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Chapter 15 Electrophoretic Fractionation of Histones Utilizing Starch Gels and Sodium Dodecyl Sulfate—Urea Gels
Part C.
Fractionation and Characterization of Histones. I I
Chapter
15
Electrophoretic Fractionation of Histones Utdieng Sturch Gels und Sodizlm Dodecyl Szllfate-Urea Gels LUBOMIR S. HNILICA, SIDNEY R. GRIMES, AND JEN-FU CHIU Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee
I. Introduction Among the techniques for protein separation and fractionation, electrophoresis in polyacrylamide gels and, to a lesser extent, in starch gels have contributed enormously to the rapid advancement of histone biochemistry and biology. Histone separation in polyacrylamide gels is clearly superior to any other method for their qualitative and quantitative analysis, and without doubt it should be standard procedure in every laboratory studying these proteins. While polyacrylamide gels containing urea in addition to the employed buffers produce the best separation of the individual histone fractions, the somewhat inferior systems containing either polyacrylamide gels with sodium dodecyl sulfate (SDS) or starch gels with urea can be useful under special circumstances.
11. Starch Gel Electrophoresis The starch gel electrophoresis was developed by Smithies ( 1 ) who showed the exceptional resolution power of this new method in his studies on pro21 1
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teins of human plasma and serum. The starch gel electrophoresis was first applied to the histones by Neelin and Neelin (2) who resolved calf thymus histone preparations into 16-22 protein bands. Since the number and intensity of several of the calf thymus histone bands varied with changing conditions of the electrophoresis (PH of the buffer, starch concentration, presence or absence of urea, etc.), it was concluded that some of the electrophoretic bands must be aggregates of some of the histone fractions and that there are probably only a few histone species. This conclusion was supported by the experiments of Johns et al. (3) who used a simplified horizontal version of starch gel electrophoresis. With 10 mM HCl as the only electrolyte, these authors separated calf thymus histones into 5 4 reproducible bands, all of which corresponded to the major histone fractions obtained by chemical fractionation procedures. Although starch gel electrophoresis of histones is now almost completely abandoned to a much more elegant, rapid, and sensitive analysis in acrylamide polymers, it is still the method of choice for metabolic studies on histones in higher animals, where low specific activities of the fractions exclude their accurate radioactivity analysis by polyacrylamide gels. It is possible to accommodate and resolve 1-2 mg of whole histone mixture in one starch gel slot as compared with 25-50 pg tolerated by each standard polyacrylamide gel. The method described here (4) was adapted from the horizontal system of Johns et al. (3). Hydrolyzed starch (Connaught Medical Research Laboratories, Toronto, Canada), 12% suspension in 10 mM HC1-0.2 mM AlCl,, is heated to 85-90" and the hot viscous mixture is poured into a plastic mold permitting the formation of at least eight strips, each measuring 15 x 6 x 250 mm. The strips are formed by septa separating each slot longitudinally. These individual troughs communicate at both ends through a horizontal cut across all the septa dividing the individual slots. Two rectangular pieces of Whatman 3 MM filter paper (double thickness) measuring approximately 120 x 180 mm are inserted into the cross cuts on both ends of the mold to serve as connectors (bridges) to electrode vessels (beakers). They are held in place by solidified starch gel. After cooling to room temperature, the elevated gel surface is removed by pulling thin molybdenum wire (3 mil) across the gel using the surface of the mold for guidance. The upper starch layer is discarded and histone samples, dissolved in 0.1 M HCl, are applied soaked into rectangular pieces (5 x 14 mm) of Whatman No. 17filter paper by inserting the rectangles into narrow cuts in the individual gels, about 3-4 cm from the anodic end. To prevent evaporation, the gel surface and filter paper bridges (wicks) connecting the beakers with electrodes are covered with a sheet of plastic (Saran Wrap). The electrode vessels are two 1000-ml beakers containing about 900 ml of 10 mM HCl and 0.2 or 0.6 mM
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FIG. 1. Frame for slicing central portions out of starch gels for quantitative evaluation. The frame is made from Plexiglas, and the individual layers serve as leading edges for the cutting wire which is drawn between them.
AICl,. The higher concentration of AICI, increases the separation of H2A and H4 histones. The separation is obtained at 40 mA of constant current for 22 hours (5 mA per slot) at room temperature. After electrophoresis, the filter-paper bridges are removed and the mold is placed in a refrigerator for about 30 minutes to facilitate transfer of the individual gel slabs, Approximately 16-cm-long pieces of gels are lifted from their troughs with a spatula and transferred into individual test tubes containing 0.1% Amido black in a mixture of methanol-water-acetic acid (552, v/v/v). After staining for about 18 hours (gentle rocking accelerates the staining), the gels are partially destained in the methanol-water-acetic acid solution without the dye approximately 2 hours. The partially destained gels are transferred into a cutting frame (Fig. l), where approximately 1-mm-thick portions are sliced off the top and bottom of each gel using a thin molybdenum wire (3 mil). The central gel portions measuring 3 x 15 x 150 mm are placed into test tubes containing methanol-water-acetic acid mixture and destained under gentle rocking motion. The destaining solution is changed 2-3 times until the gels show dark blue-stained histone bands against an almost white or pale blue background (Fig. 2). For quantitative evaluation, the individual protein bands as well as a piece of gel without any protein (ahead of the fastest H4 fraction) are cut from the gels with a razor blade. The cut pieces of gels are placed into individual test tubes and dried with acetone (three changes, each 4-6 hours), in a stream of dry air, and finally in an oven at 105' for 3-4 hours. The dry pieces are weighed and each is dissolved in 7 ml of concentrated formic acid by heating in a boiling water bath for 20 minutes. After rapid cooling in ice water, 7 ml of 1 M NaOH are added and the mixture is mixed vigorously.
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FIG. 2. Starch gel electrophoregram of calf thymus histone fractions. The electrolyte was 0.2 m M AICl, in 10 mM HCl. WH = unfractionated histone; 3 = H3 (oxidized dimer); 1 = H1; 2B = H2B; and 2A = mixture of H2A and H4.
The absorbancy determined at 630 nm is corrected for the background: where A,,,
=
corrected absorbance, A, and Ab are absorbances of the
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g 0.150
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FIG.3. The absorbancy plot of four major calf thymus histone fractions after staining with Amido black 10 B. (0) H1; (0)H2A and H4; ( A ) H2B; and (D) H3.
sample and blank gel, respectively, and W,and Wb are dry weights of the sample and blank, respectively. The absorbance remains linear for the amount of histone between 0.05 and 1.0 mg of protein in each band. The amount of dye bound to the protein in starch gel is independent from the composition of histone fractions and from the concentration of AlC1, (Fig. 3). However, since occasional batches of Amido black show differences in their binding to the H1 histones, it is recommended that each new batch of this dye be calibrated with isolated histone fractions. The radioactivity of histones labeled with [14C]- or [3H]amino acids or acetate is determined by combusting the dried gel pieces in oxygen (5-7). The method of Kalberer and Rutschmann (5)can be followed with excellent results. Dried gels are wrapped into small strips of Whatman No. 1 filter paper, placed into platinum baskets, and combusted in special flasks tilled with oxygen under atmospheric pressure. The radioactive products of combustion are trapped in a mixture of monoethanolamine in methyl cellosolve (1 :8, v/v), aliquots are mixed with a toluene-based scintillation fluid, and their radioactivity is determined in a liquid scintillation spectrometer. A combustion apparatus made by Packard Co. can be used with great convenience to process large numbers of samples in a single day. Because of the yellowish coloring of the samples, construction of quench curves to measure the extent of quenching in the individual samples is recommended.
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Usually one sample of histone is electrophoresed in at least eight slots (troughs). One-half (four gels) is used for quantitative determination of protein, and the other half (four gels) is combusted to determine the radioactivity. All values are averaged, yielding quite accurate figures of specific activities for each histone band. Histone samples with specific activities of about 100 cpm/mg or more can be analyzed with confidence. It is preferable to use chemically prefractionated histone samples for electropheresis instead of whole extracts. In a typical determination, chromatin or nuclei are first washed with 80% aqueous ethanol. The arginine-rich histones are extracted with absolute ethanol: 1.25 M HCl(4: 1, v/v) followed by extraction of the lysine-rich histones with 0.25 M HCl(8). Histones recovered from extracts either by dialysis and lyophilization or by precipitation with a 10to 15-fold excess of cold acetone are electrophoresed separately to obtain a better resolution. A modification of histone electrophoresis in starch gels was described by Sung and Smithies (9). This modification employs aluminum lactate buffer and 4 M urea for separation of histones similar to the method described here in detail.
111. Histone Electrophoresis in Polyacrylamide Gels
Containing SDS
As was already mentioned, the best method for rapid quantitative and qualitative separation of all histone fractions as well as their phosphorylated or acetylated derivatives is electrophoresis in polyacrylamide gels, which was first developed by Ornstein (2O)and Davis (11). While the application of SDS to this technique results in some loss of resolution as compared to the now classical method of Panyim and Chalkley (12),the SDS gels are valuable when the mixtures of histones and nonhistone proteins are to be analyzed. Extensive aggregation and insolubility of chromosomal nonhistone proteins in most conventional electrophoresis buffers led to the introduction of polyacylamide gel electrophoresis performed in the presence of detergents, most frequently SDS (23). Because of their interactions with this detergent, most proteins form negatively charged complexes which separate electrophoretically in polyacrylamide gels in order of their molecular weights (13,14). Indeed, the electrophoretic mobility of proteins larger than 10,000 daltons can be used for fairly accurate molecular weight determination (13,25). SDS dissociates most proteins into their subunits (unless covalently
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linked) and, consequently, the number of proteins bands observed in polyacrylamide gel electrophoresis performed in the presence of this detergent corresponds to the number of subunits and may be much larger than the true number of the in vivo protein species. On the other hand, polypeptide chains of similar or identical molecular weights will form a single protein band. Since histones are simple proteins, their electrophoretic homogeneity is not affected by exposure to SDS. However, their molecular weights just about approach the lowest range of confidence for molecular weight determinations of the SDS-protein complexes (15).The excessive electropositive charge of the carboxyterminal half of the H1 histones which results from a large accumulation of lysyl residues disturbs thedistribution of thenegatively charged SDS micelles along the H1 molecule. This disturbance is reflected in an anomalous electrophoretic mobility of H1 histones in SDS-containing gels. Consequently, the apparent molecular weight of about 34,000 calculated for HI histones from their mobilities in SDS gels differs considerably from their true molecular weights of 19,500-22,000 (average) as determined physicochemically (16). These disturbances are even greater when SDS interacts with more basic proteins of male gametes. Such complexes (e.g., of protamines or arginine-rich proteins from mammalian spermatids and spermatozoa) may become partially insoluble in electrophoretic buffers, and their electrophoretic separation in the presence of anionic detergents cannot be accomplished. The electrophoresis in SDS-containing gels can be performed both with or without urea. Since SDS eliminates virutally all the protein-protein interactions, the presence of urea is not as beneficial as in the detergent-free polyacylamide gel systems. Elimination of the positive charges on histones by SDS makes impossible the electrophoretic separation of differentially acetylated or phosphorylated histone fractions. A reasonably good separation of histones, even in the presence of chromosomal nonhistone proteins, can be obtained by a modified procedure of Maize1 and his associates (13). Polyacrylamide gels (10% polyacrylamide containing 4 M urea) can be prepared by mixing the following stock solutions: A Gel buffer: 0.2 M phosphate buffer-0.2% SDS-8 M urea @H 7.0). B. Acrylamide solution: 24% acylamide (recrystallized commercial or electrophoretic grade) and 0.6% Bis (electrophoretic grade N,N/-methylenebisacylamide) in deionized water. C. Ammonium persulfate, 90 mg in 10 ml of deionized water (must be made fresh before use). D. TEMED (N,N,N:N:-tetramethylethylenediamine), undiluted reagent. Thesolutions aremixedin theratioA:B:C:D =6:5:1:0.06.For 12-14gels
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(10 x 0.6 cm each), 30 ml of A are mixed with 25 ml of B and 5 ml of C, and the mixture is cooled on ice under reduced pressure for 15 minutes. Finally, 0.03 ml of TEMED (solution D) are added and the gels are poured immediately into stoppered glass columns to the desired height. Chilling the monomer mixture delays the polymerization and allows the time necessary for the manipulation of the individual columns. Disposable plastic syringe or Pasteur pipettes can be used conveniently to transfer the monomer mixture. The filled columns are freed of any entrapped air bubbles and carefullly overlaid each with 0.1 ml of solution A diluted 1:1with deionized water. The gels are polymerized for at least 2 hours at room temperature. In the presence of a large excess of nonhistone proteins or DNA, a 3% polyacrylamide stacking gel should be layered over the 10%gel. To prepare the stacking gel, a modified solution B containing 7.5% acrylamide and 1.2% Bis in deionized water is used instead of the regular solution. The columns are made of fire-polished 0.6-cm-bore glass tubing, cut to the desired height, washed in chromic solution, rinsed well with water, and coated with silicone (the silicone coating is not important in SDS-containing gels because these can be removed from the columns with relative ease). To prepare for electrophoresis, the tubes are closed on one end with rubber caps and placed into a polymerization rack which can be a small test tube or a special stand made of Plexiglas or other material. Samples containing 10-30 pg of unfractionated histone are dissolved in solution A, which has been made more dense by the addition of concentrated sucrose or urea, and a measured amount of the solution is carefully layered on top of each gel. The sample volume should not exceed 50 p1 to assure good resolution of the histone fractions. After filling the columns with electrolyte buffer, the rubber caps are gently removed from the columns. The columns are inserted into the upper tray of an electrophoretic apparatus (some commercial instruments do not have a separable upper and lower tray), which is then filled with electrolyte buffer (solutionA), and thesamples are electrophoresed at 8-10 mA per column for 8-10 hours. Application of a tracking dye (Bromophenol blue) to at least one of the samples helps to monitor the migration. After electrophoresis, the gels are stained in the solution of Coomassie blue or Amido black (0.2% or OS%, respectively) in methanol+icetic acid-water mixture 5 :1: 5 , or they can be first fixed in 20% aqueous sulfosalicylic acid or 15% trichloroacetic acid. This treatment also removes most of the detergent which may interfere with subsequent staining. Some batches of Coomassie blue are incompatible with trichloroacetic acid, and it may be necessary first to remove the acid by placing the fixed gels into 20% sulfosalicylic acid solution for several hours. A mixture of methanolacetic acid-water ( 5 :1:5 ) can be also used to remove the 15% trichloroacetic acid from the gels before their staining with Coomassie blue. The gels can be
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1 2 3 4 5 6 I
H1 1
H3mono+H2B
/
"** / H4
FIG. 4. Polyacrylamide gel electrophoresis in the presence of SDS of total rat thymus histones and calf thymus histone fractions. 1 = total rat thymus histone; 2 and 3 = H1 histones from rat thymus and liver, respectively; 4 = calf thymus H2B; 5 = calf thymus H2A; and 6 = calf thymus H4. The origin of migration is at the top of the gels.
destained by diffusion in an excess of methanol-acetic acid-water mixture or, much faster, by electrophoresis in 7% acetic acid. In the presence of SDS,the following histone fractions, can be resolved in order of their increasing mobilities: oxidized H3 dimer, H1, a mixture of H3 monomer and the H2B fraction, the H2A, and finally the H4 bands (Fig. 4). In addition to electrophoresis in polyacrylamide gels contained in glass tubing, electrophoresis in relatively thin gel slabs (1-5 mm) is rapidly gaining popularity. Besides the better resolution of the slab-gel electrophoresis, the uniformly thin sheets of polyacrylamide can be easily dehydrated and used for autoradiography or scanning. The stained gel can also be impregnated with 2,5-diphenyloxazolesolution and dried for fluorography (17). A good electrophoretic separation of all common histone fractions can be obtained by adaptation of the discontinuous method of Laemmli (18)
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FIG.5. Polyacrylamide gel electrophoresisin the presence of SDS in slab gels. This photograph illustrates the good resolution of all histones from chicken reticulocyte chromatin. The histone fractions are identified by their symbols. The origin of migration is at the top of the gel. (Courtesy of Dr. A. Beyer and Dr. K. Hardy.)
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22 1
using a gel of 12% or 15% polyacrylamide (Fig. 5). The gel can be formed from the following solutions: A. Acrylamide solution: 30% acrylamide and 0.8% Bis (NN-methylenebisacrylamide) in deionized water. B. 1.5 M Tris-HC1, pH 8.8 (ultrapure). C. 10% SDS. D. 0.5 M Tris-HC1, pH 6.8. E. 1.5% ammonium persulfate (made fresh). F. TEMED (N,N,N:N'-tetramethylethylenediamine), undiluted reagent. In order to prepare 50 ml of solution for a 12% polyacrylamide gel, 20 ml of solution A, 12.5 ml of B, and 16 ml of water are mixed and kept on ice under reduced pressure for 15 minutes. Finally 0.5 ml of C, 1.0 ml of E, and 12 pl of F are added. A 10-ml pipette can be used to introduce 30 ml of the mixed solution into a 14 x 15.5 cm vertical gel chamber which is formed of two glass plates separated by plastic spacers. Two spacers for the sides are 1 x 19 cm and the bottom spacer is 1 x 17 cm. The gel is overlayered with 0.1% SDS until polymerization is complete. A spacer gel of 2 or 3% polyacrylamide is prepared by mixing 1.O ml of solution A, 2.5 ml of D, 0.1 ml of C, 0.7 ml of E, 5 p1 of F, and 6.3 ml of deionized water. The overlayer solution is removed and the gel surface is rinsed with the spacer gel solution. The upper gel is poured with a 10-ml pipette, and a plastic template is inserted to form sample wells 0.7 x 2.0 cm. The slab gel can be used for a preparative separation of proteins by omitting the template so that a single sample can be applied to the entire gel width. After polymerization, the template and the bottom spacer are removed, and buffer solution [0.2 M glycine-0.025 M Tris-HC1 (pH 8.3)-0.1% SDS] is added to the upper and lower reservoirs. Proteins (usually dissolved in water) are mixed with an equal volume of sample mix (2.5 ml of solution D, 4.0 ml of C, 2.0 ml of glycerol, 1.O ml of 2-mercaptoethanol, 0.2 ml of 0.05% Bromophenol blue, and 0.3 ml of water) and heated in boiling water for 1to 2 minutes or kept at 37O for 1 hour. Samples are centrifuged at lOOOg to remove any insoluble materials. Samples containing 10-50 pg of unfractionated histone in a volume less than 100p1areplacedin to eachsamplewell. Thesamples are electrophoresed at 20 mA until the tracking dye reaches the bottom of the gel (about 6 hours). The gel is removed and stained overnight in 0.1% Coomassie blue in 25% isopropanol and 10% acetic acid as described by Fairbanks et al. (29).The gel is destained for 6 to 9 hours in 0.03% Coomassie blue in 10%isopropanol and 10% acetic acid followed by several changes in 10% acetic acid. The stained gel can be impregnated with 2,5-diphenyloxazole and dried for
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fluorography (17) or dehydrated onto cellophane sheets and cut for scanning or liquid scintillation counting. The slab gels can also be adapted for preparative elution electrophoresis by forming a channel near the lower edge of the gel for a continuous flow of buffer to carry off proteins separated by electrophoresis. A lower gel is poured up to the elution part in the 3-mm plastic side spacers. A narrow band of glycerol is added to the surface of the polymerized lower gel, and the separating gel is layered carefully over the glycerol.A template forming one large well is inserted into the upper gel during polymerization. When electrophoresis is started the glycerol is forced out of the elution port as the sweeping buffer flow is started. This method allows isolation of relatively pure protein fractions for amino acid analysis or other chemical determinations. Various commercial apparatuses permitting the analytical as well as preparative slab-gel electrophoresis can be obtained from several suppliers. Exceptionally good results were obtained with an apparatus acquired from Hoefer Scientific Instruments, San Francisco, California. The slab-gel method described here was performed with this apparatus. ACKNOWLEDGMENTS This work was supported by U.S.Public Health Service Grant CA 18389 and by NCI contract NO 1 -CP-65730. REFERENCES
1. Smithies, 0. Biochem. J. 61,629 (1955). 2. Neelin, J. M., and Neelin, E. M., Can. J. Biochem. Physiol. 38, 355 (1960). 3. Johns, E. W., Phillips, D. M. P., Simson, P., and Butler, J. A. V., Biochem. J. 80,189 (1 961). 4. Hnilica, L. S., Edwards, L. J., and Hey, A. H., Biochim. hiophys. Actu 124, 109 (1966). 5. Kalberer, F., and Rutschmann, J., Helv. Chim. Actu 44, 1956 (1961). 6. Dobbs, H. E., Anal. Chem. 35,783 (1963). 7. Baggiolini, M.,Experientia 21, 731 (1965). 8. Johns, E. W., Phillips, D. M. P., Simson, P., and Butler, J. A. V., Biochem. J. 77,631 (1960). 9. Sung, M.,and Smithies, O., Biopolymers 7 , 39 (1969). 10. Ornstein, L., Ann. N. Y. A d . Sci. 121,321 (1964). 11. Davis, B. J., Ann. N. Y.Acad. Sci. 121,404(1964). 12. Panyim. S., and Chalkley, R., Arch Biochem. Biophys. 130,337 (1969). 13. Shapiro, A. L., Vifluela, E., and Maizel, J. V., Biochem. Biophys. Res. Commun. 28,815 (1967). 14. Dunker, A. K.,and Rueckert, R. R., J. Biol. Chem. 244, 5074 (1969). 15. Williams, J. G., and Gratzer, W. B., J. Chromutogr. 57, 321 (1971). 16. Hnilica, L. S., “The Structure and Biological Functions of Histones” Chem. Rubber Publ. Co.,Cleveland, Ohio, 1972. 17. Bonner, W. M., and Laskey, R. A., Eur. J. Biochem. 46,83 (1974). 18. Laemmli, U.K., Nature (London) 227, 680 (1970). 19. Fairbanks, G., Steck, T. L., and Wallach, D. F. M., Biochemistry 10,2606 (1971).
Fractionation and Characterization of Histones. I I
Chapter
15
Electrophoretic Fractionation of Histones Utdieng Sturch Gels und Sodizlm Dodecyl Szllfate-Urea Gels LUBOMIR S. HNILICA, SIDNEY R. GRIMES, AND JEN-FU CHIU Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee
I. Introduction Among the techniques for protein separation and fractionation, electrophoresis in polyacrylamide gels and, to a lesser extent, in starch gels have contributed enormously to the rapid advancement of histone biochemistry and biology. Histone separation in polyacrylamide gels is clearly superior to any other method for their qualitative and quantitative analysis, and without doubt it should be standard procedure in every laboratory studying these proteins. While polyacrylamide gels containing urea in addition to the employed buffers produce the best separation of the individual histone fractions, the somewhat inferior systems containing either polyacrylamide gels with sodium dodecyl sulfate (SDS) or starch gels with urea can be useful under special circumstances.
11. Starch Gel Electrophoresis The starch gel electrophoresis was developed by Smithies ( 1 ) who showed the exceptional resolution power of this new method in his studies on pro21 1
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teins of human plasma and serum. The starch gel electrophoresis was first applied to the histones by Neelin and Neelin (2) who resolved calf thymus histone preparations into 16-22 protein bands. Since the number and intensity of several of the calf thymus histone bands varied with changing conditions of the electrophoresis (PH of the buffer, starch concentration, presence or absence of urea, etc.), it was concluded that some of the electrophoretic bands must be aggregates of some of the histone fractions and that there are probably only a few histone species. This conclusion was supported by the experiments of Johns et al. (3) who used a simplified horizontal version of starch gel electrophoresis. With 10 mM HCl as the only electrolyte, these authors separated calf thymus histones into 5 4 reproducible bands, all of which corresponded to the major histone fractions obtained by chemical fractionation procedures. Although starch gel electrophoresis of histones is now almost completely abandoned to a much more elegant, rapid, and sensitive analysis in acrylamide polymers, it is still the method of choice for metabolic studies on histones in higher animals, where low specific activities of the fractions exclude their accurate radioactivity analysis by polyacrylamide gels. It is possible to accommodate and resolve 1-2 mg of whole histone mixture in one starch gel slot as compared with 25-50 pg tolerated by each standard polyacrylamide gel. The method described here (4) was adapted from the horizontal system of Johns et al. (3). Hydrolyzed starch (Connaught Medical Research Laboratories, Toronto, Canada), 12% suspension in 10 mM HC1-0.2 mM AlCl,, is heated to 85-90" and the hot viscous mixture is poured into a plastic mold permitting the formation of at least eight strips, each measuring 15 x 6 x 250 mm. The strips are formed by septa separating each slot longitudinally. These individual troughs communicate at both ends through a horizontal cut across all the septa dividing the individual slots. Two rectangular pieces of Whatman 3 MM filter paper (double thickness) measuring approximately 120 x 180 mm are inserted into the cross cuts on both ends of the mold to serve as connectors (bridges) to electrode vessels (beakers). They are held in place by solidified starch gel. After cooling to room temperature, the elevated gel surface is removed by pulling thin molybdenum wire (3 mil) across the gel using the surface of the mold for guidance. The upper starch layer is discarded and histone samples, dissolved in 0.1 M HCl, are applied soaked into rectangular pieces (5 x 14 mm) of Whatman No. 17filter paper by inserting the rectangles into narrow cuts in the individual gels, about 3-4 cm from the anodic end. To prevent evaporation, the gel surface and filter paper bridges (wicks) connecting the beakers with electrodes are covered with a sheet of plastic (Saran Wrap). The electrode vessels are two 1000-ml beakers containing about 900 ml of 10 mM HCl and 0.2 or 0.6 mM
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FIG. 1. Frame for slicing central portions out of starch gels for quantitative evaluation. The frame is made from Plexiglas, and the individual layers serve as leading edges for the cutting wire which is drawn between them.
AICl,. The higher concentration of AICI, increases the separation of H2A and H4 histones. The separation is obtained at 40 mA of constant current for 22 hours (5 mA per slot) at room temperature. After electrophoresis, the filter-paper bridges are removed and the mold is placed in a refrigerator for about 30 minutes to facilitate transfer of the individual gel slabs, Approximately 16-cm-long pieces of gels are lifted from their troughs with a spatula and transferred into individual test tubes containing 0.1% Amido black in a mixture of methanol-water-acetic acid (552, v/v/v). After staining for about 18 hours (gentle rocking accelerates the staining), the gels are partially destained in the methanol-water-acetic acid solution without the dye approximately 2 hours. The partially destained gels are transferred into a cutting frame (Fig. l), where approximately 1-mm-thick portions are sliced off the top and bottom of each gel using a thin molybdenum wire (3 mil). The central gel portions measuring 3 x 15 x 150 mm are placed into test tubes containing methanol-water-acetic acid mixture and destained under gentle rocking motion. The destaining solution is changed 2-3 times until the gels show dark blue-stained histone bands against an almost white or pale blue background (Fig. 2). For quantitative evaluation, the individual protein bands as well as a piece of gel without any protein (ahead of the fastest H4 fraction) are cut from the gels with a razor blade. The cut pieces of gels are placed into individual test tubes and dried with acetone (three changes, each 4-6 hours), in a stream of dry air, and finally in an oven at 105' for 3-4 hours. The dry pieces are weighed and each is dissolved in 7 ml of concentrated formic acid by heating in a boiling water bath for 20 minutes. After rapid cooling in ice water, 7 ml of 1 M NaOH are added and the mixture is mixed vigorously.
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FIG. 2. Starch gel electrophoregram of calf thymus histone fractions. The electrolyte was 0.2 m M AICl, in 10 mM HCl. WH = unfractionated histone; 3 = H3 (oxidized dimer); 1 = H1; 2B = H2B; and 2A = mixture of H2A and H4.
The absorbancy determined at 630 nm is corrected for the background: where A,,,
=
corrected absorbance, A, and Ab are absorbances of the
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1.000
:
c.
v "
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D
D
T-
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g 0.150
0.150
0,501
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0 150
0.m
0.1
0.4
mg
1.6 I1 PlOTElN
0.1
0.2
1.1
mg
0.6
0.0
11 PnOTElN
FIG.3. The absorbancy plot of four major calf thymus histone fractions after staining with Amido black 10 B. (0) H1; (0)H2A and H4; ( A ) H2B; and (D) H3.
sample and blank gel, respectively, and W,and Wb are dry weights of the sample and blank, respectively. The absorbance remains linear for the amount of histone between 0.05 and 1.0 mg of protein in each band. The amount of dye bound to the protein in starch gel is independent from the composition of histone fractions and from the concentration of AlC1, (Fig. 3). However, since occasional batches of Amido black show differences in their binding to the H1 histones, it is recommended that each new batch of this dye be calibrated with isolated histone fractions. The radioactivity of histones labeled with [14C]- or [3H]amino acids or acetate is determined by combusting the dried gel pieces in oxygen (5-7). The method of Kalberer and Rutschmann (5)can be followed with excellent results. Dried gels are wrapped into small strips of Whatman No. 1 filter paper, placed into platinum baskets, and combusted in special flasks tilled with oxygen under atmospheric pressure. The radioactive products of combustion are trapped in a mixture of monoethanolamine in methyl cellosolve (1 :8, v/v), aliquots are mixed with a toluene-based scintillation fluid, and their radioactivity is determined in a liquid scintillation spectrometer. A combustion apparatus made by Packard Co. can be used with great convenience to process large numbers of samples in a single day. Because of the yellowish coloring of the samples, construction of quench curves to measure the extent of quenching in the individual samples is recommended.
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Usually one sample of histone is electrophoresed in at least eight slots (troughs). One-half (four gels) is used for quantitative determination of protein, and the other half (four gels) is combusted to determine the radioactivity. All values are averaged, yielding quite accurate figures of specific activities for each histone band. Histone samples with specific activities of about 100 cpm/mg or more can be analyzed with confidence. It is preferable to use chemically prefractionated histone samples for electropheresis instead of whole extracts. In a typical determination, chromatin or nuclei are first washed with 80% aqueous ethanol. The arginine-rich histones are extracted with absolute ethanol: 1.25 M HCl(4: 1, v/v) followed by extraction of the lysine-rich histones with 0.25 M HCl(8). Histones recovered from extracts either by dialysis and lyophilization or by precipitation with a 10to 15-fold excess of cold acetone are electrophoresed separately to obtain a better resolution. A modification of histone electrophoresis in starch gels was described by Sung and Smithies (9). This modification employs aluminum lactate buffer and 4 M urea for separation of histones similar to the method described here in detail.
111. Histone Electrophoresis in Polyacrylamide Gels
Containing SDS
As was already mentioned, the best method for rapid quantitative and qualitative separation of all histone fractions as well as their phosphorylated or acetylated derivatives is electrophoresis in polyacrylamide gels, which was first developed by Ornstein (2O)and Davis (11). While the application of SDS to this technique results in some loss of resolution as compared to the now classical method of Panyim and Chalkley (12),the SDS gels are valuable when the mixtures of histones and nonhistone proteins are to be analyzed. Extensive aggregation and insolubility of chromosomal nonhistone proteins in most conventional electrophoresis buffers led to the introduction of polyacylamide gel electrophoresis performed in the presence of detergents, most frequently SDS (23). Because of their interactions with this detergent, most proteins form negatively charged complexes which separate electrophoretically in polyacrylamide gels in order of their molecular weights (13,14). Indeed, the electrophoretic mobility of proteins larger than 10,000 daltons can be used for fairly accurate molecular weight determination (13,25). SDS dissociates most proteins into their subunits (unless covalently
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linked) and, consequently, the number of proteins bands observed in polyacrylamide gel electrophoresis performed in the presence of this detergent corresponds to the number of subunits and may be much larger than the true number of the in vivo protein species. On the other hand, polypeptide chains of similar or identical molecular weights will form a single protein band. Since histones are simple proteins, their electrophoretic homogeneity is not affected by exposure to SDS. However, their molecular weights just about approach the lowest range of confidence for molecular weight determinations of the SDS-protein complexes (15).The excessive electropositive charge of the carboxyterminal half of the H1 histones which results from a large accumulation of lysyl residues disturbs thedistribution of thenegatively charged SDS micelles along the H1 molecule. This disturbance is reflected in an anomalous electrophoretic mobility of H1 histones in SDS-containing gels. Consequently, the apparent molecular weight of about 34,000 calculated for HI histones from their mobilities in SDS gels differs considerably from their true molecular weights of 19,500-22,000 (average) as determined physicochemically (16). These disturbances are even greater when SDS interacts with more basic proteins of male gametes. Such complexes (e.g., of protamines or arginine-rich proteins from mammalian spermatids and spermatozoa) may become partially insoluble in electrophoretic buffers, and their electrophoretic separation in the presence of anionic detergents cannot be accomplished. The electrophoresis in SDS-containing gels can be performed both with or without urea. Since SDS eliminates virutally all the protein-protein interactions, the presence of urea is not as beneficial as in the detergent-free polyacylamide gel systems. Elimination of the positive charges on histones by SDS makes impossible the electrophoretic separation of differentially acetylated or phosphorylated histone fractions. A reasonably good separation of histones, even in the presence of chromosomal nonhistone proteins, can be obtained by a modified procedure of Maize1 and his associates (13). Polyacrylamide gels (10% polyacrylamide containing 4 M urea) can be prepared by mixing the following stock solutions: A Gel buffer: 0.2 M phosphate buffer-0.2% SDS-8 M urea @H 7.0). B. Acrylamide solution: 24% acylamide (recrystallized commercial or electrophoretic grade) and 0.6% Bis (electrophoretic grade N,N/-methylenebisacylamide) in deionized water. C. Ammonium persulfate, 90 mg in 10 ml of deionized water (must be made fresh before use). D. TEMED (N,N,N:N:-tetramethylethylenediamine), undiluted reagent. Thesolutions aremixedin theratioA:B:C:D =6:5:1:0.06.For 12-14gels
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(10 x 0.6 cm each), 30 ml of A are mixed with 25 ml of B and 5 ml of C, and the mixture is cooled on ice under reduced pressure for 15 minutes. Finally, 0.03 ml of TEMED (solution D) are added and the gels are poured immediately into stoppered glass columns to the desired height. Chilling the monomer mixture delays the polymerization and allows the time necessary for the manipulation of the individual columns. Disposable plastic syringe or Pasteur pipettes can be used conveniently to transfer the monomer mixture. The filled columns are freed of any entrapped air bubbles and carefullly overlaid each with 0.1 ml of solution A diluted 1:1with deionized water. The gels are polymerized for at least 2 hours at room temperature. In the presence of a large excess of nonhistone proteins or DNA, a 3% polyacrylamide stacking gel should be layered over the 10%gel. To prepare the stacking gel, a modified solution B containing 7.5% acrylamide and 1.2% Bis in deionized water is used instead of the regular solution. The columns are made of fire-polished 0.6-cm-bore glass tubing, cut to the desired height, washed in chromic solution, rinsed well with water, and coated with silicone (the silicone coating is not important in SDS-containing gels because these can be removed from the columns with relative ease). To prepare for electrophoresis, the tubes are closed on one end with rubber caps and placed into a polymerization rack which can be a small test tube or a special stand made of Plexiglas or other material. Samples containing 10-30 pg of unfractionated histone are dissolved in solution A, which has been made more dense by the addition of concentrated sucrose or urea, and a measured amount of the solution is carefully layered on top of each gel. The sample volume should not exceed 50 p1 to assure good resolution of the histone fractions. After filling the columns with electrolyte buffer, the rubber caps are gently removed from the columns. The columns are inserted into the upper tray of an electrophoretic apparatus (some commercial instruments do not have a separable upper and lower tray), which is then filled with electrolyte buffer (solutionA), and thesamples are electrophoresed at 8-10 mA per column for 8-10 hours. Application of a tracking dye (Bromophenol blue) to at least one of the samples helps to monitor the migration. After electrophoresis, the gels are stained in the solution of Coomassie blue or Amido black (0.2% or OS%, respectively) in methanol+icetic acid-water mixture 5 :1: 5 , or they can be first fixed in 20% aqueous sulfosalicylic acid or 15% trichloroacetic acid. This treatment also removes most of the detergent which may interfere with subsequent staining. Some batches of Coomassie blue are incompatible with trichloroacetic acid, and it may be necessary first to remove the acid by placing the fixed gels into 20% sulfosalicylic acid solution for several hours. A mixture of methanolacetic acid-water ( 5 :1:5 ) can be also used to remove the 15% trichloroacetic acid from the gels before their staining with Coomassie blue. The gels can be
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1 2 3 4 5 6 I
H1 1
H3mono+H2B
/
"** / H4
FIG. 4. Polyacrylamide gel electrophoresis in the presence of SDS of total rat thymus histones and calf thymus histone fractions. 1 = total rat thymus histone; 2 and 3 = H1 histones from rat thymus and liver, respectively; 4 = calf thymus H2B; 5 = calf thymus H2A; and 6 = calf thymus H4. The origin of migration is at the top of the gels.
destained by diffusion in an excess of methanol-acetic acid-water mixture or, much faster, by electrophoresis in 7% acetic acid. In the presence of SDS,the following histone fractions, can be resolved in order of their increasing mobilities: oxidized H3 dimer, H1, a mixture of H3 monomer and the H2B fraction, the H2A, and finally the H4 bands (Fig. 4). In addition to electrophoresis in polyacrylamide gels contained in glass tubing, electrophoresis in relatively thin gel slabs (1-5 mm) is rapidly gaining popularity. Besides the better resolution of the slab-gel electrophoresis, the uniformly thin sheets of polyacrylamide can be easily dehydrated and used for autoradiography or scanning. The stained gel can also be impregnated with 2,5-diphenyloxazolesolution and dried for fluorography (17). A good electrophoretic separation of all common histone fractions can be obtained by adaptation of the discontinuous method of Laemmli (18)
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FIG.5. Polyacrylamide gel electrophoresisin the presence of SDS in slab gels. This photograph illustrates the good resolution of all histones from chicken reticulocyte chromatin. The histone fractions are identified by their symbols. The origin of migration is at the top of the gel. (Courtesy of Dr. A. Beyer and Dr. K. Hardy.)
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22 1
using a gel of 12% or 15% polyacrylamide (Fig. 5). The gel can be formed from the following solutions: A. Acrylamide solution: 30% acrylamide and 0.8% Bis (NN-methylenebisacrylamide) in deionized water. B. 1.5 M Tris-HC1, pH 8.8 (ultrapure). C. 10% SDS. D. 0.5 M Tris-HC1, pH 6.8. E. 1.5% ammonium persulfate (made fresh). F. TEMED (N,N,N:N'-tetramethylethylenediamine), undiluted reagent. In order to prepare 50 ml of solution for a 12% polyacrylamide gel, 20 ml of solution A, 12.5 ml of B, and 16 ml of water are mixed and kept on ice under reduced pressure for 15 minutes. Finally 0.5 ml of C, 1.0 ml of E, and 12 pl of F are added. A 10-ml pipette can be used to introduce 30 ml of the mixed solution into a 14 x 15.5 cm vertical gel chamber which is formed of two glass plates separated by plastic spacers. Two spacers for the sides are 1 x 19 cm and the bottom spacer is 1 x 17 cm. The gel is overlayered with 0.1% SDS until polymerization is complete. A spacer gel of 2 or 3% polyacrylamide is prepared by mixing 1.O ml of solution A, 2.5 ml of D, 0.1 ml of C, 0.7 ml of E, 5 p1 of F, and 6.3 ml of deionized water. The overlayer solution is removed and the gel surface is rinsed with the spacer gel solution. The upper gel is poured with a 10-ml pipette, and a plastic template is inserted to form sample wells 0.7 x 2.0 cm. The slab gel can be used for a preparative separation of proteins by omitting the template so that a single sample can be applied to the entire gel width. After polymerization, the template and the bottom spacer are removed, and buffer solution [0.2 M glycine-0.025 M Tris-HC1 (pH 8.3)-0.1% SDS] is added to the upper and lower reservoirs. Proteins (usually dissolved in water) are mixed with an equal volume of sample mix (2.5 ml of solution D, 4.0 ml of C, 2.0 ml of glycerol, 1.O ml of 2-mercaptoethanol, 0.2 ml of 0.05% Bromophenol blue, and 0.3 ml of water) and heated in boiling water for 1to 2 minutes or kept at 37O for 1 hour. Samples are centrifuged at lOOOg to remove any insoluble materials. Samples containing 10-50 pg of unfractionated histone in a volume less than 100p1areplacedin to eachsamplewell. Thesamples are electrophoresed at 20 mA until the tracking dye reaches the bottom of the gel (about 6 hours). The gel is removed and stained overnight in 0.1% Coomassie blue in 25% isopropanol and 10% acetic acid as described by Fairbanks et al. (29).The gel is destained for 6 to 9 hours in 0.03% Coomassie blue in 10%isopropanol and 10% acetic acid followed by several changes in 10% acetic acid. The stained gel can be impregnated with 2,5-diphenyloxazole and dried for
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fluorography (17) or dehydrated onto cellophane sheets and cut for scanning or liquid scintillation counting. The slab gels can also be adapted for preparative elution electrophoresis by forming a channel near the lower edge of the gel for a continuous flow of buffer to carry off proteins separated by electrophoresis. A lower gel is poured up to the elution part in the 3-mm plastic side spacers. A narrow band of glycerol is added to the surface of the polymerized lower gel, and the separating gel is layered carefully over the glycerol.A template forming one large well is inserted into the upper gel during polymerization. When electrophoresis is started the glycerol is forced out of the elution port as the sweeping buffer flow is started. This method allows isolation of relatively pure protein fractions for amino acid analysis or other chemical determinations. Various commercial apparatuses permitting the analytical as well as preparative slab-gel electrophoresis can be obtained from several suppliers. Exceptionally good results were obtained with an apparatus acquired from Hoefer Scientific Instruments, San Francisco, California. The slab-gel method described here was performed with this apparatus. ACKNOWLEDGMENTS This work was supported by U.S.Public Health Service Grant CA 18389 and by NCI contract NO 1 -CP-65730. REFERENCES
1. Smithies, 0. Biochem. J. 61,629 (1955). 2. Neelin, J. M., and Neelin, E. M., Can. J. Biochem. Physiol. 38, 355 (1960). 3. Johns, E. W., Phillips, D. M. P., Simson, P., and Butler, J. A. V., Biochem. J. 80,189 (1 961). 4. Hnilica, L. S., Edwards, L. J., and Hey, A. H., Biochim. hiophys. Actu 124, 109 (1966). 5. Kalberer, F., and Rutschmann, J., Helv. Chim. Actu 44, 1956 (1961). 6. Dobbs, H. E., Anal. Chem. 35,783 (1963). 7. Baggiolini, M.,Experientia 21, 731 (1965). 8. Johns, E. W., Phillips, D. M. P., Simson, P., and Butler, J. A. V., Biochem. J. 77,631 (1960). 9. Sung, M.,and Smithies, O., Biopolymers 7 , 39 (1969). 10. Ornstein, L., Ann. N. Y. A d . Sci. 121,321 (1964). 11. Davis, B. J., Ann. N. Y.Acad. Sci. 121,404(1964). 12. Panyim. S., and Chalkley, R., Arch Biochem. Biophys. 130,337 (1969). 13. Shapiro, A. L., Vifluela, E., and Maizel, J. V., Biochem. Biophys. Res. Commun. 28,815 (1967). 14. Dunker, A. K.,and Rueckert, R. R., J. Biol. Chem. 244, 5074 (1969). 15. Williams, J. G., and Gratzer, W. B., J. Chromutogr. 57, 321 (1971). 16. Hnilica, L. S., “The Structure and Biological Functions of Histones” Chem. Rubber Publ. Co.,Cleveland, Ohio, 1972. 17. Bonner, W. M., and Laskey, R. A., Eur. J. Biochem. 46,83 (1974). 18. Laemmli, U.K., Nature (London) 227, 680 (1970). 19. Fairbanks, G., Steck, T. L., and Wallach, D. F. M., Biochemistry 10,2606 (1971).