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Polyacrylamide gel interference of the lowry method for protein determination
ANALYTICAL
BIOCHEMISTRY
Polyacrylamide
88.
196-202 (1978)
Gel Interference of the Lowry for Protein Determination
Method
P. A. FIELDS AND L. H. LARKIN Department
of Anatomy,
University of Florida College Gainesville, Florida 32610
of Medicine,
Received May 9, 1977: accepted February 3, 1978 Certain reagents utilized in the formation of polyacrylamide gels are shown to interfere in the Lowry assay for protein. Acrylamide (3-30%) and potassium ferrocyanide (O.OOlS-0.0105%) produced a linear response in color formation. Both compounds are capable of reducing the phenol reagent in the absence of copper and the interference can be compensated for by employing the appropriate blank. An extract of polymerized and electrophoresed gels also interferes in the Lowry assay, however, this increased color formation cannot be corrected by using a gel extract blank. Under the conditions studied, filtration, centrifugation, and dialysis did not sufficiently remove the acrylamide fines responsible for the interference.
Polyacrylamide disc gel electrophoresis (PAGE) has been used widely in the separation and subsequent isolation of proteins (I-5). This laboratory has employed PAGE to isolate specific fractions of the pregnancy hormone relaxin; while characteristic biological activity was present (6), however, the specific activity (units of biological activity per milligram of protein) of the hormone was considerably lower than preparations isolated by other techniques. TEMED and Tris, which are routinely used in preparing polyacrylamide gels, have been shown (7,8) to interfere with the Lowry assay (9) for proteins. Therefore, an important consideration for accurate estimation of protein concentration in polyacrylamide gel extracts is the removal of salts, buffers, and certain components of the gel from the preparation. Removal of these materials has reportedly been accomplished by one of two methods, either by preelectrophoresing the gels prior to sample addition (10) or by utilizing centrifugation and filtration to remove small gel particles and dialysis to remove TEMED and Tris (4). This paper describes analyses of materials that are used to prepare the polyacrylamide gels as well as extracts from polymerized and electrophoresed gels. Results from these studies indicate that several additional materials should be added to the growing list of reagents that interfere with the color development in the Lowry method for determining protein 0003-2697/78/0881-0196$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
196
INTERFERENCE
IN THE
LOWRY
METHOD
197
concentration. The findings are discussed in relation to what must be considered in employing the preparative PAGE system to isolate proteins for quantitative purposes. MATERIALS
AND METHODS
Reagents. The stock PAGE solutions used were as follows: (A) 15 g of acrylamide, 0.4 g of bisacrylamide, and 36 ml of distilled water; (B) 0.14 g of ammonium persulfate and 50 ml of distilled water: (C) 0.015 g of potassium ferrocyanide and 100 ml of distilled water; (D) 7.29 g of Tris, 0.16 ml of TEMED, 12 ml of 1 N HCl, and 32 ml of distilled water. The reagents were obtained as follows: Acrylamide and bisacrylamide (ultrapure) were from Polysciences; ammonium persulfate and potassium ferrocyanide were from Fisher Scientific; Tris was from Sigma; TEMED was from Canal Industrial, Inc. 1“reparation ofpolyacrylamide gel extract. The gel extract was prepared following the technique described in previous studies that involved isolation of porcine relaxin fractions (6). Potassium ferrocyanide was used in this system to inhibit rapid polymerization of the gel. Two sets of gels were prepared: Set I, 1 vol each of Solutions A, B, and D and 0.5 vol of Solution C. A second set of gels (II) was prepared in which deionized water was substituted for Solution C. Following polymerization, the gels (Set I) were run at 5 mA per gel for 2 hr in a standard electrophoretic apparatus. The gels (6 x 60 mm) were removed from the tubes and fractured by forcing the gels through a IO-cc syringe. Two volumes of distilled water was added to the gel material and the resulting slurry was allowed to incubate overnight at 4°C. The slurry was then filtered through a glass-fiber filter, Type A (Gelman Instrument Co.), utilizing a Millipore filtering system. The gel was washed twice with 1 vol of distilled water. The filtrates were combined, centrifuged at 5000 g for 30 min and the supernatant was lyophilized. Ten milliliters of distilled water was added and half the solution was dialyzed in Spectrapor TM 3 tubing (Spectrum Medical Industries, Inc.) having a molecular weight retention of 3500. The remaining one half of the acrylamide solution was dialyzed in Fisher Scientific dialysis tubing (Cat No. B-667B) having a molecular weight retention of 12,000. Both samples were dialyzed for 1 week at 4°C. The water was changed twice daily. Following dialysis, the acrylamide extracts were readjusted to the initial volume. Gels for set II were treated similarly except that they were preelectrophoresed for 24 hr and centrifuged at 60,000 g for 2 hr following fracturing. In addition, the four solutions (A, B, C, and D) utilized in the formation of the gel were dialyzed in both types of tubing for 1 week at 4°C. Measurement of interference in the Loulry assay. Color formation by the various solutions in the Lowry assay for proteins (9) was measured
198
FIELDS AND LARKIN
prior to and after dialysis. The effects of dialyzed gel extracts and the various stock solutions on the Lowry assay were also determined. Bovine serum albumin (BSA) was utilized for the protein standard curve. The samples had a volume of 1 ml and contained different concentrations of BSA with/without PAGE stock solutions. RESULTS
The effect of the PAGE preparation on the BSA standard curve is shown in Fig. 1. An increase in the color response for the PAGE extract plus BSA was demonstrated over that formed by BSA in the absence of the extract. Utilization of the appropriate blank did not correct the response brought about by the extract but in fact reduced the slope of the standard BSA curve. Figures 2 and 3 show the response of certain PAGE stock solutions in the Lowry assay. The concentrations of stock solutions utilized in the calibration curve studies were chosen, since they represent values slightly greater to slightly less than the final values of stock solutions found in polyacrylamide gels ranging from 5 to 15% acrylamide. A linear response in color development is observed with acrylamide (3-30%) and the slope is greater than that demonstrated by BSA (Fig. 2). This interference could be corrected in experimental samples by utilizing the appropriate blank. The extent to which a substance reacts directly with the Folin reagent 0.5 1
lb
5b
100
150
vg BSP FIG. 1. Color development concentrations of BSA. The curve (O-O) is a calibration (0-e) represents BSA with Standard l-ml samples were for the blank.
of the polyacrylamide gel extract in the presence of various effect of 0.2 ml (X-X) gel extract was determined. The curve for BSA in the absence of extract while the curve 0.2 ml of gel extract blanked against 0.2 ml of gel extract. utilized. Except where indicated, distilled water was used
INTERFERENCE
199
IN THE LOWRY METHOD
0.4 -
5 0 s t Y
0.3.
0.2.
; i
O.l-
IO
30
50
3
9
15
75 21
100 27
150
30
pg BSA % (W/V)
ACRYLAMIDE
FIG. 2. Standard curves for acrylamide and BSA in the presence and absence of copper in the Lowry assay. Abscissa values refer to the final concentration of acrylamide and BSA in the standard l-ml sample. The effects of acrylamide (3-30%) in the presence (X-X) and absence (V-V) of copper and of BSA (12-150 &ml) in the presence (O-O) and absence (0-O of copper were determined. Distilled water was used for the blank.
is measured by the color intensity produced in the absence of copper (9). Figure 2 demonstrates the ability of both BSA and Solution A (acrylamide solution) to reduce the Folin reagent and elicit color formation in the absence of copper. In the presence of copper, maximum absorbance had not been obtained even with the 30% acrylamide solution. In the absence of copper, this maximum was observed at 27%. Figure 3 indicates that a linear response in color development is also realized with potassium ferrocyanide (O.OOlS-0.0105%). The color development is not as dramatic as that seen with acrylamide; the interference, however, could also be corrected for by employing the appro0.4-
I 0 s
0.3.
2 $
0.2.
3 % ;
0.1.
IO
30
50
I
3
5
75 7
100 9
II
13
150
~9 MA
15
% (W/V) Potassium ferrocyantdex lO-3
FIG. 3. Standard curve for potassium ferrocyanide and BSA in the presence and absence of copper in the Lowry assay. Abscissa values refer to the final concentration in the standard l-ml sample. The effects of potassium ferrocyanide on color development in the presence (X-X) and absence (V-V) of copper and of BSA in the presence (O-O) and absence (0-O) of copper were determined. Distilled water was utilized for the blank.
200
FIELDS AND LARKIN
priate blank. Potassium ferrocyanide is also capable of reacting with the Folin reagent in the absence of copper, although the reaction is not as pronounced as that seen with Solution A. Although the ammonium persulfate results are not presented in tabular form, the ammonium persulfate had no significant effect on the color development in the presence of BSA, while the high intensity of color elicited by Solution D (Tris-TEMED buffer) made it impossible to obtain a standard curve even though the proper blanks were utilized. The three concentration ranges of Tris and TEMED in Solution D studied were as follows: (1) 10.64% Tris plus 0.21% TEMED, (2) 4.56% Tris plus 0.09% TEMED, and (3) 1.52% Tris plus 0.03% TEMED. Table 1 demonstrates the ability of dialysis to remove the gel reagents and PAGE extract that interfere with the Lowry assay. Except for potassium ferrocyanide, removal of the reagents by dialyis was essentially complete. Slightly different results were seen with the gel extract. At best, an 85% clearance was obtained. This extract was obtained from 30 g (wet weight) of electrophoresed gels (Set II) homogenized according to Gordon (lo), filtered, centrifuged, and lyophilized. The yield was 1 g (dry weight). Centrifugation of extract II at 60,OOOg for 2 hr likewise did not reduce the interference. TABLE COLOR
DEVELOPMENT AND
IN THE
UNDIALYZED
1
LOWRY GEL
METHOD
OF DIALYZED
CONSTITUENTS
Absorbance (660 nm)” Dialyzed”
Reagents Stock Solutions’ Acrylamide Potassium ferrocyanide Ammonium persulfate Tris-TEMED Polyacrylamide gel extract preparatiom
Undialyzed
Spectrapor TM3
Fischer Scientific
0.728 0.086 0 3.180
0.035 (95.2)d 0.090 (0) 0.004 0.017 (99.5)
0.008 (99) 0.045 (48) 0.002 0.015 (99.5)
0.534
0.112 (71)
0.080 (85)
a A l-ml sample was assayed for color development by the Lowry method (9). Distilled water was utilized as the blank. b A sample was dialyzed 1 week in either Spectrapor TM 3 tubing (3500 MW retention) or tubing from Fischer Scientific (12,000 MW retention). c Refer to Materials and Methods for composition. d Percentage material cleared by dialysis. e Electrophoresed gels were extracted as described under Materials and Methods and treated the same as stock solutions for dialysis.
INTERFERENCE
IN THE
LOWRY
METHOD
201
DISCUSSION
When utilizing PAGE for the isolation of proteins, the procedure used for extracting the protein from the gels should be selected with care. Several articles (7,s) have been published that describe reagents (TEMED and Tris) utilized in the preparation of acrylamide gels that interfere in the Lowry method. This current study demonstrated that acrylamide: bisacrylamide and potassium ferrocyanide also gave a positive color reaction when assayed by this method. The interference by these reagents can be compensated for by calibrating with the appropriate blank. When isolating proteins with the fractured gel technique, however, the amount of contaminating gel constitutents is unknown and the appropriate blank cannot be utilized. Gordon (10) reported that persulfate ions would be removed during electrophoresis; we did not find, however, that this compound interfered with determination of protein concentration. Although the results are not reported in tabular form, stock solutions A and D gave a positive reading with the biuret technique (11). Solution A probably reacts by a coupling of the copper ions by the large number of amine groups present in this solution. Since most of the reagents used in the gel preparation interfered with the protein assay, we looked at the effectiveness of filtration, dialysis, and centrifugation in removing the interfering compounds. Although dialysis of the stock reagents was essentially complete except for potassium ferrocyanide, this was not true for the gel extract. At best, an 85% clearance was obtained with a tubing having a molecular weight retention of 12,000. If a lower molecular weight exclusion is utilized, this percentage decreases. Even though only 15% of the original material remained, this gave an absorbance equivalent to 30 pg of protein/ml. In addition, unpublished work in our laboratory indicates a 15 to 20-fold increase in the ammonia peak from hydrolized samples obtained from PAGE when compared to the same protein (porcine relaxin) isolated by column chromatography. The omission of potassium ferrocyanide from the gel preparation should be considered because this reagent reacts with the reagents used in the Lowry assay and cannot be completely removed from the extract by dialysis. However, at best this would only reduce the interference since these studies have shown that an extract of gels formed in the absence of potassium ferrocyanide showed interference. At present the reason why potassium ferrocyanide cannot be removed by dialysis is unknown. It is obvious from this study that the smaller the protein isolated with PAGE, the less effective one will be in removing contaminating gel material by centrifuging, filtering, or dialyzing. Thus, it does not seem practical to use the fractured gel technique as a preparative technique when
202
FIELDS AND LARKIN
proteins the size of porcine relaxin (5000-6000 MW) are to be isolated. While the technique produced a biologically active fraction, it is difficult to establish the specific activity of the hormone because of the inability to measure the protein concentration accurately. Therefore, rather than utilizing the fractured gel technique for isolating proteins, a procedure of electrophoretic elution (12,13,14) following preelectrophoresis may be more practical. ACKNOWLEDGMENT This research was supported by USPHS Grant HD-8552.
REFERENCES 1. Whelan, H. A., and Wriston, Jr., J. C. (1969) Biochemistry 8, 2386-2393. 2. Tono, H., and Komberg, A. (1967) J. Biol. Chem. 242, 2375-2382. 3. Poillon, W. N., Maeno, H., Koike, K., and Feigelson, P. (1969) J. Biol. Chem. 244, 3447-3456. 4. Lewis, U. J., and Clark, M. 0. (1963) Anal. Biochem. 6, 303-315. 5. Kling, H., and Pentz, S. (1975) Comp. Biochem. Physiol. 50B, 103-104. 6. Oliver, R. M., Fields, P. A., and Larkin, L. H. (1978) J. Endocrirz.. in press. 7. Lewis, U. J. (1962)J. Biol. Chem. 237, 3141-3145. 8. Rej, R. and Richards. A. H. (1974) Anal. Biochem. 62, 240-247. 9. Lowry, 0. H., Rosebrough, N. J., Farr, A. L.. and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 10. Gordon, A. H. (1970) in Laboratory Techniques in Biochemistry and Molecular Biology. (Work, T. S., and Work, E., eds.), Vol. 1, pp. 1-144, American/Elsevier, New York. 11. Gomall, A. G., Bardawill, C. J., and David, M. M. (1949) J. Biol. Chem. 177, 75 l-766. 12. Karsnas, P. and Roos, P. (1977) Anal. Biochem. 77, 168-175. 13. Stephens, R. E. (1975) Anal. Biochem. 65, 369-379. 14. Sulitzeanu, D., Slavin, M., and Yecheskeli, E. (1967) Anal. Biochem. 21, 57-67.
BIOCHEMISTRY
Polyacrylamide
88.
196-202 (1978)
Gel Interference of the Lowry for Protein Determination
Method
P. A. FIELDS AND L. H. LARKIN Department
of Anatomy,
University of Florida College Gainesville, Florida 32610
of Medicine,
Received May 9, 1977: accepted February 3, 1978 Certain reagents utilized in the formation of polyacrylamide gels are shown to interfere in the Lowry assay for protein. Acrylamide (3-30%) and potassium ferrocyanide (O.OOlS-0.0105%) produced a linear response in color formation. Both compounds are capable of reducing the phenol reagent in the absence of copper and the interference can be compensated for by employing the appropriate blank. An extract of polymerized and electrophoresed gels also interferes in the Lowry assay, however, this increased color formation cannot be corrected by using a gel extract blank. Under the conditions studied, filtration, centrifugation, and dialysis did not sufficiently remove the acrylamide fines responsible for the interference.
Polyacrylamide disc gel electrophoresis (PAGE) has been used widely in the separation and subsequent isolation of proteins (I-5). This laboratory has employed PAGE to isolate specific fractions of the pregnancy hormone relaxin; while characteristic biological activity was present (6), however, the specific activity (units of biological activity per milligram of protein) of the hormone was considerably lower than preparations isolated by other techniques. TEMED and Tris, which are routinely used in preparing polyacrylamide gels, have been shown (7,8) to interfere with the Lowry assay (9) for proteins. Therefore, an important consideration for accurate estimation of protein concentration in polyacrylamide gel extracts is the removal of salts, buffers, and certain components of the gel from the preparation. Removal of these materials has reportedly been accomplished by one of two methods, either by preelectrophoresing the gels prior to sample addition (10) or by utilizing centrifugation and filtration to remove small gel particles and dialysis to remove TEMED and Tris (4). This paper describes analyses of materials that are used to prepare the polyacrylamide gels as well as extracts from polymerized and electrophoresed gels. Results from these studies indicate that several additional materials should be added to the growing list of reagents that interfere with the color development in the Lowry method for determining protein 0003-2697/78/0881-0196$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
196
INTERFERENCE
IN THE
LOWRY
METHOD
197
concentration. The findings are discussed in relation to what must be considered in employing the preparative PAGE system to isolate proteins for quantitative purposes. MATERIALS
AND METHODS
Reagents. The stock PAGE solutions used were as follows: (A) 15 g of acrylamide, 0.4 g of bisacrylamide, and 36 ml of distilled water; (B) 0.14 g of ammonium persulfate and 50 ml of distilled water: (C) 0.015 g of potassium ferrocyanide and 100 ml of distilled water; (D) 7.29 g of Tris, 0.16 ml of TEMED, 12 ml of 1 N HCl, and 32 ml of distilled water. The reagents were obtained as follows: Acrylamide and bisacrylamide (ultrapure) were from Polysciences; ammonium persulfate and potassium ferrocyanide were from Fisher Scientific; Tris was from Sigma; TEMED was from Canal Industrial, Inc. 1“reparation ofpolyacrylamide gel extract. The gel extract was prepared following the technique described in previous studies that involved isolation of porcine relaxin fractions (6). Potassium ferrocyanide was used in this system to inhibit rapid polymerization of the gel. Two sets of gels were prepared: Set I, 1 vol each of Solutions A, B, and D and 0.5 vol of Solution C. A second set of gels (II) was prepared in which deionized water was substituted for Solution C. Following polymerization, the gels (Set I) were run at 5 mA per gel for 2 hr in a standard electrophoretic apparatus. The gels (6 x 60 mm) were removed from the tubes and fractured by forcing the gels through a IO-cc syringe. Two volumes of distilled water was added to the gel material and the resulting slurry was allowed to incubate overnight at 4°C. The slurry was then filtered through a glass-fiber filter, Type A (Gelman Instrument Co.), utilizing a Millipore filtering system. The gel was washed twice with 1 vol of distilled water. The filtrates were combined, centrifuged at 5000 g for 30 min and the supernatant was lyophilized. Ten milliliters of distilled water was added and half the solution was dialyzed in Spectrapor TM 3 tubing (Spectrum Medical Industries, Inc.) having a molecular weight retention of 3500. The remaining one half of the acrylamide solution was dialyzed in Fisher Scientific dialysis tubing (Cat No. B-667B) having a molecular weight retention of 12,000. Both samples were dialyzed for 1 week at 4°C. The water was changed twice daily. Following dialysis, the acrylamide extracts were readjusted to the initial volume. Gels for set II were treated similarly except that they were preelectrophoresed for 24 hr and centrifuged at 60,000 g for 2 hr following fracturing. In addition, the four solutions (A, B, C, and D) utilized in the formation of the gel were dialyzed in both types of tubing for 1 week at 4°C. Measurement of interference in the Loulry assay. Color formation by the various solutions in the Lowry assay for proteins (9) was measured
198
FIELDS AND LARKIN
prior to and after dialysis. The effects of dialyzed gel extracts and the various stock solutions on the Lowry assay were also determined. Bovine serum albumin (BSA) was utilized for the protein standard curve. The samples had a volume of 1 ml and contained different concentrations of BSA with/without PAGE stock solutions. RESULTS
The effect of the PAGE preparation on the BSA standard curve is shown in Fig. 1. An increase in the color response for the PAGE extract plus BSA was demonstrated over that formed by BSA in the absence of the extract. Utilization of the appropriate blank did not correct the response brought about by the extract but in fact reduced the slope of the standard BSA curve. Figures 2 and 3 show the response of certain PAGE stock solutions in the Lowry assay. The concentrations of stock solutions utilized in the calibration curve studies were chosen, since they represent values slightly greater to slightly less than the final values of stock solutions found in polyacrylamide gels ranging from 5 to 15% acrylamide. A linear response in color development is observed with acrylamide (3-30%) and the slope is greater than that demonstrated by BSA (Fig. 2). This interference could be corrected in experimental samples by utilizing the appropriate blank. The extent to which a substance reacts directly with the Folin reagent 0.5 1
lb
5b
100
150
vg BSP FIG. 1. Color development concentrations of BSA. The curve (O-O) is a calibration (0-e) represents BSA with Standard l-ml samples were for the blank.
of the polyacrylamide gel extract in the presence of various effect of 0.2 ml (X-X) gel extract was determined. The curve for BSA in the absence of extract while the curve 0.2 ml of gel extract blanked against 0.2 ml of gel extract. utilized. Except where indicated, distilled water was used
INTERFERENCE
199
IN THE LOWRY METHOD
0.4 -
5 0 s t Y
0.3.
0.2.
; i
O.l-
IO
30
50
3
9
15
75 21
100 27
150
30
pg BSA % (W/V)
ACRYLAMIDE
FIG. 2. Standard curves for acrylamide and BSA in the presence and absence of copper in the Lowry assay. Abscissa values refer to the final concentration of acrylamide and BSA in the standard l-ml sample. The effects of acrylamide (3-30%) in the presence (X-X) and absence (V-V) of copper and of BSA (12-150 &ml) in the presence (O-O) and absence (0-O of copper were determined. Distilled water was used for the blank.
is measured by the color intensity produced in the absence of copper (9). Figure 2 demonstrates the ability of both BSA and Solution A (acrylamide solution) to reduce the Folin reagent and elicit color formation in the absence of copper. In the presence of copper, maximum absorbance had not been obtained even with the 30% acrylamide solution. In the absence of copper, this maximum was observed at 27%. Figure 3 indicates that a linear response in color development is also realized with potassium ferrocyanide (O.OOlS-0.0105%). The color development is not as dramatic as that seen with acrylamide; the interference, however, could also be corrected for by employing the appro0.4-
I 0 s
0.3.
2 $
0.2.
3 % ;
0.1.
IO
30
50
I
3
5
75 7
100 9
II
13
150
~9 MA
15
% (W/V) Potassium ferrocyantdex lO-3
FIG. 3. Standard curve for potassium ferrocyanide and BSA in the presence and absence of copper in the Lowry assay. Abscissa values refer to the final concentration in the standard l-ml sample. The effects of potassium ferrocyanide on color development in the presence (X-X) and absence (V-V) of copper and of BSA in the presence (O-O) and absence (0-O) of copper were determined. Distilled water was utilized for the blank.
200
FIELDS AND LARKIN
priate blank. Potassium ferrocyanide is also capable of reacting with the Folin reagent in the absence of copper, although the reaction is not as pronounced as that seen with Solution A. Although the ammonium persulfate results are not presented in tabular form, the ammonium persulfate had no significant effect on the color development in the presence of BSA, while the high intensity of color elicited by Solution D (Tris-TEMED buffer) made it impossible to obtain a standard curve even though the proper blanks were utilized. The three concentration ranges of Tris and TEMED in Solution D studied were as follows: (1) 10.64% Tris plus 0.21% TEMED, (2) 4.56% Tris plus 0.09% TEMED, and (3) 1.52% Tris plus 0.03% TEMED. Table 1 demonstrates the ability of dialysis to remove the gel reagents and PAGE extract that interfere with the Lowry assay. Except for potassium ferrocyanide, removal of the reagents by dialyis was essentially complete. Slightly different results were seen with the gel extract. At best, an 85% clearance was obtained. This extract was obtained from 30 g (wet weight) of electrophoresed gels (Set II) homogenized according to Gordon (lo), filtered, centrifuged, and lyophilized. The yield was 1 g (dry weight). Centrifugation of extract II at 60,OOOg for 2 hr likewise did not reduce the interference. TABLE COLOR
DEVELOPMENT AND
IN THE
UNDIALYZED
1
LOWRY GEL
METHOD
OF DIALYZED
CONSTITUENTS
Absorbance (660 nm)” Dialyzed”
Reagents Stock Solutions’ Acrylamide Potassium ferrocyanide Ammonium persulfate Tris-TEMED Polyacrylamide gel extract preparatiom
Undialyzed
Spectrapor TM3
Fischer Scientific
0.728 0.086 0 3.180
0.035 (95.2)d 0.090 (0) 0.004 0.017 (99.5)
0.008 (99) 0.045 (48) 0.002 0.015 (99.5)
0.534
0.112 (71)
0.080 (85)
a A l-ml sample was assayed for color development by the Lowry method (9). Distilled water was utilized as the blank. b A sample was dialyzed 1 week in either Spectrapor TM 3 tubing (3500 MW retention) or tubing from Fischer Scientific (12,000 MW retention). c Refer to Materials and Methods for composition. d Percentage material cleared by dialysis. e Electrophoresed gels were extracted as described under Materials and Methods and treated the same as stock solutions for dialysis.
INTERFERENCE
IN THE
LOWRY
METHOD
201
DISCUSSION
When utilizing PAGE for the isolation of proteins, the procedure used for extracting the protein from the gels should be selected with care. Several articles (7,s) have been published that describe reagents (TEMED and Tris) utilized in the preparation of acrylamide gels that interfere in the Lowry method. This current study demonstrated that acrylamide: bisacrylamide and potassium ferrocyanide also gave a positive color reaction when assayed by this method. The interference by these reagents can be compensated for by calibrating with the appropriate blank. When isolating proteins with the fractured gel technique, however, the amount of contaminating gel constitutents is unknown and the appropriate blank cannot be utilized. Gordon (10) reported that persulfate ions would be removed during electrophoresis; we did not find, however, that this compound interfered with determination of protein concentration. Although the results are not reported in tabular form, stock solutions A and D gave a positive reading with the biuret technique (11). Solution A probably reacts by a coupling of the copper ions by the large number of amine groups present in this solution. Since most of the reagents used in the gel preparation interfered with the protein assay, we looked at the effectiveness of filtration, dialysis, and centrifugation in removing the interfering compounds. Although dialysis of the stock reagents was essentially complete except for potassium ferrocyanide, this was not true for the gel extract. At best, an 85% clearance was obtained with a tubing having a molecular weight retention of 12,000. If a lower molecular weight exclusion is utilized, this percentage decreases. Even though only 15% of the original material remained, this gave an absorbance equivalent to 30 pg of protein/ml. In addition, unpublished work in our laboratory indicates a 15 to 20-fold increase in the ammonia peak from hydrolized samples obtained from PAGE when compared to the same protein (porcine relaxin) isolated by column chromatography. The omission of potassium ferrocyanide from the gel preparation should be considered because this reagent reacts with the reagents used in the Lowry assay and cannot be completely removed from the extract by dialysis. However, at best this would only reduce the interference since these studies have shown that an extract of gels formed in the absence of potassium ferrocyanide showed interference. At present the reason why potassium ferrocyanide cannot be removed by dialysis is unknown. It is obvious from this study that the smaller the protein isolated with PAGE, the less effective one will be in removing contaminating gel material by centrifuging, filtering, or dialyzing. Thus, it does not seem practical to use the fractured gel technique as a preparative technique when
202
FIELDS AND LARKIN
proteins the size of porcine relaxin (5000-6000 MW) are to be isolated. While the technique produced a biologically active fraction, it is difficult to establish the specific activity of the hormone because of the inability to measure the protein concentration accurately. Therefore, rather than utilizing the fractured gel technique for isolating proteins, a procedure of electrophoretic elution (12,13,14) following preelectrophoresis may be more practical. ACKNOWLEDGMENT This research was supported by USPHS Grant HD-8552.
REFERENCES 1. Whelan, H. A., and Wriston, Jr., J. C. (1969) Biochemistry 8, 2386-2393. 2. Tono, H., and Komberg, A. (1967) J. Biol. Chem. 242, 2375-2382. 3. Poillon, W. N., Maeno, H., Koike, K., and Feigelson, P. (1969) J. Biol. Chem. 244, 3447-3456. 4. Lewis, U. J., and Clark, M. 0. (1963) Anal. Biochem. 6, 303-315. 5. Kling, H., and Pentz, S. (1975) Comp. Biochem. Physiol. 50B, 103-104. 6. Oliver, R. M., Fields, P. A., and Larkin, L. H. (1978) J. Endocrirz.. in press. 7. Lewis, U. J. (1962)J. Biol. Chem. 237, 3141-3145. 8. Rej, R. and Richards. A. H. (1974) Anal. Biochem. 62, 240-247. 9. Lowry, 0. H., Rosebrough, N. J., Farr, A. L.. and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 10. Gordon, A. H. (1970) in Laboratory Techniques in Biochemistry and Molecular Biology. (Work, T. S., and Work, E., eds.), Vol. 1, pp. 1-144, American/Elsevier, New York. 11. Gomall, A. G., Bardawill, C. J., and David, M. M. (1949) J. Biol. Chem. 177, 75 l-766. 12. Karsnas, P. and Roos, P. (1977) Anal. Biochem. 77, 168-175. 13. Stephens, R. E. (1975) Anal. Biochem. 65, 369-379. 14. Sulitzeanu, D., Slavin, M., and Yecheskeli, E. (1967) Anal. Biochem. 21, 57-67.