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Electrophoresis of membrane proteins in concentrated formic acid solutions
ANALYTICAL
BIOCHEMISTRY
Electrophoresis
88,263-270
(1978)
of Membrane Proteins Formic Acid Solutions LEWIS
in Concentrated
C. MOKRASCH
Department of Biochemistry, Louisiana State University Medical Center, New Orleans, Louisiana 70112 Received April 30, 1977; accepted February 10, 1978 To obviate the difficulties resulting from partial solubility of membrane proteins in detergents or from the use of noxious solvent mixtures containing phenol or chloral, a simple procedure was devised for acrylamide gel electrophoresis of membrane proteins in 13 M formic acid. Polyacrylamide gels are equilibrated in 13 M formic acid and used in the electrophoresis assembly with 13 M formic acid as the electrolyte. Particulate proteinaceous preparations are dissolved in trifluoroacetic acid or in 24 M formic acid containing glycine to increase the density and to facilitate the solubilization of the protein. Protein samples (10 to 100 Kg) migrate as polycations.
Acrylamide gel zone electrophoresis of proteins insoluble in aqueous solutions presents special problems: Chemicals with higher proteinsolvent power may be used as the electrolyte, or protein derivatives with enhanced solubility can be formed for use with ordinary aqueous electrolytes. The latter procedure invariably involves extra manipulations and the possibility of irregular degrees of modification and side reactions. Sodium dodecyl sulfate, sometimes with reducing agents and urea (l-4) has been useful in solubilizing proteins, including those of membrane origins, and a useful correlation between migration distance and molecular weight exists, but solubilization is often incomplete. The combination of dilute acetic acid as the electrolyte plus solubilization by urea has proved useful for the separation of myelin proteins (5). By replacing some or all of the water of the electrolyte by more potent protein solvents such as a phenol-acetic acid mixture (6) phenolformic acid mixtures (7), or chloral (8), separations of water-insoluble proteins can be achieved. The classical techniques of (water-soluble) protein chemistry are nearly useless for separating some membrane proteins. Some separation schemes for myelin proteins involving solvent extraction and nonaqueous chromatographic procedures were devised (9- 11). Although it is possible to obtain stable aqueous solutions of some of the membrane 263
0003-2697/78/0881-0263$02.00/O Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.
264
LEWIS
C. MOKRASCH
proteins (proteolipid apoprotein) (1 l), their physical properties suggested that a high degree of aggregation exists which makes chromatographic homogeneity tests uncertain. Similarly, when electrophoresis in sodium dodecylsulfate was applied to these proteins for myelin, aggregation persisted (12) and proof of homogeneity remained elusive. Exposure of proteolipid proteins to a pH greater than 8 causes denaturation (13,14), giving a product whose solubility properties approximate those of keratin. During studies on properties of proteolipids (15-21) and on the assay of proteolipids (22-24), concentrated formic acid was found to be a useful solvent, even for denatured proteolipid. An effort was made to exploit this solvent for an electrophoretic system which would be simple and would circumvent some of the shortcomings of other electrophoretic systems. MATERIALS
Coomassie blue, acrylamide, N,N’-methylene bisacrylamide and N,N,N’,N’-tetramethylethylenediamine were of electrophoresis grade. All other reagents were analytical reagent grade or the equivalent. The Hoeffer Scientific Industries model EF301 electrophoresis unit was used with power supplies able to provide at least 200 V and 150 mA. The scanning photometer used in these studies was a Photovolt model 425 unit, modified with a motor-driven carriage and l-mm collimating slits, near the light source and over the photocell. The No. 62 filter was used for Coomassie blue stains. The output from the photocell was connected to the recorder from a Beckman Model DB spectrophotometer. The formic acid electrolyte is easily recovered by distillation; the fraction boiling between 104 and 106°C has a formic acid concentration of 12.7 to 13.5 M. METHODS
The composition of the acrylamide gels is given in Table 1. After the casting tubes (5.7 mm, i.d.) are filled with the monomer mixtures the top surface of the mixture is overlaid with butanol. Polymerization is allowed to proceed overnight. The detergent is present to facilitate the release of the gel from the casting tubes. No special treatment of the glass tubes is required, except to avoid strongly alkaline detergents which may etch the tubes and make the gel release difficult. After polymerization the gels may be extruded into 13 M formic acid for equilibration and storage. Because of swelling which takes place during the soaking in 13 M formic acid, the gels are more easily reloaded into electrophoresis tubes which are 0.1 to 0.2 mm larger in diameter than
ELECTROPHORESIS TABLE
IN FORMIC ACID
265
1
GEL COMPOSITION
“4.5%” gel
“9%” gel
Gel component
45 g/liter 2.3 g/liter 6mM 6 g/liter 0.13 g/liter 0.5 g/liter
90 g/liter 4.5 g/liter IO rnM IO giliter 0.16 g/liter 0.5 g/liter
Acrylamide NJ’-methylene bisacrylamide Tris-sulfate, pH 7.4 Triton X-100 Tetramethylethylene diamine WLMd&
the tubes in which the gels are cast. A drop of glycerol in the empty electrophoresis tubes facilitates the entry of the gels. An alternative method of equilibration is to mount the tubes of gels in the electrophoretic assembly and to equilibrate with the electrolyte by applying a potential gradient of 20 V cm-’ for 3 hr before applying the sample. Protein mixtures which are soluble in water at a concentration greater than 1% are prepared for loading onto the gels simply by adding 1 mg of tartaric acid to 1 ml of sample, to increase the density of the aqueous solution. Membrane preparations, lipid-rich preparations, and dilute protein samples are prepared for electrophoresis as follows. The solution or suspension is diluted with 10 vol of isobutanol. After centrifugation for 10 min at 2OOOg, the delipidated proteinaceous pellet is dried in vucuo over CaCl,. A solution of glycine in 98% formic acid (0.6 mg of glycineiml of formic acid) is added to the dried residue to give a protein concentration near 10 mg ml-‘. Ordinarily, heating is not necessary to effect solution, although most membrane preparations from mouse brain show no change in electrophoretic pattern after heating at 100°C in the glycineformic acid mixture for periods of less than 5 min. Another protein solvent which has been used successfully in this procedure is trifluoroacetic acid. The solutions of protein in trifluoroacetic acid, however, have a very low viscosity and tend to run out of the pipet during the sample loading operation, whereas the glycine-formic acid mixture is moderately viscous and easy to manipulate. To improve the visibility of the protein sample, methylene blue is added to a final concentration of 0.01%. Following this addition, however, the samples must be kept dark to avoid photolysis of the protein (25). The sample size is arbitrary, but 0.001 ml of a tartaric acid-saturated serum (Fig. 1A) and 0.010 ml of nerve ending preparations in glycineformic acid (Figs. 1B and 1C) with a protein concentration of approximately 10 mg ml-’ give satisfactory patterns after staining.
266
LEWIS C. MOKRASCH
The duration of the migration can be planned either by using cytochrome c or myoglobin as the visible proteins or by referring to the movement of the methylene blue added with the sample. Satisfactory patterns of membrane proteins have been obtained with migration periods three times as long as is required for the methylene blue to clear from the gel. Power settings depend partly upon the efficiency of the cooling. Satisfactory patterns have been obtained at 14 W per tube, but better patterns are obtained at 2 to 5 W per tube or less. The gels (5.7 x 100 mm) have an apparent resistance close to 13,000 R. A potential gradient of 20 V/cm results in a current of 16 mA per tube. After completion of the migration, the gels are extruded into a solution of 0.1% Coomassie blue in 0.3 M trichloroacetic acid. The stained gels are destained in acetic acid. RESULTS
In the early stages of development of this electrophoretic procedure, a comparison of the solvent power of about 30 commonly used or novel protein solvents was made; some of these are listed in Table 2. A test mixture of water-insoluble proteins from a mouse brain crude mitochondrial preparation (26,27) was delipidated, as described above. The protein residue was then finely dispersed in water and assayed for protein (22). TABLE SOLUBILITY
OF WASHED
MEMBRANE
2 PROTEINS
IN SOLVENT
MIXTURES
Solvent Distilled water 1% Sodium dodecylsulfate 1% Sodium dodecylsulfate, heated for 5 min at 100°C 1% Sodium dodecylsulfate- 1% 2-thioethanol 1% Sodium dodecylsulfate- 1% 2-thioethanol heated for 5 min at 100°C 1: 1 (v/v) Phenol and formic acid 1: 1 (v/v) Chloral and formic acid Trifluoroacetic acid Formic acid 1% 2-Thioethanol in formic acid Dichloroacetic acid 5:3 (w/w) Formic acid and glycine 96% Sulfuric acid
Percentage stainable protein dissolved 0
19 74 35 81 35 32 71 47 47 65 93 96
ELECTROPHORESIS
BRAIN
IN FORMIC ACID
267
SPINAL CORD
FIG. 1. (A) Serum, 0.001 ml, saturated with tartaric acid and applied to a 9% gel. (B) Washed membrane fraction from mouse brain crude mitochondrial fraction. One-hundredth milliliter of a solution having about 10 mg ml-’ protein dissolved in 5:3 (w/v) 98% formic acid:glycine was applied to a 9% gel. (C) Washed membrane fraction of mouse spinal cord crude mitochondrial fraction. The concentrations and conditions are the same as those given for (B). The molecular weight marks refer to the positions attained by reference solutions of cytochrome c, myogiobin, ovalbumin, serum albumin, and serum y-globulins, in order of decreasing mobility.
Aliquots of the suspensions containing 0.280 mg of protein were sedimented for 20 min at 25,OOOg. The water was drained off and replaced by 1.O ml of the test solvent. After mixing for 30 min at room temperature, the samples were filtered through fine porosity sintered glass and washed successively with 5-ml portions of the same solvent, 1 M acetic acid, 0.1% Coomassie blue in 0.3 M trichloroacetic acid, 1 M acetic acid, and 88% formic acid. The formic acid quantitatively desorbed the dye from the protein on the filter, and the color of the solution was measured at 620 nm.
268
LEWISCMOKRASCH
The electrophoretic profiles of most of the membranous subfractions of mouse brain and spinal cord were examined after centrifugal separation and preparation, as described under Methods. Generally, each fraction has its own characteristic profile, and subfractions resulting from hypoosmotic lysis show protein bands which can be correlated with bands in the parent fraction. A comparison of the “B” fraction from density gradient separations of mouse brain and spinal cord crude mitochondrial fractions is presented in Figs. 1B and 1C. The “B” fraction consists principally of nerve endings and sediments to a position just above the mitochondrial fraction (26). Superimposed on the profiles are the molecular weights of water-soluble “marker” proteins which migrate to the positions indicated under the same conditions of time, potential gradient, and temperature as for the electrophoresis of the “B” fractions. For purposes of comparison a human serum sample saturated with tartaric acid developed the pattern shown in Fig. 1A. Although there appear to be eight major bands in this scan of a 9% gel, it should be noted that the resolution of the gel exceeds the resolution of the photometer. The second and third bands from the origin (anodic surface) are doublets, and the fourth band is a triplet. DISCUSSION
Some of the nonaqueous protein solvents, phenol-acetic acid, phenolformic acid, chloral, and concentrated (22 M) formic acid are powerful vesicants. Chloral vapors may cause lung damage. Formic acid, 13 M, has at most a mild irritant action comparable to 2 M HCl and a moderately unpleasant odor which is only evident in a completely open system and at close range. This concentration of formic acid, which is approximately 50% (w/w), is not corrosive to polymethacrylate or to rubber. At higher concentrations of formic acid, an all-glass assembly, such as that of Eng et al. (29), would be recommended. The simplicity of the preparation of the electrolyte, a dilution of a commercially available reagent, makes the procedure convenient, and the recovery of the electrolyte by distillation makes it economical. Presumably the effect of glycine in enhancing the solubility of protein is similar to that of urea or guanidinium chloride in aqueous systems. In general, all of the variations of gel porosity, additional electrolytes, and protein-modifying reagents are available in the electrophoretic system described here. What has been presented is intended to be the simplest gel cylinder electrophoretic system employing an electrolyte of unusual solvent power. Adding salts to vary the ionic strength, stronger acids to increase the acidity, and sodium formate to lower the acidity of the electrolyte offer no worthwhile improvement in separations of membrane proteins. Simi-
ELECTROPHORESIS
IN FORMIC ACID
269
larly, no effect of thiols or detergents in improving separations has been observed. It is the hope that the system described herein will provide an alternate or a supplement to the much-used sodium dodecylsulfate systems of gel electrophoresis, which are not suitable for all protein mixtures. The difference in the shape and charge of the migrating proteins and in the mechanism of solution between the two systems ought to add resolution whenever both can be used. In the detergent systems, the proteins probably migrate as a micelle with a low axial ratio; in the formic acid system, coulombic repulsion probably changes the configuration of the proteins to an extended chain form with a high axial ratio (30). In addition, applying the gel concentration optimization procedure of Rodbard et al. (31) or using discontinuous gel gradients such as that of Wright et al. (32), who were able to detect about 60 serum components, would probably further improve the resolution of this system. ACKNOWLEDGMENTS The author wishes to express gratitude to Mr. Nicholas Lanson for his technical assistance and to Mr. Nicholas Nicosia for his skillful modifications of the scanning photometer. This work was supported in part by a grant from the Schlieder Foundation.
REFERENCES 1, Shapiro, A. L., Vinuela, E., and Maizel. J. V. (1967) B&hem. Biophys. Res. Commun. 28, 815-820. 2. Neville, D. M., (1971) J. Viol. Chem. 246, 6328-6334. 3. Dunker, A. K., and Ruekert, R. R. (1969) J. Biol. C/rem. 244, 5074-80. 4. Swank, R. T., and Munkres, K. D., (1971) Anal. Biochem. 39, 462-477. 5. Sammeck, R., Martenson, R. G., and Brady, R. 0. (197l)Brain Res. 34, 241-251. 6. Work, T. S., (1964) J. Mol. Biol. 10, 544-545. 7. Mehl, E., and Wolfgram, F. (1969) J. Neurochem. 16, 1091-1097. 8. Ballou, B., Sundharadas, G., and Bach, M. L. (1974) Science 185, 531-533. 9. Mokrasch, L. C. (1967) Fed. Proc. 26, 847. 10. Mokrasch, L. C. (1967) Life Sci. 6, 1905-1909. 11. Mokrasch, L. C. (1972) Prep. Biochem. 2, 1-19. 12. Chan, D. S., and Lees, M. B. (1974) Biochemistry 13, 2704-2711. 13. Webster, G. R., and Folch-Pi, J. (1961) Biochem. Biophys. Acta 49, 399-401. 14. Pritchard, E. T., and Folch-Pi, J. (1963) Biochem. Biophys. Acta 70, 481-484. 15. Mokrasch, L. C., and Manner, P. (1962) Biochem. Biophys. Acta 60, 415-417. 16. Mokrasch, L. C., (1965) Biochem. Biophys. Acta 107, 608-610. 17. Mokrasch, L. C., and Andelman, R. J. (1968) J. Neurochem. 15. 1207-1216. 18. Mokrasch, L. C. (1962) Acta Neurol. Stand. 38, (Suppl. l), 15. 19. Mokrasch, L. C., and Manner, P. (1963)J. Neurochem. 10, 541-547. 20. Mokrasch, L. C. (1966) /. Neurochem. 13, 49-58. 21. Tolani, A. J., and Mokrasch, L. C. (1967) Life Sci. 6, 1771-1774. 22. Mokrasch, L. C. (1967) Anal. Biochem. 18, 64-71. 23. Mokrasch, L. C. (1970) Anal. Biochem. 36, 273-277. 24. Mokrasch, L. C. (1974) in Methods in Enzymology (Lowenstein, J. M., ed.), Vol. 35, Part B, pp. 335-339, Academic Press, New York.
270
LEWIS C. MOKRASCH
25. Means, G. E. and Feeney, R. G. (1971) Chemical Modification of Proteins, pp. 165-169, Holden-Day, San Francisco, Calif. 26. Mokrasch, L. C. (1971) in Methods in Neurochemistry (Fried, R., ed.). Vol. 1, pp. l-29. Dekker, New York. 27. Cotman, C. W. (1974) in Methods in Enzymology (Fleischer, S., and Packer L., eds.), Vol. 31, Part A, pp. 445-452, Academic Press, New York. 28. Dounce. A. L., Witter, R. F., Monty, J. J., Pate, S., and Cottone, M. A. (1959)5. Biophys. Biochem. Cytol. 1, 139-153. 29. Eng, L. F., Bond, P., and Gerstl, B. (1971) Neurobiology 1, 58-63. 30. Thorun, W., and Mehl, E. (1965) Biochem. Biophys. Acta 160, 132-134. 31. Rodbard, D., Chambach, A., and Weiss, G. (1974) in Electrophoresis, Electrophoretic Focusing in Polyacrylamide Gel (Allen, R. C., and Maurer, H. R., eds.), pp. 62-l 13, deGruyter, Berlin. 32. Wright, G. L., Farrell, K. B., and Roberts, V. B. (1971) Clin. Chim. Acra 32, 285-296.
BIOCHEMISTRY
Electrophoresis
88,263-270
(1978)
of Membrane Proteins Formic Acid Solutions LEWIS
in Concentrated
C. MOKRASCH
Department of Biochemistry, Louisiana State University Medical Center, New Orleans, Louisiana 70112 Received April 30, 1977; accepted February 10, 1978 To obviate the difficulties resulting from partial solubility of membrane proteins in detergents or from the use of noxious solvent mixtures containing phenol or chloral, a simple procedure was devised for acrylamide gel electrophoresis of membrane proteins in 13 M formic acid. Polyacrylamide gels are equilibrated in 13 M formic acid and used in the electrophoresis assembly with 13 M formic acid as the electrolyte. Particulate proteinaceous preparations are dissolved in trifluoroacetic acid or in 24 M formic acid containing glycine to increase the density and to facilitate the solubilization of the protein. Protein samples (10 to 100 Kg) migrate as polycations.
Acrylamide gel zone electrophoresis of proteins insoluble in aqueous solutions presents special problems: Chemicals with higher proteinsolvent power may be used as the electrolyte, or protein derivatives with enhanced solubility can be formed for use with ordinary aqueous electrolytes. The latter procedure invariably involves extra manipulations and the possibility of irregular degrees of modification and side reactions. Sodium dodecyl sulfate, sometimes with reducing agents and urea (l-4) has been useful in solubilizing proteins, including those of membrane origins, and a useful correlation between migration distance and molecular weight exists, but solubilization is often incomplete. The combination of dilute acetic acid as the electrolyte plus solubilization by urea has proved useful for the separation of myelin proteins (5). By replacing some or all of the water of the electrolyte by more potent protein solvents such as a phenol-acetic acid mixture (6) phenolformic acid mixtures (7), or chloral (8), separations of water-insoluble proteins can be achieved. The classical techniques of (water-soluble) protein chemistry are nearly useless for separating some membrane proteins. Some separation schemes for myelin proteins involving solvent extraction and nonaqueous chromatographic procedures were devised (9- 11). Although it is possible to obtain stable aqueous solutions of some of the membrane 263
0003-2697/78/0881-0263$02.00/O Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.
264
LEWIS
C. MOKRASCH
proteins (proteolipid apoprotein) (1 l), their physical properties suggested that a high degree of aggregation exists which makes chromatographic homogeneity tests uncertain. Similarly, when electrophoresis in sodium dodecylsulfate was applied to these proteins for myelin, aggregation persisted (12) and proof of homogeneity remained elusive. Exposure of proteolipid proteins to a pH greater than 8 causes denaturation (13,14), giving a product whose solubility properties approximate those of keratin. During studies on properties of proteolipids (15-21) and on the assay of proteolipids (22-24), concentrated formic acid was found to be a useful solvent, even for denatured proteolipid. An effort was made to exploit this solvent for an electrophoretic system which would be simple and would circumvent some of the shortcomings of other electrophoretic systems. MATERIALS
Coomassie blue, acrylamide, N,N’-methylene bisacrylamide and N,N,N’,N’-tetramethylethylenediamine were of electrophoresis grade. All other reagents were analytical reagent grade or the equivalent. The Hoeffer Scientific Industries model EF301 electrophoresis unit was used with power supplies able to provide at least 200 V and 150 mA. The scanning photometer used in these studies was a Photovolt model 425 unit, modified with a motor-driven carriage and l-mm collimating slits, near the light source and over the photocell. The No. 62 filter was used for Coomassie blue stains. The output from the photocell was connected to the recorder from a Beckman Model DB spectrophotometer. The formic acid electrolyte is easily recovered by distillation; the fraction boiling between 104 and 106°C has a formic acid concentration of 12.7 to 13.5 M. METHODS
The composition of the acrylamide gels is given in Table 1. After the casting tubes (5.7 mm, i.d.) are filled with the monomer mixtures the top surface of the mixture is overlaid with butanol. Polymerization is allowed to proceed overnight. The detergent is present to facilitate the release of the gel from the casting tubes. No special treatment of the glass tubes is required, except to avoid strongly alkaline detergents which may etch the tubes and make the gel release difficult. After polymerization the gels may be extruded into 13 M formic acid for equilibration and storage. Because of swelling which takes place during the soaking in 13 M formic acid, the gels are more easily reloaded into electrophoresis tubes which are 0.1 to 0.2 mm larger in diameter than
ELECTROPHORESIS TABLE
IN FORMIC ACID
265
1
GEL COMPOSITION
“4.5%” gel
“9%” gel
Gel component
45 g/liter 2.3 g/liter 6mM 6 g/liter 0.13 g/liter 0.5 g/liter
90 g/liter 4.5 g/liter IO rnM IO giliter 0.16 g/liter 0.5 g/liter
Acrylamide NJ’-methylene bisacrylamide Tris-sulfate, pH 7.4 Triton X-100 Tetramethylethylene diamine WLMd&
the tubes in which the gels are cast. A drop of glycerol in the empty electrophoresis tubes facilitates the entry of the gels. An alternative method of equilibration is to mount the tubes of gels in the electrophoretic assembly and to equilibrate with the electrolyte by applying a potential gradient of 20 V cm-’ for 3 hr before applying the sample. Protein mixtures which are soluble in water at a concentration greater than 1% are prepared for loading onto the gels simply by adding 1 mg of tartaric acid to 1 ml of sample, to increase the density of the aqueous solution. Membrane preparations, lipid-rich preparations, and dilute protein samples are prepared for electrophoresis as follows. The solution or suspension is diluted with 10 vol of isobutanol. After centrifugation for 10 min at 2OOOg, the delipidated proteinaceous pellet is dried in vucuo over CaCl,. A solution of glycine in 98% formic acid (0.6 mg of glycineiml of formic acid) is added to the dried residue to give a protein concentration near 10 mg ml-‘. Ordinarily, heating is not necessary to effect solution, although most membrane preparations from mouse brain show no change in electrophoretic pattern after heating at 100°C in the glycineformic acid mixture for periods of less than 5 min. Another protein solvent which has been used successfully in this procedure is trifluoroacetic acid. The solutions of protein in trifluoroacetic acid, however, have a very low viscosity and tend to run out of the pipet during the sample loading operation, whereas the glycine-formic acid mixture is moderately viscous and easy to manipulate. To improve the visibility of the protein sample, methylene blue is added to a final concentration of 0.01%. Following this addition, however, the samples must be kept dark to avoid photolysis of the protein (25). The sample size is arbitrary, but 0.001 ml of a tartaric acid-saturated serum (Fig. 1A) and 0.010 ml of nerve ending preparations in glycineformic acid (Figs. 1B and 1C) with a protein concentration of approximately 10 mg ml-’ give satisfactory patterns after staining.
266
LEWIS C. MOKRASCH
The duration of the migration can be planned either by using cytochrome c or myoglobin as the visible proteins or by referring to the movement of the methylene blue added with the sample. Satisfactory patterns of membrane proteins have been obtained with migration periods three times as long as is required for the methylene blue to clear from the gel. Power settings depend partly upon the efficiency of the cooling. Satisfactory patterns have been obtained at 14 W per tube, but better patterns are obtained at 2 to 5 W per tube or less. The gels (5.7 x 100 mm) have an apparent resistance close to 13,000 R. A potential gradient of 20 V/cm results in a current of 16 mA per tube. After completion of the migration, the gels are extruded into a solution of 0.1% Coomassie blue in 0.3 M trichloroacetic acid. The stained gels are destained in acetic acid. RESULTS
In the early stages of development of this electrophoretic procedure, a comparison of the solvent power of about 30 commonly used or novel protein solvents was made; some of these are listed in Table 2. A test mixture of water-insoluble proteins from a mouse brain crude mitochondrial preparation (26,27) was delipidated, as described above. The protein residue was then finely dispersed in water and assayed for protein (22). TABLE SOLUBILITY
OF WASHED
MEMBRANE
2 PROTEINS
IN SOLVENT
MIXTURES
Solvent Distilled water 1% Sodium dodecylsulfate 1% Sodium dodecylsulfate, heated for 5 min at 100°C 1% Sodium dodecylsulfate- 1% 2-thioethanol 1% Sodium dodecylsulfate- 1% 2-thioethanol heated for 5 min at 100°C 1: 1 (v/v) Phenol and formic acid 1: 1 (v/v) Chloral and formic acid Trifluoroacetic acid Formic acid 1% 2-Thioethanol in formic acid Dichloroacetic acid 5:3 (w/w) Formic acid and glycine 96% Sulfuric acid
Percentage stainable protein dissolved 0
19 74 35 81 35 32 71 47 47 65 93 96
ELECTROPHORESIS
BRAIN
IN FORMIC ACID
267
SPINAL CORD
FIG. 1. (A) Serum, 0.001 ml, saturated with tartaric acid and applied to a 9% gel. (B) Washed membrane fraction from mouse brain crude mitochondrial fraction. One-hundredth milliliter of a solution having about 10 mg ml-’ protein dissolved in 5:3 (w/v) 98% formic acid:glycine was applied to a 9% gel. (C) Washed membrane fraction of mouse spinal cord crude mitochondrial fraction. The concentrations and conditions are the same as those given for (B). The molecular weight marks refer to the positions attained by reference solutions of cytochrome c, myogiobin, ovalbumin, serum albumin, and serum y-globulins, in order of decreasing mobility.
Aliquots of the suspensions containing 0.280 mg of protein were sedimented for 20 min at 25,OOOg. The water was drained off and replaced by 1.O ml of the test solvent. After mixing for 30 min at room temperature, the samples were filtered through fine porosity sintered glass and washed successively with 5-ml portions of the same solvent, 1 M acetic acid, 0.1% Coomassie blue in 0.3 M trichloroacetic acid, 1 M acetic acid, and 88% formic acid. The formic acid quantitatively desorbed the dye from the protein on the filter, and the color of the solution was measured at 620 nm.
268
LEWISCMOKRASCH
The electrophoretic profiles of most of the membranous subfractions of mouse brain and spinal cord were examined after centrifugal separation and preparation, as described under Methods. Generally, each fraction has its own characteristic profile, and subfractions resulting from hypoosmotic lysis show protein bands which can be correlated with bands in the parent fraction. A comparison of the “B” fraction from density gradient separations of mouse brain and spinal cord crude mitochondrial fractions is presented in Figs. 1B and 1C. The “B” fraction consists principally of nerve endings and sediments to a position just above the mitochondrial fraction (26). Superimposed on the profiles are the molecular weights of water-soluble “marker” proteins which migrate to the positions indicated under the same conditions of time, potential gradient, and temperature as for the electrophoresis of the “B” fractions. For purposes of comparison a human serum sample saturated with tartaric acid developed the pattern shown in Fig. 1A. Although there appear to be eight major bands in this scan of a 9% gel, it should be noted that the resolution of the gel exceeds the resolution of the photometer. The second and third bands from the origin (anodic surface) are doublets, and the fourth band is a triplet. DISCUSSION
Some of the nonaqueous protein solvents, phenol-acetic acid, phenolformic acid, chloral, and concentrated (22 M) formic acid are powerful vesicants. Chloral vapors may cause lung damage. Formic acid, 13 M, has at most a mild irritant action comparable to 2 M HCl and a moderately unpleasant odor which is only evident in a completely open system and at close range. This concentration of formic acid, which is approximately 50% (w/w), is not corrosive to polymethacrylate or to rubber. At higher concentrations of formic acid, an all-glass assembly, such as that of Eng et al. (29), would be recommended. The simplicity of the preparation of the electrolyte, a dilution of a commercially available reagent, makes the procedure convenient, and the recovery of the electrolyte by distillation makes it economical. Presumably the effect of glycine in enhancing the solubility of protein is similar to that of urea or guanidinium chloride in aqueous systems. In general, all of the variations of gel porosity, additional electrolytes, and protein-modifying reagents are available in the electrophoretic system described here. What has been presented is intended to be the simplest gel cylinder electrophoretic system employing an electrolyte of unusual solvent power. Adding salts to vary the ionic strength, stronger acids to increase the acidity, and sodium formate to lower the acidity of the electrolyte offer no worthwhile improvement in separations of membrane proteins. Simi-
ELECTROPHORESIS
IN FORMIC ACID
269
larly, no effect of thiols or detergents in improving separations has been observed. It is the hope that the system described herein will provide an alternate or a supplement to the much-used sodium dodecylsulfate systems of gel electrophoresis, which are not suitable for all protein mixtures. The difference in the shape and charge of the migrating proteins and in the mechanism of solution between the two systems ought to add resolution whenever both can be used. In the detergent systems, the proteins probably migrate as a micelle with a low axial ratio; in the formic acid system, coulombic repulsion probably changes the configuration of the proteins to an extended chain form with a high axial ratio (30). In addition, applying the gel concentration optimization procedure of Rodbard et al. (31) or using discontinuous gel gradients such as that of Wright et al. (32), who were able to detect about 60 serum components, would probably further improve the resolution of this system. ACKNOWLEDGMENTS The author wishes to express gratitude to Mr. Nicholas Lanson for his technical assistance and to Mr. Nicholas Nicosia for his skillful modifications of the scanning photometer. This work was supported in part by a grant from the Schlieder Foundation.
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