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Binding of histone to DNA
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Binding D. K. CHATTORAJ,’
162,
778-785 (1972)
of Histone HENRY
Department of Biochemistry,
to DNA
B. BULL
University
of Iowa,
AND
R. CHALKLEY
Iowa City, Iowa 6&?40
Received May 18, 1972; accepted June 27, 1972 The reconstitution of nucleohistone has been studied using a novel analytical approach. We have exploited measurements of the surface tension of histone solutions both in the presence and absence of DNA to calculate the amount of histone bound per gram of DNA as a function of protein concentrations in a variety of ionic strength solutions. Fl, F2b and F3 histones were used in these investigations. The nature of the histone-DNA binding isotherms depend significantly on the ionic strength of the medium, type of the histone fraction and the extent of DNA hydration. For F2b and F3 fractions at low ionic strengths, moles of histone present in a mole of saturated DNA-histone complex were calculated. From the measurement of surface tension of F2b histone solutions in the presence and absence of chromatin at 0.01 and 0.10 ionic strengths, the extent of additional binding of F2b histone by native nucleohistone was calculated.
Study of the reconstituted DNA-histone complex has received considerable attention in recent years because the complex is believed to display many of the properties of native chromatin (1). Using the equilibrium dialysis method, Akinrimisi, Bonner, and Ta,O (2) haven show that DNA binds histone at low ionic strengths whereas the reconstituted histone-DNA complex is extensively dissociated when the ionic strength is close to unity. These authors have also demonstrated that the various types of histone bind DNA to a different extent as a function of ionic strength. Fasman et al. (3, 4) have investigated the conformational aspects of the DNA-histone complexes using circular dichroism. According to these authors, the conformational changes associated with the histone-DNA complex are influenced by ionic strength, composition of the complex and DNA hydration. The nature of the histone-DNA binding sites in the reconstituted and native nucleohistones has been examined by Clark and Felsenfeld (5) and also bv Boublik et aZ. (6). 1 On leave of absence, Chemistry Department, Jadavpur University, Calcutta-32, India.
In the present communication, we will discuss an entirely new method which enables us to measure the amount of histone bound to DNA at a given ionic strength. The method is based on the measurement of surface tension of the histone solution in the presence and absence of DNA. MATERIALS AND METHODS Highly polymerized calf thymus DNA was ob. tained from the Worthington Biochemical Corporation. The ratio of the absorbancy of DNA dissolved in 0.01 M sodium chloride at 260 nm to that at 230 nm was 2.46 indicating absence of protein. The hyperchromic shifts of DNA solutions after denaturation by heat and alkali were 38 and 30%, respectively. The molecular weight of Worthington DNA is of the order 10’ (7). Calf thymus chromatin was prepared by the method of Panyim and Chalkley (8) and stored at an ionic strength lower than 0.9095 to inhibit proteolysis (9). Such chromatin consists of equal weights of histone and DNA together with approx 12% nonhistone protein and less than 2% RNA. There is a small amount of lipid present most likely as part of a membrane fraction. Histone fractions were isolated from calf chromatin in 0.4 N H&04 . Separation into individual fractions followed the procedures initially developed by Johns (10) as modified by Panyim (11). 778
Copyright All rights
@ 1972 by Academic Press, of reproduction in any form
Inc. reserved.
BINDING
OF HISTONE
We have used three fractions in this study: the very lysine-rich histone, Fl; a moderately lysinerich histone, F2b; and an arginine-rich histone, F3. Each histone contained less than 2% contamination with other fractions as judged by electrophoretic analysis (12). The molecular weights of F2b, F3 and Fl histones are taken as 14,400,14,000 and 21,500, respectively. Concentrations of DNA solutions were calculated from their absorbance (e, = 6600) at 260 nm (20). Vacuum-dried histone fractions were dissolved in water and from the measurement of the absorbancy at 230 nm, respective extinction co(A 230. hng/ml = 1.8), F2b efficients of Fl (Azo. lrn+l = 3.7) and F3 (AZZO, MM = 3.8) histones were calculated. All concentrations of DNA and histones used in these experiments were calculated from these values. The percentage concentrations of both DNA and histone were throughout expressed in grams of each biopolymer present in 100 ml of solution. The salt concentration of the nucleohistone solutions was adjusted to 0.005 M sodium chloride. Ten milliliters of this solution were mixed with an equal volume of 4.0 M sodium chloride solution. After allowing 24 hr for the complete dissociation of the nucleohistone at high salt concentration, the DNA concentration of the mixture was determined from the absorbancy at 260 nm. It is assumed that nucleohistone contains equal amounts of histone and DNA. DNA and histone solutions were mixed in the required proportions at a given ionic strength usually maintained by NaCl. In a few cases electrolytes such as KCl, LiCl, MgClz , NaBr and NasSOd were used instead of NaCl. At a given ionic strength, the protein concentration in the mixture was varied keeping the DNA concentration constant, provided the histone-DNA binding is low. For binding experiments at relatively higher histone concentrations, DNA concentration in the mixture was gradually lowered at a fixed concentration of histone. At ionic strength of 0.25 and below, the solution mixture in many cases became turbid presumably because of the precipitation of the nucleohistone complex. The turbidity was not observed when the ionic strength was 0.5 and above. After allowing the DNAhistone mixture to stand for 24 hr at room temperature (25”C), 25 ml of the solution were decanted into a petri dish and its surface tension was measured after 30 min of the elapse time (t) of the experiment. Histone solutions, prepared as controls side by side with the histone-DNA mixture, were also kept for 24 hr prior to the surface tension measurement. The chromatin gel was suspended in 0.001 M NaCl. However. nucleohistone solutions at ionic
TO DNA
779
strengths 0.01 and 0.10, were turbid due to the precipitation of the biopolymer. The suspension was allowed to stand for 24 hr and the surface tension of the decanted solution was measured as described above. The surface tensions of F2b histone solutions in the presence and absence of nucleohistone at ionic strengths 0.10 or 0.01 were also measured in exactly similar manner after 24 hr equilibrium of the mixture and the control solutions. The Wilhelmy balance used for the surface tension measurement consisted of a chainomatic balance from which the pans and glass were removed. Three thin and parallel microscope cover glasses (size 15 X 50 mm), 1.0 cm apart were suspended from one end of the balance. The slides dipped in the solution contained in the petri dish and the pull on the slides due to the surface tension were balanced by adding weights (m) to the other end of the balance. The entire equipment was enclosed in a Lucite cabinet. The surface tension, (r, in dynes per centimeter is equal to mg/2L where g is the acceleration due to gravity and L, the sum of the lengths of three slides plus thickness. The slight correction due to the buoyancy effect was also taken into account. The surface tension of a protein solution at a time t can be measured with an accuracy of about f0.2 to kO.3 dyn per cm. All results are expressed in terms of surface pressure, II, equal to BO - B. n and ~0 are surface tensions of the DNA or salt solutions in the presence or absence of histone respectively. The different inorganic salts used in the experiments were of analytical grade. Distilled water was treated with activated charcoal black (Darco A-60) and filtered. The surface tension of water was close to 72.0 dyn/cm at 25°C.
Frommer and Miller (13) have recently noted that DNA is unable to lower the surface tension of 1 M sodium chloride solution even when the concentration of the biopolymer is relatively high. In all our experiments, the DNA concentration was less than 0.0025%. We have confirmed that the surface tension of an electrolyte solution at a given ionic strength is the same whether DNA is present or absent. In contrast to this behavior of DNA, histone lowers the surface tension of salt solutions considerably. The magnitude of the surface pressure of a histone solution depends upon protein concentration, elapsed time (t) of the experiment and the ionic strength of the medium. In Fig. 1, surface pressure (II) is plotted as a function of time for F2b histone solutions both in the presence and absence of DNA at
780
CHATTORAJ,
BULL,
AND
CHALKLEY
%
Total
Histone
x IO4
FIG. 2. Variation of II as a function of the total F2b histone concentration (et); elapsed time is equal to 30 min. Curve A, F2b in 0.01 M NaCl; FIG. 1. Variation of surface pressure II with curve A’, F2b in 0.01M NaCl in the presence of elapsed time (t) of the experiment. Ionic strength (m) 0.0015%DNA; curve B, F2b in 0.50 M NaCl; 2.0. Curve A, 0.000610/, F2b; curve B, 0.00040% curve B’, F2b in 0.50 M NaCl in the presence of F2b; curve C, 0.00030%F2b; curve D, 0.00020% 0.0015%DNA; curve C, F2b in 2.0 M NaCl. OF2b; curve E, 0.00010% F2b. 0-F2b histone; No DNA; @-in presence0.0015%DNA. l -F2b in the presenceof 0.0015%DNA; A-F2b in the presenceof 0.00075%DNA. reaches a maximum when the ionic strength is as low as 0.01 (curves A and A’ in Fig. 2). From curve A, it is apparent that a an ionic strength of 2.0. The surface pressure of a histone solution is found to increase sig- 0.0006% histone solution exerts a surface pressure of 14 dyn per cm. From curve A’, nificanbly with time. At this high ionic we note that the same histone solution in the strength the plot of surface pressure against t.ime in Fig. 1 is insensitive to the presence of presence of 0.0015 % DNA has a surface presDNA reflecting the absence of binding of the sure practically equal to zero within the histone fraction to the DNA. From Fig. 1, it limits of our experimental error. Since all the is also apparent that at a given time the sur- histone present in such a mixture at 0.01 face pressure increases wit’h increase in his- ionic strength is completely bound to DNA, tone concent,ration and as such can be used it is apparent that reconstituted nucleohistones, like DNA, do not exert a detectable as a measure of free, unbound histone. effect on the surface tension. In Fig. 2 surface pressure is plotted against In the int.ermediate range of ionic the total concentration of F2b histone in a strengths a biopolymer mixture of DNA and series of environments differing in ionic strength and in the presence or absence of histone will contain free DNA, free histone, complex. Of these, DNA. At ionic strength of 2.0, such plots in and the DNA&stone DNA and the nucleohistone complex are unthe presence and absence of DNA do not differ from each other (curve C) as expected able to contribute to surface pressure. The because of t.he complete dissociation of the observed surface pressure of the solution is solely dependent therefore on the concentracomplex. However, when the ionic strength of the solution is reduced from 2.0 to 0.50, tion of unbound histone in the solution. At a curve B’ (for the mixture of DNA and given value of surface pressure, the observed difference between the total histone concenF2b) in Fig. 2 is now different from curve B (for the F2b histone solution which served tration in the mixture and the histone conas a control). The difference in surface centration of the control is a measure of the pressure between the mixture and the con- concentration of bound histone. For examtrol increases as the ionic strength is pro- ple, at 0.50 ionic strength (curves B and B’ in Fig. 2) and at a surface pressure of 3 dyn, gressively decrea.sed. Finally, the difference
Time
in Minutes
BINDING
OF HISTONE
781
TO DNA
the histone concentrations in the presence and absence of 0.0015% DNA are 0.00046 and 0.00016 %, respectively. The difference, 0.00030 %, then represents the concentration of bound histone. On dividing this concentration by the DXA concentration (CDNA) of the solution, we obtain the binding ratio (w), the grams of histone bound per gram DNA at an equilibrium concentration (c) of free histone. For a given value of II, the binding ratio may be expressed mathematically as : w = (Gt - c)/c,,*
(1)
The surface pressures of F’2b, F3 and Fl histones were measured at identical times after mixing (30 min) both in the presence and absence of DNA at a defined ionic strength. From these data, values of the binding ratios were calculated using Eq. 1. In this way, it is then possible to estimate the histone-DNA binding ratio for a wide range of free histone concentrations. The DNAbinding isotherms for the various histone fractions in a variety of ionic environments are shown in Figs. 3 through 5. From the curves D and F of Figs. 3 and 4, it appears that DNA has no ability to bind a histone fraction when the ionic strength of
I I
%
I
Equilibrium
%
Equilibrium
Histone
x104
Histone
-4
x104
4. F2b histone-DNA binding isoterm. Variation of binding ratio grams histone/gram DNA as function of equilibrium histone concentration. Curve A, 0.01 M NaCl; curve B, 0.10 M NaCI; curve C, 0.25 M NaCl; q , Normal experiment. n , Reversible experiment; curve D, 0.5 M NaCl; curve E, 1.0 M NaCl; curve F, 2.0 M NaCl.
1.6
% Equilibrium
3. F3 histone-DNA binding isotherm. Variation of binding ratio grams histone/gram DNA as a function of the equilibrium histone concentration (c). Curve A, 0, 0.01 M NaCl ; A, 0.10 M NaCl; curve B, 0.50 M NaCl; curve C, 1.00 M NaCl; curve D, 2.00 M NaCl.
3
FIG.
0
FIG.
I
2
3.2
Histone
4.8
x 10’
FIG. 5. Fl hi&one-DNA binding isotherm. Variation of binding ratio grams histone/gram DNA as function of equilibrium histone concentration. Curve A, 0.10 M NaCl; curve B, 0.25 M NaCl; curve C, 0.50 M NaCl.
the solution is 2.0. At 1.0 ionic strength, the binding ratio for F3 histone is about, 0.25 when the hiatone concentration is in between 0.00008 and 0.00041%. At higher histone concentrations the binding ratio increases to
782
CHATTORAJ,
BULL,
0.7. From curve E in Fig. 4, we note that the binding ratio for F2b histone at 1.0 ionic strength is relatively low even when the protein concentration is high. From curve B of Fig. 3, we note that for arginine-rich F3 histone at 0.50 ionic strength, the amount of histone bound increases sharply at low protein concentrations. At relatively higher values of C? the binding ratio seems to reach a constant value (20~)and curve B closely resembles a typical Langmuir isotherm. In contrast to this behavior of E’3 histone, the binding ratio of very lysine-rich Fl protein is observed to be very low at 0.50 JI NaCl (curve C, Fig. 5). In curve D of Fig. 4, we also note that the binding ratio of F2b histone increases slowly as a function of low concentrations of protein. When the protein concentration exceeds 0.00027 %, the binding ratio sharply increases with histone concentration. These observations parallel the affinity of those histone fractions for DNA as measured by their dissociat’ion from DNA in native chromosomal material. However, although the relative binding affinities are the same, in absolute terms they are quite different. This is particularly notable at an ionic strength of 1.0, where in this system we find that F2b has only low affinity for DNA and about 50% of F3 is bound at intermediate protein concentrations. In contrast at this ionic strength F2b is only 50 % released from native chromatin and F3 remains fully bound. Evidently, the reconstitution process has failed to develop all the histone-DNA interactions which are formed in viva and thus should be considered strictly as a model system for DNA and histone binding, recognizing its limitations. Recently we have examined several other means of reconstituting DNA and histone described by other workers, but in all cases we have failed to strictly reproduce the salt dissociation behavior of the native chromatin (R. Chalkley, unpublished observations). The binding of Fl histone at 0.25 and 0.10 ionic strength increases with increase of histone concentration (curves A and B in Fig. 5). At 0.00010 % Fl concentration, the binding ratio increases rapidly to a value as high 5.0, apparently without saturating the DNA binding sites. The binding isotherms of F3
AND
CHALKLEY TABLE
COMPOSITION
OF
THE
DNA
I
SATURATED COMPLEX
HISTONE-
Histone fraction
NaCl concentration
Moles (m,) histone bound per mole DNA
F3
0.01 0.10 0.50 0.01 0.10 0.25
1162 1162 892 1225 1632 1049
F2b
% Equilibrium
Histone
x104
FIG. 6. F2b histone-DNA binding isotherm. Variation of binding ratio grams histone/gram DNA as function of equilibrium histone concentration; ionic strength 0.50. Curve A, MgClz ; curve D, KCI; curve B, Na&Od ; curve C, N&l; curve E, NaBr; curve F, LiCI.
and F2b histones presented in Figs. 3 and 4 at ionic strengths below 0.50 have one common feature. The binding ratio in all these cases initially increases with increase of protein concentration. Finally at higher concentrations of each protein, the binding ratio tends to reach a constant value (w,) as a result of the saturation of DNA with histone. The moles of a histone fraction required to saturate 1 mole of DNA may be calculated from w,. From Table 1, we note that 900-1600 molecules of a histone fraction may be bound to a DNA molecule (M, N 10’) for its saturation at low ionic strength. In Fig. 6, the binding isotherms of F2b histone in the presence of various inorganic electrolytes are compared. The ionic strength of the electrolytes was maintained at 0.50. The features of the isotherms presented in
BINDING
OF HISTONE
Fig. 6 are similar to each other. However, at a given value of histone concentration, the value of the binding ratio depends on the nature of the inorganic electrolyte. Thus at an F2b concentration of 0.00027 % the binding ratio is found to be 0.70,0.40, 0.30, 0.20, and 0.15 for NazSOa, NaCl, KCl: NaBr and LiCl, respectively. The number of water molecules bound per nucleotide of a DNA molecule, previously reported by us (14), is 13, 6,5,3 and 0 in the presence of NazS04, NaCl, KCl, NaBr and LiCl, respectively. At 0.00021% histone concentrat.ion, the binding ratio for MgCL is higher than t’hose for all other electrolytes. This is unexpected since only 5 molecules of Hz0 remain bound per nucleotide of DNA at low MgClz concentration (14). We have also reported previously that MgCls unlike other electrolytes may be significantly bound to DNA. This may be the reason for the anomalous binding ratio of F2b histone in t,he presence of MgC&. In order to test the reversibility of the h&tone-DNA interaction, a solution of low DNA concentration was mixed wit.h excess F2b histone at an ionic strength of 0.25. The solution was distributed into several flasks. After allowing 6 hr for t,hese solutions to attain equilibirum, different amounts of a more concentrated DNA solut,ion were added to these flasks keeping the ionic strength at 0.25. After leaving these solutions 18 additional hours for equilibrium, the surface tensions of these solutions were measured. The binding ratios were t’hen calculated using equation (1). These solutions which originally contained higher histone to DNA ratio were later converted to solutions in which t’he ratio of the histone to DNA was gradually decreased by the further addition of DNA. The binding isotherms for the normal and the reversible experiments (curve D, Fig. 4) are not significantly different. In native chromatin, all five fractions of histone remain completely bound to DNA if the sodium chloride concentration is lower t.han 0.20 M (15). By analogy with the reconstituted DNA-histone complex we would expect that native chromatin at 0.01 ionic strength would have negligible surface activity. The data presented in Fig. 7 (curve A) are in agreement with this expectation: bhe observed surface pressures are only 0.03 and
783
TO DNA
24 //p
%
To+01
B
Ristone x/O4
FIG. 7. Variation of II as a function of the ct of the nucleohistone solution. ck is the histone concentration of chromatin plus the concentration of F2b histone. Curve A, chromatin, 0.01 M NaCl; curve B, chromatin in 2 M NaCl; curve C, F2b histone in 0.01 M NaCl; curve C’, chromatin in the presence of 0.0001820/, F2b histone, 0.01 M NaCl; curve D, F2b histone solution in 0.10 M NaCl; curve D’, chromatin in the presence of 0.000182’j70 F2b histone. 0.10 M NaCl.
0.62 dyn when the total histone concentrations in the chromatin are 0.0004 and 0.0006 %, respectively. For the same concentrations of the F2b hist’one, the observed surface pressures are 10 and 13 dyn, respectively. It may be pointed out here that besides histone, about 12 % no&stone proteins and some lipids are present in calf thymus chromatin. From our data we may infer that bhese components are also not extensively dissociated from chromatin in 0.01 31 NaCl. Also, as shown in curve B of Fig. 7, all histone fractions are extensively dissociated from DNA in 2 M sodium chloride, so that the lowering of surface tension of the solution is significantly larger as expected. In the native chromatin, one g of the combined histone fractions is bound per gram DNA at low ionic strength. From the various binding isotherms presented above it is apparent that one g of DNA at 0.01 and 0.10 sodium chloride concentrations is capable of binding as much as 2 g or more of a histone fraction. It seemed likely that native chromatin at low ionic strength might be able to bind additional histone and thus form saturated histone-DNA complex. We have measured the surface pressure of 0.00018% F2b
784
CHATTORAJ,
BULL,
histone solution in the presence of increasing concentration of chromatin. The results, based on measurements at 0.01 and 0.10 ionic strengths, are shown in curves C’ and D’ of Fig. 7. The corresponding curves C and D for F2b solutions are also shown for the sake of comparison. Using Eq. 1, we can calculate the total quantity (zu) of all histone fractions bound per gram DNA with the assumption that the native chromatin itself is not dissociated at the low ionic strength. From these data, the total quantity of histone required to saturate 1 g DNA in the chromatin are found to be 1.54 and 1.77 g of F2b histone at 0.10 and 0.01 M salt concentrations, respectively, presumably due to an electrostatic interaction of some kind. Native nucleohistone has an overall negative charge even though the DNA negative charges and histone positive charges are present in equal amounts and most likely neutralize one another. The residual negative charge arises from the carboxylate groups in histone since t,here is approximately one such negative charge for every three positive charges on the histone molecule. Thus when nucleohistone binds an additional quantity of histone it is probably due to the electrostatic interaction between histone and a polyanion, however the negative charge now comes from carboxylate rather than phosphate groups. Evidence of histone binding by nucleohistone has been previously reported (16). It should be pointed out, that for nucleohistone at 0.50 and higher ionic strengths, Eq. 1 should not be used for the calculation of the binding ratio, For at higher ionic strengths, the mixture will contain different amounts of the various histone proteins because of the selective dissociation of the nucleohistone and a suitable histone solution for a control in this case is not available. In contrast to the high surface act,ivity of the free histone, .both DNA and nucleohistone lack the ability to lower the surface tension of the aqueous solvent. In the previous sections, we have shown that this differential behavior may be operationally utilized for the calculation of the binding ratio. Histone molecules contain hydrophobic groups so that like other proteins, they are quite surface active. The surface activit,y of the differ-
AND CHALKLEY
ent histone fractions are not t’he same. At an ionic strength 0.10 and protein concentration 0.0005 %, the surface pressures observed for F2b, F3 and Fl histones are 16.0, 18.0 and 6.2 dyn per cm, respectively. The surface pressure of a protein solution also changes with time because of the gradual structural change taking place within the protein monolayer. In our experiments, all observations were taken 30 min after adding the solution to the surface tension apparatus though we have also noted that the binding isotherms are essentially the same if the elapsed time is taken as 20 or 40 min. The surface pressure of a histone solution also depends on the ionic strength of the medium. From curves A, B and C in Fig. 2 we note t,hat the values of the surface pressure for a 0.00034 % F2b histone solution at ionic strengths 2.0,0.50 and 0.01 are 23.7,17.2 and 9.7 dyn per cm, respectively. A similar relation between surface pressure and ionic strength is observed for egg albumin and serum albumin (17). However for 0.00033 % F3 histone solutions, the surface pressure is lower at higher ionic strengths, falling from a value of 17.0 dyn at an ionic strength of 0.1 to 2.1 dyn in 2.0 M NaCI. However molecules of F3 histone associate considerably at high ionic strength. The low value of !J is in all probability due to extensive association (and possibly precipitation) of the F3 histone at high ionic strength. In our calculations of t.he binding ratio with the help of Eq. 1, it has been assumed that the extent of association of the free istone is the same in the mixture and in the control provided the solutions are allowed to equilibrate for the same length of time. In an aqueous mixture of DNA and histone, all the histone molecules actually bound to a molecule of DNA become part of the nucleohistone. Like DNA, the nucleohistone complex does not exert surface pressure, however, unlike DNA, nucleohistone contains a large number of hydrophobic groups due to the bound histone fraction. These groups due to hydrophobic and other types of attractive interactions are presumably hidden within the interior of the nucleohistone complex. Many of the isotherms presented in Figs. 3-6 possess a tendency to as-
BINDING
OF HISTONE
sume a sigmoid shape when the?protein concentration is not high. The sigmoid shape of an isotherm indicates strong intermolecular solute-solute attraction within the bound layer (18). Thus, the outer surface of the nucleohistone most likely contains primarily the charged and uncharged hydrophilic groups. As a result of this hydrophilic envelope of the complex, the molecule may prefer to remain in solution rather than t,o move towards the surface region and contribute to surface pressure. This property of the nucleohistone may be entirelv comparable to the lack of the surface activity of the micelles formed by the bulk association of the longchain organic ions as a result of hydrophobic interactions (19). At an ionic strength 0.10 a part of nucleohistone is precipitated. This may be an additional reason for the insignificant contribution of the complex to the measured surface pressure at this ionic strength. ACKNOWLEDGMENTS This research was supported by a grant from the Division of Molecular Biology, National Science Foundation. Financial assistance of a grant (#CA-10871) from the National Institutes of Health is also acknowledged with thanks. REFERENCES 1. STELLWAGEN, R. H., AND COLE, R. D. (1969) Annu. Rev. Biochem. 38,951. 2. AKINRIMISI, F. O., BONNER, J., AND Ts’o, P. 0. (1965) J. Mol. Biol. 11.128.
TO DNA
G. D., SCH.IFFHAUSEN, B., GOLDL., AND ADLER, A. (1970) Biochemisfry 9, 2814. SHIH, T. Y., AND FASMAN, G. D. (1971) Biochemistry 10, 1675. CLARK, R. J., AND FELSENFELD, G. (1971) Nature New Biol. 229, 101. BOUBLIK, M., BRBDBURY, E. M., CRANEROBINSON, C., >\ND RATTLE, H. W. E. (1971) Nature New Biol. 229, 149. CHATTORAJ, D. K., CHOWRASHI, P., .~ND K. (1967) Biopolymers 6, 173. CHAKRAVARTI, PANYIM, S., AND CHALKLEY, R. (1971a) J. Biol. Chem. 246, 4286. BARTLEY, J. A., AND CHALKLEY, R. (1970) J. Biol. Chem. 246, 4286. JOHNS, E. W. (1964) Biochem. J. 92,55. PANYIM, S. (1971b) Ph.D. Thesis, University of Iowa. PANYIM, S., AND CH~LKLEY, R. (1969) Biochemistry 8, 3972. FROMMER, M. A., AND MILLER, I. It. (1968) J. Phys. Chem. 72, 2862. CHATTORAJ, D. K., AND BULL, H. B. (1971) Arch. Biochem. Biophys. 142, 363. SMART, J. E., AND BONNER, J. (1971) J. Mol. Biol. 68, 661. PHILLIPS, D. M. P. (1968) Experimentia 24,668. GKOSH, S., AND BULL, H. B. (1963) Biochim. Biophys. Acta 66, 150. KIPLING, J. J. (1965) Adsorption from Solutions of Non-electrolytes, p. 129, Academic Press, New York. AD~MSON, A. W. (1960) Physical Chemistry of Surfaces, pp. 91 and 374, Interscience, New York. MAHLER, H. R., KLINE, B., AND MEHROTR~L, B. D. (1964) J. Mol. Biol. 9, 801.
3. FASMAN,
SMITH,
4. 5. 6.
7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
19.
20.
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Binding D. K. CHATTORAJ,’
162,
778-785 (1972)
of Histone HENRY
Department of Biochemistry,
to DNA
B. BULL
University
of Iowa,
AND
R. CHALKLEY
Iowa City, Iowa 6&?40
Received May 18, 1972; accepted June 27, 1972 The reconstitution of nucleohistone has been studied using a novel analytical approach. We have exploited measurements of the surface tension of histone solutions both in the presence and absence of DNA to calculate the amount of histone bound per gram of DNA as a function of protein concentrations in a variety of ionic strength solutions. Fl, F2b and F3 histones were used in these investigations. The nature of the histone-DNA binding isotherms depend significantly on the ionic strength of the medium, type of the histone fraction and the extent of DNA hydration. For F2b and F3 fractions at low ionic strengths, moles of histone present in a mole of saturated DNA-histone complex were calculated. From the measurement of surface tension of F2b histone solutions in the presence and absence of chromatin at 0.01 and 0.10 ionic strengths, the extent of additional binding of F2b histone by native nucleohistone was calculated.
Study of the reconstituted DNA-histone complex has received considerable attention in recent years because the complex is believed to display many of the properties of native chromatin (1). Using the equilibrium dialysis method, Akinrimisi, Bonner, and Ta,O (2) haven show that DNA binds histone at low ionic strengths whereas the reconstituted histone-DNA complex is extensively dissociated when the ionic strength is close to unity. These authors have also demonstrated that the various types of histone bind DNA to a different extent as a function of ionic strength. Fasman et al. (3, 4) have investigated the conformational aspects of the DNA-histone complexes using circular dichroism. According to these authors, the conformational changes associated with the histone-DNA complex are influenced by ionic strength, composition of the complex and DNA hydration. The nature of the histone-DNA binding sites in the reconstituted and native nucleohistones has been examined by Clark and Felsenfeld (5) and also bv Boublik et aZ. (6). 1 On leave of absence, Chemistry Department, Jadavpur University, Calcutta-32, India.
In the present communication, we will discuss an entirely new method which enables us to measure the amount of histone bound to DNA at a given ionic strength. The method is based on the measurement of surface tension of the histone solution in the presence and absence of DNA. MATERIALS AND METHODS Highly polymerized calf thymus DNA was ob. tained from the Worthington Biochemical Corporation. The ratio of the absorbancy of DNA dissolved in 0.01 M sodium chloride at 260 nm to that at 230 nm was 2.46 indicating absence of protein. The hyperchromic shifts of DNA solutions after denaturation by heat and alkali were 38 and 30%, respectively. The molecular weight of Worthington DNA is of the order 10’ (7). Calf thymus chromatin was prepared by the method of Panyim and Chalkley (8) and stored at an ionic strength lower than 0.9095 to inhibit proteolysis (9). Such chromatin consists of equal weights of histone and DNA together with approx 12% nonhistone protein and less than 2% RNA. There is a small amount of lipid present most likely as part of a membrane fraction. Histone fractions were isolated from calf chromatin in 0.4 N H&04 . Separation into individual fractions followed the procedures initially developed by Johns (10) as modified by Panyim (11). 778
Copyright All rights
@ 1972 by Academic Press, of reproduction in any form
Inc. reserved.
BINDING
OF HISTONE
We have used three fractions in this study: the very lysine-rich histone, Fl; a moderately lysinerich histone, F2b; and an arginine-rich histone, F3. Each histone contained less than 2% contamination with other fractions as judged by electrophoretic analysis (12). The molecular weights of F2b, F3 and Fl histones are taken as 14,400,14,000 and 21,500, respectively. Concentrations of DNA solutions were calculated from their absorbance (e, = 6600) at 260 nm (20). Vacuum-dried histone fractions were dissolved in water and from the measurement of the absorbancy at 230 nm, respective extinction co(A 230. hng/ml = 1.8), F2b efficients of Fl (Azo. lrn+l = 3.7) and F3 (AZZO, MM = 3.8) histones were calculated. All concentrations of DNA and histones used in these experiments were calculated from these values. The percentage concentrations of both DNA and histone were throughout expressed in grams of each biopolymer present in 100 ml of solution. The salt concentration of the nucleohistone solutions was adjusted to 0.005 M sodium chloride. Ten milliliters of this solution were mixed with an equal volume of 4.0 M sodium chloride solution. After allowing 24 hr for the complete dissociation of the nucleohistone at high salt concentration, the DNA concentration of the mixture was determined from the absorbancy at 260 nm. It is assumed that nucleohistone contains equal amounts of histone and DNA. DNA and histone solutions were mixed in the required proportions at a given ionic strength usually maintained by NaCl. In a few cases electrolytes such as KCl, LiCl, MgClz , NaBr and NasSOd were used instead of NaCl. At a given ionic strength, the protein concentration in the mixture was varied keeping the DNA concentration constant, provided the histone-DNA binding is low. For binding experiments at relatively higher histone concentrations, DNA concentration in the mixture was gradually lowered at a fixed concentration of histone. At ionic strength of 0.25 and below, the solution mixture in many cases became turbid presumably because of the precipitation of the nucleohistone complex. The turbidity was not observed when the ionic strength was 0.5 and above. After allowing the DNAhistone mixture to stand for 24 hr at room temperature (25”C), 25 ml of the solution were decanted into a petri dish and its surface tension was measured after 30 min of the elapse time (t) of the experiment. Histone solutions, prepared as controls side by side with the histone-DNA mixture, were also kept for 24 hr prior to the surface tension measurement. The chromatin gel was suspended in 0.001 M NaCl. However. nucleohistone solutions at ionic
TO DNA
779
strengths 0.01 and 0.10, were turbid due to the precipitation of the biopolymer. The suspension was allowed to stand for 24 hr and the surface tension of the decanted solution was measured as described above. The surface tensions of F2b histone solutions in the presence and absence of nucleohistone at ionic strengths 0.10 or 0.01 were also measured in exactly similar manner after 24 hr equilibrium of the mixture and the control solutions. The Wilhelmy balance used for the surface tension measurement consisted of a chainomatic balance from which the pans and glass were removed. Three thin and parallel microscope cover glasses (size 15 X 50 mm), 1.0 cm apart were suspended from one end of the balance. The slides dipped in the solution contained in the petri dish and the pull on the slides due to the surface tension were balanced by adding weights (m) to the other end of the balance. The entire equipment was enclosed in a Lucite cabinet. The surface tension, (r, in dynes per centimeter is equal to mg/2L where g is the acceleration due to gravity and L, the sum of the lengths of three slides plus thickness. The slight correction due to the buoyancy effect was also taken into account. The surface tension of a protein solution at a time t can be measured with an accuracy of about f0.2 to kO.3 dyn per cm. All results are expressed in terms of surface pressure, II, equal to BO - B. n and ~0 are surface tensions of the DNA or salt solutions in the presence or absence of histone respectively. The different inorganic salts used in the experiments were of analytical grade. Distilled water was treated with activated charcoal black (Darco A-60) and filtered. The surface tension of water was close to 72.0 dyn/cm at 25°C.
Frommer and Miller (13) have recently noted that DNA is unable to lower the surface tension of 1 M sodium chloride solution even when the concentration of the biopolymer is relatively high. In all our experiments, the DNA concentration was less than 0.0025%. We have confirmed that the surface tension of an electrolyte solution at a given ionic strength is the same whether DNA is present or absent. In contrast to this behavior of DNA, histone lowers the surface tension of salt solutions considerably. The magnitude of the surface pressure of a histone solution depends upon protein concentration, elapsed time (t) of the experiment and the ionic strength of the medium. In Fig. 1, surface pressure (II) is plotted as a function of time for F2b histone solutions both in the presence and absence of DNA at
780
CHATTORAJ,
BULL,
AND
CHALKLEY
%
Total
Histone
x IO4
FIG. 2. Variation of II as a function of the total F2b histone concentration (et); elapsed time is equal to 30 min. Curve A, F2b in 0.01 M NaCl; FIG. 1. Variation of surface pressure II with curve A’, F2b in 0.01M NaCl in the presence of elapsed time (t) of the experiment. Ionic strength (m) 0.0015%DNA; curve B, F2b in 0.50 M NaCl; 2.0. Curve A, 0.000610/, F2b; curve B, 0.00040% curve B’, F2b in 0.50 M NaCl in the presence of F2b; curve C, 0.00030%F2b; curve D, 0.00020% 0.0015%DNA; curve C, F2b in 2.0 M NaCl. OF2b; curve E, 0.00010% F2b. 0-F2b histone; No DNA; @-in presence0.0015%DNA. l -F2b in the presenceof 0.0015%DNA; A-F2b in the presenceof 0.00075%DNA. reaches a maximum when the ionic strength is as low as 0.01 (curves A and A’ in Fig. 2). From curve A, it is apparent that a an ionic strength of 2.0. The surface pressure of a histone solution is found to increase sig- 0.0006% histone solution exerts a surface pressure of 14 dyn per cm. From curve A’, nificanbly with time. At this high ionic we note that the same histone solution in the strength the plot of surface pressure against t.ime in Fig. 1 is insensitive to the presence of presence of 0.0015 % DNA has a surface presDNA reflecting the absence of binding of the sure practically equal to zero within the histone fraction to the DNA. From Fig. 1, it limits of our experimental error. Since all the is also apparent that at a given time the sur- histone present in such a mixture at 0.01 face pressure increases wit’h increase in his- ionic strength is completely bound to DNA, tone concent,ration and as such can be used it is apparent that reconstituted nucleohistones, like DNA, do not exert a detectable as a measure of free, unbound histone. effect on the surface tension. In Fig. 2 surface pressure is plotted against In the int.ermediate range of ionic the total concentration of F2b histone in a strengths a biopolymer mixture of DNA and series of environments differing in ionic strength and in the presence or absence of histone will contain free DNA, free histone, complex. Of these, DNA. At ionic strength of 2.0, such plots in and the DNA&stone DNA and the nucleohistone complex are unthe presence and absence of DNA do not differ from each other (curve C) as expected able to contribute to surface pressure. The because of t.he complete dissociation of the observed surface pressure of the solution is solely dependent therefore on the concentracomplex. However, when the ionic strength of the solution is reduced from 2.0 to 0.50, tion of unbound histone in the solution. At a curve B’ (for the mixture of DNA and given value of surface pressure, the observed difference between the total histone concenF2b) in Fig. 2 is now different from curve B (for the F2b histone solution which served tration in the mixture and the histone conas a control). The difference in surface centration of the control is a measure of the pressure between the mixture and the con- concentration of bound histone. For examtrol increases as the ionic strength is pro- ple, at 0.50 ionic strength (curves B and B’ in Fig. 2) and at a surface pressure of 3 dyn, gressively decrea.sed. Finally, the difference
Time
in Minutes
BINDING
OF HISTONE
781
TO DNA
the histone concentrations in the presence and absence of 0.0015% DNA are 0.00046 and 0.00016 %, respectively. The difference, 0.00030 %, then represents the concentration of bound histone. On dividing this concentration by the DXA concentration (CDNA) of the solution, we obtain the binding ratio (w), the grams of histone bound per gram DNA at an equilibrium concentration (c) of free histone. For a given value of II, the binding ratio may be expressed mathematically as : w = (Gt - c)/c,,*
(1)
The surface pressures of F’2b, F3 and Fl histones were measured at identical times after mixing (30 min) both in the presence and absence of DNA at a defined ionic strength. From these data, values of the binding ratios were calculated using Eq. 1. In this way, it is then possible to estimate the histone-DNA binding ratio for a wide range of free histone concentrations. The DNAbinding isotherms for the various histone fractions in a variety of ionic environments are shown in Figs. 3 through 5. From the curves D and F of Figs. 3 and 4, it appears that DNA has no ability to bind a histone fraction when the ionic strength of
I I
%
I
Equilibrium
%
Equilibrium
Histone
x104
Histone
-4
x104
4. F2b histone-DNA binding isoterm. Variation of binding ratio grams histone/gram DNA as function of equilibrium histone concentration. Curve A, 0.01 M NaCl; curve B, 0.10 M NaCI; curve C, 0.25 M NaCl; q , Normal experiment. n , Reversible experiment; curve D, 0.5 M NaCl; curve E, 1.0 M NaCl; curve F, 2.0 M NaCl.
1.6
% Equilibrium
3. F3 histone-DNA binding isotherm. Variation of binding ratio grams histone/gram DNA as a function of the equilibrium histone concentration (c). Curve A, 0, 0.01 M NaCl ; A, 0.10 M NaCl; curve B, 0.50 M NaCl; curve C, 1.00 M NaCl; curve D, 2.00 M NaCl.
3
FIG.
0
FIG.
I
2
3.2
Histone
4.8
x 10’
FIG. 5. Fl hi&one-DNA binding isotherm. Variation of binding ratio grams histone/gram DNA as function of equilibrium histone concentration. Curve A, 0.10 M NaCl; curve B, 0.25 M NaCl; curve C, 0.50 M NaCl.
the solution is 2.0. At 1.0 ionic strength, the binding ratio for F3 histone is about, 0.25 when the hiatone concentration is in between 0.00008 and 0.00041%. At higher histone concentrations the binding ratio increases to
782
CHATTORAJ,
BULL,
0.7. From curve E in Fig. 4, we note that the binding ratio for F2b histone at 1.0 ionic strength is relatively low even when the protein concentration is high. From curve B of Fig. 3, we note that for arginine-rich F3 histone at 0.50 ionic strength, the amount of histone bound increases sharply at low protein concentrations. At relatively higher values of C? the binding ratio seems to reach a constant value (20~)and curve B closely resembles a typical Langmuir isotherm. In contrast to this behavior of E’3 histone, the binding ratio of very lysine-rich Fl protein is observed to be very low at 0.50 JI NaCl (curve C, Fig. 5). In curve D of Fig. 4, we also note that the binding ratio of F2b histone increases slowly as a function of low concentrations of protein. When the protein concentration exceeds 0.00027 %, the binding ratio sharply increases with histone concentration. These observations parallel the affinity of those histone fractions for DNA as measured by their dissociat’ion from DNA in native chromosomal material. However, although the relative binding affinities are the same, in absolute terms they are quite different. This is particularly notable at an ionic strength of 1.0, where in this system we find that F2b has only low affinity for DNA and about 50% of F3 is bound at intermediate protein concentrations. In contrast at this ionic strength F2b is only 50 % released from native chromatin and F3 remains fully bound. Evidently, the reconstitution process has failed to develop all the histone-DNA interactions which are formed in viva and thus should be considered strictly as a model system for DNA and histone binding, recognizing its limitations. Recently we have examined several other means of reconstituting DNA and histone described by other workers, but in all cases we have failed to strictly reproduce the salt dissociation behavior of the native chromatin (R. Chalkley, unpublished observations). The binding of Fl histone at 0.25 and 0.10 ionic strength increases with increase of histone concentration (curves A and B in Fig. 5). At 0.00010 % Fl concentration, the binding ratio increases rapidly to a value as high 5.0, apparently without saturating the DNA binding sites. The binding isotherms of F3
AND
CHALKLEY TABLE
COMPOSITION
OF
THE
DNA
I
SATURATED COMPLEX
HISTONE-
Histone fraction
NaCl concentration
Moles (m,) histone bound per mole DNA
F3
0.01 0.10 0.50 0.01 0.10 0.25
1162 1162 892 1225 1632 1049
F2b
% Equilibrium
Histone
x104
FIG. 6. F2b histone-DNA binding isotherm. Variation of binding ratio grams histone/gram DNA as function of equilibrium histone concentration; ionic strength 0.50. Curve A, MgClz ; curve D, KCI; curve B, Na&Od ; curve C, N&l; curve E, NaBr; curve F, LiCI.
and F2b histones presented in Figs. 3 and 4 at ionic strengths below 0.50 have one common feature. The binding ratio in all these cases initially increases with increase of protein concentration. Finally at higher concentrations of each protein, the binding ratio tends to reach a constant value (w,) as a result of the saturation of DNA with histone. The moles of a histone fraction required to saturate 1 mole of DNA may be calculated from w,. From Table 1, we note that 900-1600 molecules of a histone fraction may be bound to a DNA molecule (M, N 10’) for its saturation at low ionic strength. In Fig. 6, the binding isotherms of F2b histone in the presence of various inorganic electrolytes are compared. The ionic strength of the electrolytes was maintained at 0.50. The features of the isotherms presented in
BINDING
OF HISTONE
Fig. 6 are similar to each other. However, at a given value of histone concentration, the value of the binding ratio depends on the nature of the inorganic electrolyte. Thus at an F2b concentration of 0.00027 % the binding ratio is found to be 0.70,0.40, 0.30, 0.20, and 0.15 for NazSOa, NaCl, KCl: NaBr and LiCl, respectively. The number of water molecules bound per nucleotide of a DNA molecule, previously reported by us (14), is 13, 6,5,3 and 0 in the presence of NazS04, NaCl, KCl, NaBr and LiCl, respectively. At 0.00021% histone concentrat.ion, the binding ratio for MgCL is higher than t’hose for all other electrolytes. This is unexpected since only 5 molecules of Hz0 remain bound per nucleotide of DNA at low MgClz concentration (14). We have also reported previously that MgCls unlike other electrolytes may be significantly bound to DNA. This may be the reason for the anomalous binding ratio of F2b histone in t,he presence of MgC&. In order to test the reversibility of the h&tone-DNA interaction, a solution of low DNA concentration was mixed wit.h excess F2b histone at an ionic strength of 0.25. The solution was distributed into several flasks. After allowing 6 hr for t,hese solutions to attain equilibirum, different amounts of a more concentrated DNA solut,ion were added to these flasks keeping the ionic strength at 0.25. After leaving these solutions 18 additional hours for equilibrium, the surface tensions of these solutions were measured. The binding ratios were t’hen calculated using equation (1). These solutions which originally contained higher histone to DNA ratio were later converted to solutions in which t’he ratio of the histone to DNA was gradually decreased by the further addition of DNA. The binding isotherms for the normal and the reversible experiments (curve D, Fig. 4) are not significantly different. In native chromatin, all five fractions of histone remain completely bound to DNA if the sodium chloride concentration is lower t.han 0.20 M (15). By analogy with the reconstituted DNA-histone complex we would expect that native chromatin at 0.01 ionic strength would have negligible surface activity. The data presented in Fig. 7 (curve A) are in agreement with this expectation: bhe observed surface pressures are only 0.03 and
783
TO DNA
24 //p
%
To+01
B
Ristone x/O4
FIG. 7. Variation of II as a function of the ct of the nucleohistone solution. ck is the histone concentration of chromatin plus the concentration of F2b histone. Curve A, chromatin, 0.01 M NaCl; curve B, chromatin in 2 M NaCl; curve C, F2b histone in 0.01 M NaCl; curve C’, chromatin in the presence of 0.0001820/, F2b histone, 0.01 M NaCl; curve D, F2b histone solution in 0.10 M NaCl; curve D’, chromatin in the presence of 0.000182’j70 F2b histone. 0.10 M NaCl.
0.62 dyn when the total histone concentrations in the chromatin are 0.0004 and 0.0006 %, respectively. For the same concentrations of the F2b hist’one, the observed surface pressures are 10 and 13 dyn, respectively. It may be pointed out here that besides histone, about 12 % no&stone proteins and some lipids are present in calf thymus chromatin. From our data we may infer that bhese components are also not extensively dissociated from chromatin in 0.01 31 NaCl. Also, as shown in curve B of Fig. 7, all histone fractions are extensively dissociated from DNA in 2 M sodium chloride, so that the lowering of surface tension of the solution is significantly larger as expected. In the native chromatin, one g of the combined histone fractions is bound per gram DNA at low ionic strength. From the various binding isotherms presented above it is apparent that one g of DNA at 0.01 and 0.10 sodium chloride concentrations is capable of binding as much as 2 g or more of a histone fraction. It seemed likely that native chromatin at low ionic strength might be able to bind additional histone and thus form saturated histone-DNA complex. We have measured the surface pressure of 0.00018% F2b
784
CHATTORAJ,
BULL,
histone solution in the presence of increasing concentration of chromatin. The results, based on measurements at 0.01 and 0.10 ionic strengths, are shown in curves C’ and D’ of Fig. 7. The corresponding curves C and D for F2b solutions are also shown for the sake of comparison. Using Eq. 1, we can calculate the total quantity (zu) of all histone fractions bound per gram DNA with the assumption that the native chromatin itself is not dissociated at the low ionic strength. From these data, the total quantity of histone required to saturate 1 g DNA in the chromatin are found to be 1.54 and 1.77 g of F2b histone at 0.10 and 0.01 M salt concentrations, respectively, presumably due to an electrostatic interaction of some kind. Native nucleohistone has an overall negative charge even though the DNA negative charges and histone positive charges are present in equal amounts and most likely neutralize one another. The residual negative charge arises from the carboxylate groups in histone since t,here is approximately one such negative charge for every three positive charges on the histone molecule. Thus when nucleohistone binds an additional quantity of histone it is probably due to the electrostatic interaction between histone and a polyanion, however the negative charge now comes from carboxylate rather than phosphate groups. Evidence of histone binding by nucleohistone has been previously reported (16). It should be pointed out, that for nucleohistone at 0.50 and higher ionic strengths, Eq. 1 should not be used for the calculation of the binding ratio, For at higher ionic strengths, the mixture will contain different amounts of the various histone proteins because of the selective dissociation of the nucleohistone and a suitable histone solution for a control in this case is not available. In contrast to the high surface act,ivity of the free histone, .both DNA and nucleohistone lack the ability to lower the surface tension of the aqueous solvent. In the previous sections, we have shown that this differential behavior may be operationally utilized for the calculation of the binding ratio. Histone molecules contain hydrophobic groups so that like other proteins, they are quite surface active. The surface activit,y of the differ-
AND CHALKLEY
ent histone fractions are not t’he same. At an ionic strength 0.10 and protein concentration 0.0005 %, the surface pressures observed for F2b, F3 and Fl histones are 16.0, 18.0 and 6.2 dyn per cm, respectively. The surface pressure of a protein solution also changes with time because of the gradual structural change taking place within the protein monolayer. In our experiments, all observations were taken 30 min after adding the solution to the surface tension apparatus though we have also noted that the binding isotherms are essentially the same if the elapsed time is taken as 20 or 40 min. The surface pressure of a histone solution also depends on the ionic strength of the medium. From curves A, B and C in Fig. 2 we note t,hat the values of the surface pressure for a 0.00034 % F2b histone solution at ionic strengths 2.0,0.50 and 0.01 are 23.7,17.2 and 9.7 dyn per cm, respectively. A similar relation between surface pressure and ionic strength is observed for egg albumin and serum albumin (17). However for 0.00033 % F3 histone solutions, the surface pressure is lower at higher ionic strengths, falling from a value of 17.0 dyn at an ionic strength of 0.1 to 2.1 dyn in 2.0 M NaCI. However molecules of F3 histone associate considerably at high ionic strength. The low value of !J is in all probability due to extensive association (and possibly precipitation) of the F3 histone at high ionic strength. In our calculations of t.he binding ratio with the help of Eq. 1, it has been assumed that the extent of association of the free istone is the same in the mixture and in the control provided the solutions are allowed to equilibrate for the same length of time. In an aqueous mixture of DNA and histone, all the histone molecules actually bound to a molecule of DNA become part of the nucleohistone. Like DNA, the nucleohistone complex does not exert surface pressure, however, unlike DNA, nucleohistone contains a large number of hydrophobic groups due to the bound histone fraction. These groups due to hydrophobic and other types of attractive interactions are presumably hidden within the interior of the nucleohistone complex. Many of the isotherms presented in Figs. 3-6 possess a tendency to as-
BINDING
OF HISTONE
sume a sigmoid shape when the?protein concentration is not high. The sigmoid shape of an isotherm indicates strong intermolecular solute-solute attraction within the bound layer (18). Thus, the outer surface of the nucleohistone most likely contains primarily the charged and uncharged hydrophilic groups. As a result of this hydrophilic envelope of the complex, the molecule may prefer to remain in solution rather than t,o move towards the surface region and contribute to surface pressure. This property of the nucleohistone may be entirelv comparable to the lack of the surface activity of the micelles formed by the bulk association of the longchain organic ions as a result of hydrophobic interactions (19). At an ionic strength 0.10 a part of nucleohistone is precipitated. This may be an additional reason for the insignificant contribution of the complex to the measured surface pressure at this ionic strength. ACKNOWLEDGMENTS This research was supported by a grant from the Division of Molecular Biology, National Science Foundation. Financial assistance of a grant (#CA-10871) from the National Institutes of Health is also acknowledged with thanks. REFERENCES 1. STELLWAGEN, R. H., AND COLE, R. D. (1969) Annu. Rev. Biochem. 38,951. 2. AKINRIMISI, F. O., BONNER, J., AND Ts’o, P. 0. (1965) J. Mol. Biol. 11.128.
TO DNA
G. D., SCH.IFFHAUSEN, B., GOLDL., AND ADLER, A. (1970) Biochemisfry 9, 2814. SHIH, T. Y., AND FASMAN, G. D. (1971) Biochemistry 10, 1675. CLARK, R. J., AND FELSENFELD, G. (1971) Nature New Biol. 229, 101. BOUBLIK, M., BRBDBURY, E. M., CRANEROBINSON, C., >\ND RATTLE, H. W. E. (1971) Nature New Biol. 229, 149. CHATTORAJ, D. K., CHOWRASHI, P., .~ND K. (1967) Biopolymers 6, 173. CHAKRAVARTI, PANYIM, S., AND CHALKLEY, R. (1971a) J. Biol. Chem. 246, 4286. BARTLEY, J. A., AND CHALKLEY, R. (1970) J. Biol. Chem. 246, 4286. JOHNS, E. W. (1964) Biochem. J. 92,55. PANYIM, S. (1971b) Ph.D. Thesis, University of Iowa. PANYIM, S., AND CH~LKLEY, R. (1969) Biochemistry 8, 3972. FROMMER, M. A., AND MILLER, I. It. (1968) J. Phys. Chem. 72, 2862. CHATTORAJ, D. K., AND BULL, H. B. (1971) Arch. Biochem. Biophys. 142, 363. SMART, J. E., AND BONNER, J. (1971) J. Mol. Biol. 68, 661. PHILLIPS, D. M. P. (1968) Experimentia 24,668. GKOSH, S., AND BULL, H. B. (1963) Biochim. Biophys. Acta 66, 150. KIPLING, J. J. (1965) Adsorption from Solutions of Non-electrolytes, p. 129, Academic Press, New York. AD~MSON, A. W. (1960) Physical Chemistry of Surfaces, pp. 91 and 374, Interscience, New York. MAHLER, H. R., KLINE, B., AND MEHROTR~L, B. D. (1964) J. Mol. Biol. 9, 801.
3. FASMAN,
SMITH,
4. 5. 6.
7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
19.
20.