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Detection of copper on polyacrylamide gels
ANALYTICAL
BIOCHEMISTRY
Detection
89, 174-177 (1978)
of Copper
W. J. BRUYNINCKX,
on Polyacrylamide
S. GUTTERIDGE,~
Gels’
AND H. S. MASONS
Department of Biochemistry, University of Oregon Health Sciences Center, Portland, Oregon 97201 Received July 18, 1977; accepted April 11, 1978 A color test for the localization of copper on polyacrylamide gels is described. The test is based upon the quenching of fluorescence of bathocuproine sulfonate by CtP and is sensitive to 0.1 nmol of free or protein-bound copper. There is no false positive reaction with 10 nmol of hemeprotein, free Fe3+, Fez+, Co*+, or MtP.
Copper proteins, such as tyrosinase, ceruloplasmin, ascorbate oxidase, and cytochrome oxidase, are made up of polypeptide subunits. It is important to identify the component polypeptides to which copper is bound. In principle this can be done by separating polypeptide subunits by gel electrophoresis and localizing copper on them, providing that the conditions of electrophoresis do not mobilize the copper ions. Rubeanic acid (1,2), dianisidine-H20, (3,4), and phenazine methosulfate tetrazoleum (5) give positive tests for enzyme copper in the range of 1.5 to 8.0 nmol with varying specificities. We now describe a method based upon the quenching of the fluorescence of bathocuproine sulfonate by Cu’+ that is one to two orders of magnitude more sensitive than previously described tests and is specific with respect to interference by Fe3+, Mn2+, or heme. METHODS
Copper-protein standards. Mushroom tyrosinase, purified by a modification of the method of Bouchilloux et al. (6), contained 0.21% Cu, as determined by atomic absorption spectroscopy. Hemocyanin (Cancer magister), purified by differential centrifugation, contained 0.16% Cu. Ceruloplasmin (0.29% Cu) and superoxide dismutase (bovine erythrocyte, 0.28% Cu) were purchased from the Sigma Chemical Co. Purified hemoglobin (0.38% Fe) was kindly provided by Dr. R. T. Jones. Crystalline bovine liver catalase (0.09% Fe) and pepsin were purchased from Worth1 This study was supported in part by grants from the National Institute of Arthritis and Metabolic Diseases, AM 0718, and the American Cancer Society, BC-IN. * Fellow of the Science Research Council of Great Britain. 3 Author to whom enquiries should be directed. 0003-2697/78/0891-0174$02.00/O Copyright 6 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
174
DETECTION
OF COPPER ON GELS
175
ington Biochemical Co. Ovalbumin and bovine serum albumin were obtained from the Pentex Co. Purified p-hydroxyphenylpyruvate hydroxylase (0.07% Fe) was kindly provided by Dr. D. Winter. Electrophoresis was carried out with the Ortec slab gel system (Ortec Inc., Oakridge, Tennessee) using 8% polyacrylamide-30 mM Tris sulfate, pH 8.0, slabs run at 4°C at 150 V, 100 pulses per second for about 120 min, or until a tracking dye, bromphenol blue, reached the bottom of the slab. Proteins of known copper content were applied to the sample slots in lo- to 20-~1 volumes so that a range of protein-bound copper (0.1-2.5 nmol of copper) could be studied with indicating reagents. The sample slots had a surface of 12 mm2. Hemoglobin, catalase, and p-hydroxyphenylpyruvate hydroxylase were used to test specificity. Ten to 0.01 nmol of copper (CuSO, or CuCl,), 10 nmol of Fe3+ (FeCl,), Mn2+ (MnCl,), or Co2+ (CoCl,) were also incorporated into lo-~*.1 gels placed into sample slots. RESULTS
Gels were stained immediately after removal from the electrophoresis apparatus. They were first soaked for 1 min at 16 mM ascorbate in glacial acetic acid to denature protein and to reduce the metal ions and then for 1 min in a 0.28 mM aqueous bathocuproine sulfonate solution. Bathocuproine sulfonate (G. Frederick Smith Chemical Co., Lot NR A2) solution was stored at room temperature in a brown glass bottle. The solution was prepared every 2 weeks. The staining pattern on the gel was observed under uv light (Mineralight, Model R51, 16 W, UV Products, Los Angeles, California). The presence of copper was indicated by dark bands on a field of greenish blue fluorescence. Bathocuproine sulfonate could be replaced by biquinoline. Biquinoline (G. Frederick Smith Chemical Co., Lot NR C29) was dissolved in 16 mM ascorbate in glacial acetic acid to a final concentration of 0.28 mM. The solution was prepared freshly for each test. The gel was soaked in this solution for 2 min. The copperpositive bands were dark under uv light on a yellow-green fluorescent background. Photographs were made under uv illumination using a Polaroid Land camera with a Wratten gelatin filter, type 2A (Kodak) and type 55, 4 x 5 Land film, and 30 set- to 2-min exposures atf4.5. Positions of the dark bands were marked with small incisions on the gel. After the gels were washed with distilled water, they could be stained for protein by soaking them for 16 hr in a solution of 250 ml of isopropyl alcohol, 100 ml of acetic acid, and 50 ml of 1% Coomassie blue R-250 made up to 1 liter with water and then finally were destained in 10% acetic acid. To obtain optimal staining conditions for copper on polyacrylamide gels, the following conditions were systematically varied: incubation time in glacial acetic acid-ascorbate, substitution of hydroxylamine for ascor-
176
BRLJYNINCKX,
GUTTERIDGE,
AND MASON
FIG. I. Tyrosinase and ceruloplasmin were electrophoresed on 8% polyacrylamide gels and stained with bathocuproine sulfonate (Methods). The copper-containing polypeptides produced dark bands on a fluorescent background. (a) Tyrosinase: A, 1.5; B, 1.1; C, 0.75; D, 0.375; E, 0.19 nmol; and F, 0.06 nmol of copper per slot. (b) Ceruloplasmin: A, 1.1; B, 0.9; C, 0.67; D, 0.45; E. 0.23 nmol; and F, 0.11 nmol of copper.
bate, concentration of bathocuproine sulfonate, incubation time in the bathocuproine sulfonate solution, and gel pH after staining. The procedure described gave the most satisfactory results. A uniform background fluorescence was obtained with bathocuproine sulfonate. The nonfluorescent cuprous bathocuproine bands from 1 nmol of Cu were visible immediately after staining; at lower concentrations, e.g., 0.1 nmol per slot, the nonfluorescent bands developed over a lo-min period. There was no diffusion of the bands 90 min after staining. Under visible light, the gels showed no indication of the orange-brown color or cuprous bathocuproine sulfonate unless amounts of copper greater than 2 nmol per gel slot were employed. These weakly colored bands were not distinct. Different amounts of the copper proteins, mushroom tyrosinase, hemocyanin, ceruloplasmin, and superoxide dismutase were applied to the sample slot in a range from 0.05 to 2.5 nmol of protein-bound copper per gel slot. In all cases 0.1 nmol of protein-bound copper was readily detected (Figs. la and lb). The heme proteins hemoglobin and catalase did not quench bathocuproine sulfonate fluorescence in this procedure with 10 nmol of heme. The nonheme iron protein, p-hydroxyphenylpy-
DETECTION
OF COPPER
ON
GELS
177
ruvate hydroxylase (0.5 nmol of Fe), did not react, nor did the copperfree proteins, ovalbumin and pepsin, at levels of 100 pg of protein per slot. With free metal ions in the polyacrylamide gel, 0.1 nmol of Cu*+ was easily detected, while 10 nmol of Fe3+, Fez+, or Mn2+ did not quench bathocuproine sulfonate fluorescence. When the quantity of protein-bound copper on the gels was systematically varied, the intensity of quenching also varied. An attempt was made to quantitate this by densitometric scanning of transparent photographs (Land film, type 462) or negatives of the copper-stained gels, but the relationship between total copper and quenching intensity was not simple (see under Discussion). A scanning spectrofluorimeter, unavailable for this work, might succeed. DISCUSSION
Procedures available for localization of copper-containing polypeptides on gels have suffered from poor specificity (3,4) or sensitivity (1,2,5). The staining procedure now described uses quenching of the fluorescence of bathocuproine sulfonate or biquinoline by Cu+ formed from copper proteins on polyacrylamide gels after being denatured and then reduced. It can be applied to copper-containing proteins without interference from other protein-bound or free metal ions. The amount of protein-bound copper needed for this procedure is in the range normally run on polyacrylamide analytical gels (0.1 nmol of protein-bound copper corresponds to 3.2 pg of tyrosinase or 2.5 pg of ceruloplasmin) with the advantage that the gels can be stained for protein after staining for copper. REFERENCES 1. 2. 3. 4. 5. 6.
Whittaker, I. R. (1959) Nature (London) 184, 192- 193. Horn, E. C., and Kerr, M. S. (1969) Camp. Biochem. Physiol. 29, 493-508. Owen, I. A., and Smith, H. (1961) C/in. Chim. Acta 6, 441-444. Manwell, C., and Baker, C. M. A. (1963) C&n. Chim. Acta 8, 193-208. Gould, E., and Karolus. J. J. (1975) Anal. Biochem. 67, 515-519. Bouchilloux, S., McMahill. P., and Mason, H. S. (1963) J. Bid. Chem. 238, 1699.
BIOCHEMISTRY
Detection
89, 174-177 (1978)
of Copper
W. J. BRUYNINCKX,
on Polyacrylamide
S. GUTTERIDGE,~
Gels’
AND H. S. MASONS
Department of Biochemistry, University of Oregon Health Sciences Center, Portland, Oregon 97201 Received July 18, 1977; accepted April 11, 1978 A color test for the localization of copper on polyacrylamide gels is described. The test is based upon the quenching of fluorescence of bathocuproine sulfonate by CtP and is sensitive to 0.1 nmol of free or protein-bound copper. There is no false positive reaction with 10 nmol of hemeprotein, free Fe3+, Fez+, Co*+, or MtP.
Copper proteins, such as tyrosinase, ceruloplasmin, ascorbate oxidase, and cytochrome oxidase, are made up of polypeptide subunits. It is important to identify the component polypeptides to which copper is bound. In principle this can be done by separating polypeptide subunits by gel electrophoresis and localizing copper on them, providing that the conditions of electrophoresis do not mobilize the copper ions. Rubeanic acid (1,2), dianisidine-H20, (3,4), and phenazine methosulfate tetrazoleum (5) give positive tests for enzyme copper in the range of 1.5 to 8.0 nmol with varying specificities. We now describe a method based upon the quenching of the fluorescence of bathocuproine sulfonate by Cu’+ that is one to two orders of magnitude more sensitive than previously described tests and is specific with respect to interference by Fe3+, Mn2+, or heme. METHODS
Copper-protein standards. Mushroom tyrosinase, purified by a modification of the method of Bouchilloux et al. (6), contained 0.21% Cu, as determined by atomic absorption spectroscopy. Hemocyanin (Cancer magister), purified by differential centrifugation, contained 0.16% Cu. Ceruloplasmin (0.29% Cu) and superoxide dismutase (bovine erythrocyte, 0.28% Cu) were purchased from the Sigma Chemical Co. Purified hemoglobin (0.38% Fe) was kindly provided by Dr. R. T. Jones. Crystalline bovine liver catalase (0.09% Fe) and pepsin were purchased from Worth1 This study was supported in part by grants from the National Institute of Arthritis and Metabolic Diseases, AM 0718, and the American Cancer Society, BC-IN. * Fellow of the Science Research Council of Great Britain. 3 Author to whom enquiries should be directed. 0003-2697/78/0891-0174$02.00/O Copyright 6 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
174
DETECTION
OF COPPER ON GELS
175
ington Biochemical Co. Ovalbumin and bovine serum albumin were obtained from the Pentex Co. Purified p-hydroxyphenylpyruvate hydroxylase (0.07% Fe) was kindly provided by Dr. D. Winter. Electrophoresis was carried out with the Ortec slab gel system (Ortec Inc., Oakridge, Tennessee) using 8% polyacrylamide-30 mM Tris sulfate, pH 8.0, slabs run at 4°C at 150 V, 100 pulses per second for about 120 min, or until a tracking dye, bromphenol blue, reached the bottom of the slab. Proteins of known copper content were applied to the sample slots in lo- to 20-~1 volumes so that a range of protein-bound copper (0.1-2.5 nmol of copper) could be studied with indicating reagents. The sample slots had a surface of 12 mm2. Hemoglobin, catalase, and p-hydroxyphenylpyruvate hydroxylase were used to test specificity. Ten to 0.01 nmol of copper (CuSO, or CuCl,), 10 nmol of Fe3+ (FeCl,), Mn2+ (MnCl,), or Co2+ (CoCl,) were also incorporated into lo-~*.1 gels placed into sample slots. RESULTS
Gels were stained immediately after removal from the electrophoresis apparatus. They were first soaked for 1 min at 16 mM ascorbate in glacial acetic acid to denature protein and to reduce the metal ions and then for 1 min in a 0.28 mM aqueous bathocuproine sulfonate solution. Bathocuproine sulfonate (G. Frederick Smith Chemical Co., Lot NR A2) solution was stored at room temperature in a brown glass bottle. The solution was prepared every 2 weeks. The staining pattern on the gel was observed under uv light (Mineralight, Model R51, 16 W, UV Products, Los Angeles, California). The presence of copper was indicated by dark bands on a field of greenish blue fluorescence. Bathocuproine sulfonate could be replaced by biquinoline. Biquinoline (G. Frederick Smith Chemical Co., Lot NR C29) was dissolved in 16 mM ascorbate in glacial acetic acid to a final concentration of 0.28 mM. The solution was prepared freshly for each test. The gel was soaked in this solution for 2 min. The copperpositive bands were dark under uv light on a yellow-green fluorescent background. Photographs were made under uv illumination using a Polaroid Land camera with a Wratten gelatin filter, type 2A (Kodak) and type 55, 4 x 5 Land film, and 30 set- to 2-min exposures atf4.5. Positions of the dark bands were marked with small incisions on the gel. After the gels were washed with distilled water, they could be stained for protein by soaking them for 16 hr in a solution of 250 ml of isopropyl alcohol, 100 ml of acetic acid, and 50 ml of 1% Coomassie blue R-250 made up to 1 liter with water and then finally were destained in 10% acetic acid. To obtain optimal staining conditions for copper on polyacrylamide gels, the following conditions were systematically varied: incubation time in glacial acetic acid-ascorbate, substitution of hydroxylamine for ascor-
176
BRLJYNINCKX,
GUTTERIDGE,
AND MASON
FIG. I. Tyrosinase and ceruloplasmin were electrophoresed on 8% polyacrylamide gels and stained with bathocuproine sulfonate (Methods). The copper-containing polypeptides produced dark bands on a fluorescent background. (a) Tyrosinase: A, 1.5; B, 1.1; C, 0.75; D, 0.375; E, 0.19 nmol; and F, 0.06 nmol of copper per slot. (b) Ceruloplasmin: A, 1.1; B, 0.9; C, 0.67; D, 0.45; E. 0.23 nmol; and F, 0.11 nmol of copper.
bate, concentration of bathocuproine sulfonate, incubation time in the bathocuproine sulfonate solution, and gel pH after staining. The procedure described gave the most satisfactory results. A uniform background fluorescence was obtained with bathocuproine sulfonate. The nonfluorescent cuprous bathocuproine bands from 1 nmol of Cu were visible immediately after staining; at lower concentrations, e.g., 0.1 nmol per slot, the nonfluorescent bands developed over a lo-min period. There was no diffusion of the bands 90 min after staining. Under visible light, the gels showed no indication of the orange-brown color or cuprous bathocuproine sulfonate unless amounts of copper greater than 2 nmol per gel slot were employed. These weakly colored bands were not distinct. Different amounts of the copper proteins, mushroom tyrosinase, hemocyanin, ceruloplasmin, and superoxide dismutase were applied to the sample slot in a range from 0.05 to 2.5 nmol of protein-bound copper per gel slot. In all cases 0.1 nmol of protein-bound copper was readily detected (Figs. la and lb). The heme proteins hemoglobin and catalase did not quench bathocuproine sulfonate fluorescence in this procedure with 10 nmol of heme. The nonheme iron protein, p-hydroxyphenylpy-
DETECTION
OF COPPER
ON
GELS
177
ruvate hydroxylase (0.5 nmol of Fe), did not react, nor did the copperfree proteins, ovalbumin and pepsin, at levels of 100 pg of protein per slot. With free metal ions in the polyacrylamide gel, 0.1 nmol of Cu*+ was easily detected, while 10 nmol of Fe3+, Fez+, or Mn2+ did not quench bathocuproine sulfonate fluorescence. When the quantity of protein-bound copper on the gels was systematically varied, the intensity of quenching also varied. An attempt was made to quantitate this by densitometric scanning of transparent photographs (Land film, type 462) or negatives of the copper-stained gels, but the relationship between total copper and quenching intensity was not simple (see under Discussion). A scanning spectrofluorimeter, unavailable for this work, might succeed. DISCUSSION
Procedures available for localization of copper-containing polypeptides on gels have suffered from poor specificity (3,4) or sensitivity (1,2,5). The staining procedure now described uses quenching of the fluorescence of bathocuproine sulfonate or biquinoline by Cu+ formed from copper proteins on polyacrylamide gels after being denatured and then reduced. It can be applied to copper-containing proteins without interference from other protein-bound or free metal ions. The amount of protein-bound copper needed for this procedure is in the range normally run on polyacrylamide analytical gels (0.1 nmol of protein-bound copper corresponds to 3.2 pg of tyrosinase or 2.5 pg of ceruloplasmin) with the advantage that the gels can be stained for protein after staining for copper. REFERENCES 1. 2. 3. 4. 5. 6.
Whittaker, I. R. (1959) Nature (London) 184, 192- 193. Horn, E. C., and Kerr, M. S. (1969) Camp. Biochem. Physiol. 29, 493-508. Owen, I. A., and Smith, H. (1961) C/in. Chim. Acta 6, 441-444. Manwell, C., and Baker, C. M. A. (1963) C&n. Chim. Acta 8, 193-208. Gould, E., and Karolus. J. J. (1975) Anal. Biochem. 67, 515-519. Bouchilloux, S., McMahill. P., and Mason, H. S. (1963) J. Bid. Chem. 238, 1699.