ANALYTICAL BIOCHEMISTRY 64, 74-79 (1975)

A Method for Quantitative Determinations of Nonhistone Proteins in Chromatin J O H A N N S O N N E N B I C H L E R , I S O L D E Z E T L AND F A U S T O M A C H I C A O

Max-Planck-lnstitut fiir Biochemie, 8033 Martinsried, BRD Received June 24, 1974; accepted September 16, 1974 An electrophoretic method on cellogel strips at alkaline pH is used to determine the amount of nonhistone proteins in chromatins in a very reproducible way after isolation of the total chromatin proteins in 2 M NaC1 or 2 M CsCI q- 5 M urea. The procedure is demonstrated with calf thymus, rat liver, and pig brain chromatin. The influence of the chromatin preparation on the protein composition is discussed.

Studies on nuclear and c h r o m o s o m a l nonhistone proteins have become very important in the last years_ Quantitative estimations normally are done after separation of these proteins which m a y be performed by some modifications of a few basic methods (1-4). During these isolation procedures, however, part of the nonhistones is lost o r - a s in the case of acid extracted c h r o m a t i n s - t h e amount of nonhistone proteins is essentially falsified by denaturation processes and artefact formation (5). In general quantitative determinations based on nonhistone isolation are not very reliable, especially those done after acid treatment of chromatin. In this communication a procedure is demonstrated which allows a quantitative m e a s u r e m e n t of chromatin nonhistones in the presence of histones by cellogel-electrophoresis at p H 9.3.

MATERIALS A N D METHODS I f not otherwise stated all procedures were carried out at 0°C. Nuclei from different tissues were prepared by homogenization in 0.32 M sucrose + 1 mM MgC12 and after washing in the same solvent the nuclei were purified by ultracentrifugation in 2.2 M sucrose at 60 000g for 9 0 - 1 2 0 min. Chromatin was isolated according to the procedure of Z u b a y and D o r y (6) with slight modifications (7) as washing the crude material only once with 0.024 M E D T A ÷ 0.075 M NaC1, p H 7.0 and then in NaC1 solutions as specified in Table 2. T h e chromatin fibres were dissolved in water and brought to 2 N NaC1 ÷ 6 M urea + 0.01 M N a H S O 3 or in the case of small chromatin portions to 2 M C s C I + 5 M u r e a + 0.01 M 74 Copyright © 197s by Academic Press, Inc. Printed in the United States. All rights of reproductionin any formreserved.

NONHISTONE CHROMATIN PROTEIN

75

N a H S O 3 . Intensive stirring for 30 min or mixing with a blender was used to dissolve the material quantitatively. Subsequently the proteins are separated from the nucleic acids by ultracentrifugation with D N A concentrations 150-250 /xg/ml: in 2 M CsC1-~ 5 M urea after 70 hr at 100,000g a density gradient is formed which concentrates all the proteins in a relatively small zone in the u p p e r third of the tubes. With 2 M NaC1 + 5 M urea the proteins are distributed in the whole tube and the D N A pellet has to be redissolved and must be recentrifuged to r e m o v e more than 98% of the total proteins f r o m the D N A (7). After a short dialysis of the protein solutions against water to reduce the salt concentrations (NaC1 < 0.6 M) the proteins were precipitated with 6 vol acetone. A n a h ' s e s . The proteins were quantitatively dissolved in 1%, acetic acid, 1% mercaptoethanol + 8 M urea to concentrations of about 1 rag/100/xl. As controls purified histories were used. T h e electrophoreses were performed in 0.6 M a m m o n i u m borate buffer, 6 M urea, 0.01 M E D T A , 0.01 M mercaptoethanol, pH 10.0, on cellogel strips for 15 rain at 60 V / c m according to Machicao and Sonnenbichler (8). After staining with amido black the coloured areas were cut out and the cellogel pieces and corresponding blanks as references were dissolved in glacial acetic acid. T h e intensities measured at 630 nm parallel the protein content. The binding difference of amido black to the nonhistones and the histones was calculated repeatedly by staining definite amounts of purified histones, nonhistones, and bovine serum albumine ~ spotted on cellogel strips with and without electrophoreses_ F o r these purposes the isolation of nonhistones was performed in two ways: (a) calf thymus chromatin was stirred intensively with 0.30 M N a C I and after dialysis the extracted nonhistones were precipitated with 6 vol acetone: (b) after separation of all proteins from calf thymus chromatin (see above) the nonhistone proteins were isolated by c h r o m a t o g r a p h y on Bio-Rex 702 in 0.02 M Tris, 0.02 M j3-mercaptoethanol, 0.001 M E D T A , 5 M urea, p H 7.5, and 0.2 M NaC1. In both procedures the purity and the absence of histones was controlled by acrylamide-gel electrophoreses according to Panyim and Chalkley (9)_ All our nonhistone moieties showed equal staining abilities. Otherwise, protein was determined according to L o w r y et al. (10). Also in this case as standards calf thymus histones, nonhistones, and bovine serum albumin were used. D N A was determined according to Dische (11). Since high salt concentrations produce a considerable quenching of the diphenylamine reaction, all analyses for D N A were performed in water or in low salt conditions. ' Bovine serum albumin, purified, Behringwelke, Marburg, BRD. 2 Bio-Rex 70 1100-200 mesh) Bio-Rad Laboratolies, California.

76

SONNENBICHLER,

ZETL AND MACHICAO

RESULTS AND DISCUSSION The electrophoresis on cellogel strips at alkaline pH has proved to be a convenient method to analyze the different histone types (8). While for the characterization of histones a high resolution of the electrophoretic bands is desired, with the view to quantitative aspects as described here a discrimination of the bulk of histone and nonhistone protein will be better done at low resolution conditions. After elaboration of this paper our attention was drawn to a publication of Kleinsmith and Allfrey (12) who employed a similar method to separate phosphoproteins and histones by cellogel-electrophoresis at neutral pH. As shown in Fig. 1 in our system the histones migrate during electrophoresis relatively fast to the cathode while all nonhistones run to the anode or remain near the starting line. Opposite to our preliminary statement ( 8 ) - d e r i v e d from the buffer p H - w e now determined the actual pH on the cellogel during electrophoresis not to be 10_0 but 9.3 due to interactions of CO2 from the atmosphere. As can be seen from Fig. 1 also some nonhistone proteins apparently with pK > 9.3 migrate to the negative side but much slower than the histones do. So it is very simple to discern the bulk of histones from basic and acidic nonhistones. The presence of high molar urea avoids histone-nonhistone aggregations (13) which additionally was confirmed by analyses of artificial mixtures. The reproducibility of the staining evaluation is very satisfying as shown in Table 1. The different staining abilities of histones and nonhistones had to be determined as described in Methods. F o r the histones we calculated an average value because one can assume that in general the quantitative composition of these proteins is very similar in different chromatins. Very surprisingly the staining capacity of the nonhistone moiety is quite different from those of serum albumin and naturally histones and was therefore carefully investigated. Since the nonhistones are far more heterogeneous than the histones, different colour values for each polypeptide could not be determined. Possibly certain species of nonhistone proteins may differ widely in their staining abilities. However, our different preparations of the nonhistone moieties, also from different tissues, always

~ i :¸~

(a)

FIG. 1. L o w resolution electrophoreses on cellulose acetate sheets at pH 9.3 of (a) mixed purified histones, (b) isolated nonhistones, (c) total proteins from rat liver chromatin,

NONHISTONE

CHROMATIN

77

PROTEIN

TABLE 1 g6a 0 DATA OF CORRESPONDING STAINED AREAS ~t

E6ao of

l

2

3

4

5

6

Histone areas Nonhistone areas % Nonhistone

0.580 0.107

0.395 0.076

0.380 0.074

0.332 0.062

0.265 0.047

0.280 0.055

15.5

16.0

16.0

16.0

15_0

16.5

Average value 15.8% -+ 0.51 (without calculation of the different staining factors for histones and nonhistones) " Dissolved in glacial acetic acid after electrophoreses of the proteins from calf thymus chromatin (washed twice with 0.15 M NaCll as example for the reproducibility of the described method. Usually six electrophoreses were performed with each protein sample listed in Table 2.

TABLE 2 HISTONE AND NONHIS'IONE PORJ IONS OF SOME CttROMATINS DETERMINED BY EI.ECTROPHORETIC ANAEYSES OF "IHE TOTAl. CHROMATIN PROTEINS, AND DEPENDENCE OF THE NONHISTONE RATIOS FROM THE CHROMATIN WASH1NG PROCEDURE AFTER A PRIMARY RINSING OF TIlE MATERIAL IN

0.075 M NaCI + 0 024 M EDTA

Chromatin Calf thymus Calf thymus Calfthynms Calfthynms Calf thymus Calf thymus Call'thymus

Rat Rat Pig Pig

liver liver brain brain

Successive washing steps ill M NaCI

Parts pl otein/ 100parts DNA

% Nonhistone

9~, Histune

I x 0.15 2 x 0.15 1 :¢ 0.15 1 x 0.30 ~'~ x 0.15 1 × 0.30 2 x 0.15 2 > 0.30 1 × (I.15 '~' 0.31/ 2 - 0.15 2 / 0.30 1 × 0.35 2 × 0.15 3 × 0.30 2 x 0.15 1 × 0.15 1 × 0.30

220 210/197

29 24/23

71 76/75

205

20

80

191

18

82

189

16

84

16

84

184

12

88

228 180 228

46 36 34

54 64 66

225

28

72

78

SONNENBICHLER,

ZETL AND MACHICAO

showed identical colour values p r e s u m a b l y as a consequence of their great heterogeneity with a statistical averaging. In order to get correct protein quantities the following multiplication factors must be used: histones 1.00, nonhistones 1.69; for comparison: bovine serum albumin 1.17 (histone single values: F I : 0,76; 2al: 0.82; 2a2: 1.23, 2b: 1.06, 3: 1.02), In Table 2 from some chromatins the histone and nonhistone values are listed which were determined with the described electrophoretic method. In spite of the simple procedures which we used for chromatin purification and which will be discussed in the following, our nonhistone portions in general are somewhat lower than normally cited (14). This m a y be connected to the problems of chromatin preparation. Often contamination by absorption of n o n c h r o m o s o m a l material to chromatin during the isolation or, conversely, a loss of loosely bound c h r o m o s o m a l proteins during purification m a y falsify the analyses. Further on, often, the solubilization of the chromatin material is incomplete and a solvent dependent fractionation m a y occur. We k n o w that under salt conditions higher than 0.30 M N a C I or in the presence of detergents part of the nonhistones are released and will be enriched in the soluble fraction. With urea or with salt concentrations higher than 0.4 M NaC1 also histones dissociate (especially F1) and accumulate in the dissolved part. In addition to incomplete chromatin solubilization it must also be pointed out that in such a way euchromatic and heterochromatic portions partially m a y be selected. This argument must also be subjected to some procedures which use ultracentrifugation of chromatin in sucrose as a further purification step. T o encounter some of these difficulties our crude chromatins from the nuclei prepared in sucrose + 5 mM MgCI2 were only washed with 0.075 M NaC1 + 0.024 M E D T A in a primary washing procedure and then dissolved quantitatively by intensive stirring or mixing in water_ Further purification steps were omitted besides those listed in Table 2. T h e separation of all proteins from D N A was achieved by ultracentrifugation in 2 M N a C I + 5 M urea with addition of 0.01 M N a H S O 3 to avoid proteolysis. With small amounts of chromatin a more suitable way is density gradient centrifugation in 2 M CsC1-~- 5 m urea, where all proteins can be separated in one step and can be obtained relatively concentrated in a small volume. T o determine the influence of washing the crude chromatin with N a C I solutions > 0.15 M, we analyzed the nonhistone content of materials treated for 15 min with cold 0.30 and 0.35 M N a C I solutions, salt conditions which still are discussed for the nuclear interior (15). As can be seen from Table 2 under these conditions more nonhistones are released and their proportion decreases. The question remains what part of the nonhistones can be seen as true

NONHISTONE

CHROMATIN

PROTEIN

79

constituents of the c h r o m o s o m a l fibres. H o w e v e r , we hope to present with this contribution a method to analyze the nonhistone quantities in chromatins in a relatively simple and very reproducible way.

ACKNOWLEDGMENTS We thank F. Reichhart for valuable cooperation. We are gratefully indebted to Prof. Dr. H. Dannenberg for his general help. This work was supported by the Deutsche Forschungsgemeinschaft SFB 5 I/A-9.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. I('L 11. 12. 13. 14. 15.

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