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Determination of Sepharose-bound protein with Coomassie brilliant blue G-250
ANALYTICAL
BIOCHEMISTRY
151,
571-574 (1985)
Determination of Sepharose-Bound Protein with Coomassie Brilliant Blue G-250 R. A. ASRYANTS, I. V. DUSZENKOVA, AND N. K. NAGRAD~VA A. N. Belozersky
Laboratory of Molecular Biology and Bioorganic Moscow State University. Moscow 119899, USSR
Chemistry,
Received March 22, 1985 The calorimetric procedure of Bradford (M. M. Bradford, 1976, Anal. Biochem. 72,248-254) was found to be convenient for determining the content of a protein immobilized on Sepharose. Being simple, sensitive, and rapid, this method appears very useful in studies involving multiple analyses of immobilized protein species present at low concentrations. 0 1985 Academic RUSS, IIIC. KEY WORDS: immobilized enzymes; protein determination; Coomassie blue.
For several years our studies have focused on the preparation of Sepharose-bound oligomeric enzymes attached to the matrix via a single subunit with subsequent dissociation to obtain immobilized monomeric species. The amounts of protein attached to the matrix are usually low. The procedure requires treatment of an immobilized preparation with urea and continuous control of its protein content in order to begin removing the dissociating agent shortly after the noncovalently bound subunits have split off, thus avoiding undesirable exposure to urea of the monomer obtained. The methods for immobilized protein determination described to date in the literature ( l-4) are either not sufficiently sensitive or not rapid enough to meet the above requirements. In 1976, Bradford developed a method for the evaluation of microgram quantities of protein in solution which, while simple and rapid, is also insensitive to the presence of many compounds interfering with protein determination by other methods (5). In the course of our work we found this method suitable for the determination of protein immobilized on Sepharose. In the present paper we describe the procedure, which can be recommended for studies with low immobilized protein concentrations. 571
MATERIALS
AND METHODS
Materials. Sepharose 4B was obtained from Pharmacia Fine Chemicals, Coomassie brilliant blue G-250 and dithiothreitol were products from Sigma, and EDTA was obtained from Reanal. All other chemicals were of the best grade available. Protein preparation and coupling to Sepharose. Glyceraldehyde-3-phosphate dehydrogenase was isolated from rabbit muscle (6); the soluble enzyme concentration was estimated spectrophotometrically using Ait? equal to 1.02 (7). The enzyme was covalently linked to CNBr-activated Sepharose 4B according to the previously described procedure (89). Immobilized protein determination. Protein reagent was prepared as previously described (510). Coomassie brilliant blue G-250 (25 mg) was dissolved in 12.5 ml of 95% ethanol. Phosphoric acid [25 ml, 85% (w/v)] was added, and the resulting solution was diluted with water to a final volume of 250 ml and filtered. The reagent was stable at room temperature for a month. A suspension of Sepharose-bound protein to be analyzed was packed by centrifugation (3 min at 3008) and mixed with an 0003-2697185 $3.00 Copyright 0 1985 by Academic Press. Inc. All rights of reproduction in any form reserved.
572
ASRYANTS.
DUSZENKOVA,
equal volume of an appropriate buffer; the volume of the buffer could be increased depending on the immobilized protein content. Accurate samples (0.1 ml) were taken from the stirred suspension. The control (unactivated) Sepharose samples were prepared in a similar way. To construct a calibration curve, a series of samples was prepared containing different amounts of Sepharose-bound protein. Immobilized glyceraldehyde-3-phosphate dehydrogenase was used as a standard. The protein content of the preparation was estimated using previously described methods: a modified Lowry procedure (l), the direct spectrophotometric determination of matrix-bound protein (I), and the spectrophotometric procedure in polyethylene glycol(2); all the methods gave similar results. The standard suspension of the matrix-bound protein in an appropriate buffer was diluted with an unactivated Sepharose suspension to obtain a series of samples containing 20-200 pg protein/ml. Then 0.1 ml was taken from each sample under stirring and introduced in a test tube. Two milliliters of the dye-reagent was added to each sample. After a 2- to 3-min incubation at room temperature (20°C) under stirring, the absorbance at 595 nm was measured (under continuous stirring), using as reference a sample of unactivated Sepharose suspension. More accurate results were obtained when the samples were prepared directly in spectrophotometric cells (3-ml glass cuvettes), thus avoiding the transfer of the suspensions from test tubes to the cells. A simplified procedure can also be used to obtain a calibration curve. A series of samples containing 0.1 ml of unactivated Sepharose suspension and different amounts of the soluble protein (introduced in a volume of 5-50 ~1) is treated with the dye-reagent as described above. RESULTS
Figure 1 shows calibration curves obtained using different procedures. It is seen that the
AND NAGRADOVA
color response of immobilized protein used as a standard is identical to the response of soluble protein added to a Sepharose suspension. This justifies the employment of the simplified procedure, which is more convenient in routine work. The results of Fig. 1 demonstrate that the presence of Sepharose does not affect the linear relationship between absorbance and the amount of protein in the samples. The figure also shows that components of the buffer employed to prepare the Sepharose suspension slightly reduce the absorbance of the dye-protein complex. The effect of EDTA and dithiothreitol on Coomassie brilliant blue protein assay was previously shown to be very small (5, IO). It should be noted that in these studies the value of As95 of the free dye was measured and found to increase in the presence of EDTA
8
5
10
15
20 5 m g Protein
1
10
15
20
FIG. 1. Standard curves for the determination of protein in the presence of Sepharose 4B. (A) The reactionmixture contained 0.1 ml of Sepharose-bound protein suspension in 0.1 M sodium phosphate, pH 8.3, 5 mM EDTA, 2 mM dithiothreitol, 2 ml of the dye-reagent (0). Another series of samples contained 0.1 ml of the unactivated Sepharose suspension in the above buffer, 2.0 ml of the dye-reagent, and different amounts of soluble glyceraldehyde-3-phosphate dehydrogenase (A). Reference cells contained 0.1 ml of the unactivated Sepharose suspension plus 2.0 ml of the dye-reagent. (B) 0. I ml of the unactivated Sepharose suspension in water (line 1) or in 0.1 M sodium phosphate, pH 8.3, 5 mM EDTA, 2 mM dithiothreitol (line 2), 2.0 ml of the dye-reagent, and different amounts of the soluble enzyme. Reference cells contained 0.1 ml of the unactivated Sepharose suspension in water (line 1) or in the above buffer (line 2) plus 2.0 ml of the dye-reagent. The data are mean values from four parallel determinations. The straight lines were obtained by linear regression analysis of experimental points.
COLORIMETRIC
IMMOBILIZED
PROTEIN
DETERMINATION
573
TABLE 1 and dithiothreitol. According to our data, the effect is different if the absorbance of the dyeSEPHAROSE-BOUNDPROTEIN CONTENT DETERMINED BY DIFFERENT METHODS protein complex is determined. Figure 2 demonstrates the rate of formation pg immobilized protein/ml packed gel of the complex between Coomassie brilliant blue and the protein immobilized on SepharModified Lowry Coomassie brilliant ose. It is seen that, in accordance with the re- Sample No.” procedure b blue G-250’ sults obtained with soluble proteins (5), the color development is complete within 2 min I 156.9 158.6 of incubation. Table 1 summarizes the results 35.1 2 36.3 of Sepharose-bound protein determination 3 292.5 286.0 4 156.0 145.7 carried out with Coomassie brilliant blue and by the modified Lowry reaction. It is seen that ’ Immobilized protein suspension in 0. I M sodium the data obtained by the two methods are in phosphate, pH 8.3,5 mM EDTA, 2 mM dithiothreitol was sufficiently good agreement. However the dyepresent in all samples. I and 3, without other additions; protein complex assay obviously has some ad- 2 and 4, 3 M urea added. vantages: first, it is much easier to handle, and b Immobilized protein suspension was thoroughly second, it does not require a washing of the washed with 100-150 vol of water to remove EDTA, disamples to remove EDTA, dithiothreitol, and thiothreitol, and urea. The packed gel (0.15-0.3 ml) was made up to 1.4 ml with water. One milliliter of the mixutre urea, which interfere with protein determi(50 ml of 4% Na2COj in 0.2 M NaOH plus 1 ml of 1% nation by the Lowry procedure. CuSO., in 2% Na3C6HS07) was added. After 20 min inThis makes the Coomassie brilliant blue as- cubation under continuous stirring 0.2 ml of the Folin reagent was added. Following 2 h incubation at room say very useful in studies on immobilized oligomeric protein species. Extensive washing temperature (under stirring) the absorbance readings of the stirred suspension were taken at 750 nm. Blanks were of a preparation containing an oligomer bound composed of equal amounts of unactivated Sepharose 4B to the matrix via a single subunit may, in some suspension. cases, result in a partial dissociation of the ’ Protein determinations were performed without any noncovalently bound subunits. If, as it occurs washing of the immobilized protein samples. in our studies with glyceraldehyde-3-phos-
phate dehydrogenase, the immobilized enzyme is obtained and stored in the presence of chelating and SH- groups protecting agents, it becomes important to use a method of protein determination not requiring the removal of these agents. The insensitivity of the dyeprotein color reaction to the presence of urea (taken in 0.15 M concentration) made it possible to evaluate the immobilized protein content in samples containing rather high urea concentrations (Table 1). This appeared to be very convenient in studies involving treatment of an immobilized oligomeric enzyme with FIG. 2. The rate of complex formation between the Se- urea to obtain matrix-bound subunit forms. pharose-bound protein and Coomassie brilliant blue. One- The procedure was much simpler and more tenth milliliter immobilized glyceraldehyde-3-phosphate dehydrogenase (8.0 pg) in 0.1 M sodium phosphate buffer, rapid and sensitive than those previously employed in this laboratory (spectrophotometric pH 8.3,5 mM EDTA, 2 mM dithiothreitol was mixed with 2 ml of the dye-reagent. protein determination in polyethylene glycol
574
ASRYANTS.
DUSZENKOVA.
(2) or the modified Lowry method). The extinction coefficients (absorbance of 10 jzg of glyceraldehyde-3-phosphate dehydrogenasel ml of the sample) were calculated from calibration curves obtained in the course of protein determination by the three methods. According to the results of five experiments, they corresponded to OS3,O. 14 1, and 0.03 1 in the cases of Coomassie brilliant blue, Lowry, or spectrophotometric (in polyethylene glycol) protein assay procedures, respectively. In conclusion, the method of protein determination with Coomassie brilliant blue can be successfully used with proteins immobilized on Sepharose. Due to high sensitivity and simplicity, this method is convenient in studies involving the preparation of matrix-bound subunit forms of oligomeric proteins.
AND NAGRADOVA
REFERENCES I. Koelsch, R., Lasch, J., Marquardt. I., and Hanson, H. (1975) Anal. Biochem. 66,556-567. 2. Golovina, T. 0.. Cherednikova, T. V., Mevkh, A. T., and Nagradova, N. K. (1977) Anal. Biochem. 83, 778-781. 3. Schun, H., and Riidiger, H. (1982) Anal. Biochem. 123, 174-177. 4. Marciani, D. J., Wilkie, S. D., and Schwartz, C. L. (1983) Anal. Biochem. 128, 130-137. 5. Bradford, M. M. (1976) Anal. Biochem. 12,248-254. 6. Scheek, R. M., and Slater. E. S. (1978) Biochim. Biophys. Acta 526, 13-24. Fox, J. B., and Dandliker, W. B. (1956) J. Biol. Chem. 218, 53-57. Golovina, T. O., Muronetz, V. I., and Nagradova, N. K. (1978) Biochim. Biophys. Acta 524, 15-25. Muronetz, V. I., Ashmarina. L. I., Asryants, R. A.. and Nagradova, N. K. (1982) Biochimiya 47,977986. (in Russian) 10. Spector, T. (1978) Anal. Biochem. 86, 142-146.
BIOCHEMISTRY
151,
571-574 (1985)
Determination of Sepharose-Bound Protein with Coomassie Brilliant Blue G-250 R. A. ASRYANTS, I. V. DUSZENKOVA, AND N. K. NAGRAD~VA A. N. Belozersky
Laboratory of Molecular Biology and Bioorganic Moscow State University. Moscow 119899, USSR
Chemistry,
Received March 22, 1985 The calorimetric procedure of Bradford (M. M. Bradford, 1976, Anal. Biochem. 72,248-254) was found to be convenient for determining the content of a protein immobilized on Sepharose. Being simple, sensitive, and rapid, this method appears very useful in studies involving multiple analyses of immobilized protein species present at low concentrations. 0 1985 Academic RUSS, IIIC. KEY WORDS: immobilized enzymes; protein determination; Coomassie blue.
For several years our studies have focused on the preparation of Sepharose-bound oligomeric enzymes attached to the matrix via a single subunit with subsequent dissociation to obtain immobilized monomeric species. The amounts of protein attached to the matrix are usually low. The procedure requires treatment of an immobilized preparation with urea and continuous control of its protein content in order to begin removing the dissociating agent shortly after the noncovalently bound subunits have split off, thus avoiding undesirable exposure to urea of the monomer obtained. The methods for immobilized protein determination described to date in the literature ( l-4) are either not sufficiently sensitive or not rapid enough to meet the above requirements. In 1976, Bradford developed a method for the evaluation of microgram quantities of protein in solution which, while simple and rapid, is also insensitive to the presence of many compounds interfering with protein determination by other methods (5). In the course of our work we found this method suitable for the determination of protein immobilized on Sepharose. In the present paper we describe the procedure, which can be recommended for studies with low immobilized protein concentrations. 571
MATERIALS
AND METHODS
Materials. Sepharose 4B was obtained from Pharmacia Fine Chemicals, Coomassie brilliant blue G-250 and dithiothreitol were products from Sigma, and EDTA was obtained from Reanal. All other chemicals were of the best grade available. Protein preparation and coupling to Sepharose. Glyceraldehyde-3-phosphate dehydrogenase was isolated from rabbit muscle (6); the soluble enzyme concentration was estimated spectrophotometrically using Ait? equal to 1.02 (7). The enzyme was covalently linked to CNBr-activated Sepharose 4B according to the previously described procedure (89). Immobilized protein determination. Protein reagent was prepared as previously described (510). Coomassie brilliant blue G-250 (25 mg) was dissolved in 12.5 ml of 95% ethanol. Phosphoric acid [25 ml, 85% (w/v)] was added, and the resulting solution was diluted with water to a final volume of 250 ml and filtered. The reagent was stable at room temperature for a month. A suspension of Sepharose-bound protein to be analyzed was packed by centrifugation (3 min at 3008) and mixed with an 0003-2697185 $3.00 Copyright 0 1985 by Academic Press. Inc. All rights of reproduction in any form reserved.
572
ASRYANTS.
DUSZENKOVA,
equal volume of an appropriate buffer; the volume of the buffer could be increased depending on the immobilized protein content. Accurate samples (0.1 ml) were taken from the stirred suspension. The control (unactivated) Sepharose samples were prepared in a similar way. To construct a calibration curve, a series of samples was prepared containing different amounts of Sepharose-bound protein. Immobilized glyceraldehyde-3-phosphate dehydrogenase was used as a standard. The protein content of the preparation was estimated using previously described methods: a modified Lowry procedure (l), the direct spectrophotometric determination of matrix-bound protein (I), and the spectrophotometric procedure in polyethylene glycol(2); all the methods gave similar results. The standard suspension of the matrix-bound protein in an appropriate buffer was diluted with an unactivated Sepharose suspension to obtain a series of samples containing 20-200 pg protein/ml. Then 0.1 ml was taken from each sample under stirring and introduced in a test tube. Two milliliters of the dye-reagent was added to each sample. After a 2- to 3-min incubation at room temperature (20°C) under stirring, the absorbance at 595 nm was measured (under continuous stirring), using as reference a sample of unactivated Sepharose suspension. More accurate results were obtained when the samples were prepared directly in spectrophotometric cells (3-ml glass cuvettes), thus avoiding the transfer of the suspensions from test tubes to the cells. A simplified procedure can also be used to obtain a calibration curve. A series of samples containing 0.1 ml of unactivated Sepharose suspension and different amounts of the soluble protein (introduced in a volume of 5-50 ~1) is treated with the dye-reagent as described above. RESULTS
Figure 1 shows calibration curves obtained using different procedures. It is seen that the
AND NAGRADOVA
color response of immobilized protein used as a standard is identical to the response of soluble protein added to a Sepharose suspension. This justifies the employment of the simplified procedure, which is more convenient in routine work. The results of Fig. 1 demonstrate that the presence of Sepharose does not affect the linear relationship between absorbance and the amount of protein in the samples. The figure also shows that components of the buffer employed to prepare the Sepharose suspension slightly reduce the absorbance of the dye-protein complex. The effect of EDTA and dithiothreitol on Coomassie brilliant blue protein assay was previously shown to be very small (5, IO). It should be noted that in these studies the value of As95 of the free dye was measured and found to increase in the presence of EDTA
8
5
10
15
20 5 m g Protein
1
10
15
20
FIG. 1. Standard curves for the determination of protein in the presence of Sepharose 4B. (A) The reactionmixture contained 0.1 ml of Sepharose-bound protein suspension in 0.1 M sodium phosphate, pH 8.3, 5 mM EDTA, 2 mM dithiothreitol, 2 ml of the dye-reagent (0). Another series of samples contained 0.1 ml of the unactivated Sepharose suspension in the above buffer, 2.0 ml of the dye-reagent, and different amounts of soluble glyceraldehyde-3-phosphate dehydrogenase (A). Reference cells contained 0.1 ml of the unactivated Sepharose suspension plus 2.0 ml of the dye-reagent. (B) 0. I ml of the unactivated Sepharose suspension in water (line 1) or in 0.1 M sodium phosphate, pH 8.3, 5 mM EDTA, 2 mM dithiothreitol (line 2), 2.0 ml of the dye-reagent, and different amounts of the soluble enzyme. Reference cells contained 0.1 ml of the unactivated Sepharose suspension in water (line 1) or in the above buffer (line 2) plus 2.0 ml of the dye-reagent. The data are mean values from four parallel determinations. The straight lines were obtained by linear regression analysis of experimental points.
COLORIMETRIC
IMMOBILIZED
PROTEIN
DETERMINATION
573
TABLE 1 and dithiothreitol. According to our data, the effect is different if the absorbance of the dyeSEPHAROSE-BOUNDPROTEIN CONTENT DETERMINED BY DIFFERENT METHODS protein complex is determined. Figure 2 demonstrates the rate of formation pg immobilized protein/ml packed gel of the complex between Coomassie brilliant blue and the protein immobilized on SepharModified Lowry Coomassie brilliant ose. It is seen that, in accordance with the re- Sample No.” procedure b blue G-250’ sults obtained with soluble proteins (5), the color development is complete within 2 min I 156.9 158.6 of incubation. Table 1 summarizes the results 35.1 2 36.3 of Sepharose-bound protein determination 3 292.5 286.0 4 156.0 145.7 carried out with Coomassie brilliant blue and by the modified Lowry reaction. It is seen that ’ Immobilized protein suspension in 0. I M sodium the data obtained by the two methods are in phosphate, pH 8.3,5 mM EDTA, 2 mM dithiothreitol was sufficiently good agreement. However the dyepresent in all samples. I and 3, without other additions; protein complex assay obviously has some ad- 2 and 4, 3 M urea added. vantages: first, it is much easier to handle, and b Immobilized protein suspension was thoroughly second, it does not require a washing of the washed with 100-150 vol of water to remove EDTA, disamples to remove EDTA, dithiothreitol, and thiothreitol, and urea. The packed gel (0.15-0.3 ml) was made up to 1.4 ml with water. One milliliter of the mixutre urea, which interfere with protein determi(50 ml of 4% Na2COj in 0.2 M NaOH plus 1 ml of 1% nation by the Lowry procedure. CuSO., in 2% Na3C6HS07) was added. After 20 min inThis makes the Coomassie brilliant blue as- cubation under continuous stirring 0.2 ml of the Folin reagent was added. Following 2 h incubation at room say very useful in studies on immobilized oligomeric protein species. Extensive washing temperature (under stirring) the absorbance readings of the stirred suspension were taken at 750 nm. Blanks were of a preparation containing an oligomer bound composed of equal amounts of unactivated Sepharose 4B to the matrix via a single subunit may, in some suspension. cases, result in a partial dissociation of the ’ Protein determinations were performed without any noncovalently bound subunits. If, as it occurs washing of the immobilized protein samples. in our studies with glyceraldehyde-3-phos-
phate dehydrogenase, the immobilized enzyme is obtained and stored in the presence of chelating and SH- groups protecting agents, it becomes important to use a method of protein determination not requiring the removal of these agents. The insensitivity of the dyeprotein color reaction to the presence of urea (taken in 0.15 M concentration) made it possible to evaluate the immobilized protein content in samples containing rather high urea concentrations (Table 1). This appeared to be very convenient in studies involving treatment of an immobilized oligomeric enzyme with FIG. 2. The rate of complex formation between the Se- urea to obtain matrix-bound subunit forms. pharose-bound protein and Coomassie brilliant blue. One- The procedure was much simpler and more tenth milliliter immobilized glyceraldehyde-3-phosphate dehydrogenase (8.0 pg) in 0.1 M sodium phosphate buffer, rapid and sensitive than those previously employed in this laboratory (spectrophotometric pH 8.3,5 mM EDTA, 2 mM dithiothreitol was mixed with 2 ml of the dye-reagent. protein determination in polyethylene glycol
574
ASRYANTS.
DUSZENKOVA.
(2) or the modified Lowry method). The extinction coefficients (absorbance of 10 jzg of glyceraldehyde-3-phosphate dehydrogenasel ml of the sample) were calculated from calibration curves obtained in the course of protein determination by the three methods. According to the results of five experiments, they corresponded to OS3,O. 14 1, and 0.03 1 in the cases of Coomassie brilliant blue, Lowry, or spectrophotometric (in polyethylene glycol) protein assay procedures, respectively. In conclusion, the method of protein determination with Coomassie brilliant blue can be successfully used with proteins immobilized on Sepharose. Due to high sensitivity and simplicity, this method is convenient in studies involving the preparation of matrix-bound subunit forms of oligomeric proteins.
AND NAGRADOVA
REFERENCES I. Koelsch, R., Lasch, J., Marquardt. I., and Hanson, H. (1975) Anal. Biochem. 66,556-567. 2. Golovina, T. 0.. Cherednikova, T. V., Mevkh, A. T., and Nagradova, N. K. (1977) Anal. Biochem. 83, 778-781. 3. Schun, H., and Riidiger, H. (1982) Anal. Biochem. 123, 174-177. 4. Marciani, D. J., Wilkie, S. D., and Schwartz, C. L. (1983) Anal. Biochem. 128, 130-137. 5. Bradford, M. M. (1976) Anal. Biochem. 12,248-254. 6. Scheek, R. M., and Slater. E. S. (1978) Biochim. Biophys. Acta 526, 13-24. Fox, J. B., and Dandliker, W. B. (1956) J. Biol. Chem. 218, 53-57. Golovina, T. O., Muronetz, V. I., and Nagradova, N. K. (1978) Biochim. Biophys. Acta 524, 15-25. Muronetz, V. I., Ashmarina. L. I., Asryants, R. A.. and Nagradova, N. K. (1982) Biochimiya 47,977986. (in Russian) 10. Spector, T. (1978) Anal. Biochem. 86, 142-146.