Part D. Fractionation and Characterization of Nonhistone Chromosomal Proteins. I Chapter 15
The Isolation and Pzlr$cation of the High Mobility G r o ~(HM . G) Nonhistone Chromosomal Proteins GRAHAM H. GOODWIN
AND
ERNEST W. JOHNS
Division of Molecular Biology, Chester Bearty Research Institute, Institute of Cancer Research-Royal Cancer Hospital, London. England
I. Introduction Chromatin contains a group of nonhistone chromosomal proteins that are less firmly bound than the histones and can be extracted from the chromatin with 0.35 M NaCl (1). This nonhistone protein fraction is highly heterogenous (2); it is probably made up of structural proteins, nuclear enzymes, and possibly small amounts of regulatory proteins. The 0.35 MNaCl extract can be subdivided into two fractions based on their solubility in trichloroacetic acid (TCA) (2); (1) the many high-molecular-weight proteins which are insoluble in 2% TCA; and (2) the smaller number of lower-molecular-weight proteins which are soluble in 2% TCA. The former group we have termed the low mobility group (LMG) proteins and the latter the high mobility group (HMG) proteins because of their relative electrophoretic mobilities in polyacrylamide gels (2). We have been mostly concerned with the HMG proteins for they contain a number of interesting proteins with high contents of acidic and basic amino acids. These proteins were in fact originally discovered in perchloric acid extracts of chromatin and also in the histone 251
258
GRAHAM H. GOODWIN A N D ERNEST W. JOHNS
H2B fraction and were separated from the histone by carboxymethyl celluose (CM-cellulose) chromatography (3,4). Since then we have developed procedures for isolating fairly large quantities of three of the HMG nonhistone proteins, HMG1, 2, and 17 (5-9). [See Goodwin et al. (2) for the numbering of HMG proteins.] The HMG proteins bind to DNA and histones (lO,Zl), and they are probably chromatin structural proteins, though being present in smaller quantities than the histones they presumably have a more specific function. One of the HMG proteins, HMG2, exhibits multiple forms which could be due to sequence microheterogeneity (as is the case with histone HI) or postsynthetic modifications such as methylation or acetylation. This article describes in detail the large-scale isolation of proteins HMG1,2, and 17 and also the isolation of the HMG2 subfractions.
11. Isolation of HMG Proteins A schematic diagram for the isolation of proteins HMG1, 2, and 17 is given in Fig. 1.
A. Isolation of Total HMG Proteins from Calf Thymus The most convenient source for the large-scale preparation of nonhistone proteins is calf thymus, despite the fact that this tissue has a rather active protease(s) which partly degrades histone H1 and HMG protein causing a number of additional bands to appear in the 0.35 M NaCl extract (see below). To isolate HMG proteins the thymus tissue (either fresh or stored for up to 1 month at -20 "C) is blended with saline-ethylenediaminetetraacetic acid (EDTA) and the chromatin is washed several times with the salineEDTA. The crude chromatin is extracted with 0.35M NaCl and the LMG proteins and any contaminating nonchromosomal proteins precipitated with 2% TCA. After completely removing the precipitated material, the HMG proteins are recovered from the supernatant by acetone precipitation. To minimize the volume of acetone used here the precipitation is carried out at an alkaline pH-at pH 10 only 3 volumes of acetone are required to quantitatively precipitate the HMG proteins. The proteins are then converted to the chloride form by washing with acetone-HC1; this prevents aggregation of the proteins.
15.
HIGH MOBILITY NONHISTONE CHROMOSOMAL PROTEINS
259
Calf Thymus Chromatin
2%(w/vl trichloroacetic acid Precipitate low mobi!:ty group proteins
I
N H ~ O H / 3 v o l sacetone
I
CM-Sephadex C-25 chromatography
m Precipitate high mobility group(HMG1proteins
Fractions A
$ q $ y1 HMG 17
Redissolve and make
12% Iw/v) trichloroacetic acid
ii HMG 17
FIG.1. Schematic diagram for the isolation of HMG nonhistone chromosomal proteins.
METHOD Operations up to the acetone precipitation step are carried out at 4°C. One kilogram of minced calf thymus is divided into four portions and each is blended with approximately 700 ml of 0.075 M NaC1-0.025 M EDTA (PH7.5) for 2 minutes at full speed (12,000 rpm) in a domestic blender (Kenwood, Kenmix). Each homogenate is passed through a domestic nylon sieve to remove connective tissue and then centrifuged for 30 minutes at 2000 g. The four sediments are grouped into two and each is blended with 700 ml of NaCl-EDTA for 1 minute at full speed. The homogenates are centrifuged and the crude chromatin pellets washed three more times with
260
GRAHAM H. GOODWIN AND ERNEST W. JOHNS
NaCl-EDTA by blending for 30 seconds and centrifuging for 15 minutes. The pellet at this stage should be a cream color; if it is still pink it should be washed further. The two chromatin sediments are then each extracted 3 times with 500 ml of 0.35 M NaCl (adjusted to pH 7 with 1 M NaOH) by blending for 1 minute at half speed followed by centrifugation at 2000 g for 15 minutes. The total extract (3 liters) is made 2% (w/v) TCA by the addition of 100% (w/v) TCA. The precipitate is removed by centrifugation at 2000 g for 15 minutes, and the supernatant is passed through a No. 4 sintered glass funnel (Sinta Glass, Gallenkamp, England). The filtrate should be absolutely clear. The filtrate is made 0.01 M with respect to p-mercaptoethanol before precipitating the HMG proteins in the following manner at room temperature. First 15 ml of 0.880 ammonia solution is added to each liter of solution (raising the pH to about 10) followed rapidly by the addition of 3 volumes of acetone. After collecting the protein precipitate by centrifuging at 2000 g for 15 minutes, it is washed twice with acetone41 M HCl(6: 1 v/v) and then 3 times with acetone before drying under vacuum. The average yield of total HMG protein is about 1 gm.
B. Fractionation of Total HMG Protein by CM-Sephadex Chromatography: The Isolation of Proteins HMGl and HMG2 The gradient-sievorptive chromatography principle described by Kirkegaarde (12) is employed here for the initial part of the chromatography. This allows the HMG protein to be loaded onto the column at a fairly high ionic strength (0.15 M NaCl), thereby reducing aggregation. Because the column is preequilibrated at a low ionic strength an inherent salt gradient is set up in the column after the sample is applied. The acidic material elutes at the excluded column volume, and the inherent salt gradient that follows elutes HMGl at about 0.1 M NaCl. The other HMG proteins, being more firmly bound than HMG1, are subsequently brought off the column by conventional ion-exchange chromatography on applying a linear salt gradient rising from 0.15 M NaCl. The method described here is a modification of that described originally (7) in that the ratio of ion-exchanger to protein has been increased and the protein is loaded onto the column at a lower ionic strength (0.15 M rather than 0.2 M NaC1). These changes have made the chromatography very reproducible, and 150-200 mg of each of the purified proteins HMGl and HMG2 can be obtained from 2 gm of total HMG proteins. METHOD All operations were carried out at room temperature. Total HMG protein (usually 2 gm from the bulking of two of the above preparations) is dissolved
15.
26 1
HIGH MOBILITY NONHISTONE CHROMOSOMAL PROTEINS
in 10 ml of 7.5 mM sodium borate buffer (PH 8.8) containing 10 mM pmercaptoethanol.' After readjusting the pH to about 8.8 with I N NaOH the solution is dialyzed overnight versus the 7.5 m M borate-mercaptoethano1 buffer containing 0.15 A4 NaCl. (In this buffer and in the elution buffers described below containing NaCl, the NaCl is added to the 7.5 mM boratemercaptoethanol @H 8.8) buffer without readjusting the pH.) The dialyzed solution is clarified by centrifuging at 90,OOO g for 30 minutes and, if necessary, filtering through a small No. 4 sintered glass funnel. The sample is loaded onto a 5 x 50 cm column (approx. 1 liter column volume) of CM-Sephadex C25 equilibrated with the 7.5 mM sodium borate buffer (pH 8.8) containing 10 mM p-mercaptoethanol. Then 400 ml of the 7.5 mM borate-mercaptoethanol buffer containing 0.15 M NaCl is then pumped through the column at a flow rate of 2 ml/minute. This is followed at the same flow rate by a linear salt gradient, the two chambers of the gradientforming device each containing 1.6 liters of the 7.5 mM borate-mercaptoethanol buffer containing 0.15 M NaCl and 2.0 M NaCl respectively. The elution profile is shown in Fig. 2. Protein is precipitated from the fractions A-E by acidifying to 0.1 N HCl and adding 6 volumes of acetone (fractions D and E are first diluted to a salt concentration of 0.35 M NaCl). The precipitates are collected by centrifugation at 2000gfor 10 minutes, washed once with a c e t o n e 4 1 N HCl(6: I , v/v), then washed 3 times with acetone before drying under vacuum. The polyacrylamide gel electrophoretic analysis of the five fractions is shown in Fig. 3. Fraction A is acidic material (a mixture of proteins and
08 0.6 0.L
---I :: z -
0.2
0 FRACTION NUMBER
FIG.2. Elution profile of the CM-Sephadex C25 chromatography of total HMG protein.
-,
Transmittance at 280 nm; ----, sodium chloride concentration. Twenty-milliliter fractions were collected.
'When fractionating smaller quantities of protein than described here it is advisable to omit the mercaptoethanol in the chromatography buffer so as to allow the detection of protein in the eluate by reading the absorption at 220-230 nm.
262
GRAHAM H. GOODWIN AND ERNEST W. JOHNS
FIG. 3. Comparative polyacrylamide gel electrophoresis analysis of fractions A-E obtained by CM-Sephadex C25 chromatography of total HMG protein. The right-hand side of the gels shows fractions A-E as indicated; the left-hand side of each gel shows the total HMG protein.
nucleic acid). Fraction B is protein HMGl. The shoulder B'is possibly a modified HMGl but it also contains another contaminating protein and is therefore collected separately. Fraction C is protein HMGZ. Fraction D contains mainly HMG3 and HMG17. The latter protein can be obtained from this fraction by differential TCA precipitation (see Section 11,C below). Fraction E is mainly HMG8. We now have some evidence that HMG3 and HMG8 are degradation products of HMGl and histone HI respectively; HMG3 is probably the N-terminal two-thirds of HMGl and HMG8 the N-terminal half of H1 (J. M. Walker, G. H. Goodwin, and E. W. Johns, unpublished results). The amino acid analyses of proteins HMGl and HMG2 (fractions B and C) are given in Table I. Both proteins are characterized by the high contents of acidic and basic amino acids. Both proteins contain two cysteines which can form intramolecular disulfide bridges. The proteins can
15.
HIGH MOBILITY NONHISTONE CHROMOSOMAL PROTEINS
263
TABLE I AMINOACIDCOMPOSITION (MOLZ) AND N-TERMINAL HMGI, HMG2, AMINOACIDSOF PROTEIN AND HMG17 Amino acid
HMGl
HMG2
HMGI7
10.7 2.5 5.0
9.3 2.7 7.4
18.1
17.5
His Arg
1.8 2.2 2.9 3.6 21.3 1.7 3.9
8.9 6.5 8.1 2.3 0.0 0.4 1.3 2.o 2.0 3.O 19.4 2.0 4.1
11.2 1.7 3.2 11.2 11.9 9.9 17.1 2.2 0.0 0.0 0.1 1.2 0.0 0.0 24.7 0.2 4.9
N-terminal amino acid
Gly
G~Y
Pro
ASP Thr Ser Glu Pro GlY Ala Val Cys (8 Met Ile
Leu TYr Phe LYS
7.O 5.3 9.0 1.9 0.0 1.5
therefore exist in two forms (with or without disulphide bridge), and these are seen as closely running doublets on polyacrylamide gel electrophoresis (13). It has been noticed that occasionally a small amount of pink color coelutes with protein HMG2. This has been identified as a cytochrome. Its presence in the HMG preparation occurs if the chromatin is not sufficiently washed with saline-EDTA prior to the 0.35 MNaCl extraction. This material can be removed from the HMG2 protein by chromatography on CM-cellulose at pH 9 as in the preparation of protein HMG2 subfractions (see below) when it elutes at a higher salt concentration than HMG2.
C. Isolation of Protein HMG17 Protein HMG17 is soluble in 12% TCA while the other proteins in the CM-Sephadex fraction D are not, and hence it can be isolated by a simple TCA precipitation step.
264
GRAHAM H. GOODWIN AND ERNEST W. JOHNS
METHOD Fraction D protein is dissolved in water (4OC) at a concentration of about 15 mg/ml. Then 100% (w/v) TCA is added to a final concentration of 12% (w/v). The precipitate is removed by centrifuging at 1000 g for 10 minutes and the supernatant is clarified by filtering through a No. 4 sintered glass funnel. Protein HMG17 in the filtrate is precipitated by making the solution 0.2 M H,SO, and adding6volumes of acetone precooled to - 10°C. After washing the precipitate with acetone-HC1 (0.5 ml concd. HCl to 200 ml acetone) and then several times with acetone, it is dried under vacuum. Figure 4 shows the polyacrylamide gel electrophoretic analysis of the HMG17 protein. The amino acid analysis is given in Table I.
D. Isolation of Subfractions of HMG2 Isoelectric focusing of HMG2 reveals that it has four main subfractions focusing between pH 7 and 9. These subfractions can be isolated by CMcellulose chromatography at pH 9 using a shallow salt gradient (8).
I__--
*
FIG. 4. Comparative polyacrylamide gel electrophoresis of purified HMGI7 (left-hand side) and total HMG protein (right-hand side). From Goodwin et al. (7).
15.
HIGH MOBILITY NONHISTONE CHROMOSOMAL PROTEINS
265
METHOD All operations are carried out at room temperature. First 150 mg of protein HMG2 are disso2.ed in 15 ml of 7.5 rnM sodium borate buffer (pH 9) and readjusted to pH 9 with 1N NaOH. The solution is dialyzed overnight versus 4 liters of the 7.5 mMborate buffer (pH 9). After centrifugation at 35,000 g for 1 hour the solution is applied to a 2.5 x 15 cm CM-cellulose column equilibrated with 7.5 mM borate buffer (pH 9). An 800-ml linear salt gradient, 0-0.15 M NaCl dissolved in the borate buffer, is pumped
B
C
Fraction No.
FIG. 5. The elution profile of the CM-cellulose chromatography of total protein HMGZ. concentration. Five-milliliter fractions were collected. From Goodwin et al. (8).
-, Absorbance at 230 nm; ----, NaCl
266
GRAHAM H. GOODWIN AND ERNEST W. JOHNS
through the column at a flow rate of 0.8 ml/minute. Then 5-ml fractions are collected and the optical density at 230 nm is measured. The elution profile is shown in Fig. 5. Protein is precipitated from the fractions in the usual manner by acidifying to 0. 1 N HC1 and adding 6 volumes of acetone. The isoelectric focusing analysis of the total HMGZ and the five column fractions is shown in Fig. 6. The run-through peak (R) is a small amount of HMGl contamination in the HMG2, and peaks A, B, C, and D are the four HMGZ subfractions eluting in order of increasing basicity.
111. Alternative Procedures As mentioned in Section I the HMG proteins can also be extracted from chromatin with 5% perchloric acid (PCA). This forms the basis for an alternative procedure for isolating total HMG and protein HMGl (6). Briefly, 3.5 volumes of acetone and 0.03 volumes of concentrated HCl are added to the 5% PCA extract. This precipitates histone H1 which is removed by centrifugation leaving the HMG proteins in the supernatant. The HMG proteins are precipitated by the addition of another 2 volumes of acetone.
FIG. 6. Isoelectric focusing of total protein HMGZ and fractions R, A, B, C, and D obtained by CM-cellulose chromatographyof total protein HMG2. From Goodwin et al. (8).
15.
HIGH MOBILITY NONHISTONE CHROMOSOMAL PROTEINS
267
The HMGl can then be isolated from the mixture by a batch absorption method using CM-cellulose. The isolation of total HMG from chromatins other than those of calf thymus and chicken erythrocyte is preferably carried out by 5% PCA extraction rather than by the 0.35 M NaCl extraction method. This is because chromatins with substantial amounts of light “euchromatin” (e.g., liver chromatin) tends to be partially solubilized by 0.35M NaCl, resulting in the contamination of the HMG protein with substantial amounts of histone. The HMG protein prepared by the PCA extraction method can be fractionated by CM-Sephadex chromatography (Section II,B), and we have successfully isolated proteins HMGl and HMG2 from calf liver chromatin by this procedure. ACKNOWLEDGMENTS The authors thank R. Nicolas and V. Wright for their technical assistance and also Dr. J. Walker and R. Smith for amino acid analyses. The authors also thank the editors and publishers of Biochimim et Biophysim Acta and FEES Letters for allowing us to reproduce Figs. 4, 5, and 6. This investigation has been supported by grants to the Chester Beatty Research Institute (Institute of Cancer Research-Royal Cancer Hospital) from the Medical Research Council and the Cancer Research Campaign.
REFERENCES Johns, E. W., and Forrester, S., Eur. J. Biochem. 8, 547 (1969). Goodwin, G. H., Sanders, C., and Johns, E. W., Eur. J. Biochem. 38, 14 (1973). Johns, E. W., Biochem. J. 92, 55 (1964). Johns, E. W., Eur. J. Biochem. 4,437 (1968). 5. Goodwin, G. H., and Johns, E. W., Eur. J. Biochem. 40, 215 (1973). 6. Sanders, C., and Johns, E. W., Biochem. SOC.Trans. 2, 547 (1974). 7. Goodwin, G. H., Nicolas, R. H., and Johns, E. W., Biochim. Biophys. Acta 405, 280 1. 2. 3. 4.
(1 975).
8. Goodwin, G. H., Nicolas, R. H., and Johns, E. W., FEBS Lett. 64,412 (1976). 9. Sanders, C., Ph.D. Thesis, Univ. of London (1975). 10. Shooter, K. V., Goodwin, G. H., and Johns, E. W., Eur. J. Biochem. 47, 263 (1974). I / . Goodwin, G. H., Shooter, K. V., and Johns, E. W., Eur. J. Biochem. 54,427 (1975). 12. Kirkegaard, L. H., Biochemistry 12, 3627 (1973). 13. Walker, J. M., Goodwin, G. H., and Johns, E. W., Eur. J. Biochem. 62,461 (1976).
The Isolation and Pzlr$cation of the High Mobility G r o ~(HM . G) Nonhistone Chromosomal Proteins GRAHAM H. GOODWIN
AND
ERNEST W. JOHNS
Division of Molecular Biology, Chester Bearty Research Institute, Institute of Cancer Research-Royal Cancer Hospital, London. England
I. Introduction Chromatin contains a group of nonhistone chromosomal proteins that are less firmly bound than the histones and can be extracted from the chromatin with 0.35 M NaCl (1). This nonhistone protein fraction is highly heterogenous (2); it is probably made up of structural proteins, nuclear enzymes, and possibly small amounts of regulatory proteins. The 0.35 MNaCl extract can be subdivided into two fractions based on their solubility in trichloroacetic acid (TCA) (2); (1) the many high-molecular-weight proteins which are insoluble in 2% TCA; and (2) the smaller number of lower-molecular-weight proteins which are soluble in 2% TCA. The former group we have termed the low mobility group (LMG) proteins and the latter the high mobility group (HMG) proteins because of their relative electrophoretic mobilities in polyacrylamide gels (2). We have been mostly concerned with the HMG proteins for they contain a number of interesting proteins with high contents of acidic and basic amino acids. These proteins were in fact originally discovered in perchloric acid extracts of chromatin and also in the histone 251
258
GRAHAM H. GOODWIN A N D ERNEST W. JOHNS
H2B fraction and were separated from the histone by carboxymethyl celluose (CM-cellulose) chromatography (3,4). Since then we have developed procedures for isolating fairly large quantities of three of the HMG nonhistone proteins, HMG1, 2, and 17 (5-9). [See Goodwin et al. (2) for the numbering of HMG proteins.] The HMG proteins bind to DNA and histones (lO,Zl), and they are probably chromatin structural proteins, though being present in smaller quantities than the histones they presumably have a more specific function. One of the HMG proteins, HMG2, exhibits multiple forms which could be due to sequence microheterogeneity (as is the case with histone HI) or postsynthetic modifications such as methylation or acetylation. This article describes in detail the large-scale isolation of proteins HMG1,2, and 17 and also the isolation of the HMG2 subfractions.
11. Isolation of HMG Proteins A schematic diagram for the isolation of proteins HMG1, 2, and 17 is given in Fig. 1.
A. Isolation of Total HMG Proteins from Calf Thymus The most convenient source for the large-scale preparation of nonhistone proteins is calf thymus, despite the fact that this tissue has a rather active protease(s) which partly degrades histone H1 and HMG protein causing a number of additional bands to appear in the 0.35 M NaCl extract (see below). To isolate HMG proteins the thymus tissue (either fresh or stored for up to 1 month at -20 "C) is blended with saline-ethylenediaminetetraacetic acid (EDTA) and the chromatin is washed several times with the salineEDTA. The crude chromatin is extracted with 0.35M NaCl and the LMG proteins and any contaminating nonchromosomal proteins precipitated with 2% TCA. After completely removing the precipitated material, the HMG proteins are recovered from the supernatant by acetone precipitation. To minimize the volume of acetone used here the precipitation is carried out at an alkaline pH-at pH 10 only 3 volumes of acetone are required to quantitatively precipitate the HMG proteins. The proteins are then converted to the chloride form by washing with acetone-HC1; this prevents aggregation of the proteins.
15.
HIGH MOBILITY NONHISTONE CHROMOSOMAL PROTEINS
259
Calf Thymus Chromatin
2%(w/vl trichloroacetic acid Precipitate low mobi!:ty group proteins
I
N H ~ O H / 3 v o l sacetone
I
CM-Sephadex C-25 chromatography
m Precipitate high mobility group(HMG1proteins
Fractions A
$ q $ y1 HMG 17
Redissolve and make
12% Iw/v) trichloroacetic acid
ii HMG 17
FIG.1. Schematic diagram for the isolation of HMG nonhistone chromosomal proteins.
METHOD Operations up to the acetone precipitation step are carried out at 4°C. One kilogram of minced calf thymus is divided into four portions and each is blended with approximately 700 ml of 0.075 M NaC1-0.025 M EDTA (PH7.5) for 2 minutes at full speed (12,000 rpm) in a domestic blender (Kenwood, Kenmix). Each homogenate is passed through a domestic nylon sieve to remove connective tissue and then centrifuged for 30 minutes at 2000 g. The four sediments are grouped into two and each is blended with 700 ml of NaCl-EDTA for 1 minute at full speed. The homogenates are centrifuged and the crude chromatin pellets washed three more times with
260
GRAHAM H. GOODWIN AND ERNEST W. JOHNS
NaCl-EDTA by blending for 30 seconds and centrifuging for 15 minutes. The pellet at this stage should be a cream color; if it is still pink it should be washed further. The two chromatin sediments are then each extracted 3 times with 500 ml of 0.35 M NaCl (adjusted to pH 7 with 1 M NaOH) by blending for 1 minute at half speed followed by centrifugation at 2000 g for 15 minutes. The total extract (3 liters) is made 2% (w/v) TCA by the addition of 100% (w/v) TCA. The precipitate is removed by centrifugation at 2000 g for 15 minutes, and the supernatant is passed through a No. 4 sintered glass funnel (Sinta Glass, Gallenkamp, England). The filtrate should be absolutely clear. The filtrate is made 0.01 M with respect to p-mercaptoethanol before precipitating the HMG proteins in the following manner at room temperature. First 15 ml of 0.880 ammonia solution is added to each liter of solution (raising the pH to about 10) followed rapidly by the addition of 3 volumes of acetone. After collecting the protein precipitate by centrifuging at 2000 g for 15 minutes, it is washed twice with acetone41 M HCl(6: 1 v/v) and then 3 times with acetone before drying under vacuum. The average yield of total HMG protein is about 1 gm.
B. Fractionation of Total HMG Protein by CM-Sephadex Chromatography: The Isolation of Proteins HMGl and HMG2 The gradient-sievorptive chromatography principle described by Kirkegaarde (12) is employed here for the initial part of the chromatography. This allows the HMG protein to be loaded onto the column at a fairly high ionic strength (0.15 M NaCl), thereby reducing aggregation. Because the column is preequilibrated at a low ionic strength an inherent salt gradient is set up in the column after the sample is applied. The acidic material elutes at the excluded column volume, and the inherent salt gradient that follows elutes HMGl at about 0.1 M NaCl. The other HMG proteins, being more firmly bound than HMG1, are subsequently brought off the column by conventional ion-exchange chromatography on applying a linear salt gradient rising from 0.15 M NaCl. The method described here is a modification of that described originally (7) in that the ratio of ion-exchanger to protein has been increased and the protein is loaded onto the column at a lower ionic strength (0.15 M rather than 0.2 M NaC1). These changes have made the chromatography very reproducible, and 150-200 mg of each of the purified proteins HMGl and HMG2 can be obtained from 2 gm of total HMG proteins. METHOD All operations were carried out at room temperature. Total HMG protein (usually 2 gm from the bulking of two of the above preparations) is dissolved
15.
26 1
HIGH MOBILITY NONHISTONE CHROMOSOMAL PROTEINS
in 10 ml of 7.5 mM sodium borate buffer (PH 8.8) containing 10 mM pmercaptoethanol.' After readjusting the pH to about 8.8 with I N NaOH the solution is dialyzed overnight versus the 7.5 m M borate-mercaptoethano1 buffer containing 0.15 A4 NaCl. (In this buffer and in the elution buffers described below containing NaCl, the NaCl is added to the 7.5 mM boratemercaptoethanol @H 8.8) buffer without readjusting the pH.) The dialyzed solution is clarified by centrifuging at 90,OOO g for 30 minutes and, if necessary, filtering through a small No. 4 sintered glass funnel. The sample is loaded onto a 5 x 50 cm column (approx. 1 liter column volume) of CM-Sephadex C25 equilibrated with the 7.5 mM sodium borate buffer (pH 8.8) containing 10 mM p-mercaptoethanol. Then 400 ml of the 7.5 mM borate-mercaptoethanol buffer containing 0.15 M NaCl is then pumped through the column at a flow rate of 2 ml/minute. This is followed at the same flow rate by a linear salt gradient, the two chambers of the gradientforming device each containing 1.6 liters of the 7.5 mM borate-mercaptoethanol buffer containing 0.15 M NaCl and 2.0 M NaCl respectively. The elution profile is shown in Fig. 2. Protein is precipitated from the fractions A-E by acidifying to 0.1 N HCl and adding 6 volumes of acetone (fractions D and E are first diluted to a salt concentration of 0.35 M NaCl). The precipitates are collected by centrifugation at 2000gfor 10 minutes, washed once with a c e t o n e 4 1 N HCl(6: I , v/v), then washed 3 times with acetone before drying under vacuum. The polyacrylamide gel electrophoretic analysis of the five fractions is shown in Fig. 3. Fraction A is acidic material (a mixture of proteins and
08 0.6 0.L
---I :: z -
0.2
0 FRACTION NUMBER
FIG.2. Elution profile of the CM-Sephadex C25 chromatography of total HMG protein.
-,
Transmittance at 280 nm; ----, sodium chloride concentration. Twenty-milliliter fractions were collected.
'When fractionating smaller quantities of protein than described here it is advisable to omit the mercaptoethanol in the chromatography buffer so as to allow the detection of protein in the eluate by reading the absorption at 220-230 nm.
262
GRAHAM H. GOODWIN AND ERNEST W. JOHNS
FIG. 3. Comparative polyacrylamide gel electrophoresis analysis of fractions A-E obtained by CM-Sephadex C25 chromatography of total HMG protein. The right-hand side of the gels shows fractions A-E as indicated; the left-hand side of each gel shows the total HMG protein.
nucleic acid). Fraction B is protein HMGl. The shoulder B'is possibly a modified HMGl but it also contains another contaminating protein and is therefore collected separately. Fraction C is protein HMGZ. Fraction D contains mainly HMG3 and HMG17. The latter protein can be obtained from this fraction by differential TCA precipitation (see Section 11,C below). Fraction E is mainly HMG8. We now have some evidence that HMG3 and HMG8 are degradation products of HMGl and histone HI respectively; HMG3 is probably the N-terminal two-thirds of HMGl and HMG8 the N-terminal half of H1 (J. M. Walker, G. H. Goodwin, and E. W. Johns, unpublished results). The amino acid analyses of proteins HMGl and HMG2 (fractions B and C) are given in Table I. Both proteins are characterized by the high contents of acidic and basic amino acids. Both proteins contain two cysteines which can form intramolecular disulfide bridges. The proteins can
15.
HIGH MOBILITY NONHISTONE CHROMOSOMAL PROTEINS
263
TABLE I AMINOACIDCOMPOSITION (MOLZ) AND N-TERMINAL HMGI, HMG2, AMINOACIDSOF PROTEIN AND HMG17 Amino acid
HMGl
HMG2
HMGI7
10.7 2.5 5.0
9.3 2.7 7.4
18.1
17.5
His Arg
1.8 2.2 2.9 3.6 21.3 1.7 3.9
8.9 6.5 8.1 2.3 0.0 0.4 1.3 2.o 2.0 3.O 19.4 2.0 4.1
11.2 1.7 3.2 11.2 11.9 9.9 17.1 2.2 0.0 0.0 0.1 1.2 0.0 0.0 24.7 0.2 4.9
N-terminal amino acid
Gly
G~Y
Pro
ASP Thr Ser Glu Pro GlY Ala Val Cys (8 Met Ile
Leu TYr Phe LYS
7.O 5.3 9.0 1.9 0.0 1.5
therefore exist in two forms (with or without disulphide bridge), and these are seen as closely running doublets on polyacrylamide gel electrophoresis (13). It has been noticed that occasionally a small amount of pink color coelutes with protein HMG2. This has been identified as a cytochrome. Its presence in the HMG preparation occurs if the chromatin is not sufficiently washed with saline-EDTA prior to the 0.35 MNaCl extraction. This material can be removed from the HMG2 protein by chromatography on CM-cellulose at pH 9 as in the preparation of protein HMG2 subfractions (see below) when it elutes at a higher salt concentration than HMG2.
C. Isolation of Protein HMG17 Protein HMG17 is soluble in 12% TCA while the other proteins in the CM-Sephadex fraction D are not, and hence it can be isolated by a simple TCA precipitation step.
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GRAHAM H. GOODWIN AND ERNEST W. JOHNS
METHOD Fraction D protein is dissolved in water (4OC) at a concentration of about 15 mg/ml. Then 100% (w/v) TCA is added to a final concentration of 12% (w/v). The precipitate is removed by centrifuging at 1000 g for 10 minutes and the supernatant is clarified by filtering through a No. 4 sintered glass funnel. Protein HMG17 in the filtrate is precipitated by making the solution 0.2 M H,SO, and adding6volumes of acetone precooled to - 10°C. After washing the precipitate with acetone-HC1 (0.5 ml concd. HCl to 200 ml acetone) and then several times with acetone, it is dried under vacuum. Figure 4 shows the polyacrylamide gel electrophoretic analysis of the HMG17 protein. The amino acid analysis is given in Table I.
D. Isolation of Subfractions of HMG2 Isoelectric focusing of HMG2 reveals that it has four main subfractions focusing between pH 7 and 9. These subfractions can be isolated by CMcellulose chromatography at pH 9 using a shallow salt gradient (8).
I__--
*
FIG. 4. Comparative polyacrylamide gel electrophoresis of purified HMGI7 (left-hand side) and total HMG protein (right-hand side). From Goodwin et al. (7).
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HIGH MOBILITY NONHISTONE CHROMOSOMAL PROTEINS
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METHOD All operations are carried out at room temperature. First 150 mg of protein HMG2 are disso2.ed in 15 ml of 7.5 rnM sodium borate buffer (pH 9) and readjusted to pH 9 with 1N NaOH. The solution is dialyzed overnight versus 4 liters of the 7.5 mMborate buffer (pH 9). After centrifugation at 35,000 g for 1 hour the solution is applied to a 2.5 x 15 cm CM-cellulose column equilibrated with 7.5 mM borate buffer (pH 9). An 800-ml linear salt gradient, 0-0.15 M NaCl dissolved in the borate buffer, is pumped
B
C
Fraction No.
FIG. 5. The elution profile of the CM-cellulose chromatography of total protein HMGZ. concentration. Five-milliliter fractions were collected. From Goodwin et al. (8).
-, Absorbance at 230 nm; ----, NaCl
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GRAHAM H. GOODWIN AND ERNEST W. JOHNS
through the column at a flow rate of 0.8 ml/minute. Then 5-ml fractions are collected and the optical density at 230 nm is measured. The elution profile is shown in Fig. 5. Protein is precipitated from the fractions in the usual manner by acidifying to 0. 1 N HC1 and adding 6 volumes of acetone. The isoelectric focusing analysis of the total HMGZ and the five column fractions is shown in Fig. 6. The run-through peak (R) is a small amount of HMGl contamination in the HMG2, and peaks A, B, C, and D are the four HMGZ subfractions eluting in order of increasing basicity.
111. Alternative Procedures As mentioned in Section I the HMG proteins can also be extracted from chromatin with 5% perchloric acid (PCA). This forms the basis for an alternative procedure for isolating total HMG and protein HMGl (6). Briefly, 3.5 volumes of acetone and 0.03 volumes of concentrated HCl are added to the 5% PCA extract. This precipitates histone H1 which is removed by centrifugation leaving the HMG proteins in the supernatant. The HMG proteins are precipitated by the addition of another 2 volumes of acetone.
FIG. 6. Isoelectric focusing of total protein HMGZ and fractions R, A, B, C, and D obtained by CM-cellulose chromatographyof total protein HMG2. From Goodwin et al. (8).
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The HMGl can then be isolated from the mixture by a batch absorption method using CM-cellulose. The isolation of total HMG from chromatins other than those of calf thymus and chicken erythrocyte is preferably carried out by 5% PCA extraction rather than by the 0.35 M NaCl extraction method. This is because chromatins with substantial amounts of light “euchromatin” (e.g., liver chromatin) tends to be partially solubilized by 0.35M NaCl, resulting in the contamination of the HMG protein with substantial amounts of histone. The HMG protein prepared by the PCA extraction method can be fractionated by CM-Sephadex chromatography (Section II,B), and we have successfully isolated proteins HMGl and HMG2 from calf liver chromatin by this procedure. ACKNOWLEDGMENTS The authors thank R. Nicolas and V. Wright for their technical assistance and also Dr. J. Walker and R. Smith for amino acid analyses. The authors also thank the editors and publishers of Biochimim et Biophysim Acta and FEES Letters for allowing us to reproduce Figs. 4, 5, and 6. This investigation has been supported by grants to the Chester Beatty Research Institute (Institute of Cancer Research-Royal Cancer Hospital) from the Medical Research Council and the Cancer Research Campaign.
REFERENCES Johns, E. W., and Forrester, S., Eur. J. Biochem. 8, 547 (1969). Goodwin, G. H., Sanders, C., and Johns, E. W., Eur. J. Biochem. 38, 14 (1973). Johns, E. W., Biochem. J. 92, 55 (1964). Johns, E. W., Eur. J. Biochem. 4,437 (1968). 5. Goodwin, G. H., and Johns, E. W., Eur. J. Biochem. 40, 215 (1973). 6. Sanders, C., and Johns, E. W., Biochem. SOC.Trans. 2, 547 (1974). 7. Goodwin, G. H., Nicolas, R. H., and Johns, E. W., Biochim. Biophys. Acta 405, 280 1. 2. 3. 4.
(1 975).
8. Goodwin, G. H., Nicolas, R. H., and Johns, E. W., FEBS Lett. 64,412 (1976). 9. Sanders, C., Ph.D. Thesis, Univ. of London (1975). 10. Shooter, K. V., Goodwin, G. H., and Johns, E. W., Eur. J. Biochem. 47, 263 (1974). I / . Goodwin, G. H., Shooter, K. V., and Johns, E. W., Eur. J. Biochem. 54,427 (1975). 12. Kirkegaard, L. H., Biochemistry 12, 3627 (1973). 13. Walker, J. M., Goodwin, G. H., and Johns, E. W., Eur. J. Biochem. 62,461 (1976).
