ANALYTICAL
BIOCHEMISTRY
%,
21-23
(1979)
Determination of Protein by a Modified Lowry Procedure Presence of Some Commonly Used Detergents EVELYN Department
CADMAN, J. ROBERT BOSTWICK,ANDJOSEPH of Biophysical
Sciences>
University
of Houston,
Houston,
in the
EICHBERG Texas
77004
Received September 18, 1978
A wide variety of detergents has been employed to solubilize and purify membrane proteins (1,2). The interference caused by certain cationic and nonionic detergents in the Lowry procedure for protein determination (3) and its elimination by inclusion of sodium dodecyl sulfate (SDS)l has been previously described (4-7). However, only for Triton X-100 has this modification been shown definitively to be effective at concentrations of interfering detergent as high as 1% in the final reaction mixture over a range of protein concentrations (45). In this communication, we further document the applicability of a modified Lowry method to protein preparations containing high levels of nonionic and cationic detergents in common use. MATERIALS
AND METHODS
20% (w/v) solution of SDS and of each detergent to be tested was prepared. The protein standard consisted of 1 mg BSA/ml water. Analyses were performed essentially according to Wang and Smith (5). Aliquots of protein standard containing O-60 pg BSA were mixed with varying amounts of detergent solution and water then added to a volume of 0.70 ml. Alkaline copper solution (1 .O ml) was added and the mixture was vortexed and allowed to stand at room temperature for 15 min. SDS (0.5 ml) was added, each sample mixed thoroughly, and 0.1 ml phenol reagent was then pipetted into each tube followed by immediate mixing. After standing for 30 min to permit color development, the absorption was determined at 500 nm for each sample in a Beckman 25 spectrophotometer.
Crystalline bovine serum albumin (BSA), Triton-X-100, Tween 20, Brij 35, Lubrol PX, and cetyltrimethylammonium bromide were obtained from Sigma Chemical Company, St. Louis, Missouri. The disodium cupric salt of EDTA was purchased from ICN Pharmaceuticals, Plainview, New York. Cutscum and the Folin-Ciocalteu reagent were products of Fisher Chemical Company and Nonidet-P40 was obtained from Shell Chemical Company. Miranol H2M was a product of the Miranol Chemical Company, Irvington, New Jersey. A 1 Abbreviations used: SDS, sodium dodecyl fate; BSA, bovine serum albumin.
RESULTS AND DISCUSSION
In confirmation of the results of others (4-7), the presence of SDS (final concentration, 4.35%) had no effect on color development, but prevented the formation of a precipitate which otherwise appeared on addition of the phenol reagent to solutions containing Triton-X-100. SDS likewise acted on assay mixtures which contained Cutscum, Nonidet-P40, Tween 20, and cetyltrimethylammonium bromide. When these detergents were present at a final concentration of 1.0% (0.75% for cetyltrimethylammonium bromide), a nearly linear
sul-
21
0003-2697/79~090021-03$02.00/O Copyright &?I 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
22
CADMAN, TABLE
BOSTWICK,
1
EFFECTOFDETERGENTON REAGENTBLANK ANDCOLORFORMATION Detergent” SDS only Cetyltrimethylammonium bromide Triton X-100 Nonidet-P40 Cutscum Tween 20 Brij 35 Lubrol PX
Reagent blankb
40 pg BSA
0.015
0.146
0.042 0.045 0.131 0.058 0.151 0.043 0.031
0.174 0.186 0.270 0.202 0.318 0.206 0.203
a All detergents were present at a final concentration of 1.0% except for cetyltrimethylammonium bromide which was present at 0.75%. b Absorption determined at 500 nm.
relationship between the amount of BSA and the color generated was obtained in each case. Each of the detergents increased the reagent blank compared to an SDScontaining control, but except for Tween 20, there was no appreciable effect on color formed in the presence of BSA (Table 1). When color development was investigated as a function of the amount of each of these detergents from 0 to I%, absolute absorption values agreed within 10%. Above this amount, a precipitate could be avoided and a usable linear relationship could still be obtained up to 1.5% Cutscum and up to 2.0% Triton X-100. However, at these concentrations, the variation of slope and intercept for individual determinations was considerable. At concentrations of cetyltrimethylammonium bromide greater than 0.75%, the final reaction mixture became gelatinous. The effects of Brij 35 and Lubrol PX in the modified Lowry procedure differed from those of the other detergents examined. In the absence of SDS, the inclusion of 0. l0.5% Brij 35 in the reaction mixture resulted in a cloudy solution. When the precipitate was removed by centrifugation, the color developed was roughly linear with protein
AND EICHBERG
concentration, but had a greenish tinge. Optical densities at 500 nm were greatly reduced compared to controls. When Brij 35 was present at 0.75% or greater, no precipitate formed, but color development remained abnormally low. The addition of SDS prevented the formation of a precipitate at low concentrations of Brij 35, but brought about the appearance of a fine precipitate when 0.75% or more of the nonionic detergent was present. After removal of the precipitate by centrifugation for 5 min at 12,OOOg, the color in the clear supernatant appeared normal and was proportional to the amount of added BSA. Lubrol PX affected the assay procedure in a similar manner, but only at high levels (1.5%) did a precipitate form in the presence of SDS. The effect of the zwitterionic detergent, Miranol H2M, on the method was quite different from any of the other detergents. Although no precipitate was seen, even very low concentrations substantially increased the reagent blank and markedly decreased color development. The inclusion of SDS did not significantly reduce this interference. These findings demonstrate the applicability of a modified Lowry procedure for protein determination in the presence of high concentrations of several classes of nonionic detergents including polyoxyethylene alcohols (Brij 35, Lubroe PX), polyoxyethylene-p-t-octylphenols (Triton X100, Nonidet-P40, Cutscum) and polyoxyethylene sorbitol esters (Tween 20). In addition, the method allows assays in the presence of a cationic quatemary ammonium detergent (cetyltrimethylammonium bromide). To assure reliability, all standards and samples should contain the same final concentration of detergent. Using the Wang-Smith procedure, the determination of protein in all but very dilute solution should now be possible in the presence of a wide range of concentrations of most types of detergents employed in the extraction
PROTEIN DETERMINATION
and purification of membrane proteins, as well as in the assay of membrane-associated enzymes.
BY LOWRY
REFERENCES 1. Helenius, Biophys.
A., and Simons, K. (1975) Biochim. Acta
415,
29-79.
23
2. Tanford, C., and Reynolds, J. A. (1976) Biochim. Biophys.
Acta
457,
133-170.
3. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193,
ACKNOWLEDGMENTS This work was supported by NIH Grant NS12493 and by Grant E-675 from the Robert A. Welch Foundation.
PROCEDURE
265-275. 4.
Dulley,
J. R., and Grieve, P. A. (1975) Anal.
Biochem.
64, l36-
141.
5. Wang, C.-S., and Smith, R. L. (1975) Anal. Biothem. 63, 414-417. 6. Peterson, Ci. L. (1977)AnaI. Biochem. 83,346-356. 7. Lees, M. B., and Paxman, S., (1972) Anal. Biothem.
47,
184-192.
BIOCHEMISTRY
%,
21-23
(1979)
Determination of Protein by a Modified Lowry Procedure Presence of Some Commonly Used Detergents EVELYN Department
CADMAN, J. ROBERT BOSTWICK,ANDJOSEPH of Biophysical
Sciences>
University
of Houston,
Houston,
in the
EICHBERG Texas
77004
Received September 18, 1978
A wide variety of detergents has been employed to solubilize and purify membrane proteins (1,2). The interference caused by certain cationic and nonionic detergents in the Lowry procedure for protein determination (3) and its elimination by inclusion of sodium dodecyl sulfate (SDS)l has been previously described (4-7). However, only for Triton X-100 has this modification been shown definitively to be effective at concentrations of interfering detergent as high as 1% in the final reaction mixture over a range of protein concentrations (45). In this communication, we further document the applicability of a modified Lowry method to protein preparations containing high levels of nonionic and cationic detergents in common use. MATERIALS
AND METHODS
20% (w/v) solution of SDS and of each detergent to be tested was prepared. The protein standard consisted of 1 mg BSA/ml water. Analyses were performed essentially according to Wang and Smith (5). Aliquots of protein standard containing O-60 pg BSA were mixed with varying amounts of detergent solution and water then added to a volume of 0.70 ml. Alkaline copper solution (1 .O ml) was added and the mixture was vortexed and allowed to stand at room temperature for 15 min. SDS (0.5 ml) was added, each sample mixed thoroughly, and 0.1 ml phenol reagent was then pipetted into each tube followed by immediate mixing. After standing for 30 min to permit color development, the absorption was determined at 500 nm for each sample in a Beckman 25 spectrophotometer.
Crystalline bovine serum albumin (BSA), Triton-X-100, Tween 20, Brij 35, Lubrol PX, and cetyltrimethylammonium bromide were obtained from Sigma Chemical Company, St. Louis, Missouri. The disodium cupric salt of EDTA was purchased from ICN Pharmaceuticals, Plainview, New York. Cutscum and the Folin-Ciocalteu reagent were products of Fisher Chemical Company and Nonidet-P40 was obtained from Shell Chemical Company. Miranol H2M was a product of the Miranol Chemical Company, Irvington, New Jersey. A 1 Abbreviations used: SDS, sodium dodecyl fate; BSA, bovine serum albumin.
RESULTS AND DISCUSSION
In confirmation of the results of others (4-7), the presence of SDS (final concentration, 4.35%) had no effect on color development, but prevented the formation of a precipitate which otherwise appeared on addition of the phenol reagent to solutions containing Triton-X-100. SDS likewise acted on assay mixtures which contained Cutscum, Nonidet-P40, Tween 20, and cetyltrimethylammonium bromide. When these detergents were present at a final concentration of 1.0% (0.75% for cetyltrimethylammonium bromide), a nearly linear
sul-
21
0003-2697/79~090021-03$02.00/O Copyright &?I 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
22
CADMAN, TABLE
BOSTWICK,
1
EFFECTOFDETERGENTON REAGENTBLANK ANDCOLORFORMATION Detergent” SDS only Cetyltrimethylammonium bromide Triton X-100 Nonidet-P40 Cutscum Tween 20 Brij 35 Lubrol PX
Reagent blankb
40 pg BSA
0.015
0.146
0.042 0.045 0.131 0.058 0.151 0.043 0.031
0.174 0.186 0.270 0.202 0.318 0.206 0.203
a All detergents were present at a final concentration of 1.0% except for cetyltrimethylammonium bromide which was present at 0.75%. b Absorption determined at 500 nm.
relationship between the amount of BSA and the color generated was obtained in each case. Each of the detergents increased the reagent blank compared to an SDScontaining control, but except for Tween 20, there was no appreciable effect on color formed in the presence of BSA (Table 1). When color development was investigated as a function of the amount of each of these detergents from 0 to I%, absolute absorption values agreed within 10%. Above this amount, a precipitate could be avoided and a usable linear relationship could still be obtained up to 1.5% Cutscum and up to 2.0% Triton X-100. However, at these concentrations, the variation of slope and intercept for individual determinations was considerable. At concentrations of cetyltrimethylammonium bromide greater than 0.75%, the final reaction mixture became gelatinous. The effects of Brij 35 and Lubrol PX in the modified Lowry procedure differed from those of the other detergents examined. In the absence of SDS, the inclusion of 0. l0.5% Brij 35 in the reaction mixture resulted in a cloudy solution. When the precipitate was removed by centrifugation, the color developed was roughly linear with protein
AND EICHBERG
concentration, but had a greenish tinge. Optical densities at 500 nm were greatly reduced compared to controls. When Brij 35 was present at 0.75% or greater, no precipitate formed, but color development remained abnormally low. The addition of SDS prevented the formation of a precipitate at low concentrations of Brij 35, but brought about the appearance of a fine precipitate when 0.75% or more of the nonionic detergent was present. After removal of the precipitate by centrifugation for 5 min at 12,OOOg, the color in the clear supernatant appeared normal and was proportional to the amount of added BSA. Lubrol PX affected the assay procedure in a similar manner, but only at high levels (1.5%) did a precipitate form in the presence of SDS. The effect of the zwitterionic detergent, Miranol H2M, on the method was quite different from any of the other detergents. Although no precipitate was seen, even very low concentrations substantially increased the reagent blank and markedly decreased color development. The inclusion of SDS did not significantly reduce this interference. These findings demonstrate the applicability of a modified Lowry procedure for protein determination in the presence of high concentrations of several classes of nonionic detergents including polyoxyethylene alcohols (Brij 35, Lubroe PX), polyoxyethylene-p-t-octylphenols (Triton X100, Nonidet-P40, Cutscum) and polyoxyethylene sorbitol esters (Tween 20). In addition, the method allows assays in the presence of a cationic quatemary ammonium detergent (cetyltrimethylammonium bromide). To assure reliability, all standards and samples should contain the same final concentration of detergent. Using the Wang-Smith procedure, the determination of protein in all but very dilute solution should now be possible in the presence of a wide range of concentrations of most types of detergents employed in the extraction
PROTEIN DETERMINATION
and purification of membrane proteins, as well as in the assay of membrane-associated enzymes.
BY LOWRY
REFERENCES 1. Helenius, Biophys.
A., and Simons, K. (1975) Biochim. Acta
415,
29-79.
23
2. Tanford, C., and Reynolds, J. A. (1976) Biochim. Biophys.
Acta
457,
133-170.
3. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193,
ACKNOWLEDGMENTS This work was supported by NIH Grant NS12493 and by Grant E-675 from the Robert A. Welch Foundation.
PROCEDURE
265-275. 4.
Dulley,
J. R., and Grieve, P. A. (1975) Anal.
Biochem.
64, l36-
141.
5. Wang, C.-S., and Smith, R. L. (1975) Anal. Biothem. 63, 414-417. 6. Peterson, Ci. L. (1977)AnaI. Biochem. 83,346-356. 7. Lees, M. B., and Paxman, S., (1972) Anal. Biothem.
47,
184-192.
