan (A grade) were obtained from the Calbiochem Company, Los Angeles, CA. N-Bromosuccinimide (Practical grade) was purchased from Eastman Kodak Company, Rochester, NY, and urea (ultra pure) was obtained from Schwarz/Mann Research Laboratories. METHODS Fluorescence measurements were made with an Aminco-Bowman Spectrophotofluorometer equipped with an Aminco Ratio Photometer (American Instrument Co., Silver Spring, MD). The excitation wavelength was 288 nm and the emission was read at 352 nm. Digestion with pronase and chymotrypsin was carried out in 12 ml conical centrifuge tubes, containing the protein samples dissolved in 0.1 ml of 20 mM triethanolamine-20 mM diethanolamine buffer, pH 8.0. The tubes were covered with Parafilm and immersed for 5 min in a shallow boiling water bath to denature the protein. The tubes were cooled and 2 ~1 of a freshly prepared solution containing 0.05% (w/v) each of chymotrypsin and pronase was added. The mixtures were incubated overnight at room temperature (23”). If the solution was still turbid, an additional 2 ~1 of fresh protease mixture was added and the digestion was continued for 4-5 h. For fluorimetric analysis, the hydrolyzed samples were diluted as indicated with a solution containing 6 M urea in 20 mM diethanolamine20 mM triethanolamine buffer, pH 9.2, and 3 ml aliquots of the diluted
398
SASAKI,
ABRAMS
AND HORECKER
solution were transferred to a quartz cell for the fluorescence measurements. The concentration of protein in the solutions to be analyzed was measured by ninhydrin assay following alkaline digestion (5) and also by one of the following methods: turbidity after precipitation with 50% trichloroacetic acid (6); dry weight of lyophylized material, or ultraviolet absorption. The following factors were used to calculate protein concentrations by ultraviolet absorption: aldolase, mg/ml = A,,, X 1.10 (7); glyceraldehyde-3-phosphate dehydrogenase, mg/ml = AZ’16x 0.95 (8). Tryptophan was also determined with IV-bromosuccinimide according to Spande and Witkop (9). For these determinations the proteins were TABLE 1 EVALUATIONOFPROTEINCONTENTOFSAMPLESANALYZED Protein concentration
(mg/ml)
Protein
By weight, uv. or turbidity
By ninhydrin assay after alkaline hydrolysis”
Aldolase DNase Bovine serum albumin Bovine hemoglobin Myoglobin Cytochrome c Lysozyme RNase Alcohol dehydrogenase Trypsin Pepsin Glucagon Phosphoglucomutase Lactate dehydrogenase Pyruvate kinase Triose phosphate isomerase Glyceraldehyde-3-phosphate dehydrogenase
4.75b 5.00' 5.00' 2.50' 5.00' 5.00' 2.40c 5.00’ 1.00’ 2.90’ 5.00’ 5.00’ 0.71d 7.816 4.22” 3.89” 3.82b
4.60 4.70 5.00 2.18 5.50 4.70 2.26 4.75 0.99 2.63 4.90 4.50 0.64 7.50 4.71 3.88 3.75
a Aliquots of the protein solutions (2-20 pg) were dried and dissolved in 0.2 ml of 20% NaOH and hydrolyzed in the autoclave at 20 lb/in’ for 25 mm. Samples were cooled and neutralized with 0.2 ml of 50% acetic acid. After adding 0.2 ml of ninhydrin to each tube, the samples were placed in a boiling water bath for 15 min, diluted with 0.5 ml of 50% ethanol and the absorbance measured at 570 nm. A weighed sample of bovine serum albumin was the standard. ’ Suspensions of crystals in (NH&SO 4 were dialyzed against distilled water, and the absorbance measured as described under Methods. c Commercial samples were weighed and dissolved in distilled water. d Suspensions of crystals in (NH&SO, were dialyzed against distilled water and analyzed by the method of Bucher (6), with bovine serum albumin as the standard.
FLUOROMETRIC
DETERMINATION
OF TRYPTOPHAN
dissolved in 8 M urea, pH 4.0, to a final concentration 1 mg/ml.
399
of approximately
RESULTS
Determination of protein. The measurement of ninhydrin color value after alkaline hydrolysis proved to be a convenient and sensitive method for the determination of protein in solution (Table 1). The protein content calculated from the dry weight, turbidity after precipitation with CCl,COOH (6) or ultraviolet absorption usually was within 5-6% of that calculated from the ninhydrin color value. For some proteins, the values calculated from the weight of the sample of the turbidity method deviated from the alkaline ninhydrin value by as much as 10-I 5% ; in these cases it was assumed that the latter was correct. The protein content determined from the ninhydrin assay was used for calculation of the tryptophan content. Effect of digestion with proteins on the fluorescence emission spectrum. The effect of digestion with a mixture of pronase and chymotrypsin on the fluorescence emission spectrum of rabbit muscle aldolase and bovine hemoglobin in 6 M urea is illustrated in Fig. 1. The emission at 355 nm was significantly increased on digestion with the protease mixture, especially with some proteins that showed little or no fluorescence emission due to tryptophan in the undigested sample (see Table 1). 100
1
. HEMOGLOBIN ’
I
1
I
1
I
400
450
ALDOLASE
400 450 300 350 WAVELENGTH 1 nm 1
FIG. 1. Effect of proteolysis on fluorescence emission spectrum of representative proteins dissolved in 6 M urea. The protein samples were digested as described in Methods and diluted with 7 ml of buffered urea (see Methods) for the fluorescence measurements. For the experiment shown the following quantities of protein were digested: hemoglobin (0.07 mg); aldolase 0.067 mg. Samples containing the chymotrypsin-pronase mixture alone yielded negligible fluorescence at 352 nm.
400
SASAKI,
ABRAMS
AND
HORECKER
t I
f
z’
t 5.1
7-
to 1.0
ALDOLASE
5 TRYPTOPHAN
I
I
(69pg)
10 15 ADDED ( nmoler
1
FIG. 2. Evaluation of the tryptophan content of protein samples. Digestion was carried out as described under Methods. After dilution with 7 ml of buffered urea, pH 9.2, 3 ml ahquots were transferred to quartz curvettes and the fluorescence emission measured at 352 nm, with excitation at 288 nm. To each sample, 3 nmol quantities of tryptophan (3 ~1 of a mM solution) were added as shown and the fluorescence measured again. The quantity of tryptophan present in the protein in the 3 ml aliquot was determined by extrapolating the fluorometer readings to the X-axis, as indicated by the arrows.
Determination of tryptophan in proteins. In order to correct for possible internal quenching, aliquots of a standard tryptophan solution were added directly to the cuvette containing the unknown and the fluorescence intensity extrapolated to the base line (Fig. 2). The tryptophan content of the protein analyzed was calculated from the quantity of tryptophan in the aliquot taken for the fluorescence measurements and its protein content. Minor differences in the slopes of the lines (see Fig. 2) reflected small differences in the quenching of the protein digests, but in general these differences were small. In almost every case, digestion with the chymotrypsin-pronase mixture increased the apparent tryptophan content calculated from the fluorescence measurement (Table 2). The exceptions were alcohol dehydrogenase and bovine serum albumin, which yielded the correct values without digestion. The largest increases were observed with the heme proteins; the undigested samples showed almost no fluorescence due to tryptophan, but yielded nearly theoretical values after digestion. Several proteins, including lysozyme, trypsin and pepsin yielded low values even after digestion. We also compared the present method with that of Spande and Witkop (9) involving titration of the tryptophan residues with Nbromosuccinimide (Table 3). For a few proteins, such as trypsin and lysozyme, the results obtained with the latter method were closer to the literature values, but usually better agreement was obtained with the fluorescence method. In addition, the fluorescence method was considerably more sensitive.
FLUOROMETRIC
DETERMINATION
TABLE TRWTOPHAN
OF
401
TRYPTOPHAN
2
ANALYSIS WITH AND WITHOUT PRONASE AND CHYMOTRYPSIN
DIGESTION
Tryptophan
WITH
content (molimol protein)
Analysis” Without digestion’
Proteinb Aldolase Alcohol dehydrogenase DNase Pepsin Bovine serum albumin Glyceraldehyde-3-phosphate Cytochrome c Hemoglobin Myoglobin Trypsin Lysozyme Papain RNase
dehydrogenase
10.2 4.2 1.4 3.4 2.2 8.0 0 1.9 0.2 3.2 0.6 1.2 0
With digestiond 11.0 4.1 3.1 4.0 1.8 11.3 0.7 6.0 1.6 3.2 3.4 3.1 0.1
11.6 4.2 4.1 1.9 11.6 0.9
4.0 3.7
Literature VdUP
(10) 4 (11) 3 (12) 5 (13,14Y 2 (15)’ 12 (16)g 1 (11) 6 (11) 2 (11) 4 (11) 6 (11) 5 (11) 0 (11)
12
a The digestion, analyses, and calculations were carried out as described under Methods and in the legend to Fig. 2. Protein content was determined from the ninhydrin color values after alkaline hydrolysis (see Table 1). * For the source of the proteins used, see Materials. c The samples in triethanolamine-diethanolamine buffer were diluted with 7 ml of buffered urea (see Methods) without heating or addition of chymotrypsin and pronase. d The italicized numbers represent tryptophan values derived from the determination of the amino acid sequences of these proteins (10-12). The numbers in parenthesis refer to the literature references. e Five peptides containing tryptophan have been isolated from hog pepsin and their sequences established (13,14). f The number of tryptophan residues was determined spectrophotometrically, and confirmed by the isolation of two peptides containing tryptophan after cleavage with BrCN and reduction of the cross-linking disulfide bonds (15). B The tryptophan content for the enzyme from rabbit muscle was determined spectrophotometrically by Velick and Furfine (16); the same number of tryptophan residues has been reported in the complete sequence analysis of the enzymes from pig and lobster muscle (11).
DISCUSSION
The fluorometric method described in this paper provides a simple, sensitive and accurate method of tryptophan analysis. The strongly fluorescent amino acid, tyrosine, does not interfere because the choice of wavelengths of excitation and emission was such as to eliminate the
402
SASAKI,
ABRAMS
AND
TABLE COMPARISON
Aldolase DNase Hemoglobin Myoglobin Cytochrome c Bovine serum albumin Trypsin Pepsin Lysozyme Alcohol dehydrogenase Glyceraldehyde-3.phosphate dehydrogenase Triose phosphate isomerase Pyruvate kioase Lactate dehydrogenase Phosphoglucomutase Glucagon RNase
3
OF TRYPTOPHAN ANALYSIS BY FLUORESCENCE N-BROMOSUCCINIMIDE METHODS Fluorescmce”
Protein
HORECKER
AND
‘V-Bromosuccinimldti
Sample six tmgl
Tryptophan lmollmol protem)
Samplesire (mgl
Tryptophan lmollmol protein)
Llterifture value’
0.034 0.14 0.035 0.13 0.14 0 15 0.09 0.15 0.14 0.06 0.30
12.0 ?.I 5.7 1.7 0.8 2.0 2.6 5.0 4.1 3.8 12.0
0.91 I .oo 0.50 1.00 1.00 1.00 0.73 1.00 0.60 I .oo 0.44
I I.5 2.3 2.2 2.9 0.5 1.6 3.2 2.4 5.3 3.8 12.2
I ?” 3" 6" 2"
0 23 0.28 0.47 0.04 0.27 0.29
10.0 12.5 24.0 8.0 1.0 0.1
0.99 0.47 0.75 0 I6 0.45 0.75
I I.5 11.3 19.8 5.2 0.9 0.0
?” 4’ 5’ 6d 4" I?” IO (17)” I? 20-23tl9)’ 8 (20)' I (III” 0”
n The digestion, analyses. and calculations werr carried out as dexribed under Methods in thr legend to Fig. 2 with the modification indicated in footnote rl. Protein content was determined from thr ninhydrin color- value\ after alksline hydrolysis (see Table I). ’ According to Spade and Witkop (9). c The digestions werr carried out in one-half the usual volume with one-half the quantities of proteolytic enzymes. and the samples were diluted to only 3.5 ml with buffered urea. * The italicized numbers represent tryptophan values derived from the determination of the amino acid sequence> of these proteins (10-12). The numbers in parenthesis refer to the literature references. e The number of tryptophan residues was determined spectrophotometrically, and confirmed by the isolation of two peptides containing tryptophan after cleavage with BrCN and reduction of the cross-linkmg disulfide bonds (15). ’ Five peptides containing tryptophan have been isolated from hog pepsin and their sequences established (13.14). 0 The tryptophan content for the enzyme from rabbit muscle was determined spectrophotometrically by Velick and Furfine (16); the same number of tryptophan residues has been reported in the complete sequence analysis of the enzymes from pig and lobster muscle (11) ’ These values are based on spectrophotometric analyses (17.18). ’ Definitive data on the tryptophan content of the beef heart enzyms are not available. Pesce er ul. (191 have reported values of 20 residueslmol by a calorimetric procedure (22) and 23 residues by a spectrophotometric method 128). 1 Titration with N-bromosuccinimide in the presence and absence of urea indicates that the enzyme contains 5 accescible and 3-4 buried tryptophan raidues (20).
fluorescent contribution from this amino acid(21). Other amino acids either do not fluoresce or their fluorescence is negligible. Further, the presence of other pigments in the proteins, such as heme, did not interfere with the determinations. Undigested samples of the pigmented proteins (cytochrome c. myoglobin and hemoglobin) showed little or no fluorescence, but after digestion typical tryptophan emission curves were observed and quantitation of the tryptophan content was possible. For most of the proteins examined, digestion with the proteolytic mixture was essential. The proteases employed were selected to avoid alkaline or acid conditions, and are readily available. The combination of
FLUOROMETRIC
DETERMINATION
OF
TRYPTOPHAN
403
chymotrypsin and pronase appeared, in most cases, to provide sufficient destruction of the protein structure to eliminate internal quenching. Lysozyme, trypsin and papain, extracellular proteins that contain disulfide bridges, yielded low values for tryptophan by the present method, perhaps because the structures of these proteins were not sufficiently disrupted. Reduction before proteolysis, or the addition of other denaturating agents or reducing agents, such as sodium dodecyl sulfate or mercaptoethanol, after hydrolysis, did not improve the results with these proteins. On the other hand, pepsin and bovine serum albumin, which also contain disulfide bridges, yielded close to theoretical values. The fluorometric method described in this paper is more sensitive. less time consuming, and in most cases more accurate than other available methods for the determination of tryptophan. It offers several advantages over the fluorescence method described by Shelton and Rogers (4). Thus tryptophan is employed as the primary standard, and it is not necessary to run parallel assays with a number of standard proteins. In addition, the use of tryptophan as an internal standard eliminates the need for careful control of temperature and also for separate calibration curves. The practical limit to the sensitivity of the present method is imposed by the amount of material that can be handled quantitatively during digestion. Reducing the digestion volume to less than 50 ~1 resulted in significant losses. The digestions and assays can be carried out with 20- 150 pg of protein compared with approximately l-2 mg of protein required for the N-bromosuccinimide method (9), 0.1-5 mg for the calorimetric methods (22-25), 0.2-l mg for a chromatographic method (26) and l-3 mg for the determination by ultraviolet absorption (27,23). REFERENCES 1. Friedman. M.. and Finley. J. W. (1971) J. Agr. Food Chrm. 19, 626-631. 2. Udenfriend. S. (1962) in Fluorescence Assay in Biology and Medicine, Vol. 1. pp. 201-207, Academic Press, New York. 3. Duggan, D. E.. and Udenfriend. S. (1956) J. Bid/. Chem. 223, 313-319. 4. Shelton, K. R.. and Rogers, K. S. (1971) Anal. B&hem. 44, 134-142. 5. Hirs, C. H. W. (1955) in Methods in Enzymology (C. H. W. Hirs, ed.), Vol. Xl, pp. 325-329. Academic Press, New York. 6. Bucher, T. (1947) Biochim. Biophys. Actu 1, 292-3 14. 7. Taylor, J. T. (1955) in Methods in Enzymology (S. P. Colowick and N. 0. Kaplan, eds.), Vol. IV, pp. 310-315. Academic Press, New York. 8. Velick, S. F. (1955) in Methods in Enzymology (S. P. Colowick and N. 0. Kaplan, eds.), Vol. I, pp. 401-406, Academic Press, New York. 9. Spande, T. F., and Witkop, B. (1967) in Methods in Enzymology (C. H. W. Hirs, ed.), Vol. XI, pp. 498-506, Academic Press, New York. 10. Lai, C. Y.. Nakai, N., and Chang. D. ( 1974) Science 183, 1204- 1206. 11. Dayhoff. M. 0. (1972) Atlas of Protein Sequence and Structure. Vol. 5. p. 418, National Biomed. Research Foundation, Washington. DC.
404
SASAKI,
ABRAMS
AND HORECKER
12. Liao, T. H., Salnikow, J., Moore, S.. and Stein, W. H. (1973) J. Eiol. Chem. 248, 1489-1495. 13. Dopheide. T. A. A., and Jones. Wanda M. (1968) J. Biol. Chem. 243, 3906-3911. 14. Kostka, V., Moravek, L., and Sorm, F. (1970) Eur. J. Biochem. 13, 447-454. 1.5. King, T. P., and Spencer, M. (197O)J. Biol. Chem. 245, 6134-6148. 16. Velick, S. F., and Furfine, C. (1963) in The Enzymes (P. D. Boyer, ed.), 2nd ed., Vol. 7, p. 250, Academic Press, New York. 17. Norton, I. L., Pfuderer, Peter, Stringer, C. D., and Hartman. F. C. (1970) Biochemistry 9, 4952-4958. 18. Cottam, G. L., Hollenberg, P. F., and Coon, M. J. (1969) J. Biol. Chem. 244, 1481-1486. 19. Pesce, A., McKay, R. H., Stolzenbach, F., Cahn, R. D.. and Kaplan, N. 0. (1964) J. Biol. Chem. 239, 1753-1761. 20. Sloan, N. H., Mercer, D. W., and Danoff, M. (1964) Biochim. Biophys. Acta 92, 168-170. 2 1. Udenfriend, S. (1962) in Fluorescence Assay in Biology and Medicine. Vol. I, p. 129, Academic Press, New York. 22. Spies, J. R.. and Chambers, D. C. (1949) Anal. Chem. 21, 1249-1266. 23. Messieneo, L., and Musarra, E. (1972) Int. J. Biochem. 3, 700-704. 24. Gaitonde, M. K.. and Davey, T. (1970) Biochem. J. 117, 907-911. 25. Barman, T. E.. and Koshland, D. E., Jr. (1967) J. Biol. Chem. 242, 5771-5776. 26. Hugh, T. E., and Moore, S. (1972) J. Biol. Chem. 247, 2828-2834. 27. Edelhoch, H. (1967) Biochemistry 6, 1948-1954. 28. Goodwin, T. W., and Morton, R. A. (1946) Biochem. J. 40, 628-632.
65, 396-404
BIOCHEMISTRY
A Fluorometric
(1975)
Method
of the Tryptophan
for the Determination Content
of Proteins
T. SASAKI,' B. ABRAMS, AND B. L. HORECKER Roche institute of Molecular Biology, Nutley, New Jersey 07110 Received November
1, 1974; accepted December 13, 1974
Digestion of proteins with a mixture of chymotrypsin and pronase followed by dilution in 6 M urea eliminates the quenching effects usually observed when tryptophan fluorescence is measured in native or denatured proteins. Following proteolysis, the tryptophan content can be estimated from the fluorescence emission, using free tryptophan as an internal standard. The values obtained for a number of proteins are in agreement with the literature values. The method can be applied to as little as 40 pg of protein sample.
The determination of the tryptophan content of proteins remains difficult and uncertain, despite the existence of a number of published procedures (for a review, see Friedman (1)). Theoretically, the intrinsic fluorescence of tryptophan should provide the basis for a sensitive and rapid analytical method, but the fluorescence is strongly influenced by its microenvironment, even in denatured proteins, and may differ substantially from that of free tryptophan(2). Several attempts have been made to overcome this difficulty. The method of Duggan and Udenfriend (3) employed alkaline hydrolysis prior to the fluorescence measurements, but this treatment can lead to loss of tryptophan (1) or the formation of other fluorescent products. More recently, Shelton and Rogers (4) measured the fluorescence of proteins dissolved in sodium dodecyl sulfate and mercaptoethanol and found it necessary to introduce statistical factors to correct for the low quantum yield. In the present work, partial proteolysis of protein samples at pH 8.0 was found to eliminate the problem of quenching by the microenvironment for all but a few disulfide proteins. Coupling proteolysis with the use of an internal tryptophan standard provides a simple and sensitive method for the determination of the tryptophan content.
’ Present address: National Food Research Institute. Tokyo 13.5,Japan. 396 Copyr&ht @ 1975 by Academic Press. Inc. All rights of reproduction in any form reserved.
FLUOROMETRIC
DETERMINATION
OF
TRYPTOPHAN
397
MATERIALS Pepsin from swine stomach mucosa (twice crystallized), alcohol dehydrogenase from horse liver (twice crystallized), lysozyme from chicken egg white (twice crystallized), DNase from bovine pancreas, chymotrypsin from bovine pancreas, lactate dehydrogenase from beef heart, pyruvate kinase from rabbit muscle and papain (twice crystallized) were purchased from the Worthington Biochemical Corporation, Freehold, NJ. Triose phosphate isomerase from rabbit muscle (Type III), phosphoglucomutase from rabbit muscle, trypsin from bovine pancreas (twice crystallized), cytochrome c from horse heart (Type VI), and glucagon from bovine and porcine pancreas were purchased from the Sigma Chemical Corporation, St. Louis, MO. Glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle was obtained from the Boehringer Mannheim Corporation, New York, NY: bovine serum albumin and ribonuclease from beef pancreas (crystallized five times) were purchased from the Nutritional Biochemical Corporation, Cleveland, OH and Pentex Incorporated, Kankakee, IL, respectively. Bovine hemoglobin (twice crystallized) and horse heart myoglobin (twice crystallized) were purchased from Schwarz/Mann Research Laboratories, Orangeburg, NY. Pronase from Streptomyces griveus (B grade) and L-tryptophan (A grade) were obtained from the Calbiochem Company, Los Angeles, CA. N-Bromosuccinimide (Practical grade) was purchased from Eastman Kodak Company, Rochester, NY, and urea (ultra pure) was obtained from Schwarz/Mann Research Laboratories. METHODS Fluorescence measurements were made with an Aminco-Bowman Spectrophotofluorometer equipped with an Aminco Ratio Photometer (American Instrument Co., Silver Spring, MD). The excitation wavelength was 288 nm and the emission was read at 352 nm. Digestion with pronase and chymotrypsin was carried out in 12 ml conical centrifuge tubes, containing the protein samples dissolved in 0.1 ml of 20 mM triethanolamine-20 mM diethanolamine buffer, pH 8.0. The tubes were covered with Parafilm and immersed for 5 min in a shallow boiling water bath to denature the protein. The tubes were cooled and 2 ~1 of a freshly prepared solution containing 0.05% (w/v) each of chymotrypsin and pronase was added. The mixtures were incubated overnight at room temperature (23”). If the solution was still turbid, an additional 2 ~1 of fresh protease mixture was added and the digestion was continued for 4-5 h. For fluorimetric analysis, the hydrolyzed samples were diluted as indicated with a solution containing 6 M urea in 20 mM diethanolamine20 mM triethanolamine buffer, pH 9.2, and 3 ml aliquots of the diluted
398
SASAKI,
ABRAMS
AND HORECKER
solution were transferred to a quartz cell for the fluorescence measurements. The concentration of protein in the solutions to be analyzed was measured by ninhydrin assay following alkaline digestion (5) and also by one of the following methods: turbidity after precipitation with 50% trichloroacetic acid (6); dry weight of lyophylized material, or ultraviolet absorption. The following factors were used to calculate protein concentrations by ultraviolet absorption: aldolase, mg/ml = A,,, X 1.10 (7); glyceraldehyde-3-phosphate dehydrogenase, mg/ml = AZ’16x 0.95 (8). Tryptophan was also determined with IV-bromosuccinimide according to Spande and Witkop (9). For these determinations the proteins were TABLE 1 EVALUATIONOFPROTEINCONTENTOFSAMPLESANALYZED Protein concentration
(mg/ml)
Protein
By weight, uv. or turbidity
By ninhydrin assay after alkaline hydrolysis”
Aldolase DNase Bovine serum albumin Bovine hemoglobin Myoglobin Cytochrome c Lysozyme RNase Alcohol dehydrogenase Trypsin Pepsin Glucagon Phosphoglucomutase Lactate dehydrogenase Pyruvate kinase Triose phosphate isomerase Glyceraldehyde-3-phosphate dehydrogenase
4.75b 5.00' 5.00' 2.50' 5.00' 5.00' 2.40c 5.00’ 1.00’ 2.90’ 5.00’ 5.00’ 0.71d 7.816 4.22” 3.89” 3.82b
4.60 4.70 5.00 2.18 5.50 4.70 2.26 4.75 0.99 2.63 4.90 4.50 0.64 7.50 4.71 3.88 3.75
a Aliquots of the protein solutions (2-20 pg) were dried and dissolved in 0.2 ml of 20% NaOH and hydrolyzed in the autoclave at 20 lb/in’ for 25 mm. Samples were cooled and neutralized with 0.2 ml of 50% acetic acid. After adding 0.2 ml of ninhydrin to each tube, the samples were placed in a boiling water bath for 15 min, diluted with 0.5 ml of 50% ethanol and the absorbance measured at 570 nm. A weighed sample of bovine serum albumin was the standard. ’ Suspensions of crystals in (NH&SO 4 were dialyzed against distilled water, and the absorbance measured as described under Methods. c Commercial samples were weighed and dissolved in distilled water. d Suspensions of crystals in (NH&SO, were dialyzed against distilled water and analyzed by the method of Bucher (6), with bovine serum albumin as the standard.
FLUOROMETRIC
DETERMINATION
OF TRYPTOPHAN
dissolved in 8 M urea, pH 4.0, to a final concentration 1 mg/ml.
399
of approximately
RESULTS
Determination of protein. The measurement of ninhydrin color value after alkaline hydrolysis proved to be a convenient and sensitive method for the determination of protein in solution (Table 1). The protein content calculated from the dry weight, turbidity after precipitation with CCl,COOH (6) or ultraviolet absorption usually was within 5-6% of that calculated from the ninhydrin color value. For some proteins, the values calculated from the weight of the sample of the turbidity method deviated from the alkaline ninhydrin value by as much as 10-I 5% ; in these cases it was assumed that the latter was correct. The protein content determined from the ninhydrin assay was used for calculation of the tryptophan content. Effect of digestion with proteins on the fluorescence emission spectrum. The effect of digestion with a mixture of pronase and chymotrypsin on the fluorescence emission spectrum of rabbit muscle aldolase and bovine hemoglobin in 6 M urea is illustrated in Fig. 1. The emission at 355 nm was significantly increased on digestion with the protease mixture, especially with some proteins that showed little or no fluorescence emission due to tryptophan in the undigested sample (see Table 1). 100
1
. HEMOGLOBIN ’
I
1
I
1
I
400
450
ALDOLASE
400 450 300 350 WAVELENGTH 1 nm 1
FIG. 1. Effect of proteolysis on fluorescence emission spectrum of representative proteins dissolved in 6 M urea. The protein samples were digested as described in Methods and diluted with 7 ml of buffered urea (see Methods) for the fluorescence measurements. For the experiment shown the following quantities of protein were digested: hemoglobin (0.07 mg); aldolase 0.067 mg. Samples containing the chymotrypsin-pronase mixture alone yielded negligible fluorescence at 352 nm.
400
SASAKI,
ABRAMS
AND
HORECKER
t I
f
z’
t 5.1
7-
to 1.0
ALDOLASE
5 TRYPTOPHAN
I
I
(69pg)
10 15 ADDED ( nmoler
1
FIG. 2. Evaluation of the tryptophan content of protein samples. Digestion was carried out as described under Methods. After dilution with 7 ml of buffered urea, pH 9.2, 3 ml ahquots were transferred to quartz curvettes and the fluorescence emission measured at 352 nm, with excitation at 288 nm. To each sample, 3 nmol quantities of tryptophan (3 ~1 of a mM solution) were added as shown and the fluorescence measured again. The quantity of tryptophan present in the protein in the 3 ml aliquot was determined by extrapolating the fluorometer readings to the X-axis, as indicated by the arrows.
Determination of tryptophan in proteins. In order to correct for possible internal quenching, aliquots of a standard tryptophan solution were added directly to the cuvette containing the unknown and the fluorescence intensity extrapolated to the base line (Fig. 2). The tryptophan content of the protein analyzed was calculated from the quantity of tryptophan in the aliquot taken for the fluorescence measurements and its protein content. Minor differences in the slopes of the lines (see Fig. 2) reflected small differences in the quenching of the protein digests, but in general these differences were small. In almost every case, digestion with the chymotrypsin-pronase mixture increased the apparent tryptophan content calculated from the fluorescence measurement (Table 2). The exceptions were alcohol dehydrogenase and bovine serum albumin, which yielded the correct values without digestion. The largest increases were observed with the heme proteins; the undigested samples showed almost no fluorescence due to tryptophan, but yielded nearly theoretical values after digestion. Several proteins, including lysozyme, trypsin and pepsin yielded low values even after digestion. We also compared the present method with that of Spande and Witkop (9) involving titration of the tryptophan residues with Nbromosuccinimide (Table 3). For a few proteins, such as trypsin and lysozyme, the results obtained with the latter method were closer to the literature values, but usually better agreement was obtained with the fluorescence method. In addition, the fluorescence method was considerably more sensitive.
FLUOROMETRIC
DETERMINATION
TABLE TRWTOPHAN
OF
401
TRYPTOPHAN
2
ANALYSIS WITH AND WITHOUT PRONASE AND CHYMOTRYPSIN
DIGESTION
Tryptophan
WITH
content (molimol protein)
Analysis” Without digestion’
Proteinb Aldolase Alcohol dehydrogenase DNase Pepsin Bovine serum albumin Glyceraldehyde-3-phosphate Cytochrome c Hemoglobin Myoglobin Trypsin Lysozyme Papain RNase
dehydrogenase
10.2 4.2 1.4 3.4 2.2 8.0 0 1.9 0.2 3.2 0.6 1.2 0
With digestiond 11.0 4.1 3.1 4.0 1.8 11.3 0.7 6.0 1.6 3.2 3.4 3.1 0.1
11.6 4.2 4.1 1.9 11.6 0.9
4.0 3.7
Literature VdUP
(10) 4 (11) 3 (12) 5 (13,14Y 2 (15)’ 12 (16)g 1 (11) 6 (11) 2 (11) 4 (11) 6 (11) 5 (11) 0 (11)
12
a The digestion, analyses, and calculations were carried out as described under Methods and in the legend to Fig. 2. Protein content was determined from the ninhydrin color values after alkaline hydrolysis (see Table 1). * For the source of the proteins used, see Materials. c The samples in triethanolamine-diethanolamine buffer were diluted with 7 ml of buffered urea (see Methods) without heating or addition of chymotrypsin and pronase. d The italicized numbers represent tryptophan values derived from the determination of the amino acid sequences of these proteins (10-12). The numbers in parenthesis refer to the literature references. e Five peptides containing tryptophan have been isolated from hog pepsin and their sequences established (13,14). f The number of tryptophan residues was determined spectrophotometrically, and confirmed by the isolation of two peptides containing tryptophan after cleavage with BrCN and reduction of the cross-linking disulfide bonds (15). B The tryptophan content for the enzyme from rabbit muscle was determined spectrophotometrically by Velick and Furfine (16); the same number of tryptophan residues has been reported in the complete sequence analysis of the enzymes from pig and lobster muscle (11).
DISCUSSION
The fluorometric method described in this paper provides a simple, sensitive and accurate method of tryptophan analysis. The strongly fluorescent amino acid, tyrosine, does not interfere because the choice of wavelengths of excitation and emission was such as to eliminate the
402
SASAKI,
ABRAMS
AND
TABLE COMPARISON
Aldolase DNase Hemoglobin Myoglobin Cytochrome c Bovine serum albumin Trypsin Pepsin Lysozyme Alcohol dehydrogenase Glyceraldehyde-3.phosphate dehydrogenase Triose phosphate isomerase Pyruvate kioase Lactate dehydrogenase Phosphoglucomutase Glucagon RNase
3
OF TRYPTOPHAN ANALYSIS BY FLUORESCENCE N-BROMOSUCCINIMIDE METHODS Fluorescmce”
Protein
HORECKER
AND
‘V-Bromosuccinimldti
Sample six tmgl
Tryptophan lmollmol protem)
Samplesire (mgl
Tryptophan lmollmol protein)
Llterifture value’
0.034 0.14 0.035 0.13 0.14 0 15 0.09 0.15 0.14 0.06 0.30
12.0 ?.I 5.7 1.7 0.8 2.0 2.6 5.0 4.1 3.8 12.0
0.91 I .oo 0.50 1.00 1.00 1.00 0.73 1.00 0.60 I .oo 0.44
I I.5 2.3 2.2 2.9 0.5 1.6 3.2 2.4 5.3 3.8 12.2
I ?” 3" 6" 2"
0 23 0.28 0.47 0.04 0.27 0.29
10.0 12.5 24.0 8.0 1.0 0.1
0.99 0.47 0.75 0 I6 0.45 0.75
I I.5 11.3 19.8 5.2 0.9 0.0
?” 4’ 5’ 6d 4" I?” IO (17)” I? 20-23tl9)’ 8 (20)' I (III” 0”
n The digestion, analyses. and calculations werr carried out as dexribed under Methods in thr legend to Fig. 2 with the modification indicated in footnote rl. Protein content was determined from thr ninhydrin color- value\ after alksline hydrolysis (see Table I). ’ According to Spade and Witkop (9). c The digestions werr carried out in one-half the usual volume with one-half the quantities of proteolytic enzymes. and the samples were diluted to only 3.5 ml with buffered urea. * The italicized numbers represent tryptophan values derived from the determination of the amino acid sequence> of these proteins (10-12). The numbers in parenthesis refer to the literature references. e The number of tryptophan residues was determined spectrophotometrically, and confirmed by the isolation of two peptides containing tryptophan after cleavage with BrCN and reduction of the cross-linkmg disulfide bonds (15). ’ Five peptides containing tryptophan have been isolated from hog pepsin and their sequences established (13.14). 0 The tryptophan content for the enzyme from rabbit muscle was determined spectrophotometrically by Velick and Furfine (16); the same number of tryptophan residues has been reported in the complete sequence analysis of the enzymes from pig and lobster muscle (11) ’ These values are based on spectrophotometric analyses (17.18). ’ Definitive data on the tryptophan content of the beef heart enzyms are not available. Pesce er ul. (191 have reported values of 20 residueslmol by a calorimetric procedure (22) and 23 residues by a spectrophotometric method 128). 1 Titration with N-bromosuccinimide in the presence and absence of urea indicates that the enzyme contains 5 accescible and 3-4 buried tryptophan raidues (20).
fluorescent contribution from this amino acid(21). Other amino acids either do not fluoresce or their fluorescence is negligible. Further, the presence of other pigments in the proteins, such as heme, did not interfere with the determinations. Undigested samples of the pigmented proteins (cytochrome c. myoglobin and hemoglobin) showed little or no fluorescence, but after digestion typical tryptophan emission curves were observed and quantitation of the tryptophan content was possible. For most of the proteins examined, digestion with the proteolytic mixture was essential. The proteases employed were selected to avoid alkaline or acid conditions, and are readily available. The combination of
FLUOROMETRIC
DETERMINATION
OF
TRYPTOPHAN
403
chymotrypsin and pronase appeared, in most cases, to provide sufficient destruction of the protein structure to eliminate internal quenching. Lysozyme, trypsin and papain, extracellular proteins that contain disulfide bridges, yielded low values for tryptophan by the present method, perhaps because the structures of these proteins were not sufficiently disrupted. Reduction before proteolysis, or the addition of other denaturating agents or reducing agents, such as sodium dodecyl sulfate or mercaptoethanol, after hydrolysis, did not improve the results with these proteins. On the other hand, pepsin and bovine serum albumin, which also contain disulfide bridges, yielded close to theoretical values. The fluorometric method described in this paper is more sensitive. less time consuming, and in most cases more accurate than other available methods for the determination of tryptophan. It offers several advantages over the fluorescence method described by Shelton and Rogers (4). Thus tryptophan is employed as the primary standard, and it is not necessary to run parallel assays with a number of standard proteins. In addition, the use of tryptophan as an internal standard eliminates the need for careful control of temperature and also for separate calibration curves. The practical limit to the sensitivity of the present method is imposed by the amount of material that can be handled quantitatively during digestion. Reducing the digestion volume to less than 50 ~1 resulted in significant losses. The digestions and assays can be carried out with 20- 150 pg of protein compared with approximately l-2 mg of protein required for the N-bromosuccinimide method (9), 0.1-5 mg for the calorimetric methods (22-25), 0.2-l mg for a chromatographic method (26) and l-3 mg for the determination by ultraviolet absorption (27,23). REFERENCES 1. Friedman. M.. and Finley. J. W. (1971) J. Agr. Food Chrm. 19, 626-631. 2. Udenfriend. S. (1962) in Fluorescence Assay in Biology and Medicine, Vol. 1. pp. 201-207, Academic Press, New York. 3. Duggan, D. E.. and Udenfriend. S. (1956) J. Bid/. Chem. 223, 313-319. 4. Shelton, K. R.. and Rogers, K. S. (1971) Anal. B&hem. 44, 134-142. 5. Hirs, C. H. W. (1955) in Methods in Enzymology (C. H. W. Hirs, ed.), Vol. Xl, pp. 325-329. Academic Press, New York. 6. Bucher, T. (1947) Biochim. Biophys. Actu 1, 292-3 14. 7. Taylor, J. T. (1955) in Methods in Enzymology (S. P. Colowick and N. 0. Kaplan, eds.), Vol. IV, pp. 310-315. Academic Press, New York. 8. Velick, S. F. (1955) in Methods in Enzymology (S. P. Colowick and N. 0. Kaplan, eds.), Vol. I, pp. 401-406, Academic Press, New York. 9. Spande, T. F., and Witkop, B. (1967) in Methods in Enzymology (C. H. W. Hirs, ed.), Vol. XI, pp. 498-506, Academic Press, New York. 10. Lai, C. Y.. Nakai, N., and Chang. D. ( 1974) Science 183, 1204- 1206. 11. Dayhoff. M. 0. (1972) Atlas of Protein Sequence and Structure. Vol. 5. p. 418, National Biomed. Research Foundation, Washington. DC.
404
SASAKI,
ABRAMS
AND HORECKER
12. Liao, T. H., Salnikow, J., Moore, S.. and Stein, W. H. (1973) J. Eiol. Chem. 248, 1489-1495. 13. Dopheide. T. A. A., and Jones. Wanda M. (1968) J. Biol. Chem. 243, 3906-3911. 14. Kostka, V., Moravek, L., and Sorm, F. (1970) Eur. J. Biochem. 13, 447-454. 1.5. King, T. P., and Spencer, M. (197O)J. Biol. Chem. 245, 6134-6148. 16. Velick, S. F., and Furfine, C. (1963) in The Enzymes (P. D. Boyer, ed.), 2nd ed., Vol. 7, p. 250, Academic Press, New York. 17. Norton, I. L., Pfuderer, Peter, Stringer, C. D., and Hartman. F. C. (1970) Biochemistry 9, 4952-4958. 18. Cottam, G. L., Hollenberg, P. F., and Coon, M. J. (1969) J. Biol. Chem. 244, 1481-1486. 19. Pesce, A., McKay, R. H., Stolzenbach, F., Cahn, R. D.. and Kaplan, N. 0. (1964) J. Biol. Chem. 239, 1753-1761. 20. Sloan, N. H., Mercer, D. W., and Danoff, M. (1964) Biochim. Biophys. Acta 92, 168-170. 2 1. Udenfriend, S. (1962) in Fluorescence Assay in Biology and Medicine. Vol. I, p. 129, Academic Press, New York. 22. Spies, J. R.. and Chambers, D. C. (1949) Anal. Chem. 21, 1249-1266. 23. Messieneo, L., and Musarra, E. (1972) Int. J. Biochem. 3, 700-704. 24. Gaitonde, M. K.. and Davey, T. (1970) Biochem. J. 117, 907-911. 25. Barman, T. E.. and Koshland, D. E., Jr. (1967) J. Biol. Chem. 242, 5771-5776. 26. Hugh, T. E., and Moore, S. (1972) J. Biol. Chem. 247, 2828-2834. 27. Edelhoch, H. (1967) Biochemistry 6, 1948-1954. 28. Goodwin, T. W., and Morton, R. A. (1946) Biochem. J. 40, 628-632.
