78

Biochimica et Biophysica Acta, 383 (1975) 78--85

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 98229 PHYSICOCHEMICAL PROPERTIES OF CHROMATIN DIGESTED WITH TRYPSIN

NOBUHIKO SUGANO and SHOJI OKADAa Faculty of Pharmaceutical Sciences, University of Toyama, Gofuku 3190, Toyama and aShizuoka College of Pharmacy, Oshika 2-2-1, Shizuoka (Japan)

(Received September 17th, 1974)

Summary Chromatin was prepared from calf t h y m u s and digested with trypsin. Some physicochemical properties of chromatin were examined in connection with the time-course of the tryptic digestion. As the tryptic digestion proceeded, chromatin showed increases in viscosity and susceptibility to DNAase II and exhibited considerable alteration in thermal denaturation. A monophasic melting profile was f o u n d in the trypsic (digested) chromatin, but a biphasic one in the native (undigested) chromatin. The melting temperature descended from 78.2°C for the native chromatin to 70.2°C for the chromatin after only 10 min and further to 65.3°C after 180 min tryptic digestions. The molar percent of total basic amino acid or lysine plus arginine in the chromatin increased with the time-course of the tryptic digestion whereas that of the total hydrophobic amino acid decreased. The molar ratio of hydrophobic amino acids to basic amino acids thus descended from 1.46 for the native chromatin to 1.05 for the chromatin after a 180-min tryptic digestion. These findings suggest that the neutral or hydrophobic portions in chromatin protein might play a role in the maintenance of the tertiary structure of chromatin.

Introduction It is well established by much evidence that the chromatin proteins may be involved in the regulation of DNA template in eukaryotic cells [1,2]. The precise roles of these proteins are, however, still ambiguous. In order to understand this regulatory mechanism, structural studies on chromatin have been advanced by many workers. The electron microscopic [3] and the X-ray diffraction [4,5] techniques revealed that histones tightly fold the DNA double helix into the supercoiled structure and that the supercoils further fold themselves to give a k n o b b y fibril structure and/or chromatin fibre. Johns [6] has proposed that such supercoils will inhibit the passage of RNA polymerase along

79 the DNA double helical structure. The proteolytic digestion of chromatin will thus serve to obtain more detailed information a b o u t the chromatin structure. In this experiment, calf t h y m u s chromatin was digested with trypsin and the tryptic chromatin was examined with respect to its viscosity, susceptibility to DNAase II, thermal denaturation and amino acid composition. These results were compared with those which were derived from the undigested chromatin and discussed in relation to the chromatin structure. Materials and Methods

Preparation of chromatin The preparation of chromatin was principally performed after the procedure of Zubay and Doty [7] or Marushige and Bonner [8]. After calf thymus was homogenized and washed with 75 mM NaC1/24 mM EDTA/50 mM NaHSO3 (pH 7.8), the washed material was suspended in 50 mM Tris • HC1 (pH 7.8). The suspension was layered on 1.7 M sucrose (pH 7.8) and centrifuged in a swinging bucket rotor at 50 000 X g for 3 h. The pellet was dispersed in 0.7 mM potassium phosphate (pH 6.8) and dialysed against the same buffer. The gelatinous diffusate was homogenized in a small stainless cup (1 X 7 cm} at 100 V for 1 min and centrifuged at 70 000 X g for 1 h. The supernatant was referred to as the native chromatin in this experiment. DNA was quantitatively determined by the diphenylamine m e t h o d [9]. The protein content was measured by the Lowry procedure [ 1 0 ] . Digestion of chromatin with trypsin and DNAase H TPCK (tosylphenylalanyl chloromethyl ketone)-treated trypsin was obtained from the Worthington Biochemical Corp. The native chromatin was incubated with 1% TPCK trypsin (w/w protein) at 37 ° C and pH 7.2 for 10,30, 60 and 180 min. These tryptic (digested) chromatins were, in this experiment, called Fractions TCH-10, TCH-30, TCH-60 and TCH-180, respectively. Trypsin inhibitor (five times as much as trypsin) was added to aliquots of native and tryptic chromatins. In order to examine the susceptibility of tryptic chromatin to DNAase, the native and tryptic chromatins were digested with DNAase II (100 pg/350 pg of DNA in the chromatin) at 37°C and pH 7.2 for 90 min. Each reaction was stopped b y adding an equal volume of cold 10% perchloric acid. The resulting precipitate was removed by centrifugation at 10 000 X g. The supernatant was assayed for the quantitative determination of degraded DNA. Micrococcal DNAase II which is free of RNAase and proteolytic enzyme was obtained from Miles Laboratories Inc. Trypsin inhibitor was Type I-S from the Sigma Chemical Co. Measurements of viscosity and thermal denaturation The viscometric measurement was performed in 0.7 mM potassium phosphate (pH 6.8) at 25°C with an Ostwald-type viscometer. The thermal denaturation was measured as follows. Cold ethanol (3 vols) was added to the, native and tryptic chromatins. Each resulting fibrous precipitate was spooled out and washed with 75% cold ethanol. The washed precipitate was dissolved in 0.7 mM potassium phosphate (pH 6.8) and the measurement of thermal denatura-

80 tion was performed with a Hitachi EPS-3T spectrophotometer equipped with a temperature-controlled cell.

A m i n o acid analysis The above washed precipitate was dissolved in 2 M NaC1/5 M urea {pH 7.9). To this solution, LaC13 was added to m a k e the final concentration 13.5 mM. The dissociated DNA was thus selectively precipitated and removed by centrifugation at 10 000 × g [11]. The supernatant was then filtered on a column of Sephadex G-25 superfine with an elution of 0.01 M HC1. The peptide fraction was monitored by the absorbance at 230 nm, pooled and evaporated to dryness in a rotary evaporator. The residue was dissolved in glassdistilled HC1 and hydrolyzed at l l 0 ° C for 40 h. The hydrolysate was analyzed with a Hitachi automatic amino acid analyzer. Results

Digestion o f native or tryptic chromatin with DNAase H The ratio of mass protein to DNA for the native chromatin was 1.2 (w/w). About 18% of DNA in the native chromatin was degraded with DNAase II whereas DNA in the tryptic chromatin was more susceptible to the enzyme. About 42% of DNA was degraded in Fraction TCH-10 and eventually 58% of DNA in Fraction TCH-180 with DNAase II (Fig. 1). Viscometric and thermal denaturation profiles The change in the viscosity of chromatin was not found until the 30-min tryptic digestion. However, increases in specific viscosities of 8.3% and 34.5%

50

30

10

10 3~

6~

'

~0

Time, min Fig. I. Degradation of tryptic chromatin with D N A a s e If. T h e ordinate indicates the percentage of the nucleotide liberated with D N A a s e II c o m p a r e d to that in the tryptic chromatin before the D N A a s e II digestion. T h e abscissa indicates the time of tryptic digestion. For the quantitative assay of nucleotide liberated, the supernatant obtained after the D N A a s e II digestion (see in the text) was stayed at 9 0 ° C for 15 rain and was then assayed by the diphenylamine method. Nucleotide in the chromatin before the D N A a s e II digestion was also determined by the s a m e m e t h o d after adding an equal v o l u m e of 1 0 % perchloric acid to the chromatin. These results are the average of three experiments.

81 TABLE I VISCOSITIES OF N A T I V E A N D T R Y P T I C C H R O M A T I N S F r a c t i o n T C H - 0 s h o w s t h e r e s u l t f r o m n a t i v e e h r o m a t i n . F r a c t i o n s T C H - 1 0 , -30, -60 a n d -180 s h o w t h e results f r o m c h r o m a t i n d i g e s t e d w i t h t r y p s i n f o r 10, 30, 60 a n d 1 8 0 r a i n , r e s p e c t i v e l y , qrel is t h e r e l a t i v e viscosity, r/spc is t h e specific v i s c o s i t y . V i s c o m e t r i c m e a s u r e m e n t s w e r e p e r f o r m e d in 0.7 m M p o t a s s i t u n p h o s p h a t e ( p H 6.8) at 2 5 ° C . Chromatin fraction

r/re 1

I n c r e a s e s in ~rel*

~/spe

I n c r e a s e s in ~/spe*

TCH-0 TCH-10 TCH-30 TCH-60 TCH-180

1.84 1.85 1.85 1.91 2.13

0 1.01 1.01 1.04 1.16

0.84 0.85 0.85 0.91 1.13

0 1.01 1.01 1.08 1.35

* T h e r a t i o of relative o r specific viscosity f o u n d in t h e t r y p t i c c h r o m a t i n to t h a t in t h e n a t i v e e h r o m a t i n (Fraction TCH-0).

were found in Fractions TCH-60 and TCH-180, respectively (Table I). The thermal denaturation profiles are shown in Fig. 2. The melting temperature markedly descended after a 10-min tryptic digestion and afterwards gradually, i.e., 78.2°C for the native chromatin, 70.2°C for Fraction TCH-10, 69.2°C for TCH-30, 67.0°C for TCH-60 and 65.3°C for TCH-180. A biphasic melting profile was clearly found in the native chromatin. Monophasic and DNA-like melting profiles were found in Fractions TCH-30, TCH-60 and TCH-180. Amino acid analysis Gel filtration on a column of Sephadex G-25 was tried to obtain the mass protein or peptide which dissociated from the native or tryptic chromatin. The elution profiles are shown in Fig. 3. Only one big peak was found in the native chromatin. Several peaks appeared in all of the tryptic chromatins and the main peak shifted to the lower molecular peptide fraction. The mass amino acid analyses of these fractions were shown in Table II. As the tryptic digestion

cE 1.4 cq ~ 1'3 .a

N 1.1

1.o 50

60

70

80

90

Temperature, °C Fig. 2. T h e r m a l d e n a t u r a t i o n profiles o f t h e n a t i v e a n d t l T p t i c c h r o m a t i n s . • ~-, r e s u l t e d f r o m the n a t i v e e h r o m a t i n . Profiles o f c h z o m a t i n d i g e s t e d w i t h t r y p s i n f o r 10, 30, 6 0 a n d 1 8 0 rain w e r e s h o w n b y o o, • •, ~ ~ and • • , r e s p e c t i v e l y . All of t h e results w e r e o b t a i n e d in 0.7 m M p o t a s s i u m p h o s p h a t e ( p H 6.8).

82

0.1

~

TCH-0 i

i

5O TCH-10

0.1 E

c

100

50

0"2

TCH-30

100

E ,

50

o

.~ 0.2

0-|

0.2

TCH-60

~

100 ,

50

100 TCH-180

0.1 50 Fraction number

I00

Fig. 3. E l u t i o n p r o f i l e s o f p r o t e i n s or p e p t i d e s d i s s o c i a t e d f r o m t h e n a t i v e or t r y p t i c c h r o m a t i n o n a c o l u m n o f S e p h a d e x G - 2 5 w i t h a n e l u t i o n o f 0 . 0 1 M HC1.

TABLE II AMINO ACID COMPOSITIONS OF NATIVE AND TRYPTIC CHROMATINS Fraction TCH-0 shows the result from native chromatin. Fractions TCH-10, -30, -60 and -180 show the results from chromatin digested with trypsin for 10, 30, 60 and 180 rain, respectively. Amino acid

Lys His Arg Asp Thr Set Glu Pro Gly Ala Cyst Val Met Ile Leu Tyr Phe

mol/100 mol of total amino acids found TCH-0

TCH-10

TCH-30

TCH-60

TCH-180

15.0 2.7 6.9 5.7 6.0 5.8 8.7 3.9 9.5 13.6 0 5.9 0.7 4.1 8.3 1.8 1.5

16.0 3.8 8.6 4.1 6.4 5.5 7.5 3.5 10.3 12.1 0 6.3 0.7 4.8 7.4 1.6 1.2

16.9 3.3 9.6 3.3 6.9 5.2 6.3 4.5 11.7 12.4 0 6.0 0.4 4.3 6.8 1.4 1.2

16.6 3.3 9.9 4.3 6.5 4.5 7.0 5.0 10.4 11.7 0 6.2 0.5 4.8 7.1 i.I 1.2

17.4 3.7 10.1 4.1 6.5 4.8 6.8 4.5 10.4 12.1 0 5.9 0.5 4.1 6.2 1.8 1.0

83 TABLE In AMINO ACID COMPOSITIONS OF NATIVE AND TRYPTIC CHROMATINS

A m i n o acid

m o l / 1 0 0 tool o f t o t a l a m i n o acids f o u n d TCH-0

TCH-10

TCH-30

TCH-60

TCH-180

Basic*

24.6 (I.00)

28.4 (1.15)

29.8 (1.21)

29.8 (1.21)

31.2 (1.27)

Lys + Arg

21.9 (I.00)

24.6 (1.12)

26.5 (1.21)

26.5 (1.21)

27.5 (1.26)

Hydrophobic**

35.9 (i.00)

34.1 (0.95)

32.5 (0.91)

32.6 (0.91)

31.6 (0.88)

Neutral***

61.1 (I.00)

59.8 (0.98)

60.8 (i.00)

59.0 (0.97)

57.8 (0.95)

Acidict

14.4 (1.00)

11.6 (0.81)

9.6 (0.67)

11.3 (0.78)

10.9 (0.76)

* ** *** t

I n c l u d e s lysine, histidine a n d arginine. I n c l u d e s alanine, valine, m e t h i o n i n e , i s o l e u c i n e , l e u c i n e , t y r o s i n e a n d p h e n y l a l a n i n e . I n c l u d e s t h r e o n i n e , serine, p r o l i n e a n d g l y c i n e in a d d i t i o n t o h y d r o p h o b i c s . I n c l u d e s a s p a r t i c a n d g l u t a m i c acids. T h e n u m b e r in p a r e n t h e s i s m e a n s t h e r a t i o of t h e v a l u e f o u n d in e a c h t r y p t i c c h r o m a t i n to t h a t in t h e n a t i v e c h r o m a t i n ( F r a c t i o n T C H - 0 ) .

proceeded, the molar percent of total basic amino acid increased whereas that of hydrophobic amino acid decreased (Table III). The molar ratio of hydrophobic amino acids to basic amino acids thus descended from 1.46 for the native chromatin to 1.01 for the tryptic chromatin Fraction TCH-180. Consequently, it was found that peptides which remained in the tryptic chromatin became more basic with the time-course of the tryptic digestion (Table IV ). Discussion Although several investigations on tryptic-digested chromatin have already appeared, there are still some discrepancies among these res.ults and conclusions. Simpson [12] reported that trypsin cleaved only the n o n b o u n d regions T A B L E IV M O L A R R A T I O S OF H Y D R O P H O B I C , N E U T R A L A N D A C I D I C A M I N O A C I D S T O T H A T O F BASIC A M I N O A C I D S O R L Y S I N E P L U S A R G I N I N E F O U N D IN N A T I V E A N D T R Y P T I C C H R O M A T I N S B, H, N a n d A r e p r e s e n t t h e basic, h y d r o p h o b i c , n e u t r a l a n d acidic a m i n o acids, r e s p e c t i v e l y . T h e n u m b e r in p a r e n t h e s i s is t h e r a t i o of t h e v a l u e f o u n d in e a c h t r y p t i c c h r o m a t i n t o t h a t in t h e n a t i v e c h r o m a t i n (Fraction TCH-0). Ratios

TCH-0

TCH-10

TCH-30

TCH-60

TCH-180

H/B

].46 (1.00)

1.20 ( 0 . 8 2 )

1.09 ( 0 . 7 5 )

1.09 ( 0 . 7 5 )

1.01 ( 0 . 6 9 )

H / L y s + Arg

1.64 (1.00)

1.39 ( 0 . 8 5 )

1.23 ( 0 . 7 5 )

1.23 ( 0 . 7 5 )

1.15 (0.70)

N/B

2.48 (1.00)

2.11 ( 0 . 8 5 )

2.04 (0.82)

1.98 ( 0 . 8 0 )

1.85 ( 0 . 7 5 )

N/Lys + Arg

2.79 ( 1 . 0 0 )

2.43 ( 0 . 8 7 )

2.29 ( 0 . 8 2 )

2.23 ( 0 . 8 0 )

2.10 (0.75)

A/B

0.59 (1.00)

0.41 ( 0 . 6 9 )

0.32 (0.54)

0.38 (0.64)

0.35 (0.59)

A/Lys + Arg

0.66 ( 1 . 0 0 )

0.47 (0.71

0.36 (0.55)

0.43 ( 0 . 6 5 )

0.40 (0.61)

84 of protein to DNA with a slight alteration in the thermal denaturation of chromatin. From this result, he concluded that the n o n b o u n d regions are of little importance in the stabilization of the direct base-base interaction of DNA. Marks et al. [13] showed that the trypsin-treated chromatin exhibited far more alteration in thermal denaturation and an almost identical melting profile with that of DNA. We also observed the considerable alteration in thermal denaturation of chromatin even after a 10-min tryptic digestion, although the Tm of our native chromatin was in good agreement with that reported b y Ohba [14]. The tryptic chromatin clearly exhibited a monophasic melting profile differing from a biphasic one found in the native chromatin. Such a biphasic melting profile has been reported on the reconstituted complex on DNA-histone by several workers [15,16]. Shi and Bonner [15] explained that the first step transition is due to the melting of the free DNA segment and the second step transition to that of the histone-complexed regions. The direct base-base interaction of DNA may thus be affected by the binding of chromatin protein. In addition, the present experiment showed the increases in viscosity and susceptibility to DNAase II of the tryptic chromatin, suggesting that the partial removal of neutral of h y d r o p h o b i c portions in nuclear protein brought about the expansion of chromatin fibre. The amino acid analysis of tryptic chromatin revealed the increase in the molar percent of the total basic amino acid and the decrease in the molar ratio of neutral or hydrophobic amino acids to basic amino acids. With the X-ray diffraction study, Richard and Pardon [5] have shown that the histones, F2A2 and F3 are the simplest for the reconstruction of the DNA supercoil. The complete amino acid sequences of these t w o histone molecules have already been clarified [17--20]. The very long, nonbasic and hydrophobic sequences were thus positioned in the middle portions of F2A2 and F3. The other two histones, F2A1 and F2B also have similar sequential characteristics [21--23]. These findings together with the present results strongly suggest that such hydrophobic portions might play a role in the maintenance of the tertiary structure of chromatin and/or supercoiled structure of chromatin DNA through the bridge formed by the hydrophobic bond between histone-histone or histone-nonhistone protein. Acknowledgements The authors are indebted to Dr C. Yanaihara for the amino acid analysis, and to Professor A. Nishi, Professor K. Tsukada and Professor I. Horikoshi for their valuable advice and encouragement. References 1 2 3 4 5 6

D e L a n g e , R.J. a n d S m i t h , E.L. ( 1 9 7 1 ) A n n u . Rev. B i o c h e m . 40, 2 7 9 - - 3 1 4 Stein, G.S., Spelsberg, T.C. a n d K l e i n s m i t h , L.J. ( 1 9 7 4 ) S c i e n c e 1 8 3 , 8 1 7 - - 8 2 4 D u p r a w , E.J. ( 1 9 7 0 ) in D N A a n d C h r o m o s o m e s ( D u p r a w , E.J. ed.), pp. 1 7 2 - - 1 8 5 P a r d o n , J . F . , Wilkins, M . H . F . a n d Richards~ B.M. ( 1 9 6 7 ) N a t u r e 2 1 5 , 5 0 8 - - 5 0 9 Richards~ B.M. a n d P a r d o n , J . F . ( 1 9 7 0 ) Exp. Cell Res. 62, 1 8 4 - - 1 9 6 J o h n s , E.W. ( 1 9 6 9 ) Ciba f o u n d a t i o n s y m p o s i u m o n h o m e o s t a t i c r e g u l a t o r s , p p . 1 2 8 - - 1 4 3 , Churchill, London 7 Z u b a y , G. a n d D o t y , P. ( 1 9 5 9 ) J. Mol. Biol. 1, 1 - - 2 0 8 Marushige, K. a n d B o n n e r J. ( 1 9 6 6 ) J. Mol. Biol. 15, 1 6 0 - - 1 7 4

85 9 I0 11 12 13 14 15 16 17 18 19 20 21 22 23

Burton, K. (1956) Biochem. J. 6 2 , 3 1 5 - - 3 2 3 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 1 9 3 , 2 6 5 - - 2 7 5 Yoshida, M. and Shimura, K. (1972) Biochem. Biophys. Acta 263,690---695 Simpson, R.T. (1972) Biochemistry 11, 2 0 0 3 - - 2 0 0 8 Marks, D.B. and Schumaker, V.N. (1968) Biochem. J. 1 0 9 , 6 2 5 - - 6 3 5 Ohba, Y. (1966) Biochim. Biophys. Acta 123, 84--90 Shi, T.Y. and Bonner, J. (1970) J. Mol. Biol. 4 8 , 4 6 9 - - 4 8 7 Anscvin, A.T. and Brown, B.W. (1971) Biochemistry 10, 1133--1142 Olson, M.O.J., Sugano, N., Yeoman, L.C., Johnson, B.R. Jordan, J., Taylor, C.W., Starbuck, W.C. and Busch, H. (1972) Physiol. Chem. Phys. 4, 10--16 Sugano, N., Olson, M.O.J., Yeoman, L.C., Johnson, B.R. Taylor, C.W., Starbuck, W.C. and Busch, H. (1972) J. Biol. Chem. 247, 6018--6023 Yeoman, L.C., Olson, M.O.J., Sugano, N., Jordan, J.J., Taylor, C.W., Starbuck, W.C. and Busch, H. (1972) J. Biol. Chem. 247, 6018--6023 DeLange, R.J., Hooper, J.A. and Smith, E.L. (1972) Proc. Natl. Acad. Sci. U.S. 6 9 , 8 8 2 - - 8 8 4 DeLange, lq,.J., Fambrough, D.M., Smith, E.L. and Bonner, J. (1969) J. Biol. Chem. 2 4 4 , 3 1 9 - - 3 3 4 Ogawa, Y., Quagliarotti, G., Jordan, J., Taylor, C.W., Starbuck, W.C. and Busch, H. (1969) J. Biol. Chem. 244, 4 3 8 7 - - 4 3 9 2 Iwai, K., Ishikawa, K. and Hayashi, H. (1970) Nature 226, 1056--1058