ANALYTICAL BIOCHEMISTRY 68, 148-154 (1975)
Polyacrylamide Gel Electrophoresis of Myelin Proteins: A Caution PIERRE MORELL, RICHARD C . WIGGINS, AND MAP-dORY JONES GRAY
Biological Sciences Research Center and Department of Biochemistry and Nutrition, Division of Health Affairs, University of North Carolina, Chapel Hill, North Carolina 27515 Received December 23, 1974; accepted April 16, 1975 Some variables involved in the preparation of rat brain myelin proteins for polyacrylamide gel electrophoresis in buffers containing sodium dodecyl sulfate were studied. Under mild conditions of solubilization the resultant gel patterns
were relatively insensitive to the /~-mercaptoethanol (ME) concentration in the protein solvent used for solubilization of myelin proteins. However, if the samples were boiled in the presence of ME (a standard procedure for disruption of metastable aggregates of membrane proteins), a major myelin protein, proteolipid protein, as well as some minor proteins were preferentially excluded from the gel. This effect was proportional to the ME concentration. Characterization of the polypeptide composition of m e m b r a n e proteins b y polyacrylamide gel electrophoresis in buffers containing sodium dodecyl sulfate (SDS) has been a standard procedure since the introduction of this method (1). When using such a system, it is c u s t o m a r y to solubilize the protein sample in a protein solvent consisting of a buffer containing S D S a n d / ~ - m e r c a p t o e t h a n o l (ME) to reduce disulfide bonds, and to bring the sample to a boil to disrupt metastable aggregates (2). W e have utilized a discontinuous version of such an electrophoretic s y s t e m (2) to separate myelin proteins (3,4). In our studies we encountered problems with this boiling procedure and r e c o m m e n d e d instead initially dissolving the myelin samples in 1% S D S , placing t h e m in an ultrasonic cleaning bath for an extended period of time to affect solubilization, and only then adding M E (3). We have since quantitatively investigated the effects of boiling myelin samples in the presence or absence of M E and report our results in this communication.
METHODS
Preparation of Myelin Myelin f r o m 26-day-old L o n g - E v a n s rats was prepared essentially b y the procedure of N o r t o n and Poduslo (5), with some minor modifications (6). Myelin was lyophilized and aliquots of 10-20 mg were par148 Copyright~) 1975by AcademicPress, Inc. All rightsof reproductionin any formreserved.
M Y E L I N P R O T E I N ELECTROPHORESIS
149
tially delipidated by 5 extractions with 1 ml of ether-ethanol (3:2, v/v)/mg dry wt and dried under a stream of nitrogen (3), In some experiments, animals were injected intracranially with 200 /xCi of [4,53H]leucine (40.7 Ci/mmole) 24 hr prior to sacrifice.
Polyacrylamide Gel Electrophoresis The method for electrophoresis of myelin protein on discontinuous polyacrylamide gels has been described previously (3). Formation of gels of such a high acrylamide concentration (15% running gel, 3% spacer gel) requires special care to avoid formation of bubbles and other flaws in the gel, and some further practical details relating to the procedure are detailed below. The gel columns were cut from 8 mm o.d.-6 mm i.d. glass tubing (either Pyrex or flint glass) in 15.5 cm lengths and were treated with acid-dichromate cleaning solution, washed with detergent, rinsed with distilled water, and coated with Siliclad solution (Clay Adams, Parsippany, N J) before use. One end of the column was blocked by inserting the tube into the cavity of a solid 00 rubber safety stopper (Arthur H. Thomas, Philadelphia, PA). Gels were formed in the reverse of the usual order; initially sucrose (30% w/v) was added to a height of 2.5 cm to reserve space for the sample. A long tip Pasteur pipet was used to avoid getting sucrose on the interior surface of the cylinder. A 2 cm spacer gel was layered on top of the sucrose and overlaid with 0.1% SDS. After this had firmly polymerized (30-60 rain), the SDS solution was removed with a long tip Pasteur pipet and the column filled with running gel solution. The running gel solution must be deaerated for at least 45 min (we use house vacuum). The solution is then chilled on ice to slow polymerization which is initiated by addition of ammonium persulfate just prior to pouring the gel. After polymerization (about 30 rain) the gel tubes were topped off with 0.1% SDS, covered with Parafilm (American Can Company, Neenah, WI) to prevent the gel surface from caking, and then allowed to stand from 12 to 24 hr at room temperature before use. The gel columns could also be stored for several days in a plastic bag in the refrigerator. The sucrose was removed before use; then the columns were inverted and loaded in a Buchler Polyanalyst gel electrophoresis apparatus (with a spacer added between reservoirs to accomodate the long tubes). Air bubbles trapped beneath the running gel were removed with a bent Pasteur pipet. Myelin protein, 50-200/xg, in 0.1-0.4 ml of protein solvent (see below) was applied. Running buffer was equilibrated between the lower and upper reservoirs using an oscillating pump and an overflow tube and a constant 60 V was applied for 16 hr. The marker dye leaves the gel after 12 hr. Gels were removed by breaking the glass tubes,
150
MORELL, WIGGINS AND GRAY
fixed, stained with Fast Green and destained as described previously (3). The gels were scanned at 570 nm using a Beckman model 25 Spectrophotometer equipped with a linear transport mechanism and the areas under peaks corresponding to individual proteins quantitated (4).
Radioactivity Determination For determination of radioactivity distribution in various proteins, stained gels were frozen in an ultra low temperature freezer or on a dry ice block and firmly pressed into a razor blade stack (Hoeffer Scientific Instruments, San Francisco, Calif.) moistened with water. Each gel slice was transferred to a glass scintillation vial containing 0.3 ml of 30% hydrogen peroxide and tightly capped with a plastic lined screw cap. Following incubation for 1-2 days at 50°C, excess hydrogen peroxide was dissipated by addition of 4500 units of catalase (the C10 grade of Sigma). After standing for 15 min, 10 ml of toluene-Triton X-100 (Rohm and Haas, Philadelphia, Pa.) (4 : 1, v/v) containing 21 g/gal of Omnifluor (New England Nuclear, Boston, Mass.) was added. Vials were left loosely capped for at least 2 hr to allow for further release of oxygen, prior to being firmly tightened.
Sample Preparation Delipidated myelin was prepared for electrophoresis by suspending it in 1% SDS so that the final protein concentration was between 1 and 2 mg/ml (an approximation of the protein concentration was made from the initial dry weight of myelin assuming that it contained 28% protein). Solubilization of the protein was aided by placing the tube in an ultrasonic cleaning bath (Heat System, Ultrasonic Inc., Planview, NY) for a period of up to several hours. The temperature of the water rose to about 60°C during this time. Ten microliter aliquots were taken for protein determination (7). Our routine procedure for analysis involved diluting 3 vol of this protein solution with 1 vol of concentrated protein solvent consisting of: 4.0 ml of 0.5 M Tris-HCl at pH 6.8 [solution C(3)], 1.0 ml of 10% SDS, 0.4 ml of ME, 4.0 ml of glycerol, 0.4 ml of 0.1% bromophenol blue, and 0.2 ml of water. A volume containing about 150 /~g of protein was layered onto the spacer gel and electrophoresed. If the electrophoresis was not done on the day of sample preparation, samples were stored (either refrigerated for a few days, or frozen for up to a number of weeks), and subjected to a brief treatment in the ultrasonic cleaning bath before electrophoresis. In the present series of experiments the procedure was modified by omitting ME from the protein solvent so that the concentration of this reagent could be adjusted in individual aliquots prior to electrophoresis.
MYELIN PROTEIN ELECTROPHORESIS
15 1
FIG. 1. Photographs of p0!yacrylanfide gels following electrophoresis of 150 ~g o f myelin proteins. Gels a-f are samples Where the protein was solubilized in 1% SDS followed b y addition of ME and ~other components of ~the protein solvent. Immediately prior to electrophoresis the ME concentration was (a):0%; (b) 0.:5%; (c) 1%; (d) 2%; (e') 5% ; (f) t 0 % . G e l s a ' - f ' are duplicates ,of the:above except that following addition of ME and other components of the ,protein solvent the samples were placed in a boiling water bath for 2 rain prior to dectrophoresls.
RESULTS Figure 1 illustrates the gel electrophoresis patterns of myelin proteins in the presence of varyirrg concentrations of ME, using a protocol (see Methods) that does not involve boiling :the protein samples. Proteins are identified in order of increasing molecular weight: the two basic :proteins of rat brain myelin (8), Be and Bs; the double :band identified :as intermediate protein (4,9), I; the proteolipid protein (I0), P; the major high molecular weight protein (l 1), (W) which is just barely 'resolved as two bands in this gel system; and several other high molecular weight proteins including one identified o-n Fig. ~1by an arrow. It is clear from the first set o f gels that although some M E Ks necessary ~to give adequate resOlution of proteins (note the srrreafing o f some :of the protein 'bands in gel
152
MORELL, WIGGINS AND GRAY
a) the actual ME concentration is not very critical. In the gel without ME (gel a) and in the gel containing 10% ME (gel f), the protein bands appeared to have a higher mobility than the other gels. This phenomenon was not quantitated, since no reference point for the dye front exists (the dye is electrophoresed off the end of the gel in this system). A minor band, directly above W and just barely separated from it, appears at the higher ME concentrations. In the second set of gels, Fig. l a ' - f ' , which is identical to the first set of gels except that the samples (in small screw capped tubes) were placed in a boiling water bath for 2 min prior to electrophoresis, marked variation is observable. With increasing ME concentration, proteolipid protein is preferentially excluded from the gel and piles up at the stacking gel-running gel interface. The slower moving intermediate protein and, even more markedly, a high molecular weight protein (see arrow) are also affected by boiling at the higher ME concentration. Quantitative analysis of densitometric scans of two such complete sets of gels indicated that, under conditions of boiling with reducing reagent, 35% o f the proteolipid protein was excluded from the gel at a 1% ME concentration, and over 90% of the proteolipid protein was excluded when the ME concentration was 5%. The amount of material in the basic protein band remained constant. It was also observed that the mobility of proteolipid protein (relative to the basic proteins) was slightly decreased at increasing ME concentrations. This could be interpreted on the basis of some conformational effect of increasing ME concentration on the remaining, unaggregated proteolipid protein. However, we feel it is more likely a loading phenomenon due to the decreased amount of protein at this molecular weight entering the gel. A similar series of experiments, substituting dithiothreitol for ME, at concentrations ranging from 0. I to 5%, indicated that boiling myelin proteins for 2 min in the presence of dithiothreitol did not cause marked preferential exclusion of proteolipid protein from the gel. However, some other observations (J. Benjamins, personal communication) suggest to us that harsher treatment, such as repeated boiling for longer periods of time, in the presence of dithiothreitol will cause partial exclusion of proteolipid protein from the gel. Also, the resolution of protein was not as good, and fewer discrete bands were seen, in the presence of dithiothreitol reagent. It was considered possible that the apparent exclusion of proteolipid proteins observed in Fig. 1 might be artifactual in that the boiling in the presence of ME might somehow alter the protein so that it would no longer bind Fast Green. To control for this possibility an experiment similar to that in Fig. 1 but using radioactivity instead of densitometry to
MYELIN PROTEIN ELECTROPHORESIS
153
quantitate protein distribution was carried out (6). These studies gave results exactly analogous to those reported in Fig. 1. The presence of ME during the boiling period caused loss of radioactivity in the proteolipid protein region of the gel which was quantitatively accounted for by radioactivity at the gel origin. DISCUSSION
A standard procedure for separation of membrane proteins in SDSgels involves heating the protein sample to 100°C prior to electrophoresis in order to disrupt possible metastable aggregates (2). It is generally assumed that subjecting the sample to such vigorous denaturing conditions can only promote the desired results of reduction of sulfhydryl groups by the reducing reagent, as well as insuring uniform binding of SDS to the resultant subunits. This assumption may lead to operational difficulties in the study of certain membrane proteins. One important brain membrane protein, proteolipid protein, can be prepared in large amounts and its properties extensively studied (10). Most prominent of the peculiar properties of this protein is preferential solubility in organic solvents (10). Aqueous solutions of proteolipid protein can be obtained by stepwise dialysis from organic solvents into solutions of increasing polarity, or by the use of detergents. It is possible that under these conditions the protein is somehow not completely denatured. If the protein is subjected to more severe denaturing conditions such as repeated flash evaporation from chloroform-methanol (2:1, v/v) containing 5% (v/v) water (12) it forms a precipitate which can not be resolubilized either in organic solvent or in water even in the presence of detergents (unpublished observation in our laboratory and personal communication from Dr. W. T. Norton). It is possible that boiling in the presence of ME is sufficient to completely denature proteolipid protein which then precipitates out of the detergent containing solution and remains at the gel interface during electrophoresis. This "complete" denaturation might correspond to the dissociation of residual lipid. Another hypothesis is also based on the premise that at a lower temperature there exists an SDS-protein complex in which the protein retains some structure. At 100°C this SDS-protein complex became destabilized (sulfhydryl groups became accessible for reduction by mercaptoethanol), leading to the complete unfolding of the polypeptide chains and aggregation. Presumably, under identical conditions, dithiothreitol does not have such a marked effect because its lesser reducing potential is not sufficient to reduce the sulfhydryl groups in the destabilized S D S protein complex. It is of interest to note that neither detection method (densitometry or radioactivity scanning) gave evidence of the formation
154
MORELL, WIGGINS AND GRAY
of oligomers of molecular weight of 46,000 or 92,000 which might be derived from the 23,000 MW proteolipid protein. Whatever the theoretical basis, the practical implication of our observation to the study of myelin proteins is clear. The effect observed may not be restricted to proteolipid protein since some other proteins of myelin behave similarly. This may be a general result applying to a certain class of membrane proteins, and we therefore urge caution when utilizing the boiling procedure to disaggregate membrane protein for electrophoresis. Recently a publication describing some unusual observations relating to the SDS-polyacrylamide gel electrophoresis of whole brain protein under different conditions of application of ME has appeared (13). In that study all samples were boiled for 10 min prior to electrophoresis; and control experiments to study this variable were not done. The somewhat unusual results of these authors might, in part, be explained by our observations.
ACKNOWLEDGMENTS We thank Dr. Eugene Day for bringing to our attention the thesis work of Mary Elin Macdonald (14) where data demonstrating changes in the electrophoresis pattern of myelin proteins at different ME concentrations (all prepared by boiling) are presented. This focused our attention on the need for further investigation of this problem. This research was supported by U.S. Public Health Service Grants NS-11615 and HD-03110.
REFERENCES 1. Shapiro, A. L., Vinuela, E., and Maizel, J. V. (1967) Biochem. Biophys. Res. Commun. 28, 815-820. 2. Maizel, J. V., Jr. (1971) Meth. Virol. 5, 179-246. 3. Greenfield, S., Norton, W. T., and Morell, P. (1971)J. Neurochem. 18, 2119-2128. 4. Morell, P., Greenfield, S., Costantino-Ceccarini, E., and Wisniewski, H. (1972). J. Neurochem. 19, 2545-2554. 5. Norton, W. T., and Poduslo, S. E. (1973) J. Neurochem. 21, 749-757. 6. Benjamins, J. A., Jones, M., and Morell, P. (1975)J. Neurochem., in press. 7. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 8. Martenson, R. E., Deibler, G. E., and Kies, M. W. (1970) J. Neurochem. 17, 1329-1330. 9. Agrawal, H. C., Burton, R. M., Fishman, M. A., Mitchell, R. F., and Prensky, A. L. (I 972) J. Neurochem. 19, 2083-2089. 10. Folch, J. and Lees, M. (1951)J. Biol. Chem. 191, 807-817. 11. Wolfgram, F. (1966)J. Neurochem. 13, 461-470. 12. A'utilio, L. A., Norton, W. T., and Terry, R. D. (1964) J. Neurochem. 11, 17-27. 13. Richter-Landsberg, C., Ruchel, R., and Waehneldt, T. V. (1974) Biochem. Biophys. Res. Commun. 59, 781-788. 14. Macdonald, M~ E. (1973) Ph.D. dissertation, Duke University.
Polyacrylamide Gel Electrophoresis of Myelin Proteins: A Caution PIERRE MORELL, RICHARD C . WIGGINS, AND MAP-dORY JONES GRAY
Biological Sciences Research Center and Department of Biochemistry and Nutrition, Division of Health Affairs, University of North Carolina, Chapel Hill, North Carolina 27515 Received December 23, 1974; accepted April 16, 1975 Some variables involved in the preparation of rat brain myelin proteins for polyacrylamide gel electrophoresis in buffers containing sodium dodecyl sulfate were studied. Under mild conditions of solubilization the resultant gel patterns
were relatively insensitive to the /~-mercaptoethanol (ME) concentration in the protein solvent used for solubilization of myelin proteins. However, if the samples were boiled in the presence of ME (a standard procedure for disruption of metastable aggregates of membrane proteins), a major myelin protein, proteolipid protein, as well as some minor proteins were preferentially excluded from the gel. This effect was proportional to the ME concentration. Characterization of the polypeptide composition of m e m b r a n e proteins b y polyacrylamide gel electrophoresis in buffers containing sodium dodecyl sulfate (SDS) has been a standard procedure since the introduction of this method (1). When using such a system, it is c u s t o m a r y to solubilize the protein sample in a protein solvent consisting of a buffer containing S D S a n d / ~ - m e r c a p t o e t h a n o l (ME) to reduce disulfide bonds, and to bring the sample to a boil to disrupt metastable aggregates (2). W e have utilized a discontinuous version of such an electrophoretic s y s t e m (2) to separate myelin proteins (3,4). In our studies we encountered problems with this boiling procedure and r e c o m m e n d e d instead initially dissolving the myelin samples in 1% S D S , placing t h e m in an ultrasonic cleaning bath for an extended period of time to affect solubilization, and only then adding M E (3). We have since quantitatively investigated the effects of boiling myelin samples in the presence or absence of M E and report our results in this communication.
METHODS
Preparation of Myelin Myelin f r o m 26-day-old L o n g - E v a n s rats was prepared essentially b y the procedure of N o r t o n and Poduslo (5), with some minor modifications (6). Myelin was lyophilized and aliquots of 10-20 mg were par148 Copyright~) 1975by AcademicPress, Inc. All rightsof reproductionin any formreserved.
M Y E L I N P R O T E I N ELECTROPHORESIS
149
tially delipidated by 5 extractions with 1 ml of ether-ethanol (3:2, v/v)/mg dry wt and dried under a stream of nitrogen (3), In some experiments, animals were injected intracranially with 200 /xCi of [4,53H]leucine (40.7 Ci/mmole) 24 hr prior to sacrifice.
Polyacrylamide Gel Electrophoresis The method for electrophoresis of myelin protein on discontinuous polyacrylamide gels has been described previously (3). Formation of gels of such a high acrylamide concentration (15% running gel, 3% spacer gel) requires special care to avoid formation of bubbles and other flaws in the gel, and some further practical details relating to the procedure are detailed below. The gel columns were cut from 8 mm o.d.-6 mm i.d. glass tubing (either Pyrex or flint glass) in 15.5 cm lengths and were treated with acid-dichromate cleaning solution, washed with detergent, rinsed with distilled water, and coated with Siliclad solution (Clay Adams, Parsippany, N J) before use. One end of the column was blocked by inserting the tube into the cavity of a solid 00 rubber safety stopper (Arthur H. Thomas, Philadelphia, PA). Gels were formed in the reverse of the usual order; initially sucrose (30% w/v) was added to a height of 2.5 cm to reserve space for the sample. A long tip Pasteur pipet was used to avoid getting sucrose on the interior surface of the cylinder. A 2 cm spacer gel was layered on top of the sucrose and overlaid with 0.1% SDS. After this had firmly polymerized (30-60 rain), the SDS solution was removed with a long tip Pasteur pipet and the column filled with running gel solution. The running gel solution must be deaerated for at least 45 min (we use house vacuum). The solution is then chilled on ice to slow polymerization which is initiated by addition of ammonium persulfate just prior to pouring the gel. After polymerization (about 30 rain) the gel tubes were topped off with 0.1% SDS, covered with Parafilm (American Can Company, Neenah, WI) to prevent the gel surface from caking, and then allowed to stand from 12 to 24 hr at room temperature before use. The gel columns could also be stored for several days in a plastic bag in the refrigerator. The sucrose was removed before use; then the columns were inverted and loaded in a Buchler Polyanalyst gel electrophoresis apparatus (with a spacer added between reservoirs to accomodate the long tubes). Air bubbles trapped beneath the running gel were removed with a bent Pasteur pipet. Myelin protein, 50-200/xg, in 0.1-0.4 ml of protein solvent (see below) was applied. Running buffer was equilibrated between the lower and upper reservoirs using an oscillating pump and an overflow tube and a constant 60 V was applied for 16 hr. The marker dye leaves the gel after 12 hr. Gels were removed by breaking the glass tubes,
150
MORELL, WIGGINS AND GRAY
fixed, stained with Fast Green and destained as described previously (3). The gels were scanned at 570 nm using a Beckman model 25 Spectrophotometer equipped with a linear transport mechanism and the areas under peaks corresponding to individual proteins quantitated (4).
Radioactivity Determination For determination of radioactivity distribution in various proteins, stained gels were frozen in an ultra low temperature freezer or on a dry ice block and firmly pressed into a razor blade stack (Hoeffer Scientific Instruments, San Francisco, Calif.) moistened with water. Each gel slice was transferred to a glass scintillation vial containing 0.3 ml of 30% hydrogen peroxide and tightly capped with a plastic lined screw cap. Following incubation for 1-2 days at 50°C, excess hydrogen peroxide was dissipated by addition of 4500 units of catalase (the C10 grade of Sigma). After standing for 15 min, 10 ml of toluene-Triton X-100 (Rohm and Haas, Philadelphia, Pa.) (4 : 1, v/v) containing 21 g/gal of Omnifluor (New England Nuclear, Boston, Mass.) was added. Vials were left loosely capped for at least 2 hr to allow for further release of oxygen, prior to being firmly tightened.
Sample Preparation Delipidated myelin was prepared for electrophoresis by suspending it in 1% SDS so that the final protein concentration was between 1 and 2 mg/ml (an approximation of the protein concentration was made from the initial dry weight of myelin assuming that it contained 28% protein). Solubilization of the protein was aided by placing the tube in an ultrasonic cleaning bath (Heat System, Ultrasonic Inc., Planview, NY) for a period of up to several hours. The temperature of the water rose to about 60°C during this time. Ten microliter aliquots were taken for protein determination (7). Our routine procedure for analysis involved diluting 3 vol of this protein solution with 1 vol of concentrated protein solvent consisting of: 4.0 ml of 0.5 M Tris-HCl at pH 6.8 [solution C(3)], 1.0 ml of 10% SDS, 0.4 ml of ME, 4.0 ml of glycerol, 0.4 ml of 0.1% bromophenol blue, and 0.2 ml of water. A volume containing about 150 /~g of protein was layered onto the spacer gel and electrophoresed. If the electrophoresis was not done on the day of sample preparation, samples were stored (either refrigerated for a few days, or frozen for up to a number of weeks), and subjected to a brief treatment in the ultrasonic cleaning bath before electrophoresis. In the present series of experiments the procedure was modified by omitting ME from the protein solvent so that the concentration of this reagent could be adjusted in individual aliquots prior to electrophoresis.
MYELIN PROTEIN ELECTROPHORESIS
15 1
FIG. 1. Photographs of p0!yacrylanfide gels following electrophoresis of 150 ~g o f myelin proteins. Gels a-f are samples Where the protein was solubilized in 1% SDS followed b y addition of ME and ~other components of ~the protein solvent. Immediately prior to electrophoresis the ME concentration was (a):0%; (b) 0.:5%; (c) 1%; (d) 2%; (e') 5% ; (f) t 0 % . G e l s a ' - f ' are duplicates ,of the:above except that following addition of ME and other components of the ,protein solvent the samples were placed in a boiling water bath for 2 rain prior to dectrophoresls.
RESULTS Figure 1 illustrates the gel electrophoresis patterns of myelin proteins in the presence of varyirrg concentrations of ME, using a protocol (see Methods) that does not involve boiling :the protein samples. Proteins are identified in order of increasing molecular weight: the two basic :proteins of rat brain myelin (8), Be and Bs; the double :band identified :as intermediate protein (4,9), I; the proteolipid protein (I0), P; the major high molecular weight protein (l 1), (W) which is just barely 'resolved as two bands in this gel system; and several other high molecular weight proteins including one identified o-n Fig. ~1by an arrow. It is clear from the first set o f gels that although some M E Ks necessary ~to give adequate resOlution of proteins (note the srrreafing o f some :of the protein 'bands in gel
152
MORELL, WIGGINS AND GRAY
a) the actual ME concentration is not very critical. In the gel without ME (gel a) and in the gel containing 10% ME (gel f), the protein bands appeared to have a higher mobility than the other gels. This phenomenon was not quantitated, since no reference point for the dye front exists (the dye is electrophoresed off the end of the gel in this system). A minor band, directly above W and just barely separated from it, appears at the higher ME concentrations. In the second set of gels, Fig. l a ' - f ' , which is identical to the first set of gels except that the samples (in small screw capped tubes) were placed in a boiling water bath for 2 min prior to electrophoresis, marked variation is observable. With increasing ME concentration, proteolipid protein is preferentially excluded from the gel and piles up at the stacking gel-running gel interface. The slower moving intermediate protein and, even more markedly, a high molecular weight protein (see arrow) are also affected by boiling at the higher ME concentration. Quantitative analysis of densitometric scans of two such complete sets of gels indicated that, under conditions of boiling with reducing reagent, 35% o f the proteolipid protein was excluded from the gel at a 1% ME concentration, and over 90% of the proteolipid protein was excluded when the ME concentration was 5%. The amount of material in the basic protein band remained constant. It was also observed that the mobility of proteolipid protein (relative to the basic proteins) was slightly decreased at increasing ME concentrations. This could be interpreted on the basis of some conformational effect of increasing ME concentration on the remaining, unaggregated proteolipid protein. However, we feel it is more likely a loading phenomenon due to the decreased amount of protein at this molecular weight entering the gel. A similar series of experiments, substituting dithiothreitol for ME, at concentrations ranging from 0. I to 5%, indicated that boiling myelin proteins for 2 min in the presence of dithiothreitol did not cause marked preferential exclusion of proteolipid protein from the gel. However, some other observations (J. Benjamins, personal communication) suggest to us that harsher treatment, such as repeated boiling for longer periods of time, in the presence of dithiothreitol will cause partial exclusion of proteolipid protein from the gel. Also, the resolution of protein was not as good, and fewer discrete bands were seen, in the presence of dithiothreitol reagent. It was considered possible that the apparent exclusion of proteolipid proteins observed in Fig. 1 might be artifactual in that the boiling in the presence of ME might somehow alter the protein so that it would no longer bind Fast Green. To control for this possibility an experiment similar to that in Fig. 1 but using radioactivity instead of densitometry to
MYELIN PROTEIN ELECTROPHORESIS
153
quantitate protein distribution was carried out (6). These studies gave results exactly analogous to those reported in Fig. 1. The presence of ME during the boiling period caused loss of radioactivity in the proteolipid protein region of the gel which was quantitatively accounted for by radioactivity at the gel origin. DISCUSSION
A standard procedure for separation of membrane proteins in SDSgels involves heating the protein sample to 100°C prior to electrophoresis in order to disrupt possible metastable aggregates (2). It is generally assumed that subjecting the sample to such vigorous denaturing conditions can only promote the desired results of reduction of sulfhydryl groups by the reducing reagent, as well as insuring uniform binding of SDS to the resultant subunits. This assumption may lead to operational difficulties in the study of certain membrane proteins. One important brain membrane protein, proteolipid protein, can be prepared in large amounts and its properties extensively studied (10). Most prominent of the peculiar properties of this protein is preferential solubility in organic solvents (10). Aqueous solutions of proteolipid protein can be obtained by stepwise dialysis from organic solvents into solutions of increasing polarity, or by the use of detergents. It is possible that under these conditions the protein is somehow not completely denatured. If the protein is subjected to more severe denaturing conditions such as repeated flash evaporation from chloroform-methanol (2:1, v/v) containing 5% (v/v) water (12) it forms a precipitate which can not be resolubilized either in organic solvent or in water even in the presence of detergents (unpublished observation in our laboratory and personal communication from Dr. W. T. Norton). It is possible that boiling in the presence of ME is sufficient to completely denature proteolipid protein which then precipitates out of the detergent containing solution and remains at the gel interface during electrophoresis. This "complete" denaturation might correspond to the dissociation of residual lipid. Another hypothesis is also based on the premise that at a lower temperature there exists an SDS-protein complex in which the protein retains some structure. At 100°C this SDS-protein complex became destabilized (sulfhydryl groups became accessible for reduction by mercaptoethanol), leading to the complete unfolding of the polypeptide chains and aggregation. Presumably, under identical conditions, dithiothreitol does not have such a marked effect because its lesser reducing potential is not sufficient to reduce the sulfhydryl groups in the destabilized S D S protein complex. It is of interest to note that neither detection method (densitometry or radioactivity scanning) gave evidence of the formation
154
MORELL, WIGGINS AND GRAY
of oligomers of molecular weight of 46,000 or 92,000 which might be derived from the 23,000 MW proteolipid protein. Whatever the theoretical basis, the practical implication of our observation to the study of myelin proteins is clear. The effect observed may not be restricted to proteolipid protein since some other proteins of myelin behave similarly. This may be a general result applying to a certain class of membrane proteins, and we therefore urge caution when utilizing the boiling procedure to disaggregate membrane protein for electrophoresis. Recently a publication describing some unusual observations relating to the SDS-polyacrylamide gel electrophoresis of whole brain protein under different conditions of application of ME has appeared (13). In that study all samples were boiled for 10 min prior to electrophoresis; and control experiments to study this variable were not done. The somewhat unusual results of these authors might, in part, be explained by our observations.
ACKNOWLEDGMENTS We thank Dr. Eugene Day for bringing to our attention the thesis work of Mary Elin Macdonald (14) where data demonstrating changes in the electrophoresis pattern of myelin proteins at different ME concentrations (all prepared by boiling) are presented. This focused our attention on the need for further investigation of this problem. This research was supported by U.S. Public Health Service Grants NS-11615 and HD-03110.
REFERENCES 1. Shapiro, A. L., Vinuela, E., and Maizel, J. V. (1967) Biochem. Biophys. Res. Commun. 28, 815-820. 2. Maizel, J. V., Jr. (1971) Meth. Virol. 5, 179-246. 3. Greenfield, S., Norton, W. T., and Morell, P. (1971)J. Neurochem. 18, 2119-2128. 4. Morell, P., Greenfield, S., Costantino-Ceccarini, E., and Wisniewski, H. (1972). J. Neurochem. 19, 2545-2554. 5. Norton, W. T., and Poduslo, S. E. (1973) J. Neurochem. 21, 749-757. 6. Benjamins, J. A., Jones, M., and Morell, P. (1975)J. Neurochem., in press. 7. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 8. Martenson, R. E., Deibler, G. E., and Kies, M. W. (1970) J. Neurochem. 17, 1329-1330. 9. Agrawal, H. C., Burton, R. M., Fishman, M. A., Mitchell, R. F., and Prensky, A. L. (I 972) J. Neurochem. 19, 2083-2089. 10. Folch, J. and Lees, M. (1951)J. Biol. Chem. 191, 807-817. 11. Wolfgram, F. (1966)J. Neurochem. 13, 461-470. 12. A'utilio, L. A., Norton, W. T., and Terry, R. D. (1964) J. Neurochem. 11, 17-27. 13. Richter-Landsberg, C., Ruchel, R., and Waehneldt, T. V. (1974) Biochem. Biophys. Res. Commun. 59, 781-788. 14. Macdonald, M~ E. (1973) Ph.D. dissertation, Duke University.
