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Structure of nucleohistone. III. Interaction with toluidine blue
43~
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 95690
S T R U C T U R E OF NUCLEOHISTONE. I I I . I N T E R A C T I O N W I T H T O L U I D I N E BLUE A K I K O M I U R A * AND Y O S H I K I OHBA*"
Faculty o] Pharmaceutical Sciences, University of Tokyo, Hongo, Tokyo (Japan) (Received March 8th, 1967)
SUMMARY
The interaction of toluidine blue with calf-thymus nucleohistone and DNA was investigated spectrophotometrically with special reference to tile spectral transition occurring on formation of the complex. The dye was found to bind to nucleohistone and DNA in at least two ways (Process I, II). These binding sites could be estimated separately. It was found by Millipore filtration methods that there were 0.35 and o.32 sites per unit nucleotide of DNA and nucleohistone respectively for Process I, and I.OO and o.48 for Process II. The results indicate that in a nucleohistone molecule about half the phosphate groups are free from basic groups of the histones.
INTRODUCTION
Histones have been found in cells in association with DNA and evidently may be regarded as cellular components playing a part in the mechanism controlling the function of DNA. Studies1, 2 have been made, therefore, on what effect they have on the structure of DNA in their associated state, that is as nucleohistone. It is known that the phosphate group of DNA is responsible for DNA-histone bonding, but that there are some phosphate groups in a nucleohistone molecule which are not bound with basic groups of histones*. Only the latter would act as active genes. It seems possible to estimate the quantity of these groups by the interaction of DNA with a basic dye capable of binding with DNA phosphate. The results of such studies have been reported3, 4. KLEIN AND SZIRMAI4 studied the stoichiometric precipitation reaction between azure A and DNA or nucleohistone, and found that only half of the possible binding sites were available for the dye. Several investigators have studied the interaction of DNA with acridine dyes. According to PEACOCKE AND SKERRETa5 proflavin can bind to DNA in at least two ways. The primary strong reaction (Process I) has been studied extensively by LERMAN~, who interpreted it as intercalation, a process in which the dye molecule Present address: D e p a r t m e n t of Molecular Biology, P o r t s m o u t h College of Technology, Park Road, P o r t s m o u t h (Great Britain). ** Present address: D e p a r t m e n t of Chemistry, National i n s t i t u t e of Health, Shinagawaku, Kamiosaki, Tokyo (Japan).
Biochim. Biophys. Acts, 145 (1967) 436 445
INTERACTION OF NUCLEOHISTONE WITH TOLUIDINE BLUE
437
enters between base pairs which are normally adjacent, in a plane perpendicular to the helical axis of DNA. The secondary weak process (Process II), which has been proposed b y BRADLEY AND W O L F 7, is interpreted as the being due to the binding of dye b y stacking, or aggregation, that is by weak interaction between aminoacridine molecules and the ionic groups of polyanions such as DNA. In this study we found that toluidine blue, a cationic dye, could bind to DNA or nucleohistone in two ways as with acridine dyes. By measuring these binding sites separately, we estimated the proportion of phosphate groups which were free from histones in the nucleohistone molecule.
MATERIALS AND METHODS
Nucleohistone and other materials The procedures used for the extraction of calf thymus nucleohistone and DNA have already been described 1. Generally 0. 7 mM phosphate buffer (pH 7.0) was used as solvent. The polylysine bromide (Pilot Chemical Co.), which had a tool. wt. of 60 ooo, was used after dialysis against 0. 7 mM phosphate buffer.
Toluidine blue Toluidine blue was a single product of Merck Chemical Co. Concentrations were estimated using an extinction value of 33 200 at 632 m/~ (ref. 8). Beer's law was obeyed at 632 m# up to a dye concentration of 24/,M.
Procedures used/or binding dye and measurement o/absorption A dye solution of 80/tM to 960 #M was slowly added to DNA or nucleohistone solution, with vigorous stirring. Absorbance was measured 30 min after addition of the dye b y which time the reaction was complete. The absorbance of solutions was measured in a spectrophotometer (Hitachi SI2-B). Absorption spectra were obtained using a Cary Model-I 4. The procedure for the determination of thermal profiles was described previously 2.
Millipore /iltration A single Millipore filter (pore size, o.22 #) was used throughout. In the filtration method, the effect of possible errors occurring b y the adsorption of free dyes to the filter and the residual complex on the filter was minimized b y choosing the best condition, empirically determined, as follows. Volumes of at least 15 ml of solution of dye complex were prepared. About 3 ml of the solution was filtered first. Then the surface of the membrane was wiped with moistened, defatted cotton to eliminate the residue which could still adsorb free dye. This procedure was repeated 4 times. In this way an equilibrium was attained between free dye in the solution and dye adsorbed to the membrane. Thus the concentration of dye in the filtrate of the 5th portion was determined as the same as the concentration of free dye in the solution. Ultraviolet measurements of the filtrate, under similar conditions, proved that Biochim. Biophys. Acta, 145 (1967) 436-445
438
A. MIURA, Y. OHBA
neither leakage of DNA nor nucleohistone occurred in this filtration experiment, at least when the ratio of dye/DNA-P was greater than 0.8, or dye/nucleohistone-P greater than 0.4.
RESULTS
Spectral transition o/toluidine blue by addition o/ DNA and nucleohistone Toluidine blue showed a visible absorption spectrum of constant shape with a m a x i m u m at 632 m#, at least at concentrations of less than 24/~M in 0.7 mM phosphate buffer. Typical absorption spectra of solutions containing a constant concentration of toluidine blue and increasing amounts of DNA are shown in Fig. I, which illustrates the progressive shift of the m a x i m u m towards 548 m# (blue shift), and a marked decrease at 632 m/~. An isosbestic point was evident at 563 m#. On further increasing in the amount of DNA, a new spectral m a x i m u m appeared at 648 m/~ (red shift), with the considerable hyperchromicity of e = 41 700 on completion of the red shift, while at the same time the absorbance at 548 m/~ decreased. This spectral change which showed both red and blue shifts according to the ratio of DNA to dye m a y be explained by the presence of two types of complex. On adding increasing amounts of dye to a constant concentration of DNA (38.1/~M), the absorbance at 548 m/~ clearly increased, but there was only a little increase in absorbance at 648 m# over the range of I 2 - 2 4 # M (Fig. 2). This shows that even at a toluidine blue concentration of 12 #M, the binding sites causing the red shift are already saturated. Moreover, on increasing the amount of dye a second type of complex with a spectral m a x i m u m at 548 m# was formed. Thus, when toluI
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Fig. I. Visible a b s o r p t i o n s p e c t r a of toluidine blue in t h e presence of DNA. Concn. of toluidine blue, 24/~M. Concn. of D N A ( i n # M ) : (I) none, (2) 7.6; (3) 15.3; (4) 19.o; (5) 91.5; (6) 200; (7) 451Fig. 2. Spectral change of toluidine blue in t h e presence of a c o n s t a n t concn, of DNA, 38.1/~M. Concn. of toluidine blue (ink, M): (i) 12; (2) 18; (3) 24; (4) 30; (5) 36.
Biochim. Biophys. Acta, 145 (1967) 436-445
439
INTERACTION OF NUCLEOHISTONE WITH TOLUIDINE BLUE
idine blue was added progressively to DNA, a primary interaction occurred corresponding to the red shift, followed by that corresponding to the blue shift. These features of the spectral transitions are reminiscent of the spectral effect produced by DNA upon acridine dyes or methylene blue 9. Although the mechanism for these reversed spectral transitions was not fully elucidated, it was inferred that there were two stages in the interaction: Process I is a strong binding of individual proflavin cations (intercalation according to LERMAN6) which leads to a red shift of the absorption spectrum. In Process n, which is an extensive but weaker interaction, the spectral change of the blue shift was caused by a stacking interaction between neighbouring proflavin cations binding to phosphate groups. In Fig. I the spectral change to a red shift, as for instance, from No. 4 to No. 7, was caused by an increase in the amount of DNA, but a similar change took place on increasing the ionic strength; a complex showing the curve of No. 5 in Fig. I in 0. 7 mM phosphate buffer changed in 0.2 M NaC1 to give only one maximum at 64 ° m#. Thus, it can be said that the complex which gives a red shift is more stable to increases in ionic strength than that which gives a blue shifts, 1°. This observation supports the consideration that Process II is due to an interaction, by ionic forces, of the dye with the phosphate group.
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Fig. 3. Visible a b s o r p t i o n s p e c t r a of t o l u i d i n e blue in t h e p r e s e n c e of n u e l e o h i s t o n e . Concn. of t o l u i d i n e blue: 24/zM. Concn. of n u c l e o h i s t o n e (ill /~M): (I) n o n e ; (2) 14.4; (3) 24-3; (4) 36.0; (5) I46; (b) 270; (7) 54 TM Fig. 4. T i t r a t i o n c u r v e s of t o l u i d i n e blue w i t h D N A a n d n u c l e o h i s t o n e . Concn. of t o l u i d i n e blue: 24 #M. @ - O , D N A ; O - O , n u c l e o h i s t o n e .
This tendency for spectral transitions was also observed in the interaction of the dye with nucleohistone. In Fig. 3 the same features as in the case of DNA can been seen. Isolated calf thymus histones themselves did not give any changes in the visible absorption spectrum of toluidine blue in this concentration range. Furthermore, as shown in Fig. 4, the addition of DNA or nucleohistone to a constant concentration of dye solution resulted in a progressive alteration of the spectrum, and the titration Biochim. Biophys. Mcta, 145 (1967) 4 3 6 - 4 4 5
440
A. MIURA, Y. OHBA
curves of these, at 632 m/~, were V-shaped, consisting of two straight lines intersecting at 0.80 for DNA and at 1.56 for nucleohistones. At these critical points the same spectral transition was observed in both, in which the absorbance at 632 m# decreased to 0.29 and the spectral m a x i m u m appeared at 548 m/~ (Fig. I, No. 4 and Fig. 3, No. 4). It is very probable, therefore, that the spectral change due to nucleohistone can be attributed to the DNA moiety of nucleohistone. The spectral change m a y thus provide a convenient means of estimating the fraction of dye which binds with phosphate groups of nucleohistone. As described, however, these spectral shifts due to Process n should not take place till Process I is completed. Thus, if the number of binding sites for Process I and of the total binding sites in nucleohistone could be estimated, it would be possible to calculate the number of binding sites for Process I I which should correspond to the phosphate groups free from histones.
Estimation o/binding o/dye by Process I by spectral transition As seen in Fig. 4, on the excess DNA side of the critical point the bound dye molecules begin to become unstacked with increasing DNA concentration since they become distributed among the binding sites for Process I of the added DNA. The rate of unstacking is faster with DNA than with nucleohistone, which corresponds to the results in Fig. 5. In the figure a small difference is seen between the extent
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Fig. 5. S p e c t r a l t r a n s i t i o n w i t h t h e f o r m a t i o n of c o m p l e x I. Conch. of toluidine blue: 24/*M. 0-0, DNA; O-C), nucleohistone.
of the red shift with nucleohistone and that with DNA. This m a y simply be caused b y a difference in the amount of bound dye and not b y formation of a fundamentally different complex, because the two curves gradually approximate to each other at excess DNA concentrations. If the decreasing curves in Fig. 5. represent the extent of dye-base interaction, which corresponds to the binding capacity of Process I, and Biochim. Biophys. Acta, 145 (1967) 436-445
INTERACTION OF NUCLEOHISTONE WITH TOLUIDINE BLUE
44 1
if the interaction of nucleohistone with the dye takes place in the same way as that of DNA, a relative value for the number of sites involved in Process I with nucleohistone to that with DNA can be estimated from this figure. The absolute value for DNA had to be estimated in another way.
Estimation o/ binding o/dye in Process I by heat treatment FREIFELDER, DAVlSON AND GEIDUSCHEK11 reported that the melting temperature (Tin) of the DNA-acridine orange complex is higher than that of the DNA. According to KLEINWACHTER AND KOUDELKA 12 a n d GERSCH AND JORDAN 13 there were two types of thermal stabilization of the complex of acridine dyes with DNA. The first type of interaction causes a remarkable stabilization of the secondary structure of DNA, although the second interaction results in no further increase in T m. The stabilizing effect is probably derived from the interaction between the dye and the base ring of DNA in Process I. Thus, GERSCH AND .JORDAN 13 estimated the value of bound dye on the change-over from intercalation to external edgewise interaction on thermal denaturation. The same phenomenon appears in the case of the complex of toluidine blue and DNA. In Fig. 6, the Tm values of DNA are plotted against the progressively increasing dye concentrations. I t appears that stabilization of DNA b y toluidine blue increased until the value of the ratio reached about 0.35, when the dye molecules bound b y Process I m a y saturate the sites. Thereafter, though the blue spectral shift was obvious, no further stabilizing effect could be observed. Above this point further dye molecules might bind with the phosphate groups of DNA b y Process II. Accordingly, the value of 0.35 m a y represent the number of binding sites of Process I per unit nucleotide in DNA, where the amount of the free dye in the solution was negligible, because even at the ratio of dye/DNA-P = 0.8, 95 ~o of the dye was
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Fig. 6. Melting t e m p e r a t u r e of DNA in the presence of progressively increased a m o u n t of toluidine blue. Cohen. of DNA: 95.7/zM (Dye/DNA-P ratio < o.47 ) and 49.7/*M (Dye/DNA-P ratio ~ o.47 ). Fig. 7. Spectral change of the complex of DNA and toluidine blue on heating. ConcI1. of DNA, 5o.8/zM and toluidine blue, 28.8/zM at (I) 35°; (2) 66°; (3) 97 .80.
Biochim. Biophys. Acta, 145 (1967) 436-445
442
A. MIURA, ¥. OHBA
found as the complex by the Millipore filtlatrion method. Nucleohistone has a T,,~ value 20 ° higher than that for DNA under these conditions. Though nearly the same extent of red shift was observed as with DNA; however, no stabilizing effect due to toluidine blue was observed, perhaps because it was hidden by the extent of stabilization caused by histone moieties. If, however, the value of 0.35 for DNA is applied to the relation in Fig. 5, a value of 0.31 for dye binding in nucleohistone in Process I is obtained. These values were confirmed by the thermal dissociation of toluidine blue molecules from the complex. Fig. 7 shows the spectral changes observed on heating the toluidine blue-DNA complex. At 97.8 °, where the double helix of DNA was melted out, scarcely any shift of the spectrum to the red was seen even at a dye to DNA ratio of 0.2, because all the dye molecules were dissociated from the DNA. On increasing the amount of dye, to a ratio of dye/DNA-P = 0.57, a clear shift of the spectrum to the blue with two maxima at 59 ° m# and 645 mff was observed at room temperature and at 66 °, where the double helical structure of DNA had not yet started to melt, the blue shift was converted to a spectrum with a single maximum at 645 m/~. At this temperature, most dye molecules which had been bound by Process I I seemed to become dissociated, but the interaction by Process I could be maintained so long as the secondary structure of DNA was conserved. A similar result was obtained with nucleohistone. The observation that Process II did not occur at 66 ° provided a method for estimation of the number of sites involved in Process I. When DNA or nucleohistone was added to a constant concentration of dye at 62 °, the absorbance at 632 m/~ decreased progressively with decrease in the fraction of free dye, or with increase in the formation of the complex by Process I. Then, on saturation of the binding sites by Process I the absorbance was increased by the hyperchromic effect of the dye provoked by increased DNA (Fig. i). The saturation point, therefore, was represented by a minimum in the absorbance curve. As shown in Fig. 8, when the absorbances at 632 rake are plotted against the ratio of dye/DNA or nucleohistone-P, values of 0.35 and 0.32 for DNA and nucleohistone, respectively, are obtained. A good concordance was obtained with the result mentioned in the foregoing section.
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Biochim. Biophys. Acta, 145 (1967) 436-445
INTERACTION OF NUCLEOHISTONE WITH TOLUIDINE BLUE
443
Estimation o/dye-binding in Process I I The interaction of DNA with dyes is generally characterized b y an equilibrium constant for the binding reaction: K--
r c(n--r)
where K is related to the ratio, r, of bound dye to n, the total number of binding sites. As described above, however, the interaction was regarded as involving two processes. Thus, it must be represented as separated reactions. If toluidine blue binds b y Process I I only after saturation of the binding sites for Process I, the reaction can be represented b y the following modified equation representing particularly the equilibrium of Process II: K' =
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where m is the number of the binding sites for Process I and n - - m represents that for Process II. The amount of bound dye was estimated b y the Millipore filter method and values of m = 0.35 for DNA and m = 0.32 for nucleohistone were used. Fig. 9 I
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Fig, 9. The binding curves of toluidine blue b y DNA, nucleohistone and DNA-polylysine complexes. Concn. of toluidine blue 24/zM. O - Q , DNA; O - © , nucleohistone; × - × , DNA-polylysine complex (-NH2/DNA-P ratio, o.22); ~ - ~ , DNA-polylysine complex (-NH,/DNA-P ratio, 0.44 ).
shows the plot of (r--m)/c as a function of r both for DNA and for nucleohistone. To examine the validity of this treatment, n - - m was estimated for mixtures of polylysine and DNA in the ratios of 0.22 and o.44. The results are seen in Table I. The value of n - - m for DNA was i.oo; i.e., every phosphate group can bind one molecule of toluidine blue. With the complexes of DNA and polylysine, it was found that the experimental values corresponded to the calttlated values assuming that every amino group can bind to a phosphate groupe. A similar derivation afforded a value of o.48 for nucleohistone which should represent the quantity of free phosphate groups per nucleohistone molecule. On the other hand, judging from the metachromasia, the spectral change of the d y e - D N A complex also seems to be useful for estimation of dye binding. As Biochim. Biophys. Acta, I45 (1967) 436-445
A. M I U R A , Y. O H B A
444 TABLE
I
]ESTIMATION OF THE DYE BINDING BY THE FILTRATION METHOD Substance
m
n
n -- m
DNA DNA + polylysine*** DNA + polylysine .... Nucleohistone
0.35 o. 34"* 0 . 3 2 ** 0.32
1.35 I. i o o.84 0.80
I.OO o.76 0.52 o.48
* "* *"* ....
Calculated value*
o.78 0.56
Calculated assuming that each lysinyl residue can bind to one phosphate group of DNA. These values were assumed on tile basis of those of DNA and nucleohistone. -NHJ-P ratio, 0.22. -NH~/-P ratio, 0.44.
can be seen ill Fig. 4, the critical points are clear enough to obtain values of 1.25 for DNA and 0.64 for nucleohistone as the dye binding per unit of nucleotide. If these critical points indicate the same amount of complex formed in Process II, by subtracting the dye binding of Process I from the total binding, the binding sites of nucleohistone involved in Process I I must be 36 % of those of DNA.
DISCUSSION
The results obtained show that about 48 ~o of the DNA phosphate groups in the nucleohistone molecule are available as dye-binding sites. B y spectrophotometric titration a value of 36 % was obtained. This difference might be because the latter value reflects the extent of dye-dye association rather than that of real dye binding. Since the extrapolation of spectrophotometric titration values of nucleohistone b y azure A is difficult, KLEIN AND SZIRMAI4 used a method involving precipitation b y the dye and determined the binding of dye b y DNA and nucleohistone quantitatively. They found that about half the binding sites of nucleohistone are available for binding the dye. However, this concordance between their result and ours seems to be fortuitous, since their value indicates only the total dye binding, not that limited to Process II. According to DAVISON" AND BUTLER 3, the sum of the arginine and lysine residues of histones attached to DNA cannot neutralize more than about 78 % of the DNA phosphate groups. Our preparations have nearly the same composition as theirs. Accordingly it is concluded that about 26 % of the basic groups of histones are not bound to phosphate in the nucleohistone molecule. Recently WALKER1. reported b y electrometric and spectrophotometric titration that 2o % of the basic amino acid residues are not bound to DNA phosphate. Of course, the value of dye binding in Process I I does not always mean the net fraction of phosphate groups free from histones. Furthermore, it seemed possible that the dye molecule displaced basic groups of histones from DNA, though no metachromasia was observed when toluidine blue was mixed with isolated histories. Thus the present conclusion is only tentative. With regard to the site of Process I, the dye-binding capacity of nucleohistone was IO % less than that of DNA; thus there m a y be a slight hindrance of Process I Biochim.
Biophys.
Acla,
145 (1967) 4 3 6 - 4 4 5
INTERACTION OF NUCLEOHISTONE WITH TOLUIDINE BLUE
445
b y the presence of histones. This m i g h t be caused b y an i n t e r a c t i o n of a m i n o acid residues with D N A base. According to OHBA2 the lysine residues of histones can i n t e r a c t with D N A bases to stabilize t h e r m a l d e n a t u r a t i o n , which m a y explain the a b o v e - m e n t i o n e d difference. SONNENBERG AND ZUBAY15 reported t h a t n a t i v e calf t h y m u s nucleohistone is a relatively poor primer for R N A synthesis. However, the sonicated p r e p a r a t i o n has one t h i r d the p r i m i n g efficiency of the e q u i v a l e n t a m o u n t of calf t h y m u s DNA, a n d histones are n o t r e m o v e d d u r i n g the sonication. This observation shows t h a t 50 % of the free phosphate groups in nucleohistone occur in sequence a n d are n o t r a n d o m l y distributed. This m a y depend on the specificity of the i n t e g r a t i o n of nucleohistone i n vivo. F r o m this p o i n t of view similar studies on nucleohistones from other tissues would be valuable.
ACKNOWLEDGEMENTS The a u t h o r s wish to t h a n k Dr. D. MIZUNO for s t i m u l a t i n g discussion. T h e y are also i n d e b t e d to Dr. M. TSUBOI for his interest a n d advice.
REFERENCES I 2 3 4 5 6 7 8 9 io II 12 13
14 15 16
Y. OHBA, Biochim. Biophys. Acta, 123 (1966) 76. Y. OHBA, Biochim. Biophys. Acta, 123 (1966) 84. P. F. DAVlSOl~AND J. A. V. BUTLER, Biochim. Biophys. Acta, 21 (1956) 568. F. KLEIN AND J. A. SZlRMAI,Biochim. Biophys. Acta, 72 (1963) 48. A. R. PEACOCKEAND J. N. H. SKERRETT,Trans. Faraday Soc., 52 (1956) 261. L. S. LERMAN,J. Mol. Biol., 3 (1961) 18. D. F. BRADLEYAND M. K. WOLF, Proc. Natl. Acad. Sci. U.S., 45 (1959) 944. L. MICHAELISAND S. GRANICK,J. Am. Chem. Soc., 67 (1945) 1221. R. F. STEINERAND R. V. BEERS, Arch. Biochim. Biophys., 81 (1959) 75. D. S. DRUMMOND, V. F. W. SIMPSON--GILDEMEISTER AND A. R. PEACOCKE, Biopolymer, 3 (1956) 135. D. FREIFELDER, P. F. DAVISON AND E. P. GEIDUSCHEK, Biophys. J., I (1961) 389. V. KLEINWXCHTER AND J. KOUDELKA, Biochim. Biophys. Acta, 91 (1964) 539. N. F. GERSCH AND D. O. JORDAN, J. Mol. Biol., 13 (1965) 138. D. F. BRADLEYAND G. FELSENFELD,Nature, 184 (1959) 192o. B. P. SONNENBERGAND G. ZUBAY, Proc. Natl. Acad. Sci. U.S., 54 (1965) 415 . I. O. WALKER, J. Mol. Biol., 14 (1965) 381. Biochim. Biophys. Acta, 145 (1967) 436-445
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 95690
S T R U C T U R E OF NUCLEOHISTONE. I I I . I N T E R A C T I O N W I T H T O L U I D I N E BLUE A K I K O M I U R A * AND Y O S H I K I OHBA*"
Faculty o] Pharmaceutical Sciences, University of Tokyo, Hongo, Tokyo (Japan) (Received March 8th, 1967)
SUMMARY
The interaction of toluidine blue with calf-thymus nucleohistone and DNA was investigated spectrophotometrically with special reference to tile spectral transition occurring on formation of the complex. The dye was found to bind to nucleohistone and DNA in at least two ways (Process I, II). These binding sites could be estimated separately. It was found by Millipore filtration methods that there were 0.35 and o.32 sites per unit nucleotide of DNA and nucleohistone respectively for Process I, and I.OO and o.48 for Process II. The results indicate that in a nucleohistone molecule about half the phosphate groups are free from basic groups of the histones.
INTRODUCTION
Histones have been found in cells in association with DNA and evidently may be regarded as cellular components playing a part in the mechanism controlling the function of DNA. Studies1, 2 have been made, therefore, on what effect they have on the structure of DNA in their associated state, that is as nucleohistone. It is known that the phosphate group of DNA is responsible for DNA-histone bonding, but that there are some phosphate groups in a nucleohistone molecule which are not bound with basic groups of histones*. Only the latter would act as active genes. It seems possible to estimate the quantity of these groups by the interaction of DNA with a basic dye capable of binding with DNA phosphate. The results of such studies have been reported3, 4. KLEIN AND SZIRMAI4 studied the stoichiometric precipitation reaction between azure A and DNA or nucleohistone, and found that only half of the possible binding sites were available for the dye. Several investigators have studied the interaction of DNA with acridine dyes. According to PEACOCKE AND SKERRETa5 proflavin can bind to DNA in at least two ways. The primary strong reaction (Process I) has been studied extensively by LERMAN~, who interpreted it as intercalation, a process in which the dye molecule Present address: D e p a r t m e n t of Molecular Biology, P o r t s m o u t h College of Technology, Park Road, P o r t s m o u t h (Great Britain). ** Present address: D e p a r t m e n t of Chemistry, National i n s t i t u t e of Health, Shinagawaku, Kamiosaki, Tokyo (Japan).
Biochim. Biophys. Acts, 145 (1967) 436 445
INTERACTION OF NUCLEOHISTONE WITH TOLUIDINE BLUE
437
enters between base pairs which are normally adjacent, in a plane perpendicular to the helical axis of DNA. The secondary weak process (Process II), which has been proposed b y BRADLEY AND W O L F 7, is interpreted as the being due to the binding of dye b y stacking, or aggregation, that is by weak interaction between aminoacridine molecules and the ionic groups of polyanions such as DNA. In this study we found that toluidine blue, a cationic dye, could bind to DNA or nucleohistone in two ways as with acridine dyes. By measuring these binding sites separately, we estimated the proportion of phosphate groups which were free from histones in the nucleohistone molecule.
MATERIALS AND METHODS
Nucleohistone and other materials The procedures used for the extraction of calf thymus nucleohistone and DNA have already been described 1. Generally 0. 7 mM phosphate buffer (pH 7.0) was used as solvent. The polylysine bromide (Pilot Chemical Co.), which had a tool. wt. of 60 ooo, was used after dialysis against 0. 7 mM phosphate buffer.
Toluidine blue Toluidine blue was a single product of Merck Chemical Co. Concentrations were estimated using an extinction value of 33 200 at 632 m/~ (ref. 8). Beer's law was obeyed at 632 m# up to a dye concentration of 24/,M.
Procedures used/or binding dye and measurement o/absorption A dye solution of 80/tM to 960 #M was slowly added to DNA or nucleohistone solution, with vigorous stirring. Absorbance was measured 30 min after addition of the dye b y which time the reaction was complete. The absorbance of solutions was measured in a spectrophotometer (Hitachi SI2-B). Absorption spectra were obtained using a Cary Model-I 4. The procedure for the determination of thermal profiles was described previously 2.
Millipore /iltration A single Millipore filter (pore size, o.22 #) was used throughout. In the filtration method, the effect of possible errors occurring b y the adsorption of free dyes to the filter and the residual complex on the filter was minimized b y choosing the best condition, empirically determined, as follows. Volumes of at least 15 ml of solution of dye complex were prepared. About 3 ml of the solution was filtered first. Then the surface of the membrane was wiped with moistened, defatted cotton to eliminate the residue which could still adsorb free dye. This procedure was repeated 4 times. In this way an equilibrium was attained between free dye in the solution and dye adsorbed to the membrane. Thus the concentration of dye in the filtrate of the 5th portion was determined as the same as the concentration of free dye in the solution. Ultraviolet measurements of the filtrate, under similar conditions, proved that Biochim. Biophys. Acta, 145 (1967) 436-445
438
A. MIURA, Y. OHBA
neither leakage of DNA nor nucleohistone occurred in this filtration experiment, at least when the ratio of dye/DNA-P was greater than 0.8, or dye/nucleohistone-P greater than 0.4.
RESULTS
Spectral transition o/toluidine blue by addition o/ DNA and nucleohistone Toluidine blue showed a visible absorption spectrum of constant shape with a m a x i m u m at 632 m#, at least at concentrations of less than 24/~M in 0.7 mM phosphate buffer. Typical absorption spectra of solutions containing a constant concentration of toluidine blue and increasing amounts of DNA are shown in Fig. I, which illustrates the progressive shift of the m a x i m u m towards 548 m# (blue shift), and a marked decrease at 632 m/~. An isosbestic point was evident at 563 m#. On further increasing in the amount of DNA, a new spectral m a x i m u m appeared at 648 m/~ (red shift), with the considerable hyperchromicity of e = 41 700 on completion of the red shift, while at the same time the absorbance at 548 m/~ decreased. This spectral change which showed both red and blue shifts according to the ratio of DNA to dye m a y be explained by the presence of two types of complex. On adding increasing amounts of dye to a constant concentration of DNA (38.1/~M), the absorbance at 548 m/~ clearly increased, but there was only a little increase in absorbance at 648 m# over the range of I 2 - 2 4 # M (Fig. 2). This shows that even at a toluidine blue concentration of 12 #M, the binding sites causing the red shift are already saturated. Moreover, on increasing the amount of dye a second type of complex with a spectral m a x i m u m at 548 m# was formed. Thus, when toluI
I
I
1.0
0.8
0.8[
~ 0.6
0.6
o
E
Z~
< 0.4
~0.4
0.2
0 500
t
550 600 Wavelength (m/J)
650
700
0
I
I
550 600 Wavelength (m/J)
i
650
Fig. I. Visible a b s o r p t i o n s p e c t r a of toluidine blue in t h e presence of DNA. Concn. of toluidine blue, 24/~M. Concn. of D N A ( i n # M ) : (I) none, (2) 7.6; (3) 15.3; (4) 19.o; (5) 91.5; (6) 200; (7) 451Fig. 2. Spectral change of toluidine blue in t h e presence of a c o n s t a n t concn, of DNA, 38.1/~M. Concn. of toluidine blue (ink, M): (i) 12; (2) 18; (3) 24; (4) 30; (5) 36.
Biochim. Biophys. Acta, 145 (1967) 436-445
439
INTERACTION OF NUCLEOHISTONE WITH TOLUIDINE BLUE
idine blue was added progressively to DNA, a primary interaction occurred corresponding to the red shift, followed by that corresponding to the blue shift. These features of the spectral transitions are reminiscent of the spectral effect produced by DNA upon acridine dyes or methylene blue 9. Although the mechanism for these reversed spectral transitions was not fully elucidated, it was inferred that there were two stages in the interaction: Process I is a strong binding of individual proflavin cations (intercalation according to LERMAN6) which leads to a red shift of the absorption spectrum. In Process n, which is an extensive but weaker interaction, the spectral change of the blue shift was caused by a stacking interaction between neighbouring proflavin cations binding to phosphate groups. In Fig. I the spectral change to a red shift, as for instance, from No. 4 to No. 7, was caused by an increase in the amount of DNA, but a similar change took place on increasing the ionic strength; a complex showing the curve of No. 5 in Fig. I in 0. 7 mM phosphate buffer changed in 0.2 M NaC1 to give only one maximum at 64 ° m#. Thus, it can be said that the complex which gives a red shift is more stable to increases in ionic strength than that which gives a blue shifts, 1°. This observation supports the consideration that Process II is due to an interaction, by ionic forces, of the dye with the phosphate group.
ii m. E
~u 0.6
cJ 0.6
<
./"
#3 £0
o
0.4
~ o.5 8 .Q
0.2
.o'o
o / " o....o
o.4
i
8
°"
o.3 <
~oo
s-~o
6~o
Wavelength (mju)
GAo
7oo
o
~
~
~
~
~
DNA-P or nudeohistcne-P/toluidine
blue
Fig. 3. Visible a b s o r p t i o n s p e c t r a of t o l u i d i n e blue in t h e p r e s e n c e of n u e l e o h i s t o n e . Concn. of t o l u i d i n e blue: 24/zM. Concn. of n u c l e o h i s t o n e (ill /~M): (I) n o n e ; (2) 14.4; (3) 24-3; (4) 36.0; (5) I46; (b) 270; (7) 54 TM Fig. 4. T i t r a t i o n c u r v e s of t o l u i d i n e blue w i t h D N A a n d n u c l e o h i s t o n e . Concn. of t o l u i d i n e blue: 24 #M. @ - O , D N A ; O - O , n u c l e o h i s t o n e .
This tendency for spectral transitions was also observed in the interaction of the dye with nucleohistone. In Fig. 3 the same features as in the case of DNA can been seen. Isolated calf thymus histones themselves did not give any changes in the visible absorption spectrum of toluidine blue in this concentration range. Furthermore, as shown in Fig. 4, the addition of DNA or nucleohistone to a constant concentration of dye solution resulted in a progressive alteration of the spectrum, and the titration Biochim. Biophys. Mcta, 145 (1967) 4 3 6 - 4 4 5
440
A. MIURA, Y. OHBA
curves of these, at 632 m/~, were V-shaped, consisting of two straight lines intersecting at 0.80 for DNA and at 1.56 for nucleohistones. At these critical points the same spectral transition was observed in both, in which the absorbance at 632 m# decreased to 0.29 and the spectral m a x i m u m appeared at 548 m/~ (Fig. I, No. 4 and Fig. 3, No. 4). It is very probable, therefore, that the spectral change due to nucleohistone can be attributed to the DNA moiety of nucleohistone. The spectral change m a y thus provide a convenient means of estimating the fraction of dye which binds with phosphate groups of nucleohistone. As described, however, these spectral shifts due to Process n should not take place till Process I is completed. Thus, if the number of binding sites for Process I and of the total binding sites in nucleohistone could be estimated, it would be possible to calculate the number of binding sites for Process I I which should correspond to the phosphate groups free from histones.
Estimation o/binding o/dye by Process I by spectral transition As seen in Fig. 4, on the excess DNA side of the critical point the bound dye molecules begin to become unstacked with increasing DNA concentration since they become distributed among the binding sites for Process I of the added DNA. The rate of unstacking is faster with DNA than with nucleohistone, which corresponds to the results in Fig. 5. In the figure a small difference is seen between the extent
,@
15-~NNN
o\
ID W
o
0
i
0.1
f
0.2
0.3
Toluidine blue/DNA-P or nucleohistone- P
Fig. 5. S p e c t r a l t r a n s i t i o n w i t h t h e f o r m a t i o n of c o m p l e x I. Conch. of toluidine blue: 24/*M. 0-0, DNA; O-C), nucleohistone.
of the red shift with nucleohistone and that with DNA. This m a y simply be caused b y a difference in the amount of bound dye and not b y formation of a fundamentally different complex, because the two curves gradually approximate to each other at excess DNA concentrations. If the decreasing curves in Fig. 5. represent the extent of dye-base interaction, which corresponds to the binding capacity of Process I, and Biochim. Biophys. Acta, 145 (1967) 436-445
INTERACTION OF NUCLEOHISTONE WITH TOLUIDINE BLUE
44 1
if the interaction of nucleohistone with the dye takes place in the same way as that of DNA, a relative value for the number of sites involved in Process I with nucleohistone to that with DNA can be estimated from this figure. The absolute value for DNA had to be estimated in another way.
Estimation o/ binding o/dye in Process I by heat treatment FREIFELDER, DAVlSON AND GEIDUSCHEK11 reported that the melting temperature (Tin) of the DNA-acridine orange complex is higher than that of the DNA. According to KLEINWACHTER AND KOUDELKA 12 a n d GERSCH AND JORDAN 13 there were two types of thermal stabilization of the complex of acridine dyes with DNA. The first type of interaction causes a remarkable stabilization of the secondary structure of DNA, although the second interaction results in no further increase in T m. The stabilizing effect is probably derived from the interaction between the dye and the base ring of DNA in Process I. Thus, GERSCH AND .JORDAN 13 estimated the value of bound dye on the change-over from intercalation to external edgewise interaction on thermal denaturation. The same phenomenon appears in the case of the complex of toluidine blue and DNA. In Fig. 6, the Tm values of DNA are plotted against the progressively increasing dye concentrations. I t appears that stabilization of DNA b y toluidine blue increased until the value of the ratio reached about 0.35, when the dye molecules bound b y Process I m a y saturate the sites. Thereafter, though the blue spectral shift was obvious, no further stabilizing effect could be observed. Above this point further dye molecules might bind with the phosphate groups of DNA b y Process II. Accordingly, the value of 0.35 m a y represent the number of binding sites of Process I per unit nucleotide in DNA, where the amount of the free dye in the solution was negligible, because even at the ratio of dye/DNA-P = 0.8, 95 ~o of the dye was
1.0
v
I
I
,
90
to~e '~o~° 80
O.e
0.6
./
~Te ___6q
O.,C
i
I
0.5 Toluidine blue/ONA- P
I
t
1.0
55o
6oo
65o
"
Wovelength (rn~u~
Fig. 6. Melting t e m p e r a t u r e of DNA in the presence of progressively increased a m o u n t of toluidine blue. Cohen. of DNA: 95.7/zM (Dye/DNA-P ratio < o.47 ) and 49.7/*M (Dye/DNA-P ratio ~ o.47 ). Fig. 7. Spectral change of the complex of DNA and toluidine blue on heating. ConcI1. of DNA, 5o.8/zM and toluidine blue, 28.8/zM at (I) 35°; (2) 66°; (3) 97 .80.
Biochim. Biophys. Acta, 145 (1967) 436-445
442
A. MIURA, ¥. OHBA
found as the complex by the Millipore filtlatrion method. Nucleohistone has a T,,~ value 20 ° higher than that for DNA under these conditions. Though nearly the same extent of red shift was observed as with DNA; however, no stabilizing effect due to toluidine blue was observed, perhaps because it was hidden by the extent of stabilization caused by histone moieties. If, however, the value of 0.35 for DNA is applied to the relation in Fig. 5, a value of 0.31 for dye binding in nucleohistone in Process I is obtained. These values were confirmed by the thermal dissociation of toluidine blue molecules from the complex. Fig. 7 shows the spectral changes observed on heating the toluidine blue-DNA complex. At 97.8 °, where the double helix of DNA was melted out, scarcely any shift of the spectrum to the red was seen even at a dye to DNA ratio of 0.2, because all the dye molecules were dissociated from the DNA. On increasing the amount of dye, to a ratio of dye/DNA-P = 0.57, a clear shift of the spectrum to the blue with two maxima at 59 ° m# and 645 mff was observed at room temperature and at 66 °, where the double helical structure of DNA had not yet started to melt, the blue shift was converted to a spectrum with a single maximum at 645 m/~. At this temperature, most dye molecules which had been bound by Process I I seemed to become dissociated, but the interaction by Process I could be maintained so long as the secondary structure of DNA was conserved. A similar result was obtained with nucleohistone. The observation that Process II did not occur at 66 ° provided a method for estimation of the number of sites involved in Process I. When DNA or nucleohistone was added to a constant concentration of dye at 62 °, the absorbance at 632 m/~ decreased progressively with decrease in the fraction of free dye, or with increase in the formation of the complex by Process I. Then, on saturation of the binding sites by Process I the absorbance was increased by the hyperchromic effect of the dye provoked by increased DNA (Fig. i). The saturation point, therefore, was represented by a minimum in the absorbance curve. As shown in Fig. 8, when the absorbances at 632 rake are plotted against the ratio of dye/DNA or nucleohistone-P, values of 0.35 and 0.32 for DNA and nucleohistone, respectively, are obtained. A good concordance was obtained with the result mentioned in the foregoing section.
"~"0.8 I
r
I
I
I
0.7
/
o.6[~
~
8
8
o,,"
o
.2"8 •
.""
"~-':~-..o..o.~.o.~.~•
<0.4
I
I
I
I
0 0.5 1.0 Toluidine b l u e / D N A - P or nucleohistone-P
Fig. 8. A b s o r b a n c e of toluidine blue in the presence of D N A and nucleohistone at 62 ° a t 632 ]n~u. Conch. of toluidine blue: I8/~M. 0 - 0 , D N A ; O - O nucleohistone.
Biochim. Biophys. Acta, 145 (1967) 436-445
INTERACTION OF NUCLEOHISTONE WITH TOLUIDINE BLUE
443
Estimation o/dye-binding in Process I I The interaction of DNA with dyes is generally characterized b y an equilibrium constant for the binding reaction: K--
r c(n--r)
where K is related to the ratio, r, of bound dye to n, the total number of binding sites. As described above, however, the interaction was regarded as involving two processes. Thus, it must be represented as separated reactions. If toluidine blue binds b y Process I I only after saturation of the binding sites for Process I, the reaction can be represented b y the following modified equation representing particularly the equilibrium of Process II: K' =
r--m c[(n--rn)-- (r--m)l
r--m c(n--r)
(r > m )
where m is the number of the binding sites for Process I and n - - m represents that for Process II. The amount of bound dye was estimated b y the Millipore filter method and values of m = 0.35 for DNA and m = 0.32 for nucleohistone were used. Fig. 9 I
n
\
,\
I
•
\
\.O
\
o\.
i \,
X
OXo \ O"
0
0
0.5
P
1.o
L 1.5
Fig, 9. The binding curves of toluidine blue b y DNA, nucleohistone and DNA-polylysine complexes. Concn. of toluidine blue 24/zM. O - Q , DNA; O - © , nucleohistone; × - × , DNA-polylysine complex (-NH2/DNA-P ratio, o.22); ~ - ~ , DNA-polylysine complex (-NH,/DNA-P ratio, 0.44 ).
shows the plot of (r--m)/c as a function of r both for DNA and for nucleohistone. To examine the validity of this treatment, n - - m was estimated for mixtures of polylysine and DNA in the ratios of 0.22 and o.44. The results are seen in Table I. The value of n - - m for DNA was i.oo; i.e., every phosphate group can bind one molecule of toluidine blue. With the complexes of DNA and polylysine, it was found that the experimental values corresponded to the calttlated values assuming that every amino group can bind to a phosphate groupe. A similar derivation afforded a value of o.48 for nucleohistone which should represent the quantity of free phosphate groups per nucleohistone molecule. On the other hand, judging from the metachromasia, the spectral change of the d y e - D N A complex also seems to be useful for estimation of dye binding. As Biochim. Biophys. Acta, I45 (1967) 436-445
A. M I U R A , Y. O H B A
444 TABLE
I
]ESTIMATION OF THE DYE BINDING BY THE FILTRATION METHOD Substance
m
n
n -- m
DNA DNA + polylysine*** DNA + polylysine .... Nucleohistone
0.35 o. 34"* 0 . 3 2 ** 0.32
1.35 I. i o o.84 0.80
I.OO o.76 0.52 o.48
* "* *"* ....
Calculated value*
o.78 0.56
Calculated assuming that each lysinyl residue can bind to one phosphate group of DNA. These values were assumed on tile basis of those of DNA and nucleohistone. -NHJ-P ratio, 0.22. -NH~/-P ratio, 0.44.
can be seen ill Fig. 4, the critical points are clear enough to obtain values of 1.25 for DNA and 0.64 for nucleohistone as the dye binding per unit of nucleotide. If these critical points indicate the same amount of complex formed in Process II, by subtracting the dye binding of Process I from the total binding, the binding sites of nucleohistone involved in Process I I must be 36 % of those of DNA.
DISCUSSION
The results obtained show that about 48 ~o of the DNA phosphate groups in the nucleohistone molecule are available as dye-binding sites. B y spectrophotometric titration a value of 36 % was obtained. This difference might be because the latter value reflects the extent of dye-dye association rather than that of real dye binding. Since the extrapolation of spectrophotometric titration values of nucleohistone b y azure A is difficult, KLEIN AND SZIRMAI4 used a method involving precipitation b y the dye and determined the binding of dye b y DNA and nucleohistone quantitatively. They found that about half the binding sites of nucleohistone are available for binding the dye. However, this concordance between their result and ours seems to be fortuitous, since their value indicates only the total dye binding, not that limited to Process II. According to DAVISON" AND BUTLER 3, the sum of the arginine and lysine residues of histones attached to DNA cannot neutralize more than about 78 % of the DNA phosphate groups. Our preparations have nearly the same composition as theirs. Accordingly it is concluded that about 26 % of the basic groups of histones are not bound to phosphate in the nucleohistone molecule. Recently WALKER1. reported b y electrometric and spectrophotometric titration that 2o % of the basic amino acid residues are not bound to DNA phosphate. Of course, the value of dye binding in Process I I does not always mean the net fraction of phosphate groups free from histones. Furthermore, it seemed possible that the dye molecule displaced basic groups of histones from DNA, though no metachromasia was observed when toluidine blue was mixed with isolated histories. Thus the present conclusion is only tentative. With regard to the site of Process I, the dye-binding capacity of nucleohistone was IO % less than that of DNA; thus there m a y be a slight hindrance of Process I Biochim.
Biophys.
Acla,
145 (1967) 4 3 6 - 4 4 5
INTERACTION OF NUCLEOHISTONE WITH TOLUIDINE BLUE
445
b y the presence of histones. This m i g h t be caused b y an i n t e r a c t i o n of a m i n o acid residues with D N A base. According to OHBA2 the lysine residues of histones can i n t e r a c t with D N A bases to stabilize t h e r m a l d e n a t u r a t i o n , which m a y explain the a b o v e - m e n t i o n e d difference. SONNENBERG AND ZUBAY15 reported t h a t n a t i v e calf t h y m u s nucleohistone is a relatively poor primer for R N A synthesis. However, the sonicated p r e p a r a t i o n has one t h i r d the p r i m i n g efficiency of the e q u i v a l e n t a m o u n t of calf t h y m u s DNA, a n d histones are n o t r e m o v e d d u r i n g the sonication. This observation shows t h a t 50 % of the free phosphate groups in nucleohistone occur in sequence a n d are n o t r a n d o m l y distributed. This m a y depend on the specificity of the i n t e g r a t i o n of nucleohistone i n vivo. F r o m this p o i n t of view similar studies on nucleohistones from other tissues would be valuable.
ACKNOWLEDGEMENTS The a u t h o r s wish to t h a n k Dr. D. MIZUNO for s t i m u l a t i n g discussion. T h e y are also i n d e b t e d to Dr. M. TSUBOI for his interest a n d advice.
REFERENCES I 2 3 4 5 6 7 8 9 io II 12 13
14 15 16
Y. OHBA, Biochim. Biophys. Acta, 123 (1966) 76. Y. OHBA, Biochim. Biophys. Acta, 123 (1966) 84. P. F. DAVlSOl~AND J. A. V. BUTLER, Biochim. Biophys. Acta, 21 (1956) 568. F. KLEIN AND J. A. SZlRMAI,Biochim. Biophys. Acta, 72 (1963) 48. A. R. PEACOCKEAND J. N. H. SKERRETT,Trans. Faraday Soc., 52 (1956) 261. L. S. LERMAN,J. Mol. Biol., 3 (1961) 18. D. F. BRADLEYAND M. K. WOLF, Proc. Natl. Acad. Sci. U.S., 45 (1959) 944. L. MICHAELISAND S. GRANICK,J. Am. Chem. Soc., 67 (1945) 1221. R. F. STEINERAND R. V. BEERS, Arch. Biochim. Biophys., 81 (1959) 75. D. S. DRUMMOND, V. F. W. SIMPSON--GILDEMEISTER AND A. R. PEACOCKE, Biopolymer, 3 (1956) 135. D. FREIFELDER, P. F. DAVISON AND E. P. GEIDUSCHEK, Biophys. J., I (1961) 389. V. KLEINWXCHTER AND J. KOUDELKA, Biochim. Biophys. Acta, 91 (1964) 539. N. F. GERSCH AND D. O. JORDAN, J. Mol. Biol., 13 (1965) 138. D. F. BRADLEYAND G. FELSENFELD,Nature, 184 (1959) 192o. B. P. SONNENBERGAND G. ZUBAY, Proc. Natl. Acad. Sci. U.S., 54 (1965) 415 . I. O. WALKER, J. Mol. Biol., 14 (1965) 381. Biochim. Biophys. Acta, 145 (1967) 436-445