Detection of Hemoprotein Peroxidase Activity on Polyvinylidene Difluoride Membrane Chhanda Dutta and Helen L. Henry Department of Biochemistry, University of California, Riverside, California 92521 Received



The feasibility of detecting hemoproteins after electroblotting was examined. Hemoproteins were subjected to lithium dodecyl sulfate-polyacrylamide gel electrophoresis (LDS-PAGE) and then electroblotted and peroxidase activity was detected with 3,3’,5,5’-tetramethylbenzidine. The sensitivity and specificity of tetramethylbenzidine staining of LDS-PAGE gels was retained when proteins were electroblotted. Subsequent staining of the membrane with Coomassie blue R250 revealed a protein pattern similar to that in the polyacrylamide gel. Thus electroblotting of hemoproteins does not affect resolution of the electrophoretic pattern and heme-associated peroxidase activity. Additionally, the ability to stain hemoproteins on polyvinylidene difluoride membranes offers the advantage of utilizing the same membrane for further biochemical and immunological characterizations. D 1990 Academia press, IDC.

Electrophoresis and electroblotting are commonly used in the characterization of a variety of proteins. Electrophoretie separation of proteins on polyacrylamide gels is useful in determining the molecular weight and the purity of a specific protein. However, further analysis of proteins immobilized in gels is limited. On the other hand, once proteins are transferred from a gel to a membrane, the mechanical difficulty of handling fragile gels is avoided. Furthermore, the same blot can be subjected to a variety of protein and immunological staining procedures (1). Due to such advantages of using blots, the current investigation was undertaken to examine the feasibility of applying this technique to the analysis of hemoproteins. Earlier studies have shown that following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGEI), hemoproteins can be identified by a staining ‘Abbreviations used: SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; TMBZ, 3,3’,5,5’-tetramethylbenzidine; LDS, lithium dodecyl sulfate; DTT, dithiothreitol; PVDF, polyvinylidene dikoride.


procedure which takes advantage of the heme-associated peroxidase activity (2-4). In this procedure a substrate specific for heme, 3,3’,5,5’-tetramethylbenzidine (TMBZ), is incubated with the polyacrylamide gel and hemoproteins are then detected with the addition of hydrogen peroxide. Unfortunately, SDS-polyacrylamide gel electrophoresis can also hinder the analysis of hemoproteins since the noncovalently bound heme can dissociate from hemoproteins and then bind nonspecifically to other proteins (5). Since the dissociated heme retains its peroxidase activity, TMBZ staining of such gels could yield unreliable results. The use of lithium dodecyl sulfate (LDS) and solubilization of hemoproteins at O-4% for electrophoresis has been reported to prevent the loss of heme (6). Due to this advantage, LDS-polyacrylamide gel electrophoresis was utilized in the current analysis of hemoproteins. The objective of this study was to demonstrate that hemoproteins of varying molecular weights can be electroblotted without loss of heme-associated peroxidase activity. It is also shown that following heme staining, the blotted membrane can undergo staining with Coomassie brilliant blue R-250 to yield an electrophoretic pattern similar to that seen in polyacrylamide gels.




Chemicals. Electrophoretic grade reagents from BioRad (Richmond, CA) were used for electrophoresis and electroblotting. Emulgen 911 was obtained from Karlan Chemical Corp. (Torrance, CA). All other chemicals, including cytochrome c (equine heart) and 3,3’,5,5’-tetramethylbenzidine were purchased from Sigma Chemical Co. (St. Louis, MO). Isolation of kidney submitochondrial particles. All procedures were carried out at 0-4°C. Mitochondria were prepared from kidney tissue obtained from White Leghorn cockerels (Lakeview Farms, CA), by the 0003~2697/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.







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FIG. 1. Sensitivity of TMBZ staining following electrotransfer from LDS-PAGE onto PVDF membrane. Duplicate lanes of various amounts of cytochrome c underwent LDS-PAGE. One-halfof the gel was then electroblotted onto a PVDF membrane. The gel and PVDF membrane were subjected to TMBZ staining as described under Materials and Methods.

method of Cunningham et al. (7). Following differential centrifugation, submitochondrial particles were resuspended in solubilization buffer (50 mM potassium phosphate, 0.1 mM EDTA, 1.0 mM DTT, 20% glycerol, pH 7.4). This fraction underwent detergent solubilization (0.5% sodium cholate and 0.2% Emulgen 911) and ammonium sulfate precipitation (40%). Ammonium sulfate-precipitated material was then redissolved in phosphate buffer (10 mM potassium phosphate, 0.1 mM EDTA, 1.0 mM DTT, 500 mM NaCl, 20% glycerol, pH 7.0), prior to electrophoretic analysis. LDS-polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis was performed by the method of Laemmli (8), except sodium dodecyl sulfate was replaced by lithium dodecyl sulfate in the buffers. Protein samples and molecular weight standards were prepared for electrophoresis by the addition of sample buffer (312 mM Tris-HCl, 0.4% LDS, 5 mM DTT, 50% glycerol, pH 6.8). A low concentration of DTT was used in the sample buffer, since it can diminish the intensity of TMBZ staining (9). The molecular weight standards were placed in boiling water for 5 min and then loaded onto the polyacrylamide gel. Previous experiments had shown that heat treatment of submitochondrial particles prior to electrophoresis, destroyed the heme-associated peroxidase activity. Thus the protein samples were directly applied to the polyacrylamide gels following the addition of sample buffer. Cytochrome c and submitochondrial samples were electrophoresed on 15 and 7% polyacrylamide gels (12 cm long, 1.5 mm thick), respectively. The gels were run overnight, at 4°C and at constant current (8 mA/gel). Following electrophoresis the gels were cut in half. One-half of the gel was directly placed in TMBZ staining solution and the other half was subjected to electroblotting. Electroblotting procedure. A modified procedure of Pluskal et al. (1) was used to transfer the protein samples




from the polyacrylamide gels onto polyvinylidene difluoride (PVDF) membranes (Millipore Corp., Bedford, MA). PVDF filters were prewetted in 100% methanol prior to use in electrotransfer. Following electrophoresis, the gels were equilibrated in blotting buffer (0.25 M Tris, 0.01% LDS, pH 8.3). The composition of the blotting buffer differed with the molecular weight of the protein being electroblotted. For efficient transfer of lowmolecular-weight proteins, 10% methanol was added to the blotting buffer. The membrane and gel were then placed between pieces of wet Whatman 3MM paper. This sandwich was then placed in a semi-dry blotting apparatus (Rhino Associates, Riverside, CA) and constant current (120 mA) was applied for l-2 h. Blotting buffer was added intermittently during the blotting process, to keep the Whatman papers moist. Heme and protein staining. Staining of hemoproteins on polyacrylamide gels was performed by the method of Thomas et al. (2), with the modification that the TMBZ staining was carried out at 4°C. Hemoproteins in the kidney submitochondrial preparation could not be detected when the staining protocol was done at room temperature. Following incubation (3-4 h) of the gels with the TMBZ solution (6.3 mM TMBZ final concentration), heme-staining

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FIG. 2. Detection of hemoproteins in kidney submitochondrial preparation with TMBZ and Coomassie blue staining. A kidney submitochondrial preparation (450 pg) was subjected to LDS-PAGE (A, C) and electroblotting (B, D). Both the gel and PVDF membrane were stained with TMBZ and then with Coomassie blue R-250 to correlate heme staining with protein bands.




bands were detected with the addition of Hz02 (final concentration 30 mM). The gels were then placed in 0.25 M sodium acetate (pH 5.0) with 3% isopropanol, to remove background staining. The borders of the heme-stained band were marked with India ink and the gels were then placed in the destaining solution (70 IIIM sodium sulfite). Destaining was carried out at room temperature. Next the gels underwent three washes (20 min each) with 30% isopropanol prior to being processed for protein staining with Coomassie brilliant blue R-250. TMBZ staining of PVDF membrane was similar to the procedure used for gels, with some modifications. The membranes were incubated with TMBZ solution for 3 h (in darkness at 4°C) and following the addition of Hz02, the hemoproteins became visible within 1 h. The location of the heme-stained bands on the PVDF membrane were marked with a felt-tipped pen and the membrane was then placed in distilled water. Storage of the membrane in staining solution (following addition of H202) for periods longer than 3 h resulted in increased background staining which was irreversible. In contrast to the lengthy destaining of polyacrylamide gels, the PVDF membrane can be quickly processed for protein staining. Heme-stained filters were washed twice with 100% methanol and were destained within 5 min. Subsequently the membranes were placed in Coomassie stain (0.5% Coomassie brilliant blue R-250,50% methanol, 10% acetic acid) for 10 min and then rapidly destained with 90% methanol.




To examine the sensitivity of TMBZ staining following electroblotting, various amounts of cytochrome c were subjected to LDS-PAGE and then transferred onto PVDF membrane. In Fig. 1, TMBZ staining of cytochrome c in a polyacrylamide gel (Fig. 1A) and in membrane (Fig. 1B) is compared. In the polyacrylamide gel, 50-400 pmol of cytochrome c was easily detectable. Upon heme staining of the PVDF membrane (Fig. lB), 100-400 pmol of cytochrome c yielded intensely stained bands. Although a faint band was obtained with 50 pmol of cytochrome c, the electroblotting and heme staining of lower amounts of cytochrome c was not reliably obtained. Furthermore, with a molecular weight of 12.8K, amounts of cytochrome c 50 pmol and lower correspond to protein concentrations which are well below the detection limit of Coomassiestaining of PVDF membrane (1). In the analysis of proteins, quite often it is of interest to correlate activity with a specific protein band. Previous studies have shown that TMBZ staining of polyacrylamide gels can be utilized to identify heme-associated peroxidase activity in a complex mixture of proteins (2,3). To determine whether hemoproteins could be


identified from a crude tissue preparation transferred onto PVDF membrane, kidney submitochondrial particles were subjected to LDS-PAGE, electroblotting, and TMBZ staining. The results are shown in Fig. 2. TMBZ staining of polyacrylamide gel and PVDF membrane (Figs. 2A and 2B, respectively) both showed two hemestaining bands. Subsequent staining for proteins with Coomassie brilliant blue R-250 revealed the apparent molecular weights of the hemoproteins (Figs. 2C and 2D) to be 59K and 56K. Additionally the specificity of the heme staining procedure remained unaltered with electroblotting, since the presence of the molecular weight markers was evident only after staining with Coomassie blue (Fig. 2B). Since the blotting conditions (exclusion of methanol in buffer and length of blotting time) were optimized for proteins 50K-70K, the lower molecular weight proteins were transferred inefficiently. Accordingly, uniform Coomassie staining of these proteins was not obtained. The LDS gels were photographed within 1 h of detection of the heme-stained bands and could be stored in the sodium acetate and isopropanol mixture (in darkness, 4°C) for 48 h. Longer periods of storage resulted in diffuse and faded heme-stained bands. The hemestained bands on the PVDF membrane were found to be very light sensitive. Loss in the intensity of the hemestained band was evident during photography of the membranes. When protected from light and stored in distilled water, heme-stained bands on membranes were stable for 12 h at 4°C. It has been previously shown that with electrophoresis in the presence of small amounts of SDS, hemeproteins can lose their heme and subsequently bind to other proteins (2). Furthermore, ovalbumin and albumin have been reported to have a high afhnity for the dissociated heme (10). Although bovine serum albumin (66.2K) and hen egg white ovalbumin (42.7K) were constituents of the molecular weight standards usedin this study, heme staining of these proteins was not evident with the use of LDS. Thus the results confirm the findings of Guikema et al. (6) that hemoproteins remain intact with LDS electrophoresis. It was also shown that following electroblotting onto PVDF membrane, hemoproteins of varying molecular weights could be detected with TMBZ staining. Staining of hemoproteins on the membrane was found to be highly sensitive and specific. Subsequent staining of these membranes with Coomassiebrilliant blue enabled the correlation of the heme-associated peroxidase activity with a protein band. Additional studies indicate that following Coomassiestaining, the samePVDF filter can undergo Western blot analysis. Furthermore, hemoproteins blotted onto membranes can undergo amino acid sequence determination without further manipulations, as PVDF membranescan be directly placed into Applied Biosystems sequencers(11). In conclu-





sion, the feasibility of detecting hemoproteins on a blotted membrane is a powerful tool in the biochemical characterization of such proteins.

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Detection of hemoprotein peroxidase activity on polyvinylidene difluoride membrane.

The feasibility of detecting hemoproteins after electroblotting was examined. Hemoproteins were subjected to lithium dodecyl sulfate-polyacrylamide ge...
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