[97] Isolation and purification of nuclear proteins

[97]

NUCLEAR PROTEINS

65

concentrations of Mg ~ and Mn ++ precipitate nucleohistone from solution26 A second important condition which must be fulfilled if nucleohistone is to remain soluble in the RNA synthesis reaction mixture is that the nueleohistone must be relatively free of contaminating nonchromosomal protein. It is for this reason among others that the sucrose density gradient centrifugation step described in Section II has been introduced into the procedure for the preparation of soluble nucleohistones. At the sucrose density gradient step, a maior portion of the nonchromosoma] protein contained in preparations of crude chromatin is removed. 1 Crude chromatin, if sheared directly, yields nucleohistone containing contaminating nonchromosomal protein. The presence of such contaminants, which are separable from nucleohistone not only by sucrose density gradient centrifugation but also by zone electrophoresis (Section XI) leads to aggregation of the nucleohist~ne under a variety of experimental circumstances. Thus thymus nucleohistone prepared by the method of Zubay and Dory 3 and without purification by the sucrose density gradient step, aggregates in reaction mixture of the composition given above. Acknowledgment l=teport of work supported in part by the Herman Frasch Foundation, in part by the U.S. Public Health Service, Grant Nos. GM-5143, GM-3977, AM-3102, GM-10991, and by U.S. Public Health Service Training Grant no. 2G-86. ~J. Bonner and R. C. C. Huang, Biochem. Biophys. Res. Commun. 22, 211 (1966).

[97]

I s o l a t i o n a n d P u r i f i c a t i o n of N u c l e a r P r o t e i n s 1 By HARMS BUSCH

The nuclear proteins are divisible into histones, acidic nuclear and nucleolar proteins, and nuclear and nucleolar enzymes. 2 Nucleoproteins are deoxyribonucleoprotein complexes containing mainly DNA and histones, but also some RNA and acidic proteins. The problems involved in the isolation of nuclear proteins are extensions of the problems of isolation of nuclei. Although, as indicated elsewhere (Vol. XII, Part A [51]), none of the methods are completely satisfactory, both the sucrose1Studies on this subject have been supported in this laboratory by grants from the U.S. Public Health Service (CA 8182), the American Cancer Society (P-339 and P-369), the National Science Foundation and the Jane Childs Fund.

~H. Busch, "Histories and Other Nuclear Proteins." Academic Press, New York, 1965.

66

PREPARATION OF NUCLEOPROTEINS

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calcium procedure and the nonaqueous procedure have been employed. In addition, tissues such as t h y m u s have been used for isolation of nucleoproteins because they are rich in nuclei as compared to liver. Other problems in the isolation of these proteins are those of isolation of gels, particles, and soluble fractions from the nuclei in native states, the insolubility of acidic nuclear proteins, and the possibility t h a t biological activities m a y be altered. Finally, for comparison from tissue to tissue there is the problem of extraction of nuclear proteins in satisfactory yield sufficient to p e r m i t subfractionation into individual molecular species. Isolation o] N u c l e a r Sub]ractions. The procedures for isolation of nuclear components are summarized in T a b l e I. Methods are being improved continually for isolation of nucleoli, chromosomes, nuclear ribosomes, and deoxyribonucleoproteins from which structural proteins and enzymes m a y be isolated. F o r the isolation of nuclear ribonucleoproteins in the "nuclear sap," the nuclei are extracted with either dilute salt solutions or with Tris saline buffer. T h e deoxyribonucleoproteins TABLE I A SUMMARY OF PROCEDURES FOR ~SOLATION OF NUCLEAR COMPONENTSa

A. Following initial isolation of nuclei 1. Isolation of nudeoli: Either by sonication of nuclei prepared in 0.0033-0.005 M CaC12 and 0.25 M sucrose or by compression and rapid decompression of nuclear preparations in the French pressure cell; purification by differential centrifugation (see Vol. XII, Part A [52]). 2. Isolation of chromosomes: By gentle procedures employing ceils in metaphase; aspiration and ejection from syringes followed by centrifugation in sucrose solutions 0.0005 M with respect to Mg++ and Ca ++. 3. Isolation of nuclear ribonucleoproteins: Extraction from nuclei of calf thymus by 0.15 M NaC1 or 0.01 M Tris buffers containing 0.001 M MgCl~ followed by differential centrifugation. 4. Isolation of dcoxyribonucleoprateins: Either by extraction of nuclei with 2 M NaGl or with water followed by precipitation of the deoxyribonucleoproteins from solutions 0.10-0.20 M with respect to NaC1. 5. Nucleochromosomal apparatus: A residual fraction obtained after successive extraction of nuclei with 0.15 M NaG1 and 2 M NAG1; bears many similarities to the nuclear ribonucleoprotein network, but probably contains other components including the nuclear membrane. B. Without initial isolation of nuclei 1. Nucleoli: Obtained from tumors without extensive nuclear preparation inasmuch as it is not possible to remove cytoplasmic components in the presence of calcium ions at concentrations required to maintain the integrity of the nucleoli. Techniques are the same as in A, 1, above (see Vol. XII, Part A [52]). 2. Deoxyribonucleoproteins: Obtained as a residue following prolonged treatment of nuclear preparations with isotonic saline solutions. a From H. Busch and W. J. Steele, Advan. Cancer Res. 8, 41 (1964).

[97]

NUCLEAR PROTEINS

67

m a y then be extracted with 2 M NaC1. The residual fraction t h a t remains after extraction of the ribonucleoprotein and deoxyribonucleoprotein fractions is referred to as the "nucleolochromosomal apparatus" by Zbarsky and his colleagues2 ~5 As indicated in the chapter on isolation of nucleoli (Vol. XII, P a r t A [52])1 this fraction contains the nuclear ribonucleoprotein network. 6 Isolation of Deoxyribormcleoprotein When they use the term "nucleoproteins," most authors are generally referring to the deoxyribonucleoprotein ( D N P ) complexes. For the isolation of histones, the methods for purification of deoxyribonucleoproteins are of greatest importance. At present, three methods are available for isolation of deoxyribonucleoproteins, (a) direct extraction of nuclei with water or dilute buffer solutions such as 0 . 7 M phosphate buffer; ~ (b) extraction of nuclei with 0.14 M NaC1 containing 0.01 M sodium citrate to remove most of the nuclear components, and (c) extraction of nuclei with 1-2 M NaC1 containing 0.01 M trisodium citrate. M e t h o d a. Direct Extraction o] Deoxyribonucleoprotein with Water or Dilute Buf]ers. Lilienfeld s and later H a m m a r s t e n 9 reported a procedure for extraction of nuclei with water to obtain nucleoprotein preparations. The product has recently been studied by Commerford et al., TM who showed that the nucleoprotein contained about 5% R N A / The nueleoprotein was readily soluble in water containing ethylenediaminetetraacetic acid ( E D T A ) . More than 90% of the nucleoprotein was extracted in 20 minutes. The molecular weight of the D N P was aproximately two to three million. Their product was probably markedly degraded from its native state, since this value is very low by comparison to others ;~, 1~-1:) ~I. B. Zbarsky and N. P. Dmitrieva, Acta Unio Intern. Contra Cancrum 18, 123 (1962). 4I. B. Zbarsky, N. P. I)mitrieva, and L. P. Yermolayeva, Exptl. Cell Res. 27, 573 (1962). 5I. B. Zbarsky and G. P. Georgiev, Biochim. Biophys. Acta 32, 301 (1959). K. Smetana, W. J. Steele, and H. Busch, Exptl. Cell Res. 31, 198 (1963). 7G. Zubay and P. Doty, J. Mol. Biol. 1, 1 (1959). a L. Lilienfeld, Z. Physiol. Chem. 18, 472 (1893). 9E. Hammaxsten, Bioehem. Z. 144, 383 (1924). 10S. L. Commerford, M. J. Hunter, and J. L. Oncley, J. Biol. Chem. 238, 2123, (1963). l~j. A. V. Butler and P. F. Davison, Advan. Enzymol. 18, 161 (1957). 12j. A. V. Butler, P. F. Davison, D. W. F. James, and K. V. Shooter, Biochim. Biophys. Acta 13, 224 (1954). ~ K. S. Kirby, Biochem. J. 66, 495 (1957). ~4K. V. Shooter, P. F. Davison, and J. A. V. Butler, Biochim. Biophys. Acta 13, 192 (1954). ~5A. E. Mirsky, Advan. Enzymol. 3, 1 (1943).

68

PREPARATION OF NUCLEOPROTEINS

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values of 18.5 million have been found. 7 Commerford et al. 1° attempted to determine the molarity of salt at which histone dissociated from DNA. At 1 M NaC1 the histone and DNA remained associated, but with 6 M CsCl they were completely dissociated as determined by ultracentrifugation. As carried out in this laboratory, nuclei or nuclear preparations are first extracted with dilute saline solutions containing either EDTA (0.025 M), citrate (0.01 M), or Tris buffer (0.01-0.10 M) at pH 7-8 such that the final ionic strength approximates 0.15. These additions are made to inhibit DNase. Sufficient volumes of the extractant are added so that the ratio of volume to weight of original tissue from which the nuclei are derived approximates 20:1. The optimal procedure for this extraction is by homogenization, preferably in the continuous tissue homogenizer discussed in this volume with reference to isolation of nuclei (Vol. XII, Part A [51]). After homogenization, the suspension is stirred approximately 20-30 minutes in the cold laboratory and is then centrifuged at 6000 to 10,000 g for 10-20 minutes. The suspension and sedimentation is then repeated with the same buffer or may be repeated with bicarbonate buffer (0.05 M NaHC03 to 0.10 M NaC1) with an ionic strength of 0.15. The sediment is now extracted with distilled water or 0.7 mM phosphate buffer (pH 6.8-7.6) in a volume of 1 to 3 ml per original gram of tissue which is sufficient to extract the deoxyribonucleoprotein. Although a number of workers have used at this point the Waring blendor or blendors of this type, it is a most dangerous procedure inasmuch as the destruction of the DNA strands is certain to be most marked. The destruction of RNA is such that in this laboratory no 45 or 55 S RNA has ever been obtained in preparations treated at this stage with the Waring blendor. After the sediment is homogenized in water, it is stirred for 20-30 minutes and then is centrifuged for 30 minutes at 15,000 g in a Servall centrifuge. The supernatant suspension contains the deoxyribonueleoprotein, which forms a stable gel at temperatures below freezing or remains as a viscous solution at 4 °. This procedure is repeated twice for complete extraction of the deoxyribonucleoprotein, although 90% or more of the deoxyribonucleoprotein may be extracted in the first step. The deoxyribonucleoprotein obtained by this procedure contains approximately 5% ribonucleoprotein, l°,1° M e t h o d b. Direct Extraction of Cells with Dilute Saline Solutions. In the second method for isolation of the nucleoproteins, nuclei are not isolated. 11,12 Instead, the cells are directly extracted with buffered dilute saline solutions by stirring in a mixer. As carried out in this laboratory, the tissues are dissected in the cold room to remove any connective and u W. J. Steele and H. Busch, Cancer Res. 23, 1153 (1968).

[97]

NUCLEAR PROTEINS

69

necrotic tissue and then are cut into small pieces and dispersed in a Waring blendor at full speed for 2 minutes in 5 volumes of a solution containing 0.14 M NaC1 and 0.01 M trisodium citrate. 17 The suspension is strained through eight layers of cheesecloth and centrifuged at 1500 g for 40 minutes. To decrease foaming, a few drops of octanol are added after each blending. After centrifugation, the sediment is suspended in 5 volumes of the NaCl-citrate solution by blending at reduced speed (7080 volts) for 30 seconds. To prevent enzymatic hydrolysis of the nuclear proteins, sufficient diisopropyl fluorophosphate is added to make a final concentration of 0.1 raM. The suspension is then centrifuged for 30 minutes at 1000 g. This washing procedure is repeated four times or more until the supernatant fluid is clear and virtually colorless. The final sediment, used for further fractionation procedures, is white and is designated as crude deoxyribonucleoprotein. M e t h o d c. Extraction of Nuclei with 2 M NaCl. The technique employing concentrated saline solutions is based upon the finding of Bensley TM and Pollister and Mirsky TM that nucleoproteins were soluble in concentrated salt solutions, i.e., about 5-10~o NaC1 or approximately 12 M NaC1. Although this technique has been subjected to a large number of variations, one of the presently employed procedures ~6 for fractionation of nuclei is shown in Table II, which demonstrates the key steps. As carried out in this laboratory, the procedure is as follows: Initially, soluble nuclear proteins and ribosomes are extracted twice with 10 volumes of 0.14 M NaCl and once with 5 volumes of 0.1 M Tris buffer pH 7.6, in that order, by homogenization and stirring for 20 minutes. A precipitate is obtained in each case by centrifugation at 6000 g for 10 minutes. After extraction with Tris, the precipitate is extracted with 1-2 volumes of 2.0 M NaC1 for each gram of wet tissue to obtain deoxyribonucleoproteins. In the initial extraction with 2 M NaC1, the sample is blended for 20 seconds at 40 volts with the aid of a variable transformer, and several drops of octanol are added to control foaming. The extract is centrifuged at 25,000 g for 1 hour, and the clear supernatant solution is decanted and stored overnight in a deep freezer. The precipitate is stirred with one-fourth of the initial volume of 2 M NaC1 overnight with the aid of a magnetic stirrer, and reextracted for 1 hour the next morning. The suspensions are combined and centrifuged at 20,000 g for 20 minutes; the supernatant solutions are decanted and saved. As modified recently, soluble nuclear proteins are extracted with saline (0.14 M) containing 0.05 M Tris buffer (pH 7.6), 1 mM Mg ++ and 17L. S. Hnilica and H. Busch, J. Biol. Chem. 238, 918 (1963). Bensley, Sc/ence 96, 389 (1942). I~A. W. Pollister and A. E. Mirsky, J. Gen. Physiol. 30, 101 (1946).

18 R. R.

70

[97]

PREPARATION OF NUCLEOPROTEINS TABLE II CHEMICAL PROCEDURE FOR ISOLATION OF NUCLEAR PROTEIN FRACTIONS a

Isolated nuclei

I

0.14 M NaC1, 2 times

I precipitate

"nuclear sap" proteins

0.1 M Tris p H 7.6

preel ~itate

"nuclear sap" proteins

2.0 M NaC1

preci ,itate

deoxyribonucleoproteins-1 /

0.5 N H,S04; extract | twice with 0.2 N H,SO4

2.0 M NaCl

precipitate

~

deoxyribonucleoproteins-2

precipitate

histones

0.05 N NaOH, 3 times

"residual proteins"

acidic ribonucleoproteins

a From W. J. Steele and H. Busch, Cancer Res. 23, 1153 (1963).

polyvinyl sulfate (150 ~g/ml), by homogenization with a loose-fitting Teflon pestle (10 strokes). This procedure is repeated once. The deoxyribonueleoprotein is extracted by treating the precipitate with 10 volumes per gram of wet tissue of 2 M NaC1 by homogenizing with a tight pestle (0.003-0.005 inch clearance) ten strokes and centrifuging immediately for 1 hour at 40,000 g. This step removes more than 95% of the DNA, and almost all the remainder is removed by a second wash with 2 M NaC1 and centrifugation at 20,000 g for 10 minutes. In this procedure, blending is omitted. Although the proteins extracted with dilute saline solution are referred to as the proteins of the "nuclear sap," it is equally likely that these include proteins of the nucleolus and of the chromatin since there is no satisfactory way at present to exclude this possibility. 2° Similarly, it is not possible to be precisely certain that all the proteins present in the deoxyribo~ucleoprotein extract (with 2 M NaC1) originate in the chromatin. Some may originate in the nucleolus and the nuclear RNP network2 Others may represent portions of the soluble entities of the nucleus that are left behind when the initial saline extraction has been completed.21,22 The deoxyribonucleoprotein extracted in 2 M NaC1 contains histones, D. Grogan, R. Desjardins, and H. Busch, Cancer Res. 26, 775 (1966). G. P. Georgiev and V. L. Mantieva, Biokhimiya 27, 949 (1962). G. P. Georgiev and V. L. Mantieva, Biochim. Biophys. Acta 61, 153 (1962).

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NUCLEAR PROTEINS

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acidic proteins, RNA, and DNA; the histones and DNA are dissociated in this medium. Thus, a native deoxyribonucleoprotein from mammaliazl cells is not isolated with this procedure, although the components are both present. Composition o] the Products Obtained by Nuclear Extractions. Table III shows the composition of the products obtained in the various steps of the extraction procedure of Table II. 18 Approximately 15% of the nucleoprotein is extracted with dilute saline solution, and approximately another 5-15% is extracted with the Tris solution. More was extracted from the nuclei of tumor cells. The largest percentage, almost 60%, of the nucleoprotein is extracted with 2 M NaC1. In the liver, less of the total nucleoprotein is extracted with 0.05 N NaOH than was extracted in the tumor. Part of this greater extraction of components with Tris and NaOH in the tumor may be related to the partial adherence of cytoplasmic contaminants to the tumor nuclear preparation. 1 Table III also shows the composition of the various fractions. In the liver, the nucleic acids compose only 5% of the total mass of the fraction extracted with dilute salt solution and 18% of the fraction extracted with Tris. In the tumor extracts, a larger percentage of the Tris fraction is composed of RNA. A much larger percentage of the fraction extracted with 2 M NaC1 was composed of nucleic acids, i.e., about one-third; in the first extract, the amount of DNA is about 6 times that of RNA. In the other fractions, the amount of RNA is considerably greater than the amount of DNA. In both the liver and tumor fractions, there is some DNA in the Tris extract.

Isolation o] Deoxyribonucleoprotein Complexes with the Phenol Procedure. The success of the phenol procedure of Kirby is for the isolation of DNA naturally led to the attempt to utilize the same methods for isolation of the deoxyribonucleoproteins. 2~ For homogenization of the tissue, the medium utilized was either sodium benzoate or sodium phenolphthalein diphosphate (15 mM at pH 8) in a ratio of 10-20 volumes of the medium per gram of tissue. Extraction with 90% (w/v) phenol was then carried out to purify deoxyribonucleoprotein complexes by removing other proteins. After precipitation of the deoxyribonucleoproteins, they were redissolved in 10 mM sodium acetate. The samples in the aqueous phases were centrifuged for 5 hours at 20,000 rpm in a Spinco Model L centrifuge to fractionate the nucleoproteins when sodium benzoate was used for the initial homogenization and for 12 horn's when phenolphthalein diphosphate was used; in the latter casc, 30,000 rpm were required. In the deoxyribonucleoprotein product of the precipitate, ~:~ large amounts of acidic amino acids were found, i.c., 18-34% of the total ~P. M. Frearson and K. S. Kirby, Biochem. J. 90, 578 (1964).

72

PREPA~ATION OF NUCLEOPROTEINS

Z

O ~9 O

~9

o~

O

O

[97]

[97]

NUCLEAR PROTEINS

73

residues were glutamic and aspartic acid. The proteins in the supernatant fraction contained large amounts of lysine, i.e., 13-28% of the torn! amino acid residues. The content of acidic amino acids ranged from 16 to 48% of the total amino acid residues. However, the differences were not great in the amino acid composition of the supernatant and sedimentable fractions obtained from a transplantable tumor. In the sediment from other tissues, the concentration of acidic amino acids was greater than in the supernatant fraction. Although the authors ~ suggested that they had separated a DNA fr~ction containing a lysine-rich histone fraction from another DNA fraction, further studies are required te establish the nature of the histones in both of these fractions. Extraction of Histones The standard method for separation of histones from deoxyribonucleoproteins is extraction with HC1, ~ stemming from the early experiments of Kossel. ~4 There have been attempts to substitute an alcohol extraction procedure for the acid extraction and also to substitute the use of H2S04 for HC1, but thus far, these other agents have not been widely used. As shown in Table I, the methods for extraction of histones have been limited to extraction with acid or acid-ethanol, which leave DNA as a residue. For complete extraction of histones, 10-20 volumes of 0.25 N HC1 per volume of deoxyribonucleoprotein is sufficient. The procedure employing acid extraction has received important support from the X-ray diffraction studies of Zubay and Wilkins. ~ In studying the histones isolated by the acid extraction procedure and the histones isolated by the procedure employing ethanol precipitation of deoxyribonucleic acid, it was shown that the former had the natural or ~-configuration of the proteins linked to DNA and the latter were largely in the fl-form; the /%configuration suggests that they were partially or completely denatured in the course of the extraction procedure. Fractionation of the Histones Improvements introduced by Johns et al. 26~9 are used in the procedure shown in Table IV, which presents the steps involved in the isolation of histories of the Walker tumor, rat liver, and calf thymus. 17 In this ~A. Kossel, "The Protamines and ttistones." Longmans, Green, London, 1928. G. Zubay and M. It. F. Wilkins, J. Mol. Biol. 4, 444 (1962). E. W. Johns, Biochem. J. 92, 55 (1964). ~,E. W. Johns and J. A. V. Butler, Biochem. J. @2, 15 (1962). E. W. Johns, D. M. P. Phillips, P. Simson, and J. A. V. Butler, Biochem. J. 77, 631 (19~0). E. W. Johns, D. M. P. Phillips, P. Simson, and J. A. V. Butler, Biochem. J. 80, 189 (196D.

74

PREPARATION OF NUCLEOPROTEINS

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TABLE IV SCHEME FOR FRACTIONATION OF HISTONES OF THE WALKER

TUMORa

Crude deoxyribonucleoprotein

I

extraction with 10 volumes of 80% ethanol

I

sediment

l

supernatant (discarded)

I I

three extractions with 10 volumes of 80°/o ethanol -1- 20% i. 25 N HC1, 6--8 hours each

I

I

extracts (fractions 1, 2a, 3)

residue

volume reduced to one-third

three extractions with 0.25 N HCI, 6-8 hours each

dialysis against H20 [ precipitation with trichloroacetic acid, 5% final concentration [

] } extracts residue (alkali-soluble (fractions 1, 2b, 3) and residual proteins) ] dialysis against H~O

I

supernatant

]

I

precipitate precipitation with trichloroacetic acid, (fractions 1, 2a, 3) 5% final concentration

I

i

chromatography on ]' [ CM-cellulose supernatant precipitate columns I (fractions 2b and 3) precipitation with I trichloroacetic acid, chromatography 20% final concentration on CM-cellulose I columns

I

supernatant

I

precipitate (fraction 1T)

• H. Busch, "Histones and Other Nuclear Proteins." Academic Press, New York, 1965. scheme, the critical step is the initial separation of histones into two groups by exCraeion with a solution which is 0.25 N HC1 in 80% ethanol. This extract contains a mixture of fractions designated as 2a and 3. The residue contains a mixture of fractions 1, 2b, and 3. T h e crude deoxyribonucleoprotein is blended at reduced speed (70-80 volts) in a Waring blendor for 30 seconds with 10 volumes of 8 0 ~ ethanol; the suspension is ~hen centrifuged for 30 minutes at 1500 g. The supernatan~ fraction

[97]

NUCLEAR PROTEINS

75

is discarded, and the sediment is extracted three times with a solution of acidified ethanol (95% ethanol-l.25 N HC1, 4:1) for 6-8 hours. The volume of the extract is reduced by one-third at 4 ° , and the extract is then dialyzed against distilled water; trichloroacetic acid is added to a final concentration of 5%. The proteins precipitated from this solution contain the crude fractions 2a and 3. The supernatant solution generally contains small amounts of fraction 1, which is precipitated by adding trichloroacetic acid to a concentration of 20%. The protein insoluble in acidified ethanol, is extracted with 0.25 N HC1 for three periods of 6-8 hours. The combined extracts are dialyzed against distilled water and clarified by centrifugation at 20,000 g for 30 minutes. To precipitate fraction 2b, triehloroacetie acid is added to a concentration of 5%. To precipitate fraction 1, trichloroacetic acid is added to the supernatant solution to a final concentration of 20%. The proteins precipitated with trichloroacetic acid are converted to hydrochlorides by suspension in acetone containing 1% HC1; the proteins are then washed with acetone and ether and then dried. Chromatography o] Histones. All the fractions, with the exception of fraction 1 are purified by chromatography on carboxymethyl cellulose 0.4-0.7 meq/g). For small-scale experiments, the columns used are 2 cm in diameter and contain 20 g of the carboxymethyl cellulose. A maximum of 20 mg of protein per gram of cellulose is added to the column; all the procedures are carried out at 4 °. The histones are added to the column in 0.1 M acetate buffers at pH 4.2. The elution of the F1 histone peak is carried out with 0.2 M acetate buffer at pH 4.2 containing 0.3 M KC1. Approximately 125 fractions are collected, each containing ~ ml of eluent. When the F1 peak and its trailing edge are eluted, 0.01 N HC1 is added and depending on the fraction applied, either the F2a or F2b histone fraction is eluted in a large peak emerging approximately 100 fractions following the initial addition of 0.01 HC1. When the peak is completely eluted, an additional 20-30 fractions are collected and then 0.02 N HC1 is added. The F3 peak is then eluted in 20-30 fractions and an additional 20-30 fractions are collected to obtain the trailing edge. The flow rate of these columns is 0.1 ml/min per gram of cellulose. For large-scale fractionation, columns 30 cm in height and 5.6 cm in diameter have been effectively used. Amounts of histones up to 2 grams can be fractionated with such columns. The fractions in the peaks are pooled and the proteins are precipitated with either 5% or 20% trichloroacetic acid. The amino acid composition of the histone fractions obtained by this procedure is shown in Table V along with the nomenclature usually used. Johns 26 has recently modified the extraction procedures to provide

76

PREPARATION" OF NUCLEOPROTEINS

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TABLE V AMINO ACID COMPOSITION OF REPRESENTATIVE HISTONE SUBFRACTIONSa'b

Composition: Chemical fractions (20-22) : Amino acid Alanine Arginine Aspartic acid Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tyrosine Valine

Very lysine-rich F1

Slightly lysine-rich N-acetylalanine F2a

Slightly lysine-rich N-prollne F2b

"Argininerich" F3

23.5 2.5 2.8 6.0 6.8 0.5 1.6 4.4 26.3 -0.8 7.9 6.2 5.5 0.5 5.0

10.5 11.5 6.0 8.5 12.5 2.0 4.5 10.5 10.5 -1.6 3.0 3.1 5.6 3.0 7.0

10.5 7.5 5.5 9.0 7.0 2.5 5.0 6.0 14.5 0.7 2.0 4.5 9.0 6.5 3.1 6.8

12.5 13.0 5.0 11.0 6.5 2.1 5.0 8.5 9.0 0.7 2.5 4.5 4.5 7.0 2.0 6.0

a From H. Busch, "Histones and Other Nuclear Proteins." Academic Press, New York, 1965. The values are percentages of total moles of amino acids recovered in the particular amino acid. The data are averages from representative studies. relatively simple chemical methods t h a t separate the histenes into the four fractions. T o extract the more soluble fraction 1, crude D N P is treated with 5?9 HC10~ and the protein extracted is precipitated with 18% trichloroacetic acid. T h e residue remaining a f t e r the extraction with H C I 0 4 is then treated successively with 0.25 N HC1 in 80% ethanol to extract fractions 2a and 3 and with 0.25 N HC1 to extract fraction 2b. T h e last par~ of the procedure is similar bo t h a t shown in T a b l e IV. The yields obtained in this modified procedure are very similar to those o b ~ i n e d in the other procedure developed by Johns et al. (26-29). Starch Gel Electrophoresis o] Histone Fractions. Electrophoresis of the histories is carried out in the horizontal starch gel system described by Johns et al. (26-29). The 12% starch suspensions are prepared in 0.01 N HCI, p H 2.5, and the a p p a r a t u s used permits the formation of eight blocks, 5 m m X 15 m m X 250 mm. Samples are applied with the aid of thick filter p a p e r inserted into a narrow cut across the gel near the positive pole. Histones are dissolved in 0.1 N HC1 and applied in amounts of 0.8--1.0 mg; for fraction 1, 0.5 mg is sufficient for later staining. A voltage

[97]

NUCLEAR PROTEINS

77

gradient of 4 volts/cm is applied for 18 hours at room temperature to provide reproducible patterns. Both electrode vessels contain 0.01 N HC1 and are connected to the gel by bridges of Whatman 3MM filter paper. The tray is covered with Saran Wrap to prevent evaporation. At the end of the electrophoresis, the gels are sliced longitudinally and stained with 0.1% amido black in methanol-water-acetic acid. The technique employed for fractionation of the histones does not provide single zones on starch gel electrophoresis. For example, there are several bands in fraction 3. There is one dense and one light band in fraction 1 and small amounts of more diffuse contaminants. At least two zones are found in fraction 2b; the major one is slower moving and the other moves more rapidly. A similar number of zones is found in fraction 2a. On staining, the 2b and 2a zones are dense and the contaminants are very pale by comparison. When urea is added to the gels, there is a separation of some of the components of the F1 band. However, it is not certain whether these components are subunits of the main band, individual protein fractions, or products of denaturaion of the protein of this band. Recently, there has been a good deal of use of acrylamide electrophoresis of the histones utilizing the procedure of McAllister et al. 3° The conditions for this fractionation are very similar to those of the starch gel electrophoresis. Although the bands appear quite sharply separated, they had not been correlated with the chemical fractions in as satisfactory manner as the starch gel electrophoretic patterns until recently. NH2-Terminal Acids o] the Histone Fractions. One of the criteria of purity of these proteins is their content of various NH3-terminal amino acids (Table VI). In the fractions from the Walker tumor, 17 there are several NH2-terminal amino acids in each of the fractions, but the amounts vary from fraction to fraction. The purest fraction appears to be the 2b fraction, in which proline accounts for 80.8% or more of the NH2-terminals. In fraction 2a, two-thirds of the NH2-terminals are alanine residues and approximately 20% are glycine residues. In fraction 1 and fraction 3, alanine is also present as an NH~-terminal amino acid. Similar results are obtained for fractions of calf thymus histone. 28-~9 These results are subject to some corrections in view of the finding of Phillips ~1,32 that some of the NH2-terminal amino acids of the histones may be acetylated and the methods for determination of the NH2-terminal amino acid do not detect such substituted amino acids. He notes that NH~-terminal amino acids of the very lysine-rich histones include N H. C. McAllister, Jr., Y. C. Wan, and J. L. Irvin, Anal. Biochem. 5, 321 (1963). ~ D . M. P. Phillips, Biochem. J. 86, 397 (1963). 3: D. M. P. Phillips, Biochem. J. 87, 258 (1963).

78

PREPARATION OF NUCLEOPROTEINS

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proline, alanine, lysine, serine, and glychne. The NH2-terminal amino acids of fraction 2a are alanine, glycine, proline, and lysine. In view of the fact that the major NH~-terminal of fraction 3, the "arginine-rich" histone, is alanine and the major terminal of the N-proline histone (fraction 2b) is proline, the possibility exists that both the very lysinerich histones and fraction 2a are contaminated in part with these fractions. On the basis of the total NH2-terminals, including the acetylated NH~-terminals, the minimal molecular weights of the proteins in fractions 1 and 2a ranged from 9000 to 12,000 to 14,000, respectively. On the same basis, the minimal molecular weights of the proteins of fraction 2b and 3 are approximately 10,000. These findings are in accord with some earlier sedimentation studies28 However, these molecular weights are significantly smaller than the values derived from studies on hTH2terminals in which the degree of acetylation is not taken into account. Subfractionation of Various Histone Groups

The fact that most of the histone fractions obtained by combined chemical and chromatographic fractionation are mixtures has led to attempts to isolate pure proteins. Thus far, these attempts have not been completely successful although some progress has been reported by Phillips and Johns 3" in fractionation of the major components of the 2a fraction. In their procedure, 1 gram of the 2a fraction is dissolved at room temperature in 63 ml of 0.01 N HOl, and 2 ml of 11 N HC1 is then added followed by 179 ml of acetone (2.75 volume), with stirring. The flocculent precipitate (fraction F2al) is centrifuged down and washed with 100 ml of an identical HCl-acetone solvent and then twice more with acetone. It is dried to a white powder by evacuation of the centrifuge cup, with release of the vacuum periodically to enable the larger lumps to be broken up with a glass rod. The yield of F2al is approximately 450 mg (45?~). The supernatant from F2al is mixed with the first washing and the t~tal volume is measured; 0.9 of this volume of acetone is added. The precipitate that forms (F2a2) is centrifuged down, washed with an identical HCl-acetone solvent, and dried as described above. The yield of F2a2 is approximately 400 mg (40~). The subfractions are repreeipitated as follows. Each fraction is dissolved in 0.01 N HC1 to give about 20 mg of protein per milliliter, and 11 N HC1 is added to give a final concentration of 0.35 N. The volume is measured, and F2al is precipitated by the addition of 2.3 volume of acetone. The supernatant is discarded. Subfraction F2a2 is treated R. Trautman and C. F. Crampton, J. A m . Chem. ~Soc. 81, 4036 (1959). HD. M. P. Phillips and E. W. Johns, Biochem. J. 94, 127 (1965).

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similarly, but a n y precipitate appearing during the addition of up to 3 volumes of acetone is discarded. T h e F2a2 is then precipitated by adding acetone to give a final proportion of 6 volumes of acetone per volume of the original acid solution of F2a2. T h e yields of subfractions F 2 a l and F2a2 on reprecipitation are 80-90% of the original weight. 34 T h e 2al fraction differs from the 2a2 fraction in t h a t it contains more acetyl terminal groups and contains more giycine, valine, and arginine and less alanine and proline t h a n the 2a2 fraction (Table V I ) . TABLE VI COMPOSITION OF 2a HISTONB FRACTIONSa'b

Amino acid Alanine Arginine Aspartic acid Glutamic acid Glycine Histidine Isoleucine Leueine Lysine Methionine Proline Phenylalanine Serine Threonine Tyrosine Valine

F2al by the recommended method (average of 3)

F2a2 by the recommended method (average of 7)

7.4 12.8 5.9 7.4 15.1 2.6 5.7 8.3 10.1 0.7 1.6 2.2 2.4 6.4 3.4 8.4

12.4 9.9 6.4 9.8 10.2 2.9 4.3 11.5 11.1 0.2 4.5 1.2 3.2 4. O 2.1 6.1

a From D. M. P. Phillips and E. W. Johns, Biochem. J. 94, 127 (1965). b The amino acids are expressed as moles/100 moles of all amino acids found, and no corrections have been made for hydrolytic losses.

Separation o] Fractions o] the Very Lysine-Rich Histories. Johns 26 chromatographed fraction 1 on c a r b o x y m e t h y l cellulose and obtained three fractions. The samples were added to the columns in a borate buffer at p H 9, and the proteins were eluted with a linear gradient of NaC1 in the same buffer. About 50% of the protein was in the third fraction eluted, which had an amino acid composition v e r y similar to t h a t of the crude fraction 1. T h e amino acid compositions of the proteins in the other two peaks were v e r y similax but were r e m a r k a b l e for the v e r y large amounts of acidic amino acids present. Fractionation o] F3 Histories. Bellair and Mauritzen 3~ have sub~J. T. Bellair and C. M. Mauritzen, Australian J. Biol. Sci. 18, 160 (1965).

80

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PREPARATION OF NUCLEOPROTEINS

fractionated the histones of fraction 3 ("arginine-rich" histones) with the aid of exclusion c h r o m a t o g r a p h y on Sephadex G-200. Perspex (Lucite) columns, 180 by 6 cm, packed-bed volume 4.4 liters, are filled with Sephadex G-200 which is washed to remove fines, degassed under reduced pressure, and equilibrated with 0.3 N HC1 in 4 M urea, the same TABLE VII AMINO ACID COMPOSITION OF FRACTIONS OF F3 HISTONES OBTAINED BY EXCLUSION CHROMATOGRAPHY ON SEPHADEX G - 2 0 0 a'b

Fraction No. Parameter

7

10

Fraction as a percentage of total recovered Starch gel components present in fraction Amino acid Alanine Arginine Aspartic acid ½-Cystine Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tyrosine Valine

15.0

36.4

5 (6)

7 (6)

14.4 12.6 4.3 0.4 12.8 6.0 1.5 5.1 9.8 8.7 1.2 3.1 4.1 4.1 6.8 2.2 4.8

8.4 13.9 5.2 7.5 14.9 1.9 5.5 8.5 10.0 0.9 2.0 1.7 2.5 6.4 3.5 7.4

o From J. T. Bellair and C. M. Mauritzen, Australian J. Biol. Sc/. 18, 160 (1965). b Values as molar percentage of total amino acids. No correction has been made for the destruction of serine or threonine on hydrolysis. solution being used for elution. T h e columns are operated under 20-25 cm of solvent. T h e F3 histone is dissolved in a 4 M urea at a concentration of between 5 and 7 ~ w / v . A time-operated fraction collector is used to collect fractions of a p p r o x i m a t e l y 15 ml. Histone concentration is determined by measuring the absorption of the effluent fractions, diluted where necessary, at 230 and 280 n ~ . T h e fractions are separated into two peaks, one a m a j o r and one a

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81

smaller peak. Significant differences are found in the amino acid compositions of pooled fractions 7 and 10 as indicated in Table VII.

Nucleoproteins Chromatography of Nuc~eoproteins. Attempts have been made by a number of workers to subfractionate whole deoxyribonucleoproteins, but little success has been obtained. 2 Recently, Ohba 36 has reported that nueleohistones have been fractionated on ECTEOLA columns and differences in the composition of the various peaks have been found. The deoxyribonucleoprotein is dissolved in 0.001 M Tris buffer, pH 7.65, at a concentration of 10 mg/ml and mixed with the suspension of ECTEOLA cellulose in appropriate volume of the Tris buffer. The ratio of the amounts of deoxyribonucleoprotein to ECTEOLA is 1:60. In each experiment 16-200 mg of deoxyribonucleoprotein is applied. The mixture is stirred for 10 minutes (10 minutes are enough for deoxyribonucleoprotein to be adsorbed completely). The mixed suspension is poured into a tube of 1.5 X 50 cm, the bottom end of which is sealed with a coarse sinteredglass funnel. The column is washed with about 400 ml of 0.05 M Tris buffer (pH 7.6). Elution is carried out with a concentration gradient of sodium chloride; the reservoir contains 2 M NaC1 with 0.05 M Tris buffer at pH 7.6 and the mixing chamber contains 2 liters of the Tris buffer. The column is washed with 2 M NaC1 containing 1 M NaOH or 0.2 M Tris buffer at pH 9.8. It is not clear that the method rules out the possibility of enzymatic degradation which produces so much difficulty in the fractionation of DNA. Fractionation of Acidic Nuclear Proteins Thus far, two types of acidic nuclear proteins have been studied with respect to subfractionation, i.e., the soluble acidic proteins of nuclear ribonucleoproteins extractable with dilute saline or Tris buffer (Table II) and the proteins of the deoxyribonueleoprotein complex or the nuclear ribonucleoprotein network that are soluble in alkaline media. For reasons that are not entirely clear, the nuclear proteins soluble in isotonic saline solutions and the proteins of their ribonucleoproteins have been mainly the objective of studies with free boundary electrophoresis. 37,~s Evidence has been obtained that a variety of types of proteins are present in liver nuclei which may be grouped into basic, near-neutral, weakly acidic, highly acidic, and strongly polyanionic28 Of this group, eighteen hypothetical classes have been suggested to exist. Y. Ohba, J. Biochem. 56, 37 (1964). A. D. Barton, Z. Zelllorsch. 64, 74 (1964). 28B. Bakay and S. Sorof, Cancer Res. 24, 1814 (1964).

82

PREPARATION OF NUCLEOPROTEINS

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Changes in the distribution of these proteins have been reported following the administration of carcinogens to animals ~7,as and on starvation, ~7 but no chemical evidence has been presented on either their composition or purification. Patel and Wang ~9 have also investigated these proteins by starch gel electrophoresis and agree that a number of classes of these proteins exist. Studies on the acidic proteins of the deoxyribonucleoproteins*e,*° have been made, but other than demonstrating that these proteins contain approximately 14% glutamic acid and 10~ aspartic acid, little is known of their number or structure or function. Studies on the NH2-terminal amino acids of these proteins have shown that they have alanine, serine, and glycine as the major terminals and that smaller amounts of aspartic acid, glutamic acid, lysine, leucine, and threonine are also present. At pH 9.2 in borate buffer, Dounce and Hilgartner 4° separated two fractions of the acidic proteins that migrated in opposite directions on electrophoresis on cellulose acetate paper. Fractionation of the Nucleolar Proteins The methods used for the isolation of nucleoti have permitted studies on the nucleolar proteins. ~ For subfractionation of the nucleolar proteins, isolated nucleoli are extracted 3-5 times with 0.14M NaCl containing 0.01 M sodium citrate by homogenization of the sample in the saline solution. After each extraction, the samples are centrifuged at 17,000 g for 15 minutes. The pellets are washed once with 80% ethanol. The washed nucleolar residues are extracted twice with 0.25 N HC1 for 30 minutes and the suspensions are centrifuged for 15 minutes at 17,000 g. The acid-soluble proteins are dialyzed 48 hours against two changes of 20 liters of distilled water. After dialysis, the samples are centrifuged at 100,000 g for 30 minutes and the supernatant is lyophilized to obtain the nucleolar histories. The 0.14M NaC1 extract is treated in two ways: (1) the sample is adjusted to 20% with respect to trichloroacetic acid; or (2) it is dialyzed against 0.25 N HC1. In the first procedure, the precipitate is collected by centrifugation for 10 minutes at 17,000 g and then lyophilized. In the second procedure, the NaC1 extract is dialyzed against 0.25 N HC1 for 24 hours at 3 °. The precipitate formed during dialysis against 0.25N HC1 is separated from the supernatant by centrifugation for 10 minutes at 17,000 g. The clear supernatant is then dialyzed 24 hours against distilled water to remove the HC1. Both fractions are lyophilized. In a similar manner, the residue remaining after the extraction with 0.14 M G. Patel and T.-Y. Wang, Exptl. Cell Res. 34, 120 (1964). 4°A. L. Dounce and C. A. Hilgartner, Exptl. Cell Res. 36, 228 (1964).

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NUCLEAR PROTEINS

83

NaC1-0.01 M sodium citrate and 0.25 N HC1 extraction is lyophilized to obtain the dry powder. T h e nucleolar histones resemble the whole nuclear histenes both in amino acid analysis and starch gel electrophoretic behavior. Although the nucleolar proteins soluble in 0.14 M NaC1 are soluble in dilute acid and separate into 7 bands on starch gel electrophoresis at p H 2, they are acidic proteins, as indicated in T a b l e V I I I . T h e y differ in composition TABLE VIII AMINO ACID CONTENT OF PROTEINS OBTAINED BY FRACTIONATING LIVER NUCLEOLIa'b

Nucleoli

Fraction

NaC1-HC1soluble

HC1 residue, "residual nucleolar proteins"

Alanine Arginine Aspartic acid ½-Cystine Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tyrosine Valine Acidic: basic

9.2 3.6 11.0 1.9 11.7 8.5 2.3 3.7 8.5 8.7 1.6 3.3 6.2 6.3 4.7 1.3 6.4 1.6

7.9 5.5 9.1 1.4 12.8 8.6 2.1 4.4 9.5 7.8 1.4 3.5 5.8 7.7 4.4 1.6 6.5 1.4

a From D. Grogan, R. Desjardins, and H. Busch, Cancer Res. 26, 775 (1966). b The values are percentages of total moles of amino acid recovered and are averages of 3 to 5 experiments. The value for glycine was corrected for nucleic acid present, The average standard deviation from the mean is 8.0%. from the "residual nucleolar proteins" insoluble in 0.25 N HC1 and 0.15 M NaC1. I n normal liver nucleoli, the histones, proteins soluble in 0.14 M NaC1, and the residual nueleolar proteins each account for approximately one-third of the nucleolar proteins. In the nucleoli of the W a l k e r tumor, the residual proteins account for 60% of the total nucleolar proteins.

84

PREPARATION OF NUCLEOPROTEINS

[97]

Note Added in Proof The pace of research on fractionation of the histones is increasing rapidly, and two major breakthroughs have been made in the last six months. In the first of these, the "arginine-rich" histones have been separated by exclusion chromatography on large-scale columns (12-14 liters) containing Sephadex G-100 in bead form into 5 peaks which contain histones differing in amino acid composition, peptide fingerprint patterns, and mobility on polyacrylamide gels. 41 Of these histone fractions the histone rich in lysine and arginine (lysine-arginine rich or GAR histone) which has some similarities to histone F2al isolated by the procedure of Phillips and Johns s~ was obtained in almost pure form as indicated by the "fingerprint" and the high purity of most of the peptides now being isolated by chromatographic procedures. Recently, the separation of very lysine-rich histones (VLR histones) was reported by Kinkade and Cole. 42 In this procedure VLR histones were isolated by extraction with trichloroacetic acid and chromatographed on Amberlite IRC-50 with a 7 - 1 4 ~ gradient of guanidinium chloride 0.1 M phosphate (pH 6.8). Evidence for fractionation of the VLR histones included studies with polyacrylamide gel electrophoresis and structural analysis based upon fractionation of the tryptie digests on Dowex 50-X8. It is hoped that definition of the linear sequences of amino acids in the histones will clarify some of the currently unsolved problems of genetic control. Acknowledgment The author wishes to express his sincere appreciation for helpful criticism and comments to Drs. Donald E. Grogan, Wesley C. Starbuck, and William J. Steele and to Mr. Charles W. Taylor.

" H . Busch and C. M. Mauritzen, "Methods in Cancer Research" (H. Busch, ed.), Vol. III. Academic Press, New York, 1967. '~J. M. Kinkade, Jr. and R. D. Cole, J. Biol. Chem. 241, 5798 (1966).