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Interaction of histones and nucleic acids in vitro
49 °
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96402
I N T E R A C T I O N OF H I S T O N E S AND NUCLEIC ACIDS EV V I T R O
M. SLUYSER
AND
N. H. SNELLEN-JURGENS
Department o[ Biochemistry, Antoni van Leeuwenhoekhuis, The Netherlands Cancer Institute, Amsterdam (The Netherlands) (Received September 8th, 1969)
SUMMARY
I. Peptides derived from very lysine-rich histone b y digestion with chymotrypsin interacted with DNA. 2. The solubility of complexes formed between histones or other polybases and nucleic acid depends largely on the degree of cross-linking within these complexes. At low concentration of salt or polybase, soluble complexes were formed. Higher concentrations of salt or polybase caused the formation of insoluble cross-linked complexes. At still higher concentrations of salt or polybase, relatively soluble linear complexes between nucleic acid and polybase were formed, although these sometimes gave rise to turbidity. 3- Evidence is presented to suggest that the very lysine-rich histones more easily form cross-links between nucleic acid molecules than do the arginine-rich histones. This m a y reflect the way these histones interact with DNA in chromatin.
INTRODUCTION
The mode of interaction between histones and DNA is of interest in view of a possible role of histones in regulating gene activity. The initial finding b y PHILLIPS AND SIMSON1 that the spacing of basic amino acid residues along the histone chain is not regular suggested that the salt-linkage structure of nucleohistone is irregular in the sense that loops of various lengths of non-basic amino acid residues form bulges adjacent to the salt linkages. The elucidation of the amino acid sequence of the glycine-rich-arginine-rich histone revealed a polarity of charge with the hydrophobic and acidic amino acid residues mainly clustered towards the C-terminus 2,5. In the very lysine-rich histone, the polarity of charge appears to be the reverse, since in these proteins the basic amino acids are located mainly at the C-terminus 6. Information on the structure of nucleohistone might be obtained b y investigating the interaction of DNA and histone in vitro, for instance b y determining the amount of DNA precipitated b y adding histone under various experimental conditions. In such studies, JOHNS AND FORRESTER7 found that when lysine-rich histone is added to DNA in water, low or high concentrations of histone cause more precipitation of DNA than does a medium concentration of histone. These authors repeated Biochim. Biophys. Acta, 199 (t97 o) 490-499
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an earlier proposal 8 t h a t the explanation of this effect might be the same as that of a similar phenomenon in antigen-antibody reactions. The following experiments were undertaken to determine whether this effect could also be obtained with peptides derived from lysine-rich histone b y enzymic digestion, and with other basic polypeptides such as arginine-rich histones and poly-L-lysine. Comparisons of precipitation curves in salt solutions and distilled water have therefore been made, using various concentrations of polypeptides and nucleic acids.
MATERIALS AND METHODS
Lysine-rich histone was prepared from fresh calf thymus as follows. The fI protein fraction was obtained according to Method 2 of JOHNS9. The fI was dissolved in distilled water and 6.1 M trichloroacetic acid was added to a final concentration of 0. 3 M. Following centrifugation for 30 min at 3000 ×g, the sediment was discarded and 6.1 M trichloroacetic acid was added to the supernatant to a final concentration of I . I M. After centrifugation, the sediment was suspended in 20 ml acetone-o.I ml concentrated HCI and submitted to centrifugation. The sediment was washed 3 times with acetone 1°, dissolved in a pH- 9 buffer containing 7 mM boric acid and 3 mM NaOH, and applied to a column of carboxymethylceUulose (CAM Nr. 132, obtained from Schleicher and Schiill, Dassel, Germany). After washing the column with the same buffer, the basic non-histone protein was removed b y eluting with 0.4 M NaC1 in the borate buffer. The lysine-rich histone was then obtained b y eluting with borate buffer containing 1. 5 M NaC1. This procedure was used because it has been shown 9,n that the fI protein fraction contains contaminating basic non-histone proteins which can be separated from the histone by chromatography on carboxymethylcellulose. The non-histone proteins are eluted with borate buffer or o. 4 M NaC1 in borate buffer. Trichloroacetic acid was added to a final concentration of 0. 3 M to the solution of lysine-rich histone. After centrifugation the sediment was discarded and trichloroacetic acid was added to the supernatant to a final concentration of I . I M. The precipitate was removed b y centrifugation, dissolved in distilled water and dialyzed against distilled water. The solution was freeze-dried yielding purified lysine-rich histone. Arginine-rich histone was isolated from calf thymus according to Method 2 of JOHNS (f3 histone) ~. DNA was prepared from calf thymus according to the procedure of KAY et al. TM. ~-Chymotrypsin was obtained from Boehringer, Mannheim, Germany Soluble RNA (Type IV) from calf liver and poly-L-lysine (HBr) were obtained from Sigma Chemical Co., St. Louis, Mo., U.S.A. The poly-L-lysines used were Type I (approximate tool. wt. 230 ooo) and Type I I (approximate tool. wt. 2600). Amino acid analysis was carried out with a Beckman Model Unichrom analyzer 13. Sephadex was a product of Pharmacia, Uppsala, Sweden.
Precipitation o/nucleic acids by adding basic polypeptides Solutions of basic polypeptides were added to solutions of DNA or RNA. The mixtures were submitted to centrifugation for I h at 3o0o ×g. The amount of nucleic acid precipitated was calculated b y measuring the absorbance at 260 n m of the supernatants. Bioehim. Biophys. Aeta, 199 (197o) 49o-499
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M. S L U Y S E R , N. H . S N E L L E N - J U R G E N S
RESULTS
Fig. I a shows the result obtained when a preparation of lysine-rich histone was submitted to gel filtration in o.oI M HC1 on a column of Sephadex G-Ioo. The protein was collected in Tubes 14-26. 50 mg lysine-rich histone was then dissolved in 2.5 ml phosphate buffer of p H 7.8 and ionic strength o.I, and dialysed overnight against a large volume of the phosphate buffer, o.I mg chymotrypsin in o.2 ml phosphate buffer was added to the histone solution, and the mixture was incubated for 3 h at 37 °. Precipitation of part of the material was observed. The precipitate was removed b y centrifugation. The supernatant (designated lysine-rich peptides) represented 85-95 % of the total material based on the recovery during amino acid analysis. When a portion of the lysine-rich peptides was submitted to gel filtration on Sephadex G-Ioo in o.oI M HC1, the material was collected in Tubes 31-4o, indicating that no undigested lysine-rich histone was present (Fig. Ib). In control experiments, the lysine-rich histone was incubated for 3 h at 37 ° with o.2 ml phosphate buffer in the absence of chymotrypsin. Under these conditions no precipitation of material was observed. A280 nm (a) 0,15
O.lO
0.05
(b)
0.15-
0.10.
0.05"
(c) 0.40. 0.30' 0.20 o.10
1()
20
30 40 -----a,,.- Tube number
F i g . I. G e l f i l t r a t i o n o n a c o l u m n (I c m × 7 ° c m ) o f S e p h a d e x G - l o o . E l u t i o n w i t h o . o z M H C I . F r a c t i o n s of z m l w e r e c o l l e c t e d , a. 1o m g ] y s i n e - r i c h h i s t o n e , b. 8 m g l y s i n e - r i c h p e p t i d e s , c. zo m g s a l t - f r e e ] y s i n e - r i c h p e p t i d e s .
Biochim. Biophys. dcta, 1 9 9 (197 o) 4 9 0 - 4 9 9
HISTONE-NUCLEIC
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Precipitation of DNA Fig. za illustrates the results obtained when various amounts of lysine-rich histone or lysine-rich peptides were added to portions of DNA in salt solutions. The DNA was precipitated when lysine-rich histone was added, whereas no precipitation of DNA occurred when lysine-rich peptides were added. However, an effect of lysinerich peptides on the solubility of DNA was revealed by the following experiment. Lysine-rich peptides were added to a salt solution containing DNA. The mixtures 7. DNA .reci.itated
-
mg
hi*,one
or peptides
tied
--c
lotal
“OI”ln~
(ml
1
Fig. 2. Precipitation of DNA following the addition of basic polypeptides. a and b. Solutions of 50 pg calf-thymus DNA in I ml o.rq M NaCl were incubated for 5 min at room temperature with 0.5 ml phosphate buffer (pH 7.8, I = 0.1) or with IOO ,ug lysine-rich peptides in 0.5 ml phosphate buffer. Various amounts of lysine-rich histone, lysine-rich peptides or arginine-rich histone in 0.5 ml phosphate buffer were then added. Lysine-rich histone (0-O). lysine-rich peptides added to DNA preincubated with phosphate. Lysine(0-e ) and arginine-rich h’is t one (f--n) rich histone (A- A) and arginine-rich histone (m-m) added to DNA preincubated with IOO pg lysine-rich peptides in phosphate. c. Lysine-rich histone (0-O) or lysine-rich peptides (o-0) in I ml phosphate buffer were added to I ml solutions containing 0.14 M NaCl and I mg calf2 mg lysine-rich peptides (o-0 ) or 0.6 mg thymus DNA. d. 2 mg lysine-rich histone (0-O). lysine-rich histone (A- - - A) in I ml phosphate buffer were added to portions of I mg calf-thymus DNA dissolved in various volumes of phosphate buffer. Total volumes were as indicated in the figure. Biochim.
Biophyys. Acta,
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were kept for 5 min at room temperature and then various amounts of lysine-rich histone were added. The amount of lysine-rich histone required to precipitate DNA in this case was lower than that required in the absence of lysine-rich peptides (Fig. 2a). In a similar experiment, arginine-rich histone was added to DNA and to DNA in the presence of lysine-rich peptides. Fig. zb shows that smaller amounts of arginine-rich histone were required to precipitate DNA in the presence of lysinerich peptides than in the absence of lysine-rich peptides. Precipitation curves were also studied using higher concentrations of lysinerich histones, lysine-rich peptides and DNA. Fig. 2c shows that under these conditions precipitation of DNA occurred when lysine-rich peptides were added. However, larger amounts of lysine-rich peptides than of lysine-rich histone were required to precipitate the DNA. Fig. 2d illustrates the results obtained when constant amounts of basic polypeptides and DNA were mixed in different volumes of phosphate buffer. When 2 mg lysine-rich histone was mixed with I mg DNA in a total volume of 2 ml, complete precipitation of DNA occurred. Complete precipitation also took place when mixing was carried out in larger volumes (up to 21 ml). Adding 0.6 mg lysine-rich histone or 2 mg lysine-rich peptides to I mg DNA in a 2-ml volume caused precipitation of of about 30 o/oof the DNA. When mixing was carried out in larger volumes, the percentage of DNA precipitated decreased markedly. Thus, when mixing was carried out in a zI-ml volume, no DNA was precipitated by 0.6 mg lysine-rich histone, and only IO o/o DNA was precipitated by z mg lysine-rich peptides. These experiments therefore revealed that in the presence of excess lysine-rich histone, changes in volume had no effect on the amount of DNA precipitated. By contrast, under experimental conditions whereby only about 30 y/oof the DNA was precipitated, increases in volume caused less DNA to be precipitated. Effect of ionic strength
Fig. 3a shows that when lysine-rich histone was mixed with DNA in distilled water, no precipitation of DNA occurred even when excess histone was added. By contrast, when lysine-rich peptides were mixed with DNA in distilled water, complete precipitation of DNA was observed. Fig. 3b shows the effect of ionic strength on the precipitation of DNA by poly-L-lysines. When poly-L-lysines Types I or II were mixed with DNA in 0.1 M phosphate, complete precipitation of DNA took place, whereas no precipitation occurred when mixing was carried out in distilled water. It seemed possible that the ability of lysine-rich peptides to precipitate DNA in distilled water (Fig. 3a) was due to the fact that these peptides had been prepared in salt solution, since digestion of lysine-rich histone with chymotrypsin had been carried out in 0.1 M phosphate buffer. The lysine-rich peptides would therefore be present as peptide salts, and precipitation of DNA by the peptide salts would consequently be partly due to a salting-out effect. In order to test this assumption, “salt-free” lysine-rich peptides were prepared from lysine-rich histone in the following way. 50 mg lysine-rich histone were dissolved in 2.5 ml distilled water, the pH of which had been raised to 7.8 by carefully adding dilute alkali. After dialysis against a large volume of water (pH 7.8), 0.1 mg chymotrypsin in 0.2 ml water (pH 7.8) was added and the mixture incubated for 3 h at 37”. The resulting precipitate was removed by centrifugation and a portion of the supernatant studied by gel Biochim.
Biophys.
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(1970)
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495
7. D N A precipitated
(b)
,.-t A?~=,,-D=P=D~D5Q=n=B 0 -
0.4 mg histone
OP peptides
0.6 odded
1
0.4
0 -
0.6 mg
polylysine
added
Fig. 3. Precipitation of DNA following the addition of basic polypeptides. Various amounts of polypeptides were added to solutions containing 0.1 mg calf-thymus DNA. Total volume 1.1 ml. a. In distilled water: O-0, lysine-rich histone; 0-0, lysine-rich peptides; l - - -m, salt-free lysine-rich peptides. b. In 0.1 M phosphate: W-U, poly-r.-lysine Type I; A- A, poly-L-lysine Type II. In distilled water: n-0, poly-r_-lysine Type I; A-A, poly-L-lysine Type II.
filtration on a column of Sephadex G-100. Fig. IC shows that all the material was eluted after Tube 27. Since undigested lysine-rich histone was completely eluted before tube 27 (Fig. ra), the soluble material obtained from lysine-rich histone by digestion with chymotrypsin in distilled water (pH 7.8) apparently did not contain undigested histone. Since the material collected after Tube 27 in Fig. IC was obtained in a practically salt-free medium, it will hereafter for convenience be designated salt-free lysine-rich peptides. Fig. 3a shows the result when various amounts of saltfree lysine-rich peptides were mixed with DNA in distilled water. When approximately 0.4 mg salt-free lysine-rich peptides was added to 0.1 mg DNA, almost complete precipitation of the DNA occurred. When an excess of salt-free lysine-rich peptides was added, the solution became slightly turbid but no DNA could be removed by centrifugation for I h at 3000 xg (see MATERIALSAND METHODS). Precipitation
of denatured DNA
CVETKOVIC AND SAVIC~*reported that heat-treated DNA was precipitated to
a smaller extent by fr histone than was native DNA. Since the fr fraction contains lysine-rich histone which is contaminated with other basic proteinseF1l,we examined the effect of purified lysine-rich histone on the solubility of heat-denatured DNA. Calf-thymus DNA in phosphate buffer was heated for IO min in a boiling-water bath, and then rapidly cooled by immersion in a solid CO,-acetone mixture. It was then allowed to attain room temperature. Another portion of DNA in phosphate buffer was heated for IO min in a boiling-water bath and then slowly cooled by standing at room temperature. The hypochromicity of these DNA solutions was B&him.
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496 %D NA precipitated
I-
(0) t 60
(b)
: ,/ 60
i0
40
60
80
20
100 A
tag histone
40 added
60
100
80
Fig. 4. Precipitation of native and denatured DNA by histones. Various amounts of lysine-rich histone (a) or arginine-rich histone (b) in 0.1 ml phosphate buffer (pH 7.8, I = 0.1) were added to ~oopg DNA in I ml phosphate. l - - -0, native calf-thymus DNA; A-A, DNA heated for IO min at 100’ and then rapidly cooled; o-0, DNA heated for IO min at 100’ and then slowly cooled. Corrections were made for hypochromicity.
%R N A precipitated (a)
(b)
L.
2.:
0 -
mg
0.4 polypeptide
0.6 added
Fig. 5. Precipitation of RNA following the addition of basic polypeptides. Solutions of poly-Llysine Type I (0-O). poly-L-lysine Type II (a-0). lysine-rich histone (n-n) or arginine-rich histone (A-A) were added to solutions containing 0.1 mg calf-liver RNA. Total volume r.r ml of phosphate buffer (pH 7.8, I = 0.1 (a)), phosphate buffer (pH 7.8, 2 = 0.01, (b)) and distilled water (c), All experiments were carried out in duplicate. Biochim.
Biophys.
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INTERACTION
1.75 and 1.37, respectively, compared to native DNA. Fig. qa shows that denatured DNA was less easily precipitated by lysine-rich histone than was native DNA. Rapidly cooled denatured DNA was precipitated to practicallly the same extent as slowly-cooled denatured DNA. In Fig. qb the results of similar experiments with arginine-rich histone are presented. Again heat-denatured DNA was precipitated to a lesser extent by histone than was native DNA. Precipitatiolz of RNA Poly-L-lysines Types I and II, lysine-rich histone and arginine-rich histone were mixed with calf-liver RNA in phosphate of ionic strength 0.1 (Fig. 5a), in phosphate of ionic strength 0.01 (Fig. 5b) or in distilled water (Fig. 5~). Practically all of the RNA remained in solution when mixing was carried out in distilled water. The amounts of RNA precipitated in phosphate buffer depended both on the ionic strength of the buffer and the type of basic polypeptide used. The solubility of RNA in the presence of basic polypeptides diminished with increasing ionic strenth of the phosphate. This effect was especially notable at high polypepticle/RNA concentration ratios. Changes in ionic strength of the phosphate or in the amount of histone added markedly affected the precipitation of RNA by arginine-rich histone but had a much smaller effect on the precipitation of RNA by lysine-rich histone.
DISCUSSION
Interactions between polybases and nucleic acids yield multiple possibilities of cross-linking, giving rise to many different products. Various aspects of these interactions have been discussed by other authors 7,8,14,15,1e,23.When conditions are unfavorable for cross-linking, i.e. when the macromolecules are too far apart, linear complexes are formed in which molecules of the polybase are aligned lengthwise on single DNA molecules15. The data presented here are consistent with the scheme shown in Fig. 6. Starting with a DNA solution of a certain concentration (I), the addition of 3NA DNA
)NF
I soluble
soluble
Fig. 6. Complexes formed when different containing DNA ().
insoluble
amounts
soluble
of histone
(- - -) are added
to a solution
histones or other polybases yields complexes of varied solubility. At low histone concentrations, the degree of cross-linking is insufficient to cause precipitation of the histone (II). At higher histone concentrations, cross-linked complexes areprecipitated from solution (III). When more histone is added, the degree of cross-linking Biochim.
Biophys.
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M. SLUYSER, N. H. SNELLEN-JURGENS
decreases but the complex still remains insoluble (IV). Finally, at very high histone concentrations relatively soluble linear complexes between DNA and histone are formed (V), although these complexes may cause turbidity of the solution. The formation of the latter complex is due to the fact that at high histone concentrations there is little likelihood that a histone molecule will attach to combining sites on two different DNA molecules at the same time, since one of these sites will already be occupied by histone. The degree to which each of these complexes exists depends on the concentration of the interacting macromolecules, the type and size of these macromolecules, and the ionic strength of the medium. The finding that lysine-rich histone more easily precipitates DNA than RNA 17 can be used as an analytical tool for removing contaminating RNA from preparations of DNA 18. When low concentrations of DNA and lysine-rich peptides are mixed, soluble Complex II is formed. By contrast, when similar concentrations of DNA and lysinerich histones are mixed, insoluble Complex III is formed (Fig. Ia). This is probably due to the DNA molecules being too greatly separated for cross-linking by lysinerich peptides to occur. High concentrations of lysine-rich peptides and DNA give rise to insoluble Complex III (Fig. ic). When lysine-rich histones or arginine-rich histones are added to the soluble Complex II formed between low concentrations of lysine-rich peptides and DNA, insoluble Complex III is formed, due to additional cross-linking by the histones (Figs. Ia and b). When the DNA is denatured, the histones apparently form cross-links less easily between the DNA molecules than they do with native DNA (Fig. 4). It may be assumed that the cross-links between DNA are formed by salt bridges. In distilled water, no cross-linking can take place (Fig. 3). A similar phenomenon is observed with RNA (Fig. 5). The effect of salt on the solubility of the complex is strongest at high polybase/nucleic acid concentration ratios. This is clearly seen in Figs. 5a and 5b, where lowering of the concentration of phosphate causes the precipitation curves of the poly-L-lysines to become bell shaped. This may be explained by the fact that when the polylysine/RNA ratio is IO, complexes of Type IV are formed, whereas when the polylysine/RNA ratio is 2, complexes of Type III are formed. In Complex III more cross-links exist between the RNA molecules than in Complex IV, and therefore Complex III tends to remain more insoluble, even when the ionic strength of the medium is lowered. A similar effect is observed it~ Fig. 3 a with lysine-rich peptides and DNA. Removal of salt from these peptides causes the precipitation plot to attain a bell-shaped curve. In general, increasing salt concentrations will convert soluble Complex II to insoluble Complexes III and IV and finally to soluble Complex V. Formation of the latter is probably due to salt ions screening off the combining groups 15. The finding that smaller amounts of lysine-rich histone than of arginine-rich histone are required to precipitate DNA irom salt solution 17,19 suggested that the lysine-rich histones tend to form cross-links between DNA molecules, whereas the arginine-rich histories tend to align themselves along separate DNA molecules 19. This is consistent with evidence presented by LITTAU et al. ~° that lysine-rich histories form cross-links between DNA strands in calf-thymus chromatin, whereas arginine-rich histones are arranged along the DNA chain. The data presented in Figs. 5a and 5b are also consistent with this view. When the ionic strength of the medium was lowered, the precipitation of nucleic acid by a high concentration of arginine-rich histone was markedBiochim. Biophys. Acta~ 199 (197 o) 49o-499
HISTONE-NUCLEIC
ACID INTERACTION
vitro is not certain.
REFERENCES D. M. P. PHILLIPS AND P. SIMSON, Biochem. J., 82 (1962) 236. R. J. DE LANGE, D. M. FAMBROUGH, E. L. SMITH AND J. BONNER, J. Biol. Chem.,
243 (1968)
5906. P. SAUTIERE, W. C. STARBUCK, C. ROTH AND H. BUSCH, J. Biol. Chem., 243 (1968) 5899. L. DESAI, Y. OGAWA, C. M. MAURITZEN, C. W. TAYLOR AND W. C. STARBUCK, Biochim. Biophys. Acta, 181 (1969) 146. R. J. DE LANGE, D. M. FAMBROUGH, E. L. SMITH AND J. BONNER, J. Biol. Chem., 244 (1969)
319.
IO II
12 I3 I4 I5 16 I7
18 Ig 20 21
22 23 24
M. BUSTIN, S. C. RALL, R. H. STELLWAGEN AND R. D. COLE, Science, 163 (1969) 391. E. W. JOHNS AND S. FORRESTER, B&hem. J., III (1969) 371. A. R. PEACOCKE, Progv. Biophys. Biophys. Chew, IO (1960) 105. E. W. JOHNS, B&hem. J., g2 (1964) 55. C. DICK AND E. W. JOHNS, Biochim. Biophys. Acta, 174 (1969) 380. M. SLUYSER, PH. R~MKE AND A. HEKMAN, Immunochemistry, 6 (1969) 494. E. KAY, N. SIMMONS AND A. DOUNCE, J. Am. Chem. Sot., 74 (1952) ‘724. D. H. SPACKMAN, W. H. STEIN AND S. MOORE, Anal. Chem., 30 (1958) 1190. M. D. CVETKOVIC AND R. M. SAVIC, Biochem. J., 112 (1969) 801. P. SPITNIK, R. LIPSHITZ AND E. CHARGAFF, J. Biol. Chem., 215 (1955) 765. G. ZUBAY, in J. BONNER AND P. 0. P. Ts’O, The nucleohistones, Holden Day, San FranciscoLondon-Amsterdam, 1964, p. 95. J. A. V. BUTLER AND E. W. JOHNS, Biochem. J., 91 (1964) 15C. M. SLUYSER AND L. DEN ENGELSE, Anal. Biochem., 25 (1968) 444. M. SLUYSER, in V. V. KONINGSBERGER AND L. BOSCH, Regulation of nucleic acid and protein synthesis, Elsevier, Amsterdam, 1967, p. 225. V. C. LITTAU, C. J. BURDICK, V. G. ALLFREY AND A. E. MIRSKY, J. Cell Biol., 27 (1965) 124A. A. J. HAYDON AND A. R. PEACOCKE, Biochem. ,J., IIO (1968) 243. M. SLUYSER, Biochim. Riophys. Acta, 182 (1969) 235. K. B. BJORNESJO AND T. TEORELL, Arkiv. Kemi Min. Geol., IDA, No. 34 (1945). E. M. BRADBURY, C. CRANE-ROBINSON, D. M. P. PHILLIPS, E. W. JOHNS AND K. MURRAY, Nature, 205 (1965) 1315.
Biochim.
Biophys.
Acta,
Igg
(1970)
490-499
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96402
I N T E R A C T I O N OF H I S T O N E S AND NUCLEIC ACIDS EV V I T R O
M. SLUYSER
AND
N. H. SNELLEN-JURGENS
Department o[ Biochemistry, Antoni van Leeuwenhoekhuis, The Netherlands Cancer Institute, Amsterdam (The Netherlands) (Received September 8th, 1969)
SUMMARY
I. Peptides derived from very lysine-rich histone b y digestion with chymotrypsin interacted with DNA. 2. The solubility of complexes formed between histones or other polybases and nucleic acid depends largely on the degree of cross-linking within these complexes. At low concentration of salt or polybase, soluble complexes were formed. Higher concentrations of salt or polybase caused the formation of insoluble cross-linked complexes. At still higher concentrations of salt or polybase, relatively soluble linear complexes between nucleic acid and polybase were formed, although these sometimes gave rise to turbidity. 3- Evidence is presented to suggest that the very lysine-rich histones more easily form cross-links between nucleic acid molecules than do the arginine-rich histones. This m a y reflect the way these histones interact with DNA in chromatin.
INTRODUCTION
The mode of interaction between histones and DNA is of interest in view of a possible role of histones in regulating gene activity. The initial finding b y PHILLIPS AND SIMSON1 that the spacing of basic amino acid residues along the histone chain is not regular suggested that the salt-linkage structure of nucleohistone is irregular in the sense that loops of various lengths of non-basic amino acid residues form bulges adjacent to the salt linkages. The elucidation of the amino acid sequence of the glycine-rich-arginine-rich histone revealed a polarity of charge with the hydrophobic and acidic amino acid residues mainly clustered towards the C-terminus 2,5. In the very lysine-rich histone, the polarity of charge appears to be the reverse, since in these proteins the basic amino acids are located mainly at the C-terminus 6. Information on the structure of nucleohistone might be obtained b y investigating the interaction of DNA and histone in vitro, for instance b y determining the amount of DNA precipitated b y adding histone under various experimental conditions. In such studies, JOHNS AND FORRESTER7 found that when lysine-rich histone is added to DNA in water, low or high concentrations of histone cause more precipitation of DNA than does a medium concentration of histone. These authors repeated Biochim. Biophys. Acta, 199 (t97 o) 490-499
HISTONE-NUCLEIC
491
ACID INTERACTION
an earlier proposal 8 t h a t the explanation of this effect might be the same as that of a similar phenomenon in antigen-antibody reactions. The following experiments were undertaken to determine whether this effect could also be obtained with peptides derived from lysine-rich histone b y enzymic digestion, and with other basic polypeptides such as arginine-rich histones and poly-L-lysine. Comparisons of precipitation curves in salt solutions and distilled water have therefore been made, using various concentrations of polypeptides and nucleic acids.
MATERIALS AND METHODS
Lysine-rich histone was prepared from fresh calf thymus as follows. The fI protein fraction was obtained according to Method 2 of JOHNS9. The fI was dissolved in distilled water and 6.1 M trichloroacetic acid was added to a final concentration of 0. 3 M. Following centrifugation for 30 min at 3000 ×g, the sediment was discarded and 6.1 M trichloroacetic acid was added to the supernatant to a final concentration of I . I M. After centrifugation, the sediment was suspended in 20 ml acetone-o.I ml concentrated HCI and submitted to centrifugation. The sediment was washed 3 times with acetone 1°, dissolved in a pH- 9 buffer containing 7 mM boric acid and 3 mM NaOH, and applied to a column of carboxymethylceUulose (CAM Nr. 132, obtained from Schleicher and Schiill, Dassel, Germany). After washing the column with the same buffer, the basic non-histone protein was removed b y eluting with 0.4 M NaC1 in the borate buffer. The lysine-rich histone was then obtained b y eluting with borate buffer containing 1. 5 M NaC1. This procedure was used because it has been shown 9,n that the fI protein fraction contains contaminating basic non-histone proteins which can be separated from the histone by chromatography on carboxymethylcellulose. The non-histone proteins are eluted with borate buffer or o. 4 M NaC1 in borate buffer. Trichloroacetic acid was added to a final concentration of 0. 3 M to the solution of lysine-rich histone. After centrifugation the sediment was discarded and trichloroacetic acid was added to the supernatant to a final concentration of I . I M. The precipitate was removed b y centrifugation, dissolved in distilled water and dialyzed against distilled water. The solution was freeze-dried yielding purified lysine-rich histone. Arginine-rich histone was isolated from calf thymus according to Method 2 of JOHNS (f3 histone) ~. DNA was prepared from calf thymus according to the procedure of KAY et al. TM. ~-Chymotrypsin was obtained from Boehringer, Mannheim, Germany Soluble RNA (Type IV) from calf liver and poly-L-lysine (HBr) were obtained from Sigma Chemical Co., St. Louis, Mo., U.S.A. The poly-L-lysines used were Type I (approximate tool. wt. 230 ooo) and Type I I (approximate tool. wt. 2600). Amino acid analysis was carried out with a Beckman Model Unichrom analyzer 13. Sephadex was a product of Pharmacia, Uppsala, Sweden.
Precipitation o/nucleic acids by adding basic polypeptides Solutions of basic polypeptides were added to solutions of DNA or RNA. The mixtures were submitted to centrifugation for I h at 3o0o ×g. The amount of nucleic acid precipitated was calculated b y measuring the absorbance at 260 n m of the supernatants. Bioehim. Biophys. Aeta, 199 (197o) 49o-499
492
M. S L U Y S E R , N. H . S N E L L E N - J U R G E N S
RESULTS
Fig. I a shows the result obtained when a preparation of lysine-rich histone was submitted to gel filtration in o.oI M HC1 on a column of Sephadex G-Ioo. The protein was collected in Tubes 14-26. 50 mg lysine-rich histone was then dissolved in 2.5 ml phosphate buffer of p H 7.8 and ionic strength o.I, and dialysed overnight against a large volume of the phosphate buffer, o.I mg chymotrypsin in o.2 ml phosphate buffer was added to the histone solution, and the mixture was incubated for 3 h at 37 °. Precipitation of part of the material was observed. The precipitate was removed b y centrifugation. The supernatant (designated lysine-rich peptides) represented 85-95 % of the total material based on the recovery during amino acid analysis. When a portion of the lysine-rich peptides was submitted to gel filtration on Sephadex G-Ioo in o.oI M HC1, the material was collected in Tubes 31-4o, indicating that no undigested lysine-rich histone was present (Fig. Ib). In control experiments, the lysine-rich histone was incubated for 3 h at 37 ° with o.2 ml phosphate buffer in the absence of chymotrypsin. Under these conditions no precipitation of material was observed. A280 nm (a) 0,15
O.lO
0.05
(b)
0.15-
0.10.
0.05"
(c) 0.40. 0.30' 0.20 o.10
1()
20
30 40 -----a,,.- Tube number
F i g . I. G e l f i l t r a t i o n o n a c o l u m n (I c m × 7 ° c m ) o f S e p h a d e x G - l o o . E l u t i o n w i t h o . o z M H C I . F r a c t i o n s of z m l w e r e c o l l e c t e d , a. 1o m g ] y s i n e - r i c h h i s t o n e , b. 8 m g l y s i n e - r i c h p e p t i d e s , c. zo m g s a l t - f r e e ] y s i n e - r i c h p e p t i d e s .
Biochim. Biophys. dcta, 1 9 9 (197 o) 4 9 0 - 4 9 9
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Precipitation of DNA Fig. za illustrates the results obtained when various amounts of lysine-rich histone or lysine-rich peptides were added to portions of DNA in salt solutions. The DNA was precipitated when lysine-rich histone was added, whereas no precipitation of DNA occurred when lysine-rich peptides were added. However, an effect of lysinerich peptides on the solubility of DNA was revealed by the following experiment. Lysine-rich peptides were added to a salt solution containing DNA. The mixtures 7. DNA .reci.itated
-
mg
hi*,one
or peptides
tied
--c
lotal
“OI”ln~
(ml
1
Fig. 2. Precipitation of DNA following the addition of basic polypeptides. a and b. Solutions of 50 pg calf-thymus DNA in I ml o.rq M NaCl were incubated for 5 min at room temperature with 0.5 ml phosphate buffer (pH 7.8, I = 0.1) or with IOO ,ug lysine-rich peptides in 0.5 ml phosphate buffer. Various amounts of lysine-rich histone, lysine-rich peptides or arginine-rich histone in 0.5 ml phosphate buffer were then added. Lysine-rich histone (0-O). lysine-rich peptides added to DNA preincubated with phosphate. Lysine(0-e ) and arginine-rich h’is t one (f--n) rich histone (A- A) and arginine-rich histone (m-m) added to DNA preincubated with IOO pg lysine-rich peptides in phosphate. c. Lysine-rich histone (0-O) or lysine-rich peptides (o-0) in I ml phosphate buffer were added to I ml solutions containing 0.14 M NaCl and I mg calf2 mg lysine-rich peptides (o-0 ) or 0.6 mg thymus DNA. d. 2 mg lysine-rich histone (0-O). lysine-rich histone (A- - - A) in I ml phosphate buffer were added to portions of I mg calf-thymus DNA dissolved in various volumes of phosphate buffer. Total volumes were as indicated in the figure. Biochim.
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were kept for 5 min at room temperature and then various amounts of lysine-rich histone were added. The amount of lysine-rich histone required to precipitate DNA in this case was lower than that required in the absence of lysine-rich peptides (Fig. 2a). In a similar experiment, arginine-rich histone was added to DNA and to DNA in the presence of lysine-rich peptides. Fig. zb shows that smaller amounts of arginine-rich histone were required to precipitate DNA in the presence of lysinerich peptides than in the absence of lysine-rich peptides. Precipitation curves were also studied using higher concentrations of lysinerich histones, lysine-rich peptides and DNA. Fig. 2c shows that under these conditions precipitation of DNA occurred when lysine-rich peptides were added. However, larger amounts of lysine-rich peptides than of lysine-rich histone were required to precipitate the DNA. Fig. 2d illustrates the results obtained when constant amounts of basic polypeptides and DNA were mixed in different volumes of phosphate buffer. When 2 mg lysine-rich histone was mixed with I mg DNA in a total volume of 2 ml, complete precipitation of DNA occurred. Complete precipitation also took place when mixing was carried out in larger volumes (up to 21 ml). Adding 0.6 mg lysine-rich histone or 2 mg lysine-rich peptides to I mg DNA in a 2-ml volume caused precipitation of of about 30 o/oof the DNA. When mixing was carried out in larger volumes, the percentage of DNA precipitated decreased markedly. Thus, when mixing was carried out in a zI-ml volume, no DNA was precipitated by 0.6 mg lysine-rich histone, and only IO o/o DNA was precipitated by z mg lysine-rich peptides. These experiments therefore revealed that in the presence of excess lysine-rich histone, changes in volume had no effect on the amount of DNA precipitated. By contrast, under experimental conditions whereby only about 30 y/oof the DNA was precipitated, increases in volume caused less DNA to be precipitated. Effect of ionic strength
Fig. 3a shows that when lysine-rich histone was mixed with DNA in distilled water, no precipitation of DNA occurred even when excess histone was added. By contrast, when lysine-rich peptides were mixed with DNA in distilled water, complete precipitation of DNA was observed. Fig. 3b shows the effect of ionic strength on the precipitation of DNA by poly-L-lysines. When poly-L-lysines Types I or II were mixed with DNA in 0.1 M phosphate, complete precipitation of DNA took place, whereas no precipitation occurred when mixing was carried out in distilled water. It seemed possible that the ability of lysine-rich peptides to precipitate DNA in distilled water (Fig. 3a) was due to the fact that these peptides had been prepared in salt solution, since digestion of lysine-rich histone with chymotrypsin had been carried out in 0.1 M phosphate buffer. The lysine-rich peptides would therefore be present as peptide salts, and precipitation of DNA by the peptide salts would consequently be partly due to a salting-out effect. In order to test this assumption, “salt-free” lysine-rich peptides were prepared from lysine-rich histone in the following way. 50 mg lysine-rich histone were dissolved in 2.5 ml distilled water, the pH of which had been raised to 7.8 by carefully adding dilute alkali. After dialysis against a large volume of water (pH 7.8), 0.1 mg chymotrypsin in 0.2 ml water (pH 7.8) was added and the mixture incubated for 3 h at 37”. The resulting precipitate was removed by centrifugation and a portion of the supernatant studied by gel Biochim.
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HISTONE-NUCLEICACID INTERACTION
495
7. D N A precipitated
(b)
,.-t A?~=,,-D=P=D~D5Q=n=B 0 -
0.4 mg histone
OP peptides
0.6 odded
1
0.4
0 -
0.6 mg
polylysine
added
Fig. 3. Precipitation of DNA following the addition of basic polypeptides. Various amounts of polypeptides were added to solutions containing 0.1 mg calf-thymus DNA. Total volume 1.1 ml. a. In distilled water: O-0, lysine-rich histone; 0-0, lysine-rich peptides; l - - -m, salt-free lysine-rich peptides. b. In 0.1 M phosphate: W-U, poly-r.-lysine Type I; A- A, poly-L-lysine Type II. In distilled water: n-0, poly-r_-lysine Type I; A-A, poly-L-lysine Type II.
filtration on a column of Sephadex G-100. Fig. IC shows that all the material was eluted after Tube 27. Since undigested lysine-rich histone was completely eluted before tube 27 (Fig. ra), the soluble material obtained from lysine-rich histone by digestion with chymotrypsin in distilled water (pH 7.8) apparently did not contain undigested histone. Since the material collected after Tube 27 in Fig. IC was obtained in a practically salt-free medium, it will hereafter for convenience be designated salt-free lysine-rich peptides. Fig. 3a shows the result when various amounts of saltfree lysine-rich peptides were mixed with DNA in distilled water. When approximately 0.4 mg salt-free lysine-rich peptides was added to 0.1 mg DNA, almost complete precipitation of the DNA occurred. When an excess of salt-free lysine-rich peptides was added, the solution became slightly turbid but no DNA could be removed by centrifugation for I h at 3000 xg (see MATERIALSAND METHODS). Precipitation
of denatured DNA
CVETKOVIC AND SAVIC~*reported that heat-treated DNA was precipitated to
a smaller extent by fr histone than was native DNA. Since the fr fraction contains lysine-rich histone which is contaminated with other basic proteinseF1l,we examined the effect of purified lysine-rich histone on the solubility of heat-denatured DNA. Calf-thymus DNA in phosphate buffer was heated for IO min in a boiling-water bath, and then rapidly cooled by immersion in a solid CO,-acetone mixture. It was then allowed to attain room temperature. Another portion of DNA in phosphate buffer was heated for IO min in a boiling-water bath and then slowly cooled by standing at room temperature. The hypochromicity of these DNA solutions was B&him.
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496 %D NA precipitated
I-
(0) t 60
(b)
: ,/ 60
i0
40
60
80
20
100 A
tag histone
40 added
60
100
80
Fig. 4. Precipitation of native and denatured DNA by histones. Various amounts of lysine-rich histone (a) or arginine-rich histone (b) in 0.1 ml phosphate buffer (pH 7.8, I = 0.1) were added to ~oopg DNA in I ml phosphate. l - - -0, native calf-thymus DNA; A-A, DNA heated for IO min at 100’ and then rapidly cooled; o-0, DNA heated for IO min at 100’ and then slowly cooled. Corrections were made for hypochromicity.
%R N A precipitated (a)
(b)
L.
2.:
0 -
mg
0.4 polypeptide
0.6 added
Fig. 5. Precipitation of RNA following the addition of basic polypeptides. Solutions of poly-Llysine Type I (0-O). poly-L-lysine Type II (a-0). lysine-rich histone (n-n) or arginine-rich histone (A-A) were added to solutions containing 0.1 mg calf-liver RNA. Total volume r.r ml of phosphate buffer (pH 7.8, I = 0.1 (a)), phosphate buffer (pH 7.8, 2 = 0.01, (b)) and distilled water (c), All experiments were carried out in duplicate. Biochim.
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INTERACTION
1.75 and 1.37, respectively, compared to native DNA. Fig. qa shows that denatured DNA was less easily precipitated by lysine-rich histone than was native DNA. Rapidly cooled denatured DNA was precipitated to practicallly the same extent as slowly-cooled denatured DNA. In Fig. qb the results of similar experiments with arginine-rich histone are presented. Again heat-denatured DNA was precipitated to a lesser extent by histone than was native DNA. Precipitatiolz of RNA Poly-L-lysines Types I and II, lysine-rich histone and arginine-rich histone were mixed with calf-liver RNA in phosphate of ionic strength 0.1 (Fig. 5a), in phosphate of ionic strength 0.01 (Fig. 5b) or in distilled water (Fig. 5~). Practically all of the RNA remained in solution when mixing was carried out in distilled water. The amounts of RNA precipitated in phosphate buffer depended both on the ionic strength of the buffer and the type of basic polypeptide used. The solubility of RNA in the presence of basic polypeptides diminished with increasing ionic strenth of the phosphate. This effect was especially notable at high polypepticle/RNA concentration ratios. Changes in ionic strength of the phosphate or in the amount of histone added markedly affected the precipitation of RNA by arginine-rich histone but had a much smaller effect on the precipitation of RNA by lysine-rich histone.
DISCUSSION
Interactions between polybases and nucleic acids yield multiple possibilities of cross-linking, giving rise to many different products. Various aspects of these interactions have been discussed by other authors 7,8,14,15,1e,23.When conditions are unfavorable for cross-linking, i.e. when the macromolecules are too far apart, linear complexes are formed in which molecules of the polybase are aligned lengthwise on single DNA molecules15. The data presented here are consistent with the scheme shown in Fig. 6. Starting with a DNA solution of a certain concentration (I), the addition of 3NA DNA
)NF
I soluble
soluble
Fig. 6. Complexes formed when different containing DNA ().
insoluble
amounts
soluble
of histone
(- - -) are added
to a solution
histones or other polybases yields complexes of varied solubility. At low histone concentrations, the degree of cross-linking is insufficient to cause precipitation of the histone (II). At higher histone concentrations, cross-linked complexes areprecipitated from solution (III). When more histone is added, the degree of cross-linking Biochim.
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decreases but the complex still remains insoluble (IV). Finally, at very high histone concentrations relatively soluble linear complexes between DNA and histone are formed (V), although these complexes may cause turbidity of the solution. The formation of the latter complex is due to the fact that at high histone concentrations there is little likelihood that a histone molecule will attach to combining sites on two different DNA molecules at the same time, since one of these sites will already be occupied by histone. The degree to which each of these complexes exists depends on the concentration of the interacting macromolecules, the type and size of these macromolecules, and the ionic strength of the medium. The finding that lysine-rich histone more easily precipitates DNA than RNA 17 can be used as an analytical tool for removing contaminating RNA from preparations of DNA 18. When low concentrations of DNA and lysine-rich peptides are mixed, soluble Complex II is formed. By contrast, when similar concentrations of DNA and lysinerich histones are mixed, insoluble Complex III is formed (Fig. Ia). This is probably due to the DNA molecules being too greatly separated for cross-linking by lysinerich peptides to occur. High concentrations of lysine-rich peptides and DNA give rise to insoluble Complex III (Fig. ic). When lysine-rich histones or arginine-rich histones are added to the soluble Complex II formed between low concentrations of lysine-rich peptides and DNA, insoluble Complex III is formed, due to additional cross-linking by the histones (Figs. Ia and b). When the DNA is denatured, the histones apparently form cross-links less easily between the DNA molecules than they do with native DNA (Fig. 4). It may be assumed that the cross-links between DNA are formed by salt bridges. In distilled water, no cross-linking can take place (Fig. 3). A similar phenomenon is observed with RNA (Fig. 5). The effect of salt on the solubility of the complex is strongest at high polybase/nucleic acid concentration ratios. This is clearly seen in Figs. 5a and 5b, where lowering of the concentration of phosphate causes the precipitation curves of the poly-L-lysines to become bell shaped. This may be explained by the fact that when the polylysine/RNA ratio is IO, complexes of Type IV are formed, whereas when the polylysine/RNA ratio is 2, complexes of Type III are formed. In Complex III more cross-links exist between the RNA molecules than in Complex IV, and therefore Complex III tends to remain more insoluble, even when the ionic strength of the medium is lowered. A similar effect is observed it~ Fig. 3 a with lysine-rich peptides and DNA. Removal of salt from these peptides causes the precipitation plot to attain a bell-shaped curve. In general, increasing salt concentrations will convert soluble Complex II to insoluble Complexes III and IV and finally to soluble Complex V. Formation of the latter is probably due to salt ions screening off the combining groups 15. The finding that smaller amounts of lysine-rich histone than of arginine-rich histone are required to precipitate DNA irom salt solution 17,19 suggested that the lysine-rich histones tend to form cross-links between DNA molecules, whereas the arginine-rich histories tend to align themselves along separate DNA molecules 19. This is consistent with evidence presented by LITTAU et al. ~° that lysine-rich histories form cross-links between DNA strands in calf-thymus chromatin, whereas arginine-rich histones are arranged along the DNA chain. The data presented in Figs. 5a and 5b are also consistent with this view. When the ionic strength of the medium was lowered, the precipitation of nucleic acid by a high concentration of arginine-rich histone was markedBiochim. Biophys. Acta~ 199 (197 o) 49o-499
HISTONE-NUCLEIC
ACID INTERACTION
vitro is not certain.
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M. BUSTIN, S. C. RALL, R. H. STELLWAGEN AND R. D. COLE, Science, 163 (1969) 391. E. W. JOHNS AND S. FORRESTER, B&hem. J., III (1969) 371. A. R. PEACOCKE, Progv. Biophys. Biophys. Chew, IO (1960) 105. E. W. JOHNS, B&hem. J., g2 (1964) 55. C. DICK AND E. W. JOHNS, Biochim. Biophys. Acta, 174 (1969) 380. M. SLUYSER, PH. R~MKE AND A. HEKMAN, Immunochemistry, 6 (1969) 494. E. KAY, N. SIMMONS AND A. DOUNCE, J. Am. Chem. Sot., 74 (1952) ‘724. D. H. SPACKMAN, W. H. STEIN AND S. MOORE, Anal. Chem., 30 (1958) 1190. M. D. CVETKOVIC AND R. M. SAVIC, Biochem. J., 112 (1969) 801. P. SPITNIK, R. LIPSHITZ AND E. CHARGAFF, J. Biol. Chem., 215 (1955) 765. G. ZUBAY, in J. BONNER AND P. 0. P. Ts’O, The nucleohistones, Holden Day, San FranciscoLondon-Amsterdam, 1964, p. 95. J. A. V. BUTLER AND E. W. JOHNS, Biochem. J., 91 (1964) 15C. M. SLUYSER AND L. DEN ENGELSE, Anal. Biochem., 25 (1968) 444. M. SLUYSER, in V. V. KONINGSBERGER AND L. BOSCH, Regulation of nucleic acid and protein synthesis, Elsevier, Amsterdam, 1967, p. 225. V. C. LITTAU, C. J. BURDICK, V. G. ALLFREY AND A. E. MIRSKY, J. Cell Biol., 27 (1965) 124A. A. J. HAYDON AND A. R. PEACOCKE, Biochem. ,J., IIO (1968) 243. M. SLUYSER, Biochim. Riophys. Acta, 182 (1969) 235. K. B. BJORNESJO AND T. TEORELL, Arkiv. Kemi Min. Geol., IDA, No. 34 (1945). E. M. BRADBURY, C. CRANE-ROBINSON, D. M. P. PHILLIPS, E. W. JOHNS AND K. MURRAY, Nature, 205 (1965) 1315.
Biochim.
Biophys.
Acta,
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(1970)
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