ANALYTICAL
BIOCHEMISTRY
88,
388-398 (1978)
A Preparative and Analytical Microscale Procedure Nonhistone Chromosomal Proteins
for
CHRISTIAN KRUGERAND K. POLLOW Freie Vniversitiit Berlin, lnstitut fiir Molekularbiologie und Biochemie, Arnimallee 1000 Berlin 33, Germany
22,
Received April 4, 1977; accepted February 10, 1978 A nonhistone chromosomal protein (NHC proteins) fractionation scheme is described, which enables us to obtain separations of these proteins on a preparative scale in milligram quantities. The following experimental procedure was applied: differential dissociation of chromatin, cation exchange chromatography on BioRex 70, preparative fractionation of those NHC proteins which are not absorbed on Bio-Rex 70, and determination of the isoelectric point range in a zone convection-focusing apparatus. In a subsequent continuous (O-40% acrylamide) microgradient SDS-gel electrophoresis system, we could detect more than 440 different NHC protein components of cow’s udder.
It has been demonstrated that nonhistone chromosomal (NHC) proteins are involved in the control of specific gene transcription for the globin gene (l-3) and for the histone genes (4), but investigating their precise role, chemical and functional properties, and the production of antibodies against specific NHC proteins requires their isolation and fractionation. Several methods recover only a portion of chromosomal proteins (5) or utilize strange chemical conditions (6-8) for their isolation. The chromatin dissociation in DNA, histones, and NHC proteins is usually carried out in salt or salt/urea solutions (9- 14) or by gel filtration (15,16), and the histones and NHC proteins are fractionated by ion-exchange chromatography (ll20). Some investigators use hydroxylapatite chromatography for the fractionation of all three components in one step (21,22). Because of the limited resolution of the present methodology, the total number of NHC proteins in any one tissue is not known. Several authors used the two-dimensional gel electrophoresis technique for determining the heterogeneity of NHC proteins (6,23-25). The most promising study on this problem came from Peterson and McConkey (26), who applied a two-dimensional polyacrylamide technique, first introduced by O’Farrell (27), to NHC proteins of HeLa cells. They found about 400 NHC protein components. A separation of NHC proteins on a preparative scale and a molecular
0003-2697/78/0882-0388$02.00/O Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
388
PREPARATION
OF NONHISTONES
389
weight fractionation of these proteins on a linear polyacrylamide pore gradient in a microscale are described in this paper. This approach is very similar to that of Sevalevic and Stamenkovic (28), and it also bears some similarity to the procedure of MacGillivray and Rickwood (29), but it overcomes precipitation problems of NHC proteins found when the isoelectric focusing procedure is carried out in a sucrose or glycerol-stabilized pH gradient (30). We therefore constructed a zone convection electrofocusing apparatus (31), because in this apparatus the precipitates remain in the compartment where they are first collected. We wish to communicate that the microgradient SDS-gel electrophoresis (32) of prefractionated NHC proteins in lo-$ capillaries as an analytical end point analysis is a powerful method for determining the heterogeneity of NHC proteins. Lactating cow’s udder was chosen, because of its relatively high protein synthetic activity (33) and because it is a hormone-sensitive tissue (34). MATERIALS
AND METHODS
Tissue. Cow’s udders from the 18th week of lactation were obtained from the Max-Planck-Institut fur Tierzucht und Tierernahrung (Trenthorst, Germany). Chromatin fractionation. To avoid proteolytic processes, 1 to 5 mM of the protease inhibitor sodium hydrogensulfite (35) was added to all buffers used in isolating nuclei and in preparing the chromatin. Triton X-loo-treated nuclei from cow’s udder were purified over three different sucrose (0.25,0.8,2 M) centrifugation steps by the slightly modified method of Blobel and Potter (36). The informofers were extracted by the method of Samarina et al. (37), and the chromatin was prepared by the saline EDTA procedure of Graziano and Huang (17). The degree of purity of the nuclei and chromatin was checked by electron microscopy. The chromatin was first dissociated (1 mg of chromatin DNA/ml of dissociation buffer) in a 0.1 M sodium phosphate buffer (pH 7.0) containing 0.35 M guanidine hydrochloride, 6 M urea, 1.5 M sodium hydrogensulfite, 2 mM EDTA, and 2 mM dithiothreitol(O.35 M guanidine dissociation buffer) (14). The partially dissociated chromatin DNA was sedimented at 176,000g for 48 hr. Crystalline reagents were added to the sediment to a final concentration of 3 M NaCl, 6 M urea, 1.5 mM sodium hydrogensulfite, 2 mM EDTA, and 2 mM dithiothreitol, 1 mM Tris-HCl buffer (pH 8.3) (3 M NaCl dissociation buffer) at a chromatin DNA concentration of 1 mg/ml. The suspension was sheared in an Ultra-Turrax for 3 min at 60 V, and centrifugation at 254,000g for 60 hr was performed to sediment the residual DNA. The supernatants containing the 0.35 M guanidine or 3 M NaCl-dissociated chromosomal proteins, respectively, were removed. The 3 M NaCl-dissociated chromosomal proteins were dialyzed against 0.35 M
390
KRUGER
AND
POLLOW
guanidine dissociation buffer for 8 hr at 4°C. The chromosomal proteins were then fractionated on the cationic exchanger Bio-Rex 70 (running buffer: 0.35 M guanidine dissociation buffer) into a strongly basic fraction which is adsorbed on Bio-Rex 70 under the running buffer conditions (histone fraction) and NHC proteins, which are not adsorbed by the resin (14). The histone fractions were eluted with 0.1 M sodium phosphate buffer (pH 7) containing 4 M guanidine hydrochloride, 6 M urea, and 1 mM sodium hydrogen sulfite. The basic proteins were made 50 mM in mercaptoethanol, reduced for 3 hr at 37”C, precipitated in a dialysis bag against 4 M ammonium sulfate, and dialyzed extensively against 0.1 N acetic acid, 1 mM mercaptoethanol. The chromosomal proteins not adsorbed by Bio-Rex 70 were dialyzed and quantitatively precipitated for 35 hr at 4°C in a dialysis bag against 4 M ammonium sulfate and 2 mM mercaptoethanol. The precipitated proteins were extensively dialyzed against 50 mM dithiothreitol and 7 M urea, reduced for 3 hr at 37°C and again dialyzed against 7 M urea, 2 mM dithiothreitol. Isoelectric focusing and microgradient gel electrophoresis. The outer chambers of the lower part of the zone convection isoelectric focusing apparatus, first described by E. Valmet (31), were filled with electrode solutions (cathode: 50 mM sodium/bicarbonate, pH 10, 12% sucrose; anode: 1% acetic acid, pH 2.8, 12% sucrose), and the following two chambers were filled with electrode protection solution containing 12% sucrose and 4% ampholine (pH 3- 10). The remaining chambers were filled with 88 mg (0.35 M Guanidine dissociation buffer), or 62 mg (3 M NaCl dissociation buffer) of NHC proteins containing 2 mM dithiothreitol, 7 M urea, and 3% ampholine (pH 3- 10). The focusing procedure was carried out for 60 hr at a constant wattage (3 W). The pH gradient was determined with a glass microelectrode inside the chambers. The solutions of two to three chambers were pooled, and the protein content was determined by the method of Lowry (38), and DNA and RNA contamination were determined by the method of Burton (40) and Ogur (43). All fractions were first dialyzed for 25 hr (changed four times) against 5 mM mercaptoethanol, 7M urea and then against 5 mM mercaptoethanol for 25 hr (changed four times) and lyophilized. In the last step of fractionation, the slightly modified method of continuous microgradient acrylamide (O-40%) gel electrophoresis of Rtichel et al, (32) under reducing SDS-electrode buffer conditions [0.05 M Tris-glycine, pH 8.4; 0.1% SDS, 0.1% (v/v) thioglycolic acid] was used. To obtain maximal dodecylsulfate charging, we incubated 1 mg of each NHC protein fraction with 1 ml of the following sample solution: 0.035 M Tris-sulfate buffer (pH 8.6), 1% dithiothreitol, 1% SDS, and IO% glycerol for 2 min at 100°C according to the method of Laemmli (39). The sample solution, l-2 ~1, was applied on top of a 0 to 40% microgradient gel in a lo-p1 capillary. Electrophoresis was carried out in the first 5 min at 40 V, at 80 V until the tracking dye was leaving the gel, and
PREPARATION Preparation Cell
pocdure
with
for the chromosomal
prote#nr
nuclei
Lysls
Dlfferentel 1,
391
OF NONHISTONES
0.35
M
Gus
chromatxn
of
the
inner
envelope
dissocaatoan
-dirsocntlon-buffer
2.
with
3M
NaCl
Extract.
3M
NaCl
BID-Rex
70
w-exchange
dlssoclatlon-buffer
I t
1
mm Extract:
0.35
M Gus.
chromosomal
proteins
chramosomal
protans
I
BIO-Rex
2 D
70
xx-exchange
chromatography
gelelectro-
3M
Fractionauon
phoresls
by
0 35 M Gus. Fr.
1.
2.
lxelectric
focusing
4.
5.
3 M 6.
B
7.
9.
v/t Amino
acid
analysts
NHC
fraction
I” a Valmet-focusing
NHC-prote,ns 3.
NaCl
NaCl 10
chromatography
3 M NaCl
apparatus
2 D
NHC-pro,e,ns 11.
12.
fract,on
gelelectro-
phoresas 13
14.
+\v Fractlonatlon
by
molecular
weight
in micro-gradlent-acrylamidege14
SCHEME
Amkno
acid
analysts
I
afterward at 100 V for 30 min. Immediately after electrophoresis the gels were expelled from the capillaries by pressure on the 40% end with a stainless steel wire. The dodecylsulfate proteins were stained for 10 min at 50°C in a 0.18% (w/v) Coomassie brilliant blue R-250 solution containing 7% acetic acid and 93% methanol/water (1: 1). The gels were destained and stored in 7% acetic acid. The molecular weights of the NHC protein components were determined by comparing the equivalent position of the marker proteins phosphorylase a, aldolase, and cytochrome c in the gradient gel. The overall preparation scheme of the chromosomal proteins is shown in Scheme 1. Amino acid analysis. Samples of about 10 nmol of the various preparatively prepared chromosomal proteins were hydrolyzed in 400 ~1 of quartzdistilled 6 N HCl in sealed tubes under Nz for 24 hr at 110°C. Amino acid composition was determined with a Durrum amino acid analyzer. Apparatus and reagents. The details of the homemade zone convection electrofocusing apparatus are described in the thesis of Kruger (30). The power supply and stands for capillary microelectrophoresis and forceps were obtained from E. Schtitt (Gottingen, Germany), and lo-p1 capillaries (32 mm) were from Brand (Wertheim, Germany). The Ultra-Turrax was obtained from Janke & Kunkel KG (Staufen im Breisgau, Germany), BioRex 70 (100-200 mesh) was from Bio-Rad Laboratories (Miinchen, Germany), and guanidine hydrochloride and sucrose were from Merck
KRUGER
392
AND PGLLOW
(Darmstadt, Germany). Acrylamide and NJ’-methylenebisacrylamide, urea, and sodiumdodecylsufate were obtained from Serva (Heidelberg) and prepared as described (28). Triton X-100, Coomassie brillant blue R-250, and thioglycolic acid were also obtained from Serva. Ampholine (pH 3-10) was purchased from LKB (Bromma, Sweden). The proteins used for molecular weight calibrations of the microgradient gels were phosphorylase a, (MW 92,500), aldolase (MW 39,500), and cytochrome c (MW 12,500) (all from Boeringer, Mannheim). RESULTS
We define in this communication the NHC proteins as those chromosomal proteins which are not adsorbed by the cationic exchanger Bio-Rex 70 under the running buffer conditions. This is not quite exact, because there are about 30 strongly basic minor NHC protein components, which are adsorbed together with the histones on Bio-Rex 70 under the running buffer conditions, as demonstrated by two-dimensional gel electrophoresis (30), and which will be published elsewhere. The NHC proteins of cow’s udder comprise nearly 50% of the total chromosomal proteins by weight. Eighty-two percent of the total dissociated nonhistones were peeled off from the chromatin by the 0.35 M guanidine dissociation step; about 18% of the nonhistones dissociate under the 3 M NaCl dissociation conditions. The results from the preparative isoelectric focusing procedure (Fig. 1) clearly indicate that the complex NHC protein mixture of the 0.35 M guanidine dissociated as well as that of the 3 M NaCl-dissociated NHC proteins has quantitative maxima between the isoelectric points (IP) of pH 20
1 % NHC proteins
16
12 aa
4
0
FIG. 1. Isoelectric point range and percentage (related to the total dissociated NHC proteins) of the differentially dissociated and preparatively isoelectrically focused NHC proteins of cow’s udder (For details see under Materials and Methods). (a) 0.35 M Guanidine dissociation. The chromosomal proteins are dissociated in 0.35 M guanidine hydrochloride, 2 mM dithiothreitol, 1.5 mM sodium disulfite, 2 mM EDTA, 0.1 M sodium phosphate buffer (pH 7). (b) The remaining DNA: NHC protein complex was sheared and dissociated in 3 M NaCl, 6 M urea, 2 mM dithiothreitol, 2 mM EDTA, 1.5 mM sodium disulfite, 1 mM TrisiHCl buffer (pH 8.3).
PREPARATION 0.35
M
OF NONHISTONES
393
. Gua . Dtssoclatlan
a
-
Phosphorylase
Aldolase
a
d_
-
92500
-
39500--
-
-
FIG. 2. (a) Microgradjent acrylamide gel electrophoresis, showing the heterogeneity of the different NHC protein fractions of cow’s udder. (b) Reproduction of the microgradient gels shown in (a) by visual detection of the Coomassie-stained bands. The marker proteins in the SDS microelectrophoresis in continuous polyacrylamide gradient gels are phosphorylase a, aldolase, and cytochrome c.
5.5 and 6.0 and that the NHC proteins not adsorbed by Bio-Rex 70 have IPs over the total pH range of ampholines used. With the zone convection focusing procedure, we obtain preparatively NHC protein fractions which comprise as little as 1% of the total NHC protein mass. We could not detect
394
KRUGER
AND POLLOW
DNA or RNA in this fraction. That the differentially dissociated NHC proteins are really different components is shown in Fig. 2 and Table 1. The occurrence of high molecular weight protein components in the 3 M NaCldissociated fraction is obvious. We could detect visually a total of ca. 440 NHC protein components from 14 different preparatively focused fractions: 247 NHC protein components have a molecular weight greater than 90,000; 111 have molecular weights between 40,000 and 90,000; and 84 have molecular weights between 10,000 and 40,000. The NHC proteins with molecular weights smaller than 6000 pass through the 40% gel under the experimental conditions, as has been proved for insulin. TABLE QUANTITATIVE
COMFQSITION FRACTIONS
NHC
proteins
of the 0.35 M guanidine
Number of bands
MW
IF
>9O,OC4 4o,coo-9o,ooo* 10$0040,ooo
3.5-4.3
+
1
OF THE OF
NHC
16 5 3
I.8
4.1-4.9
17 7 8
7.0
5.0-5.4
16.1
5.6-5.9
>90,00+ 4o,muLsO,OaO lO.Oc&40,000
+
I9 9 6
3.7
>9o,ooo 40.00&90,OKl~ 10.000-40.000
+
6.0-6.7
3.2
-
23 9 4
290.tmo 4o,OOc-swOO lO,OOO-40,ooo
+
24 7 4
3.0
8.2-8.7
>9o.m 40300-90,ooo 10.000-40,000
-9 + +
-
8.8-9.5
>9o,ooo -+ 4o.o00-90,000 -t lO,coO-4O,OOll-
>90,000 4o,ooo-swJoo lO,tM-40,ooO
+ + -
18 I3 8
17.1
5.9-6.0
>90,004 4o,lmo-90,m 10,tmO-40,@30
-+
I7 I2 9
ND
7.0-8.
6.1-6.7
>9o,m 4o,ooo-90,@30 lO,Ow-40,cmO
-3 + -t
I7 I2 9
II.6
290,m 40.000-90,cw 10,ln3o-40#00
+
17 IO I2
12.2
>9o,cunl 40,00&90,ooo 10,Ow-40,000
+ -
20 IO 9
15.8
8.6-9.7
a The 0.35 M guanidine-dissociated L The 3 M N&I-dissociated NHC r Isoelectric point. d Not determined.
1.7
2.8
+
I
ND I7 8 5
I5 4 6
6.9-8.
Percentage of the total NHC proteins
-f +
*
5.5-5.8
dissociation’
Number of bands
MW
>9o,ooo
>9o,m 4o,cnm-9o,ooo+ lO,OUO-40,OlM
4.8-5.3
of the 3 M NaCl
40,000-90,000 10.000-40,000
* -+
proteins
IP
>9o,ooo 4o,ooo-9o.ooo~ lO,WO-40,000
4.4-4.7
PROTEIN
UDDER
dissociation” Percentage of the total NHC proteins
NHC
DIFFERENT
Cow’s
I
NHC proteins are 82% of the total dissociated proteins are 18% of the total dissociated NHC
-t
NHC proteins. proteins.
7
20 5 I
2.4
0.9
21.6
6.9
4.0
Asp, Glu
Lys. Arg, His
Acidic* BaSlCS
2.6
II.8
31.1
1.9
13.2
25.4
3.0 8.5 2.2 3.1 1.8 5.3 6.1
10.3 6.2 7.4 15.1 5.5 9.9 8.2 5.8
5.5-5.8
‘I Moles per lo0 moles of total amino acids found. (i Decomposltwn of purines might have occurred. which
3.3
8.5
27.9
5.7 7.3 2.6
3.9 9.7 4.3 4.3 0.7 3.4 4.4
5.2 6.6
14.1 5.0 7.9 17.0 5.3 5.6 10.5 6.1
13.4 5.8 6.3 14.5 5.4 9.3 7.3 6.1
12.4 12.6 6.2 15.2 5.3 8.6 8.7 5.2 3.2 8.2 2.7 3.2 I.1 3.0 2.8
ASP Thr Ser au Pro GIY Ala Vd Met Be LW TY~ Phe His LYS AW
4.8-5.3
accounts
1.9
12.4
23.8
10.9 5.7 6.3 12.9 6.4 10.7 7.8 5.6 4.4 8.6 2.8 3.5 1.6 5.3 5.5
5.9-6.0
IP range
THE
I.0
of glycine.
0.5
25.8
Total basics 18.5
5.0 6.2 5.8 8.8 4.2 8.1 10.9 6.1 0.7 4.6 7.3 2.9 2.0 1.9 14.0 9.9
Histone fraction
HISTONE
13.8
AND
acidics 19.3
Total
8.0 4.9 5.5 II.3 6.3 11.0 10.4 5.9 4.3 9.0 2.1 2.2 1.8 9.3 7.4
8.6-9.7
high confenf
1.3
15.9
20.8
9.0 5.1 6.1 II.8 6.0 9.9 8.9 6.0 0.5 4.1 8.9 2.4 3.0 1.8 7.2 6.9
6.9-8.1
for the relatively
I.5
15.0
22.7
9.6 5.6 6.6 13.1 5.3 9.5 8.1 6.1 0.8 4.3 9.2 2.6 3.2 2.0 6.4 6.6
6.1-6.7
2
PREPARATIVELY
FOCUSING
DIFFERENT
ISOELECTRIC
OF
dissociation
AFTER
COMPOSITION”
0.35 M Guanidine
4.4-4.7
acid
ACID
3.5-4.3
Amino
AMINO
TABLE
2.3
II.6
26.8
3.8 8.7 2.7 3.4 1.3 4.6 5.7
II.7 4.9 6.8 15.1 6.0 II.7 7.6 5.3
5.0-5.4
FRACTIONS
PREPARED
1.9
13.0
25.4
3.7 9.3 2.7 3.0 1.3 5.2 6.5
10.4 4.5 7.1 15.0 4.8 13.0 7.9 5.0
5.6-5.9
NHC
1.7
12.5
20.9
8.9 4.6 5.8 12.0 8.5 20.9 8.5 4.0 2.9 6.3 2.0 2.6 2.0 4.8 5.7
6.0-6.7
Dissociation
FRACTIONS
I.8
9.3
16.5
6.9 3.0 4.8 9.6 11.7 31.30 II.7 2.3 1.9 4.2 0.8 1.6 0.9 2.8 5.6
7.0-8.1
IP range
3 M NaCl
PROTEIN
1.7
8.6
I.7
8.3
14.4
1.2 0.7 2.5 5.1
1.3 0.7 2.5 5.4
14.3
1.5 3.1
5.9 2.2 4.5 8.5 12.0 36.2O 13.0 2.4
8.8-9.5
1.6 3.2
6.0 2.2 4.1 8.3 12.3 36.3b 12.8 2.2
8.2-8.7
0.5
25.6
14.4
5.3 5.9 7.4 9.1 4.2 8.2 10.6 5.9 4.6 7.2 2.8 2.0 2.0 13.7 9.9
Histone fraction
396
KRUGER
AND
POLLOW
The most heterogeneous NHC protein fraction in relation to the isoelectric point range is the one with the isoelectric points between 5.5 and 6.5. The most heterogeneous NHC protein fraction, in relation to the total NHC protein mass, comprises proteins of the 3 M NaCl-dissociated fraction with isoelectric points between pH 8.8 and 9.5. The total heterogeneity of NHC proteins is further supported by the quantitative differences in the amino acid composition of the different fractions. (Table 2) DISCUSSION
Attempts to characterize chemically the NHC proteins are impeded by the major obstacle that we did not have any functional criteria for supposing that an isolated NHC protein functioned on the chromosome, in the cytoplasm, on both, or on the nuclear matrix. With the chromosomal protein preparation procedure reported herein, we could isolate distinct NHC protein classes under conditions which normally do not irreversibly denature proteins (9). The combination of preparative isoelectric focusing and microgradient gel electrophoresis allows for a high degree of resolution and the enhancement of the sensitivity for rare chromosomal proteins. The appearence of low molecular weight proteins in the alkaline end of the pH gradient in the Valmet-focusing procedure from the 0.35 M Guanidine-solubilized protein fraction (Figs. 2a and 2b, pH 6.9-8.1; 8.6-9.7) suggests the possibility that the Bio-Rex 70 ion exchange procedure for separation of histones from NHC proteins might not work efficiently enough under the experimental conditions, because the histones would migrate to similar positions in the microgradient gel. At the same time the ratio of histones to NHC proteins would not reflect the original ratio of this two types of proteins in the chromatin. The molecular weight determination of the NHC proteins might be misleading sometimes, especially in the high molecular weight range, because tightly bound nucleic acids not detectable by the method of Burton (40) might retard the migration of NHC protein-nucleic acid complexes (42). From the results shown in Fig. 2, we could observe several bands of identical molecular weight in adjacent pH fractions. It could be possible that some protein components “smeared” over a wide pH range. This type of cross-contamination would reduce the total number of protein components resolved by the system. The potential of our fractionation scheme for determining the NHC protein heterogeneity will be tested on material from five different areas of the brain. This work is now in preparation. The heterogeneity of NHC proteins and their frequency of occurrence with different molecular weights and different isoelectric points raise important questions regarding the structure of chromatin, the interaction of these proteins with different
PREPARATION
DNA sequence families, mRNA transcription.
397
OF NONHISTONES
and the basic principle
of gene regulation
and
ACKNOWLEDGMENTS We thank Dr. H. Endou from the University of Tokyo for technical assistance with the microgradient gels. This work was supported by funds of the Deutsche Forschungsgemeinschaft.
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398
KRUGER
AND POLLOW
32. Rtichel, R., Mesecke, S., Wolfrum, D. I., and Neuhoff, V. (1973) Hoppe-Seyler’s Z. Physiol. Chem. 354, 1351-1368. 33. Witt, M., Flock, D., and Pfleiderer, U. E. (1969)Z. Tierziicht. Zlchtungsbiol. 86, l-26. 34. Gardner, D. G., and Wittliff, J. L. (1973) Biochemisfry 16, 3090-3096. 35. Garrels, J. I., Elgin, S. C. R., and Bonner, J. (1972) Biochem. Biophys. Res. Commun. 46, 545-551. 36. Blobel, G., and Potter, Van R. (1966) Science 154, 1662-1665. 37. Samarina, 0. P., Lukanidin, E. M., Molnar, J., and Georgiev, G. P. (1968)J. Mol. Biol. 33, 251-263. 38. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, J. R. (1951)J. Biol. Chem. 193, 265-275. 39. Laemmli, U. K. (1970) Nature (London) 227, 680-685. 40. Burton, K. (1956) Biochem. J. 62, 315-323. 41. Liew, C. C., and Chan, P. K. (1976) Proc. Nat. Acad. Sci. USA 73, 3458-3462. 42. Bhojee, J. S., and Pederson, T. (1976) Anal. Biochem. 71, 393-404. 43. Ogur, M., and Rosen, R. (1950) Arch. Biochem. Biophys. 25, 262-276.
BIOCHEMISTRY
88,
388-398 (1978)
A Preparative and Analytical Microscale Procedure Nonhistone Chromosomal Proteins
for
CHRISTIAN KRUGERAND K. POLLOW Freie Vniversitiit Berlin, lnstitut fiir Molekularbiologie und Biochemie, Arnimallee 1000 Berlin 33, Germany
22,
Received April 4, 1977; accepted February 10, 1978 A nonhistone chromosomal protein (NHC proteins) fractionation scheme is described, which enables us to obtain separations of these proteins on a preparative scale in milligram quantities. The following experimental procedure was applied: differential dissociation of chromatin, cation exchange chromatography on BioRex 70, preparative fractionation of those NHC proteins which are not absorbed on Bio-Rex 70, and determination of the isoelectric point range in a zone convection-focusing apparatus. In a subsequent continuous (O-40% acrylamide) microgradient SDS-gel electrophoresis system, we could detect more than 440 different NHC protein components of cow’s udder.
It has been demonstrated that nonhistone chromosomal (NHC) proteins are involved in the control of specific gene transcription for the globin gene (l-3) and for the histone genes (4), but investigating their precise role, chemical and functional properties, and the production of antibodies against specific NHC proteins requires their isolation and fractionation. Several methods recover only a portion of chromosomal proteins (5) or utilize strange chemical conditions (6-8) for their isolation. The chromatin dissociation in DNA, histones, and NHC proteins is usually carried out in salt or salt/urea solutions (9- 14) or by gel filtration (15,16), and the histones and NHC proteins are fractionated by ion-exchange chromatography (ll20). Some investigators use hydroxylapatite chromatography for the fractionation of all three components in one step (21,22). Because of the limited resolution of the present methodology, the total number of NHC proteins in any one tissue is not known. Several authors used the two-dimensional gel electrophoresis technique for determining the heterogeneity of NHC proteins (6,23-25). The most promising study on this problem came from Peterson and McConkey (26), who applied a two-dimensional polyacrylamide technique, first introduced by O’Farrell (27), to NHC proteins of HeLa cells. They found about 400 NHC protein components. A separation of NHC proteins on a preparative scale and a molecular
0003-2697/78/0882-0388$02.00/O Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
388
PREPARATION
OF NONHISTONES
389
weight fractionation of these proteins on a linear polyacrylamide pore gradient in a microscale are described in this paper. This approach is very similar to that of Sevalevic and Stamenkovic (28), and it also bears some similarity to the procedure of MacGillivray and Rickwood (29), but it overcomes precipitation problems of NHC proteins found when the isoelectric focusing procedure is carried out in a sucrose or glycerol-stabilized pH gradient (30). We therefore constructed a zone convection electrofocusing apparatus (31), because in this apparatus the precipitates remain in the compartment where they are first collected. We wish to communicate that the microgradient SDS-gel electrophoresis (32) of prefractionated NHC proteins in lo-$ capillaries as an analytical end point analysis is a powerful method for determining the heterogeneity of NHC proteins. Lactating cow’s udder was chosen, because of its relatively high protein synthetic activity (33) and because it is a hormone-sensitive tissue (34). MATERIALS
AND METHODS
Tissue. Cow’s udders from the 18th week of lactation were obtained from the Max-Planck-Institut fur Tierzucht und Tierernahrung (Trenthorst, Germany). Chromatin fractionation. To avoid proteolytic processes, 1 to 5 mM of the protease inhibitor sodium hydrogensulfite (35) was added to all buffers used in isolating nuclei and in preparing the chromatin. Triton X-loo-treated nuclei from cow’s udder were purified over three different sucrose (0.25,0.8,2 M) centrifugation steps by the slightly modified method of Blobel and Potter (36). The informofers were extracted by the method of Samarina et al. (37), and the chromatin was prepared by the saline EDTA procedure of Graziano and Huang (17). The degree of purity of the nuclei and chromatin was checked by electron microscopy. The chromatin was first dissociated (1 mg of chromatin DNA/ml of dissociation buffer) in a 0.1 M sodium phosphate buffer (pH 7.0) containing 0.35 M guanidine hydrochloride, 6 M urea, 1.5 M sodium hydrogensulfite, 2 mM EDTA, and 2 mM dithiothreitol(O.35 M guanidine dissociation buffer) (14). The partially dissociated chromatin DNA was sedimented at 176,000g for 48 hr. Crystalline reagents were added to the sediment to a final concentration of 3 M NaCl, 6 M urea, 1.5 mM sodium hydrogensulfite, 2 mM EDTA, and 2 mM dithiothreitol, 1 mM Tris-HCl buffer (pH 8.3) (3 M NaCl dissociation buffer) at a chromatin DNA concentration of 1 mg/ml. The suspension was sheared in an Ultra-Turrax for 3 min at 60 V, and centrifugation at 254,000g for 60 hr was performed to sediment the residual DNA. The supernatants containing the 0.35 M guanidine or 3 M NaCl-dissociated chromosomal proteins, respectively, were removed. The 3 M NaCl-dissociated chromosomal proteins were dialyzed against 0.35 M
390
KRUGER
AND
POLLOW
guanidine dissociation buffer for 8 hr at 4°C. The chromosomal proteins were then fractionated on the cationic exchanger Bio-Rex 70 (running buffer: 0.35 M guanidine dissociation buffer) into a strongly basic fraction which is adsorbed on Bio-Rex 70 under the running buffer conditions (histone fraction) and NHC proteins, which are not adsorbed by the resin (14). The histone fractions were eluted with 0.1 M sodium phosphate buffer (pH 7) containing 4 M guanidine hydrochloride, 6 M urea, and 1 mM sodium hydrogen sulfite. The basic proteins were made 50 mM in mercaptoethanol, reduced for 3 hr at 37”C, precipitated in a dialysis bag against 4 M ammonium sulfate, and dialyzed extensively against 0.1 N acetic acid, 1 mM mercaptoethanol. The chromosomal proteins not adsorbed by Bio-Rex 70 were dialyzed and quantitatively precipitated for 35 hr at 4°C in a dialysis bag against 4 M ammonium sulfate and 2 mM mercaptoethanol. The precipitated proteins were extensively dialyzed against 50 mM dithiothreitol and 7 M urea, reduced for 3 hr at 37°C and again dialyzed against 7 M urea, 2 mM dithiothreitol. Isoelectric focusing and microgradient gel electrophoresis. The outer chambers of the lower part of the zone convection isoelectric focusing apparatus, first described by E. Valmet (31), were filled with electrode solutions (cathode: 50 mM sodium/bicarbonate, pH 10, 12% sucrose; anode: 1% acetic acid, pH 2.8, 12% sucrose), and the following two chambers were filled with electrode protection solution containing 12% sucrose and 4% ampholine (pH 3- 10). The remaining chambers were filled with 88 mg (0.35 M Guanidine dissociation buffer), or 62 mg (3 M NaCl dissociation buffer) of NHC proteins containing 2 mM dithiothreitol, 7 M urea, and 3% ampholine (pH 3- 10). The focusing procedure was carried out for 60 hr at a constant wattage (3 W). The pH gradient was determined with a glass microelectrode inside the chambers. The solutions of two to three chambers were pooled, and the protein content was determined by the method of Lowry (38), and DNA and RNA contamination were determined by the method of Burton (40) and Ogur (43). All fractions were first dialyzed for 25 hr (changed four times) against 5 mM mercaptoethanol, 7M urea and then against 5 mM mercaptoethanol for 25 hr (changed four times) and lyophilized. In the last step of fractionation, the slightly modified method of continuous microgradient acrylamide (O-40%) gel electrophoresis of Rtichel et al, (32) under reducing SDS-electrode buffer conditions [0.05 M Tris-glycine, pH 8.4; 0.1% SDS, 0.1% (v/v) thioglycolic acid] was used. To obtain maximal dodecylsulfate charging, we incubated 1 mg of each NHC protein fraction with 1 ml of the following sample solution: 0.035 M Tris-sulfate buffer (pH 8.6), 1% dithiothreitol, 1% SDS, and IO% glycerol for 2 min at 100°C according to the method of Laemmli (39). The sample solution, l-2 ~1, was applied on top of a 0 to 40% microgradient gel in a lo-p1 capillary. Electrophoresis was carried out in the first 5 min at 40 V, at 80 V until the tracking dye was leaving the gel, and
PREPARATION Preparation Cell
pocdure
with
for the chromosomal
prote#nr
nuclei
Lysls
Dlfferentel 1,
391
OF NONHISTONES
0.35
M
Gus
chromatxn
of
the
inner
envelope
dissocaatoan
-dirsocntlon-buffer
2.
with
3M
NaCl
Extract.
3M
NaCl
BID-Rex
70
w-exchange
dlssoclatlon-buffer
I t
1
mm Extract:
0.35
M Gus.
chromosomal
proteins
chramosomal
protans
I
BIO-Rex
2 D
70
xx-exchange
chromatography
gelelectro-
3M
Fractionauon
phoresls
by
0 35 M Gus. Fr.
1.
2.
lxelectric
focusing
4.
5.
3 M 6.
B
7.
9.
v/t Amino
acid
analysts
NHC
fraction
I” a Valmet-focusing
NHC-prote,ns 3.
NaCl
NaCl 10
chromatography
3 M NaCl
apparatus
2 D
NHC-pro,e,ns 11.
12.
fract,on
gelelectro-
phoresas 13
14.
+\v Fractlonatlon
by
molecular
weight
in micro-gradlent-acrylamidege14
SCHEME
Amkno
acid
analysts
I
afterward at 100 V for 30 min. Immediately after electrophoresis the gels were expelled from the capillaries by pressure on the 40% end with a stainless steel wire. The dodecylsulfate proteins were stained for 10 min at 50°C in a 0.18% (w/v) Coomassie brilliant blue R-250 solution containing 7% acetic acid and 93% methanol/water (1: 1). The gels were destained and stored in 7% acetic acid. The molecular weights of the NHC protein components were determined by comparing the equivalent position of the marker proteins phosphorylase a, aldolase, and cytochrome c in the gradient gel. The overall preparation scheme of the chromosomal proteins is shown in Scheme 1. Amino acid analysis. Samples of about 10 nmol of the various preparatively prepared chromosomal proteins were hydrolyzed in 400 ~1 of quartzdistilled 6 N HCl in sealed tubes under Nz for 24 hr at 110°C. Amino acid composition was determined with a Durrum amino acid analyzer. Apparatus and reagents. The details of the homemade zone convection electrofocusing apparatus are described in the thesis of Kruger (30). The power supply and stands for capillary microelectrophoresis and forceps were obtained from E. Schtitt (Gottingen, Germany), and lo-p1 capillaries (32 mm) were from Brand (Wertheim, Germany). The Ultra-Turrax was obtained from Janke & Kunkel KG (Staufen im Breisgau, Germany), BioRex 70 (100-200 mesh) was from Bio-Rad Laboratories (Miinchen, Germany), and guanidine hydrochloride and sucrose were from Merck
KRUGER
392
AND PGLLOW
(Darmstadt, Germany). Acrylamide and NJ’-methylenebisacrylamide, urea, and sodiumdodecylsufate were obtained from Serva (Heidelberg) and prepared as described (28). Triton X-100, Coomassie brillant blue R-250, and thioglycolic acid were also obtained from Serva. Ampholine (pH 3-10) was purchased from LKB (Bromma, Sweden). The proteins used for molecular weight calibrations of the microgradient gels were phosphorylase a, (MW 92,500), aldolase (MW 39,500), and cytochrome c (MW 12,500) (all from Boeringer, Mannheim). RESULTS
We define in this communication the NHC proteins as those chromosomal proteins which are not adsorbed by the cationic exchanger Bio-Rex 70 under the running buffer conditions. This is not quite exact, because there are about 30 strongly basic minor NHC protein components, which are adsorbed together with the histones on Bio-Rex 70 under the running buffer conditions, as demonstrated by two-dimensional gel electrophoresis (30), and which will be published elsewhere. The NHC proteins of cow’s udder comprise nearly 50% of the total chromosomal proteins by weight. Eighty-two percent of the total dissociated nonhistones were peeled off from the chromatin by the 0.35 M guanidine dissociation step; about 18% of the nonhistones dissociate under the 3 M NaCl dissociation conditions. The results from the preparative isoelectric focusing procedure (Fig. 1) clearly indicate that the complex NHC protein mixture of the 0.35 M guanidine dissociated as well as that of the 3 M NaCl-dissociated NHC proteins has quantitative maxima between the isoelectric points (IP) of pH 20
1 % NHC proteins
16
12 aa
4
0
FIG. 1. Isoelectric point range and percentage (related to the total dissociated NHC proteins) of the differentially dissociated and preparatively isoelectrically focused NHC proteins of cow’s udder (For details see under Materials and Methods). (a) 0.35 M Guanidine dissociation. The chromosomal proteins are dissociated in 0.35 M guanidine hydrochloride, 2 mM dithiothreitol, 1.5 mM sodium disulfite, 2 mM EDTA, 0.1 M sodium phosphate buffer (pH 7). (b) The remaining DNA: NHC protein complex was sheared and dissociated in 3 M NaCl, 6 M urea, 2 mM dithiothreitol, 2 mM EDTA, 1.5 mM sodium disulfite, 1 mM TrisiHCl buffer (pH 8.3).
PREPARATION 0.35
M
OF NONHISTONES
393
. Gua . Dtssoclatlan
a
-
Phosphorylase
Aldolase
a
d_
-
92500
-
39500--
-
-
FIG. 2. (a) Microgradjent acrylamide gel electrophoresis, showing the heterogeneity of the different NHC protein fractions of cow’s udder. (b) Reproduction of the microgradient gels shown in (a) by visual detection of the Coomassie-stained bands. The marker proteins in the SDS microelectrophoresis in continuous polyacrylamide gradient gels are phosphorylase a, aldolase, and cytochrome c.
5.5 and 6.0 and that the NHC proteins not adsorbed by Bio-Rex 70 have IPs over the total pH range of ampholines used. With the zone convection focusing procedure, we obtain preparatively NHC protein fractions which comprise as little as 1% of the total NHC protein mass. We could not detect
394
KRUGER
AND POLLOW
DNA or RNA in this fraction. That the differentially dissociated NHC proteins are really different components is shown in Fig. 2 and Table 1. The occurrence of high molecular weight protein components in the 3 M NaCldissociated fraction is obvious. We could detect visually a total of ca. 440 NHC protein components from 14 different preparatively focused fractions: 247 NHC protein components have a molecular weight greater than 90,000; 111 have molecular weights between 40,000 and 90,000; and 84 have molecular weights between 10,000 and 40,000. The NHC proteins with molecular weights smaller than 6000 pass through the 40% gel under the experimental conditions, as has been proved for insulin. TABLE QUANTITATIVE
COMFQSITION FRACTIONS
NHC
proteins
of the 0.35 M guanidine
Number of bands
MW
IF
>9O,OC4 4o,coo-9o,ooo* 10$0040,ooo
3.5-4.3
+
1
OF THE OF
NHC
16 5 3
I.8
4.1-4.9
17 7 8
7.0
5.0-5.4
16.1
5.6-5.9
>90,00+ 4o,muLsO,OaO lO.Oc&40,000
+
I9 9 6
3.7
>9o,ooo 40.00&90,OKl~ 10.000-40.000
+
6.0-6.7
3.2
-
23 9 4
290.tmo 4o,OOc-swOO lO,OOO-40,ooo
+
24 7 4
3.0
8.2-8.7
>9o.m 40300-90,ooo 10.000-40,000
-9 + +
-
8.8-9.5
>9o,ooo -+ 4o.o00-90,000 -t lO,coO-4O,OOll-
>90,000 4o,ooo-swJoo lO,tM-40,ooO
+ + -
18 I3 8
17.1
5.9-6.0
>90,004 4o,lmo-90,m 10,tmO-40,@30
-+
I7 I2 9
ND
7.0-8.
6.1-6.7
>9o,m 4o,ooo-90,@30 lO,Ow-40,cmO
-3 + -t
I7 I2 9
II.6
290,m 40.000-90,cw 10,ln3o-40#00
+
17 IO I2
12.2
>9o,cunl 40,00&90,ooo 10,Ow-40,000
+ -
20 IO 9
15.8
8.6-9.7
a The 0.35 M guanidine-dissociated L The 3 M N&I-dissociated NHC r Isoelectric point. d Not determined.
1.7
2.8
+
I
ND I7 8 5
I5 4 6
6.9-8.
Percentage of the total NHC proteins
-f +
*
5.5-5.8
dissociation’
Number of bands
MW
>9o,ooo
>9o,m 4o,cnm-9o,ooo+ lO,OUO-40,OlM
4.8-5.3
of the 3 M NaCl
40,000-90,000 10.000-40,000
* -+
proteins
IP
>9o,ooo 4o,ooo-9o.ooo~ lO,WO-40,000
4.4-4.7
PROTEIN
UDDER
dissociation” Percentage of the total NHC proteins
NHC
DIFFERENT
Cow’s
I
NHC proteins are 82% of the total dissociated proteins are 18% of the total dissociated NHC
-t
NHC proteins. proteins.
7
20 5 I
2.4
0.9
21.6
6.9
4.0
Asp, Glu
Lys. Arg, His
Acidic* BaSlCS
2.6
II.8
31.1
1.9
13.2
25.4
3.0 8.5 2.2 3.1 1.8 5.3 6.1
10.3 6.2 7.4 15.1 5.5 9.9 8.2 5.8
5.5-5.8
‘I Moles per lo0 moles of total amino acids found. (i Decomposltwn of purines might have occurred. which
3.3
8.5
27.9
5.7 7.3 2.6
3.9 9.7 4.3 4.3 0.7 3.4 4.4
5.2 6.6
14.1 5.0 7.9 17.0 5.3 5.6 10.5 6.1
13.4 5.8 6.3 14.5 5.4 9.3 7.3 6.1
12.4 12.6 6.2 15.2 5.3 8.6 8.7 5.2 3.2 8.2 2.7 3.2 I.1 3.0 2.8
ASP Thr Ser au Pro GIY Ala Vd Met Be LW TY~ Phe His LYS AW
4.8-5.3
accounts
1.9
12.4
23.8
10.9 5.7 6.3 12.9 6.4 10.7 7.8 5.6 4.4 8.6 2.8 3.5 1.6 5.3 5.5
5.9-6.0
IP range
THE
I.0
of glycine.
0.5
25.8
Total basics 18.5
5.0 6.2 5.8 8.8 4.2 8.1 10.9 6.1 0.7 4.6 7.3 2.9 2.0 1.9 14.0 9.9
Histone fraction
HISTONE
13.8
AND
acidics 19.3
Total
8.0 4.9 5.5 II.3 6.3 11.0 10.4 5.9 4.3 9.0 2.1 2.2 1.8 9.3 7.4
8.6-9.7
high confenf
1.3
15.9
20.8
9.0 5.1 6.1 II.8 6.0 9.9 8.9 6.0 0.5 4.1 8.9 2.4 3.0 1.8 7.2 6.9
6.9-8.1
for the relatively
I.5
15.0
22.7
9.6 5.6 6.6 13.1 5.3 9.5 8.1 6.1 0.8 4.3 9.2 2.6 3.2 2.0 6.4 6.6
6.1-6.7
2
PREPARATIVELY
FOCUSING
DIFFERENT
ISOELECTRIC
OF
dissociation
AFTER
COMPOSITION”
0.35 M Guanidine
4.4-4.7
acid
ACID
3.5-4.3
Amino
AMINO
TABLE
2.3
II.6
26.8
3.8 8.7 2.7 3.4 1.3 4.6 5.7
II.7 4.9 6.8 15.1 6.0 II.7 7.6 5.3
5.0-5.4
FRACTIONS
PREPARED
1.9
13.0
25.4
3.7 9.3 2.7 3.0 1.3 5.2 6.5
10.4 4.5 7.1 15.0 4.8 13.0 7.9 5.0
5.6-5.9
NHC
1.7
12.5
20.9
8.9 4.6 5.8 12.0 8.5 20.9 8.5 4.0 2.9 6.3 2.0 2.6 2.0 4.8 5.7
6.0-6.7
Dissociation
FRACTIONS
I.8
9.3
16.5
6.9 3.0 4.8 9.6 11.7 31.30 II.7 2.3 1.9 4.2 0.8 1.6 0.9 2.8 5.6
7.0-8.1
IP range
3 M NaCl
PROTEIN
1.7
8.6
I.7
8.3
14.4
1.2 0.7 2.5 5.1
1.3 0.7 2.5 5.4
14.3
1.5 3.1
5.9 2.2 4.5 8.5 12.0 36.2O 13.0 2.4
8.8-9.5
1.6 3.2
6.0 2.2 4.1 8.3 12.3 36.3b 12.8 2.2
8.2-8.7
0.5
25.6
14.4
5.3 5.9 7.4 9.1 4.2 8.2 10.6 5.9 4.6 7.2 2.8 2.0 2.0 13.7 9.9
Histone fraction
396
KRUGER
AND
POLLOW
The most heterogeneous NHC protein fraction in relation to the isoelectric point range is the one with the isoelectric points between 5.5 and 6.5. The most heterogeneous NHC protein fraction, in relation to the total NHC protein mass, comprises proteins of the 3 M NaCl-dissociated fraction with isoelectric points between pH 8.8 and 9.5. The total heterogeneity of NHC proteins is further supported by the quantitative differences in the amino acid composition of the different fractions. (Table 2) DISCUSSION
Attempts to characterize chemically the NHC proteins are impeded by the major obstacle that we did not have any functional criteria for supposing that an isolated NHC protein functioned on the chromosome, in the cytoplasm, on both, or on the nuclear matrix. With the chromosomal protein preparation procedure reported herein, we could isolate distinct NHC protein classes under conditions which normally do not irreversibly denature proteins (9). The combination of preparative isoelectric focusing and microgradient gel electrophoresis allows for a high degree of resolution and the enhancement of the sensitivity for rare chromosomal proteins. The appearence of low molecular weight proteins in the alkaline end of the pH gradient in the Valmet-focusing procedure from the 0.35 M Guanidine-solubilized protein fraction (Figs. 2a and 2b, pH 6.9-8.1; 8.6-9.7) suggests the possibility that the Bio-Rex 70 ion exchange procedure for separation of histones from NHC proteins might not work efficiently enough under the experimental conditions, because the histones would migrate to similar positions in the microgradient gel. At the same time the ratio of histones to NHC proteins would not reflect the original ratio of this two types of proteins in the chromatin. The molecular weight determination of the NHC proteins might be misleading sometimes, especially in the high molecular weight range, because tightly bound nucleic acids not detectable by the method of Burton (40) might retard the migration of NHC protein-nucleic acid complexes (42). From the results shown in Fig. 2, we could observe several bands of identical molecular weight in adjacent pH fractions. It could be possible that some protein components “smeared” over a wide pH range. This type of cross-contamination would reduce the total number of protein components resolved by the system. The potential of our fractionation scheme for determining the NHC protein heterogeneity will be tested on material from five different areas of the brain. This work is now in preparation. The heterogeneity of NHC proteins and their frequency of occurrence with different molecular weights and different isoelectric points raise important questions regarding the structure of chromatin, the interaction of these proteins with different
PREPARATION
DNA sequence families, mRNA transcription.
397
OF NONHISTONES
and the basic principle
of gene regulation
and
ACKNOWLEDGMENTS We thank Dr. H. Endou from the University of Tokyo for technical assistance with the microgradient gels. This work was supported by funds of the Deutsche Forschungsgemeinschaft.
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