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H2O2 staining is not specific for heme proteins separated by gel electrophoresis
ANALYTICAL
BIOCHEMISTRY
140,
577-580
(1984)
3,3’,5,5’-TetramethyIbenzidine/H202 for Heme Proteins Separated DAVID J. MILLER
Staining Is Not Specific by Gel Electrophoresis
AND D. J. D. NICHOLAS’
Department of Agricultural Biochemistry, Waite Agricultural Research Institute. University of Adelaide, Glen Osmond, South Australia 5064, Australia Received February 7, 1984 Staining of sodium dodecyl sulfate or lithium dodecyl sulfate gels with 3,3’,5,5’-tetramethylbenzidine (TMBZ)/H202 after electrophoresis has frequently been used as a specific method of detecting heme proteins. That TMBZ is an electron donor for O2 reduction by the nonhemesoluble cytochrome oxidase/nitrite reductase from Nitrosomonas europaea is now shown; this protein is detected by the TMBZ/H202 method. A method for the determination of TMBZ oxidase activity is given; hence, the detection of artifactual staining due to proteins of this type is possible. KEY WORDS: heme staining; oxidases; copper proteins; nitrite reductases; Nitrosomonas europaea; nitrifying bacteria.
TMBZ/H202 staining has been used to characterize the cytochrome composition of Anacystis nidulans (5), Rhodopseudomonas sphaeroides (6), and Nitrosomonas europaea (7); TMBZ/H202 staining after SDS-PAGE has been used in many investigations on the distribution and characteristics of c-type cytochromes (see, for example, Kasprzak and Steenkamp (8)). The assumption throughout these studies was that the staining method employed was specific for heme proteins. The soluble cytochrome oxidase/nitrite reductase from N. europaea was found to be a copper protein, devoid of heme (9). The present communication shows that the protein utilizes TMBZ as an electron donor for O2 reduction, and thus stains using the TMBZ/ H202 system; hence the method is not specific for heme proteins, as had been assumed previously.
Under mildly acid conditions heme-contaming proteins, such as cytochrome P-420, possess peroxidase activity (1,2). Welton and Aust (3) developed a specific staining method for heme proteins after PAGE2 based on this peroxidase activity, using benzidine as a chromogenic electron donor and Hz02 as an electron acceptor. Recently this method has been modified (4); thus, use of 3,3’,5,5’-tetramethylbenzidine (TMBZ) as the chromogen led to improvements in the sensitivity, resolution, and stability of stained gels. Recently, it has been shown (5) that if the lithium salt of dodecyl sulfate was substituted for SDS in Laemmli-type electrophoresis, sample denaturation and electrophoresis could be conducted near 0°C. Under these conditions many cytochromes containing noncovalently bound heme retained heme and hence stained with TMBZ/H202. LDS-PAGE followed by ’ To whom correspondence should be addressed. 2 Abbreviations used: PAGE, polyacrylamide gel electrophoresis; TMBZ, 3,3’,5,5’-tetramethylbenzidine; SDS, sodium dodecyl sulfate; LDS, lithium dodecyl sulfate.
MATERIALS
AND METHODS
N. europaea ATCC 19 178 was grown and harvested by published methods (10). The 577
0003-2697184 $3.00 Copyright 0 1984 by Academic F’res, Inc. All rights of reproduction in any form reserved.
578
MILLER
AND NICHOLAS
protein assay was by the microtannin method ( 11). SDS-PAGE was conducted as described by Laemmli ( 12). Details of LDS-PAGE and TMBZ/HIOI staining were as described previously (6). The soluble cytochrome oxidase/nitrite reductase from N. europaea was purified by the published method (9), except that elution from the first ion-exchange column was achieved with a 400-ml linear gradient of O-O.25 M NaCl in 10 mM Tris-HCl buffer, pH 8.0. The criterion of purity for the N. europaea protein was a single band on Laemmli-type SDSPAGE (Fig. 1A). Electrophoretically homogenous nitrite reductase from R. sphaeroides forma sp. denitrificans was kindly supplied by Dr. W. Mich-
alski of this department. This copper-containing nonheme nitrite reductase also has cytochrome oxidase activity (13). Because of their analogous properties, nitrite reductase from R. sphaeroides denitrijcans was used for comparison with the Nitrosomonas protein.
RESULTS
Gel Electrophoresis
When gels loaded with N. europaea cytochrome oxidase/nitrite reductase were submitted to TMBZ/H202 staining after SDSPAGE a single green band was observed, corresponding in mobility to the 35,000 M, for each of four subunits as reported previously (9). LDS-PAGE of the purified Nitrosomonas protein followed by TMBZ staining gave a (B) 63 (A) strong green band in the high-molecular.* weight region prior to the addition of H202. This band faded slightly after Hz02 treatment, sbut was still clearly visible after gels had been fully stained and destained as described by Thomas et al. (4) (Fig. 1B). When TMBZ/ HzOz-stained gels were bleached in sodium sulfite and then stained with Coomassie blue (4), a single protein-staining band was found to correspond in mobility to the TMBZ/HzOzstaining band (Fig. 1C). In 8%-acrylamide LDS-PAGE the mobility of this single band, Dcompared with those of the following hemecontaining proteins, horse heart cytochrome FIG. 1. Gel electrophoresis of the soluble cytochrome c (iWr 11,700), N. europaea cytochrome ~554 oxidase/nitrite reductase from N. ewopaea. Gel tracks: oxidase (A) after SDS-PAGE followed by staining with Coomassie (Mr 28,000 (14)), and hydroxylamine blue (protein loaded = 40 w); (B) after LDS-PAGE fol(Mr 200,000), was found to be approximately lowed by staining with TMBZ/H*Or (protein loaded = 12 130,000. Thus under LDS-PAGE this protein pg); (C) the same gel track as in (B), but bleached with did not produce subunits to any significant sodium sulfite and then stained with Coomassie blue as extent. described under Materials and Methods. Electrophoresis was conducted throughout in 15% (w/v) acrylamide-reNitrite reductase from R. sphaeroides denisolving gels with 5% (w/v) stacking gels. For (A) sample trijkans did not give a TMBZ/HzOz-staining treatment was as described by Wood ( 17), and electro- band after LDS-PAGE. Protein staining of phoresis was conducted for 4 h at 30 mA and ambient LDS gels indicated that, as with the Nitrotemperature. For (B, C) sample preparation and electrosomonas protein, separation to subunits had phoresis conditions were as described previously (6). S, Start of resolving gel; D, dye front. not occurred.
579
3,3’,5,5’-TETRAMETHYLBENZIDINE/HrOr
Oxidase Assays Using TMBZ Dihydrochloride TMBZ itself is relatively insoluble in aqueous buffers ( 15), but the dihydrochloride form is sufficiently water soluble to permit its use as an electron donor in aqueous buffers for 02-uptake and spectrophotometric studies. Results of such experiments with the N. europaea cytochrome oxidase/nitrite reductase confirmed t at TMBZ was an electron donor for O2 reduc. ion (Fig. 2). Neither endogenous peroxidase (16), present in impure preparations, nor exogenous catalase significantly affected the spectrophotometric assay for TMBZ oxidation. DISCUSSION
The soluble cytochrome oxidase/nitrite reductase from IV. europaea gave an intense band on TMBZ/HzOz staining after LDS-
TMBZ
protsin I
I
-
T
0.2 ’A
1
/
(A)
TMEZprot*in I +
20
min
--y
J
I C
2 min
PAGE, a reaction which had been assumed to be specific for heme proteins. This band was visible even prior to addition of HzOz. It is unlikely that its detection was due to contaminating heme protein(s), since the preparation was shown to be absolutely homogenous on SDS-PAGE. The soluble cytochrome oxidase/nitrite reductase from IV. europaea has been shown to utilize a variety of electron donors for reduction of O2 (9): TMBZ is also a donor for the oxidase reaction with this protein. 3,3’-Diaminobenzidine is frequently employed as an electron donor for oxidases, so it is to be expected that the structurally similar compound TMBZ can also support O2 reduction with some oxidases. The above results indicate that TMBZ/HzO&aining bands after LDSPAGE are not specific for heme-containing proteins. Thus TMBZ oxidase activity should be checked on all preparations which are to be subjected to TMBZ/H102 staining. Neither the N. europaea cytochrome oxidase/nitrite reductase nor the Rhodopseudomonas nitrite reductase produced subunits under the conditions used for LDS solubilization and electrophoresis. This result was not affected by variations in the protein:LDS ratio (data not shown). Several authors have reported changes in relative molecular weights of proteins with variation in solubilization temperatures (5,6), but the complete retention of polymeric-form proteins in LDS-PAGE is without precedent.
-i
FIG. 2. Oxidase activity of the N. europaea soluble cytochrome oxidase/nitrite reductase using TMBZ aselectron donor. (A) Oxygen uptake with TMBZ oxidation. The O2 uptake was monitored in a Clark oxygen-electrode cuvette containing 0.25 M sodium acetate buffer, pH 5.0 (2 ml). TMBZ dihydrochloride (180 pM in 50 pl HrO) and purified enzyme (5 pg) were added as shown. (B) Spectrophotometric determination of TMBZ oxidation. Absorbance at 650 nm was monitored using a PerkinElmer X5 recording spectrophotometer. Cuvettes contained 2 ml of 0.25 M sodium acetate buffer, pH 5.0. TMBZ dihydrochloride (180 PM) and purified enzyme ( 10 pg) were added as shown. Both O2 uptake and spectrophotometric assayswere conducted at 22’C.
ACKNOWLEDGMENTS We thank the Australian Research Grants Scheme for a research grant. D. J. Miller acknowledges with thanks the award of a research associateship by the Australian Research Grants Scheme.
REFERENCES 1. Hrycay, E. Biochem. 2. Hrycay, E. Biochem.
G., and Biophys. G., and Biophys.
O’Brien, P. J. (1971) Arch. 147, 14-27. O’Brien, P. J. (1971) Arch. 147, 28-35.
580
MILLER
AND NICHOLAS
3. Welton, A. F., and Aust, S. D. (1974) Biochem. Bi& phys. Res. Comm. 56, 898-906. 4. Thomas, P. E., Ryan, D., and Levin, W. (1976) A#ai. B&hem. 75, 168-176. 5. Guikema, J. A., and Sherman, L. A. (1981) Biochem. Biophys. Acta 637, 189-20 1. 6. Ward, J. A., Hunter, C. N., and Jones, 0. T. G. (1983) Biochem. J. 212, 783-790. 7. Miller, D. J., and Wood, P. M. (1983) FEMS Left. 20.323-326. 8. Kasprzak, A. A., and Steenkamp, D. J. (1983) J. Bacteriol. 156, 348-353. 9. Miller, D. J., and Wood, P. M. (1983) J. Gen. Microbiol. 129, 1645-1650. 10. Nicholas, D. J. D., and Rao, P. S. ( 1964) Biochim. Biophys. Acta 82, 394-397.
11. Mejbaum-Katzenellenbogen, W. (1955) But/. Acad Pal. Sci., Cl. 2 3, 171-173. 12 Laemmli, U. K. (1970) Nature (London) 227, 680685. 13. Sawada, E., Satoh, T., and Kitamura, H. (1978) Planf Cell Physiol. 19, 1339-1351. 14. Miller, D. J., and Wood, P. M. (1982) Eiochem. J. 207, 51 l-517. 15. Liem, H. H., Cardenas, F., Tavassoli, M., Poh-Fitzpatrick, M. B., and Muller-Eberhard, U. (1979) Anal. B&hem. 98,388-393. 16. Anderson, J. R., Strumeyer, D. H., and Pramer, D. (1968) J. Baczeriol. 96, 93-97. 17. Wood, P. M. (1981) Anal. Biochem. 111, 235-239.
BIOCHEMISTRY
140,
577-580
(1984)
3,3’,5,5’-TetramethyIbenzidine/H202 for Heme Proteins Separated DAVID J. MILLER
Staining Is Not Specific by Gel Electrophoresis
AND D. J. D. NICHOLAS’
Department of Agricultural Biochemistry, Waite Agricultural Research Institute. University of Adelaide, Glen Osmond, South Australia 5064, Australia Received February 7, 1984 Staining of sodium dodecyl sulfate or lithium dodecyl sulfate gels with 3,3’,5,5’-tetramethylbenzidine (TMBZ)/H202 after electrophoresis has frequently been used as a specific method of detecting heme proteins. That TMBZ is an electron donor for O2 reduction by the nonhemesoluble cytochrome oxidase/nitrite reductase from Nitrosomonas europaea is now shown; this protein is detected by the TMBZ/H202 method. A method for the determination of TMBZ oxidase activity is given; hence, the detection of artifactual staining due to proteins of this type is possible. KEY WORDS: heme staining; oxidases; copper proteins; nitrite reductases; Nitrosomonas europaea; nitrifying bacteria.
TMBZ/H202 staining has been used to characterize the cytochrome composition of Anacystis nidulans (5), Rhodopseudomonas sphaeroides (6), and Nitrosomonas europaea (7); TMBZ/H202 staining after SDS-PAGE has been used in many investigations on the distribution and characteristics of c-type cytochromes (see, for example, Kasprzak and Steenkamp (8)). The assumption throughout these studies was that the staining method employed was specific for heme proteins. The soluble cytochrome oxidase/nitrite reductase from N. europaea was found to be a copper protein, devoid of heme (9). The present communication shows that the protein utilizes TMBZ as an electron donor for O2 reduction, and thus stains using the TMBZ/ H202 system; hence the method is not specific for heme proteins, as had been assumed previously.
Under mildly acid conditions heme-contaming proteins, such as cytochrome P-420, possess peroxidase activity (1,2). Welton and Aust (3) developed a specific staining method for heme proteins after PAGE2 based on this peroxidase activity, using benzidine as a chromogenic electron donor and Hz02 as an electron acceptor. Recently this method has been modified (4); thus, use of 3,3’,5,5’-tetramethylbenzidine (TMBZ) as the chromogen led to improvements in the sensitivity, resolution, and stability of stained gels. Recently, it has been shown (5) that if the lithium salt of dodecyl sulfate was substituted for SDS in Laemmli-type electrophoresis, sample denaturation and electrophoresis could be conducted near 0°C. Under these conditions many cytochromes containing noncovalently bound heme retained heme and hence stained with TMBZ/H202. LDS-PAGE followed by ’ To whom correspondence should be addressed. 2 Abbreviations used: PAGE, polyacrylamide gel electrophoresis; TMBZ, 3,3’,5,5’-tetramethylbenzidine; SDS, sodium dodecyl sulfate; LDS, lithium dodecyl sulfate.
MATERIALS
AND METHODS
N. europaea ATCC 19 178 was grown and harvested by published methods (10). The 577
0003-2697184 $3.00 Copyright 0 1984 by Academic F’res, Inc. All rights of reproduction in any form reserved.
578
MILLER
AND NICHOLAS
protein assay was by the microtannin method ( 11). SDS-PAGE was conducted as described by Laemmli ( 12). Details of LDS-PAGE and TMBZ/HIOI staining were as described previously (6). The soluble cytochrome oxidase/nitrite reductase from N. europaea was purified by the published method (9), except that elution from the first ion-exchange column was achieved with a 400-ml linear gradient of O-O.25 M NaCl in 10 mM Tris-HCl buffer, pH 8.0. The criterion of purity for the N. europaea protein was a single band on Laemmli-type SDSPAGE (Fig. 1A). Electrophoretically homogenous nitrite reductase from R. sphaeroides forma sp. denitrificans was kindly supplied by Dr. W. Mich-
alski of this department. This copper-containing nonheme nitrite reductase also has cytochrome oxidase activity (13). Because of their analogous properties, nitrite reductase from R. sphaeroides denitrijcans was used for comparison with the Nitrosomonas protein.
RESULTS
Gel Electrophoresis
When gels loaded with N. europaea cytochrome oxidase/nitrite reductase were submitted to TMBZ/H202 staining after SDSPAGE a single green band was observed, corresponding in mobility to the 35,000 M, for each of four subunits as reported previously (9). LDS-PAGE of the purified Nitrosomonas protein followed by TMBZ staining gave a (B) 63 (A) strong green band in the high-molecular.* weight region prior to the addition of H202. This band faded slightly after Hz02 treatment, sbut was still clearly visible after gels had been fully stained and destained as described by Thomas et al. (4) (Fig. 1B). When TMBZ/ HzOz-stained gels were bleached in sodium sulfite and then stained with Coomassie blue (4), a single protein-staining band was found to correspond in mobility to the TMBZ/HzOzstaining band (Fig. 1C). In 8%-acrylamide LDS-PAGE the mobility of this single band, Dcompared with those of the following hemecontaining proteins, horse heart cytochrome FIG. 1. Gel electrophoresis of the soluble cytochrome c (iWr 11,700), N. europaea cytochrome ~554 oxidase/nitrite reductase from N. ewopaea. Gel tracks: oxidase (A) after SDS-PAGE followed by staining with Coomassie (Mr 28,000 (14)), and hydroxylamine blue (protein loaded = 40 w); (B) after LDS-PAGE fol(Mr 200,000), was found to be approximately lowed by staining with TMBZ/H*Or (protein loaded = 12 130,000. Thus under LDS-PAGE this protein pg); (C) the same gel track as in (B), but bleached with did not produce subunits to any significant sodium sulfite and then stained with Coomassie blue as extent. described under Materials and Methods. Electrophoresis was conducted throughout in 15% (w/v) acrylamide-reNitrite reductase from R. sphaeroides denisolving gels with 5% (w/v) stacking gels. For (A) sample trijkans did not give a TMBZ/HzOz-staining treatment was as described by Wood ( 17), and electro- band after LDS-PAGE. Protein staining of phoresis was conducted for 4 h at 30 mA and ambient LDS gels indicated that, as with the Nitrotemperature. For (B, C) sample preparation and electrosomonas protein, separation to subunits had phoresis conditions were as described previously (6). S, Start of resolving gel; D, dye front. not occurred.
579
3,3’,5,5’-TETRAMETHYLBENZIDINE/HrOr
Oxidase Assays Using TMBZ Dihydrochloride TMBZ itself is relatively insoluble in aqueous buffers ( 15), but the dihydrochloride form is sufficiently water soluble to permit its use as an electron donor in aqueous buffers for 02-uptake and spectrophotometric studies. Results of such experiments with the N. europaea cytochrome oxidase/nitrite reductase confirmed t at TMBZ was an electron donor for O2 reduc. ion (Fig. 2). Neither endogenous peroxidase (16), present in impure preparations, nor exogenous catalase significantly affected the spectrophotometric assay for TMBZ oxidation. DISCUSSION
The soluble cytochrome oxidase/nitrite reductase from IV. europaea gave an intense band on TMBZ/HzOz staining after LDS-
TMBZ
protsin I
I
-
T
0.2 ’A
1
/
(A)
TMEZprot*in I +
20
min
--y
J
I C
2 min
PAGE, a reaction which had been assumed to be specific for heme proteins. This band was visible even prior to addition of HzOz. It is unlikely that its detection was due to contaminating heme protein(s), since the preparation was shown to be absolutely homogenous on SDS-PAGE. The soluble cytochrome oxidase/nitrite reductase from IV. europaea has been shown to utilize a variety of electron donors for reduction of O2 (9): TMBZ is also a donor for the oxidase reaction with this protein. 3,3’-Diaminobenzidine is frequently employed as an electron donor for oxidases, so it is to be expected that the structurally similar compound TMBZ can also support O2 reduction with some oxidases. The above results indicate that TMBZ/HzO&aining bands after LDSPAGE are not specific for heme-containing proteins. Thus TMBZ oxidase activity should be checked on all preparations which are to be subjected to TMBZ/H102 staining. Neither the N. europaea cytochrome oxidase/nitrite reductase nor the Rhodopseudomonas nitrite reductase produced subunits under the conditions used for LDS solubilization and electrophoresis. This result was not affected by variations in the protein:LDS ratio (data not shown). Several authors have reported changes in relative molecular weights of proteins with variation in solubilization temperatures (5,6), but the complete retention of polymeric-form proteins in LDS-PAGE is without precedent.
-i
FIG. 2. Oxidase activity of the N. europaea soluble cytochrome oxidase/nitrite reductase using TMBZ aselectron donor. (A) Oxygen uptake with TMBZ oxidation. The O2 uptake was monitored in a Clark oxygen-electrode cuvette containing 0.25 M sodium acetate buffer, pH 5.0 (2 ml). TMBZ dihydrochloride (180 pM in 50 pl HrO) and purified enzyme (5 pg) were added as shown. (B) Spectrophotometric determination of TMBZ oxidation. Absorbance at 650 nm was monitored using a PerkinElmer X5 recording spectrophotometer. Cuvettes contained 2 ml of 0.25 M sodium acetate buffer, pH 5.0. TMBZ dihydrochloride (180 PM) and purified enzyme ( 10 pg) were added as shown. Both O2 uptake and spectrophotometric assayswere conducted at 22’C.
ACKNOWLEDGMENTS We thank the Australian Research Grants Scheme for a research grant. D. J. Miller acknowledges with thanks the award of a research associateship by the Australian Research Grants Scheme.
REFERENCES 1. Hrycay, E. Biochem. 2. Hrycay, E. Biochem.
G., and Biophys. G., and Biophys.
O’Brien, P. J. (1971) Arch. 147, 14-27. O’Brien, P. J. (1971) Arch. 147, 28-35.
580
MILLER
AND NICHOLAS
3. Welton, A. F., and Aust, S. D. (1974) Biochem. Bi& phys. Res. Comm. 56, 898-906. 4. Thomas, P. E., Ryan, D., and Levin, W. (1976) A#ai. B&hem. 75, 168-176. 5. Guikema, J. A., and Sherman, L. A. (1981) Biochem. Biophys. Acta 637, 189-20 1. 6. Ward, J. A., Hunter, C. N., and Jones, 0. T. G. (1983) Biochem. J. 212, 783-790. 7. Miller, D. J., and Wood, P. M. (1983) FEMS Left. 20.323-326. 8. Kasprzak, A. A., and Steenkamp, D. J. (1983) J. Bacteriol. 156, 348-353. 9. Miller, D. J., and Wood, P. M. (1983) J. Gen. Microbiol. 129, 1645-1650. 10. Nicholas, D. J. D., and Rao, P. S. ( 1964) Biochim. Biophys. Acta 82, 394-397.
11. Mejbaum-Katzenellenbogen, W. (1955) But/. Acad Pal. Sci., Cl. 2 3, 171-173. 12 Laemmli, U. K. (1970) Nature (London) 227, 680685. 13. Sawada, E., Satoh, T., and Kitamura, H. (1978) Planf Cell Physiol. 19, 1339-1351. 14. Miller, D. J., and Wood, P. M. (1982) Eiochem. J. 207, 51 l-517. 15. Liem, H. H., Cardenas, F., Tavassoli, M., Poh-Fitzpatrick, M. B., and Muller-Eberhard, U. (1979) Anal. B&hem. 98,388-393. 16. Anderson, J. R., Strumeyer, D. H., and Pramer, D. (1968) J. Baczeriol. 96, 93-97. 17. Wood, P. M. (1981) Anal. Biochem. 111, 235-239.