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Amino acid analysis with fluorescamine of stained protein bands from polyacrylamide gels
ANALYTICAL
BIOCHEMISTRY
Amino
Acid Protein S. STEIN,
Roche Institute
60,
272-277 (1974)
Analysis Bands
with from
Fluorescamine Polyacrylamide
Gels
C. H. CHANG, P. BOHLEN, AND S. UDENFRIEND
of Molecular
Received December
Biology, Nutleg,
of Stained
K. IMAI,
New Jersey 07110
10, 1973; accepted February
11, 1974
Bands in stained polyacrylamide gels, containing microgram quantities of protein, have been subjected to hydrolysis and subsequent amino acid analysis with fluorescamine. Analyses on these protein bands agree with determinations performed on aqueous solutions of the protein and with published results.
Polyacrylamide gel electrophoresis is one of the most widely used methods in protein biochemistry. In many cases, essentially complete resolution of protein components can be achieved on analytical gels. To visualize the bands, gels are generally stained with dyes such as Coomassie Blue. Unfortunately, after staining, the protein is not easily extractable for further chemical study. A previous attempt has been made to determine the amino acid composition of protein bands in stained polyacrylamide gels on a commercial analyzer utilizing ninhydrin (1). A serious difficulty associated with this procedure is the large quantity of ammonia released from the gel during hydrolysis. Fluorescamine, which has recently been applied to the automated determination of amino acids in protein hydrolysatep (2,3) has properties which make it suitable for this type of assay. The reagent not only has high sensitivity toward amino acids, but also gives lower fluorescence with ammonia (2). This report describes initial attempts to take advantage of these favorable features for determining the amino acid compositions of microgram quantities of proteins in sections cut out of stained polyacrylamide gels. MATERIALS
AND METHODS
Electrophoresis. Sodium
dodecyl sulfate (SDS) polyacrylamide gels (7% gels of 6 mm diam) were prepared and run according to the procedure of Shapiro et al. (4). Bovine serum albumin (Pentex, Kankakee, IL.) was incubated in 0.1 M phosphate buffer, pH 7.1 containing 1% SDS and 1% 2-mercaptoethanol at 37°C for 2 hr before application of 10 pg 272 Copyright @ 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
AMINO
ACID
ANALYSIS
OF
STAINED
PROTEIN
273
of protein to the gels. After electrophoresis with 0.1 M phosphate buffer, pH 7.1 containing 0.1% SDS, the gels were stained for several hours with 0.25% Coomassie Blue in 12.5% trichloroacetic acid and 1% methanol. The stained gels were then washed with several 1 liter changes of 12.5% trichloroacetic acid over a period of 2 days at room temperature. All reagents were of grades typically used for electrophoresis. Preparation of sample. Protein bands l-2 mm in height were cut out of each gel with a razor blade.*Each gel slice was placed in a l-ml ampoule (Bellco Glass, Vineland, NJ) and several ampoules were placed in a lyophilizing bottle. Lyophilization was necessary so as not to dilute the 6 N hydrochloric acid used for hydrolysis. After lyophilizing, a constriction was made in the ampoules to facilitate sealing. Each gel slice was covered with 200 pliters of constant boiling hydrochloric acid (Pierce Chem. Co., Rockford, IL), made 1% with respect to thioglycollic acid (Fisher Sci., Springfield, NJ). This reducing agent was included to prevent destruction of tyrosine and histidine during hydrolysis (1). The ampoules were flushed with nitrogen and evacuated several times before they were sealed in zlucuo. Hydrolysis was carried out at 115°C for 22 hr. After cooling, the sealed ampoules were centrifuged to collect all the liquid at the bottom. The hydrolysate, as well as 100 ,&ters of water used to rinse the gel, was transferred to a 1 ml tube. The contents were dried on a Buchi flash evaporator and dissolved in 300 ,uliters of pH 2.2 buffer. A 50-pliter aliquot was applied to the amino acid analyzer column. Gel slices containing no protein were carried through the entire procedure and served as blanks, For comparison, a 1 mg/ml solution of bovine serum albumin in water was prepared. To each ampoule was added 7.5 yliters of the protein solution and 200 pliters of 6 N hydrochloric acid containing 1% thioglycollic acid. The ampoule was sealed and hydrolysis was carried out as described above. Amino acid analysis. Analysis was performed on the instrumentation utilizing fluorescamine (Hoffmann-La Roche, Nutley, NJ) as previously 1described with certain modifications (2). An Autolab System IV in+ grator (Spectra Physics, Parsippany, NJ) was used for measuring peak areas, A one-column system was employed. For elution of the basic amino acids, the third buffer was 0.50~ sodium citrate, 0.70 N sodium chloride (total sodium concn of 1.2 N), pH 5.36. In order to perform proline analysis, the thiodiglycol was eliminated from the first buffer (3). Thiodiglycol was added to the samples to a final concentration of 0.2% (3). For proline analysis N-chlorosuccinimide was added to the column eluate after the elution of the glutamic acid peak and was stopped prior
274
STEIN
ET
AL.
too go80 -
20 -
2
3 TIME
I 4
(hours)
FIG. 1. Chromatography of a hydrolysate of a protein band from a stained polyacrylamide gel. An equivalent of about 1.5 fig of bovine serum albumin was applied to the column. Norleucine (750 pmoles) was added to the hydrolysate as an internal standard.
100 90 80 5
70-
z :: 60Y 22 50: z 405 =
3020IO -
FIG. 2. Determination of proline in a hydrolysate equivalent to that presented in Fig. 1. Addition of N-chlorosuccinimide produces a baseline drop, while termination of this oxidant results in a return to the original baseline level.
AMINO
ACID
ANALYSIS
OF
STAINED
275
PROTEIN
to the glycine peak, as previously described (3). In the presence of the reducing agent in the gel hydrolysates, cysteine rather than the more commonly measured cystine appeared on the chromatogram. Because of the close proximity of the cysteine and proline peaks under conditions using standard buffers, there was insufficient time to begin the addition of N-chlorosuccinimide and establish a baseline without interfering with cysteine analysis. Thus, duplicate runs were performed with and without addition of N-chlorosuccinimide for proline and cysteine assay, respeetively (Figs. 1 and 2). RESULTS
AND
DISCUSSION
The procedure presented in this report is practicable for obtaining the amino acid compositions of proteins in bands cut out of stained polyacrylamide gels. This is demonstrated by the fact that the composition of bovine serum albumin, determined in a gel band, was in agreement with that determined on an aqueous solution of this protein and with previous results (Table 1). The major problem of hydrolyzing in the
Amino Amino
acid
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Cysteine Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
Acid
TABLE I Composition of Bovine
Solution” 9.03 2.93 3.72 8.21 4.99 3.90 12.2 5.02 n.d.d 2.62 7.23 5.72 0.67 2.08 10.0 3.16 4.41
(.lO) C.09) (.02) (.62) (.07) (.12) (.6) (.42) (.08) (.18) (.lO) (.19) (0.1) (0.05) (.12)
Serum Gel*
8.86 2.72 3.74 8.32 5.15 3.89 12.5 3.82 4.15 2.80 7.37 6.04 0.70 2.19 10.0 3.11 4.29
(.13) (.09) (.12) (.35) (.19) (.29) (.13) (.31) (.15) (0.1) (.12) (.17) (.20) (.ll) (.02) (.06)
Albumin Report,e& 9.3 2.8 3.6 8.7 Ti 2 4.2 11.9 0‘8 4.3 2.5 7 5 .5 4 0 .6 2.1 10.0 3 0 4.2
a Determined on the hydrolysate of 7.5 pg of protein. * Determined on a band containing 10 ~g of protein from a stained polyacrylamide gel. c A Laboratory Manual of Analytical Methods of Protein Chemistry (1966) (Alexander, P. and Lundgren, H. P., eds.) Pergamon Press, New York. d Not determined. All values represent the averages obtained from assays of three separate gels. The ranges are indicated by the average deviations given in parentheses. Leucine was arbitrarily set equal to 10.
276
STEIN
ET
AL.
presence of polyacrylamide gel has been the copious quantity of ammonia produced (1). Ammonia yields lower fluorescence with fluorescamine and, although a strong peak is observed, there is no interference with the determination of lysine or histidine (Fig. 1). In the ninhydrin analyzer, the ammonia peak would be at least one order of magnitude greater with respect to the amino acids, thus causing difficulty with lysine and histidine measurements. Additionally, ammonia in such high concentrations does not form an insoluble product with fluorescamine as it does with ninhydrin (1)) avoiding the danger of particles clogging the tubing. Furthermore, there was no selective binding of any amino acids to the gel residue, since the compositions determined in the presence and absence of gel were the same (Table 1). Amino acid recoveries from the gel slices were essentially quantitative. It should be noted, however, that the amino acid composition of a protein is generally calculated on a relative basis, and variations in recovery (if uniform for all amino acids) do not effect the results. The data presented was determined on proteins run on SDS-acrylamide gels with phosphate buffer. Analyses have been performed on protein bands from gels run in Tris-glycine buffer by the method of Ornstein and Davis (5). Virtually all of the free glycine was removed by washing these gels for a few days in 12.5% trichloroacetic acid. Polyacrylamide gels containing urea can also be used for analysis. The presence of urea would be inconsequential, since this compound does not yield a fluorophor with fluorescamine. Although data are presented for 10 pg of protein, the sensitivity is sufficient to determine the amino acid composition of as little as 1 pg of protein (2). A limitation is the presence of amino acid contamination in the blank gels (Fig. 3) at levels higher than those found previously for hydrochloric acid alone (2). With 10 pg of protein in a gel band this contamination was negligible and no corrections were made. By utilizing smaller pieces of gel for analysis this contamination, which is not readily removed by dialysis, can be minimized. Analysis of cysteine presents some problem. Cysteine fluorescence is unusually low, the relative fluorescence for this amino acid being only about one-fourth of that for glutamic acid. Furthermore, proline and cysteine analysis require duplicate runs, as explained in Materials and Methods. Additionally, the cysteine analyses were low and variable (Table 1). This may be explained by the formation, during hydrolysis, of a mixed disulfide of cysteine with thioglycollic acid. Perhaps, if a nonthiol reducing agent were used during hydrolysis, cysteine could be oxidized to cystine before application to the column, thereby avoiding these difficulties.
AMINO
ACID
ANALYSIS
OF
TIME
STAINED
277
PROTEIN
(hours)
FIG. 3. Chromatography of a hydrolysate polyacrylamide gel. Norleucine (500 pmoles) internal standard.
of a blank gel slice from a stained was added to the hydrolysate as an
The purpose of this report is to demonstrate the feasibility of determining the amino acid composition of protein bands in stained polyacrylamide gels. Analysis by fluorescamine can readily be performed in the presence of polyacrylamide gel hydrolysis products with only microgram quantities of protein. Several investigators in this laboratory are currently applying this procedure to specific problems. As fluorescamine analysis becomes more popular, this new methodology should become a valuable tool in protein research. REFERENCES 1. Housm~, L. L. (1971) Anal. Biochem. 44, S-88. 2. STEIN, S., B~HLEN, P., STONE, J., DAIRMAN, W., AND UDENFRIEND, Biochem. Riophys. 155, 202-212. 3. FELIX. A. M., AND TERKELSEN, G. (1973) Arch. Biochem. Btiphys. 4. SHAPIRO, A. L., VINUELA, E., AND MAIZEL, J. V. (1967) Biochem. Commun. 28, 815-820. 5. DAVIS, B. J. (1964) Ann. hr. I’. Aead. Sci. 121, 404.
S. (1973)
Arch.
157, 177-182. Biophys. Res.
BIOCHEMISTRY
Amino
Acid Protein S. STEIN,
Roche Institute
60,
272-277 (1974)
Analysis Bands
with from
Fluorescamine Polyacrylamide
Gels
C. H. CHANG, P. BOHLEN, AND S. UDENFRIEND
of Molecular
Received December
Biology, Nutleg,
of Stained
K. IMAI,
New Jersey 07110
10, 1973; accepted February
11, 1974
Bands in stained polyacrylamide gels, containing microgram quantities of protein, have been subjected to hydrolysis and subsequent amino acid analysis with fluorescamine. Analyses on these protein bands agree with determinations performed on aqueous solutions of the protein and with published results.
Polyacrylamide gel electrophoresis is one of the most widely used methods in protein biochemistry. In many cases, essentially complete resolution of protein components can be achieved on analytical gels. To visualize the bands, gels are generally stained with dyes such as Coomassie Blue. Unfortunately, after staining, the protein is not easily extractable for further chemical study. A previous attempt has been made to determine the amino acid composition of protein bands in stained polyacrylamide gels on a commercial analyzer utilizing ninhydrin (1). A serious difficulty associated with this procedure is the large quantity of ammonia released from the gel during hydrolysis. Fluorescamine, which has recently been applied to the automated determination of amino acids in protein hydrolysatep (2,3) has properties which make it suitable for this type of assay. The reagent not only has high sensitivity toward amino acids, but also gives lower fluorescence with ammonia (2). This report describes initial attempts to take advantage of these favorable features for determining the amino acid compositions of microgram quantities of proteins in sections cut out of stained polyacrylamide gels. MATERIALS
AND METHODS
Electrophoresis. Sodium
dodecyl sulfate (SDS) polyacrylamide gels (7% gels of 6 mm diam) were prepared and run according to the procedure of Shapiro et al. (4). Bovine serum albumin (Pentex, Kankakee, IL.) was incubated in 0.1 M phosphate buffer, pH 7.1 containing 1% SDS and 1% 2-mercaptoethanol at 37°C for 2 hr before application of 10 pg 272 Copyright @ 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
AMINO
ACID
ANALYSIS
OF
STAINED
PROTEIN
273
of protein to the gels. After electrophoresis with 0.1 M phosphate buffer, pH 7.1 containing 0.1% SDS, the gels were stained for several hours with 0.25% Coomassie Blue in 12.5% trichloroacetic acid and 1% methanol. The stained gels were then washed with several 1 liter changes of 12.5% trichloroacetic acid over a period of 2 days at room temperature. All reagents were of grades typically used for electrophoresis. Preparation of sample. Protein bands l-2 mm in height were cut out of each gel with a razor blade.*Each gel slice was placed in a l-ml ampoule (Bellco Glass, Vineland, NJ) and several ampoules were placed in a lyophilizing bottle. Lyophilization was necessary so as not to dilute the 6 N hydrochloric acid used for hydrolysis. After lyophilizing, a constriction was made in the ampoules to facilitate sealing. Each gel slice was covered with 200 pliters of constant boiling hydrochloric acid (Pierce Chem. Co., Rockford, IL), made 1% with respect to thioglycollic acid (Fisher Sci., Springfield, NJ). This reducing agent was included to prevent destruction of tyrosine and histidine during hydrolysis (1). The ampoules were flushed with nitrogen and evacuated several times before they were sealed in zlucuo. Hydrolysis was carried out at 115°C for 22 hr. After cooling, the sealed ampoules were centrifuged to collect all the liquid at the bottom. The hydrolysate, as well as 100 ,&ters of water used to rinse the gel, was transferred to a 1 ml tube. The contents were dried on a Buchi flash evaporator and dissolved in 300 ,uliters of pH 2.2 buffer. A 50-pliter aliquot was applied to the amino acid analyzer column. Gel slices containing no protein were carried through the entire procedure and served as blanks, For comparison, a 1 mg/ml solution of bovine serum albumin in water was prepared. To each ampoule was added 7.5 yliters of the protein solution and 200 pliters of 6 N hydrochloric acid containing 1% thioglycollic acid. The ampoule was sealed and hydrolysis was carried out as described above. Amino acid analysis. Analysis was performed on the instrumentation utilizing fluorescamine (Hoffmann-La Roche, Nutley, NJ) as previously 1described with certain modifications (2). An Autolab System IV in+ grator (Spectra Physics, Parsippany, NJ) was used for measuring peak areas, A one-column system was employed. For elution of the basic amino acids, the third buffer was 0.50~ sodium citrate, 0.70 N sodium chloride (total sodium concn of 1.2 N), pH 5.36. In order to perform proline analysis, the thiodiglycol was eliminated from the first buffer (3). Thiodiglycol was added to the samples to a final concentration of 0.2% (3). For proline analysis N-chlorosuccinimide was added to the column eluate after the elution of the glutamic acid peak and was stopped prior
274
STEIN
ET
AL.
too go80 -
20 -
2
3 TIME
I 4
(hours)
FIG. 1. Chromatography of a hydrolysate of a protein band from a stained polyacrylamide gel. An equivalent of about 1.5 fig of bovine serum albumin was applied to the column. Norleucine (750 pmoles) was added to the hydrolysate as an internal standard.
100 90 80 5
70-
z :: 60Y 22 50: z 405 =
3020IO -
FIG. 2. Determination of proline in a hydrolysate equivalent to that presented in Fig. 1. Addition of N-chlorosuccinimide produces a baseline drop, while termination of this oxidant results in a return to the original baseline level.
AMINO
ACID
ANALYSIS
OF
STAINED
275
PROTEIN
to the glycine peak, as previously described (3). In the presence of the reducing agent in the gel hydrolysates, cysteine rather than the more commonly measured cystine appeared on the chromatogram. Because of the close proximity of the cysteine and proline peaks under conditions using standard buffers, there was insufficient time to begin the addition of N-chlorosuccinimide and establish a baseline without interfering with cysteine analysis. Thus, duplicate runs were performed with and without addition of N-chlorosuccinimide for proline and cysteine assay, respeetively (Figs. 1 and 2). RESULTS
AND
DISCUSSION
The procedure presented in this report is practicable for obtaining the amino acid compositions of proteins in bands cut out of stained polyacrylamide gels. This is demonstrated by the fact that the composition of bovine serum albumin, determined in a gel band, was in agreement with that determined on an aqueous solution of this protein and with previous results (Table 1). The major problem of hydrolyzing in the
Amino Amino
acid
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Cysteine Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
Acid
TABLE I Composition of Bovine
Solution” 9.03 2.93 3.72 8.21 4.99 3.90 12.2 5.02 n.d.d 2.62 7.23 5.72 0.67 2.08 10.0 3.16 4.41
(.lO) C.09) (.02) (.62) (.07) (.12) (.6) (.42) (.08) (.18) (.lO) (.19) (0.1) (0.05) (.12)
Serum Gel*
8.86 2.72 3.74 8.32 5.15 3.89 12.5 3.82 4.15 2.80 7.37 6.04 0.70 2.19 10.0 3.11 4.29
(.13) (.09) (.12) (.35) (.19) (.29) (.13) (.31) (.15) (0.1) (.12) (.17) (.20) (.ll) (.02) (.06)
Albumin Report,e& 9.3 2.8 3.6 8.7 Ti 2 4.2 11.9 0‘8 4.3 2.5 7 5 .5 4 0 .6 2.1 10.0 3 0 4.2
a Determined on the hydrolysate of 7.5 pg of protein. * Determined on a band containing 10 ~g of protein from a stained polyacrylamide gel. c A Laboratory Manual of Analytical Methods of Protein Chemistry (1966) (Alexander, P. and Lundgren, H. P., eds.) Pergamon Press, New York. d Not determined. All values represent the averages obtained from assays of three separate gels. The ranges are indicated by the average deviations given in parentheses. Leucine was arbitrarily set equal to 10.
276
STEIN
ET
AL.
presence of polyacrylamide gel has been the copious quantity of ammonia produced (1). Ammonia yields lower fluorescence with fluorescamine and, although a strong peak is observed, there is no interference with the determination of lysine or histidine (Fig. 1). In the ninhydrin analyzer, the ammonia peak would be at least one order of magnitude greater with respect to the amino acids, thus causing difficulty with lysine and histidine measurements. Additionally, ammonia in such high concentrations does not form an insoluble product with fluorescamine as it does with ninhydrin (1)) avoiding the danger of particles clogging the tubing. Furthermore, there was no selective binding of any amino acids to the gel residue, since the compositions determined in the presence and absence of gel were the same (Table 1). Amino acid recoveries from the gel slices were essentially quantitative. It should be noted, however, that the amino acid composition of a protein is generally calculated on a relative basis, and variations in recovery (if uniform for all amino acids) do not effect the results. The data presented was determined on proteins run on SDS-acrylamide gels with phosphate buffer. Analyses have been performed on protein bands from gels run in Tris-glycine buffer by the method of Ornstein and Davis (5). Virtually all of the free glycine was removed by washing these gels for a few days in 12.5% trichloroacetic acid. Polyacrylamide gels containing urea can also be used for analysis. The presence of urea would be inconsequential, since this compound does not yield a fluorophor with fluorescamine. Although data are presented for 10 pg of protein, the sensitivity is sufficient to determine the amino acid composition of as little as 1 pg of protein (2). A limitation is the presence of amino acid contamination in the blank gels (Fig. 3) at levels higher than those found previously for hydrochloric acid alone (2). With 10 pg of protein in a gel band this contamination was negligible and no corrections were made. By utilizing smaller pieces of gel for analysis this contamination, which is not readily removed by dialysis, can be minimized. Analysis of cysteine presents some problem. Cysteine fluorescence is unusually low, the relative fluorescence for this amino acid being only about one-fourth of that for glutamic acid. Furthermore, proline and cysteine analysis require duplicate runs, as explained in Materials and Methods. Additionally, the cysteine analyses were low and variable (Table 1). This may be explained by the formation, during hydrolysis, of a mixed disulfide of cysteine with thioglycollic acid. Perhaps, if a nonthiol reducing agent were used during hydrolysis, cysteine could be oxidized to cystine before application to the column, thereby avoiding these difficulties.
AMINO
ACID
ANALYSIS
OF
TIME
STAINED
277
PROTEIN
(hours)
FIG. 3. Chromatography of a hydrolysate polyacrylamide gel. Norleucine (500 pmoles) internal standard.
of a blank gel slice from a stained was added to the hydrolysate as an
The purpose of this report is to demonstrate the feasibility of determining the amino acid composition of protein bands in stained polyacrylamide gels. Analysis by fluorescamine can readily be performed in the presence of polyacrylamide gel hydrolysis products with only microgram quantities of protein. Several investigators in this laboratory are currently applying this procedure to specific problems. As fluorescamine analysis becomes more popular, this new methodology should become a valuable tool in protein research. REFERENCES 1. Housm~, L. L. (1971) Anal. Biochem. 44, S-88. 2. STEIN, S., B~HLEN, P., STONE, J., DAIRMAN, W., AND UDENFRIEND, Biochem. Riophys. 155, 202-212. 3. FELIX. A. M., AND TERKELSEN, G. (1973) Arch. Biochem. Btiphys. 4. SHAPIRO, A. L., VINUELA, E., AND MAIZEL, J. V. (1967) Biochem. Commun. 28, 815-820. 5. DAVIS, B. J. (1964) Ann. hr. I’. Aead. Sci. 121, 404.
S. (1973)
Arch.
157, 177-182. Biophys. Res.