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The binding of calcium with deoxyribonucleic acid and deoxyribonucleic acid-protein complexes
BIOCHIMICAET BIOPHYSICAACTA
127
BBA 95861
T H E B I N D I N G OF CALCIUM W I T H D E O X Y R I B O N U C L E I C ACID AND D E O X Y R I B O N U C L E I C A C I D - P R O T E I N COMPLEXES
KIM YONG CHANG* AND CHARLES W. CARR Department o/ Biochemistry, University of Minnesota Medical School, Minneapolis, Minn. (U.S.A.)
(Received November I7th, 1967)
SUMMARY
The binding of Ca ~+ to highly polymerized DNA in the presence of several different small cations has been studied b y the technique of equilibrium dialysis. In the neutral range of pH, the cations Na +, diamine *+, La 2+, Th 4+, spelmidine 3+ and sperimine 4+ compete with Ca ~+ for the phosphate binding sites. The competitive effect for a given ion is almost entirely a function of the magnitude of the cationic charge. The effect of p H on the binding of Ca z+ also shows the electrostatic nature of the binding process because the binding curve is closely correlated with the H + titration curve in the p H range 2.0-11.0. In studies with DNA-protein complexes, competition between Ca 2÷ and cationic proteins is also shown. When the Ca ~+ concentration is below 5 raM, cationic proteins such as protamine and histones will block DNA binding sites for Ca 2+ in direct proportion to the concentration of cationic groups present. When the concentration of Ca ~÷ is greater than 20 mM, Ca 2+ is bound to its full-capacity to D N A , indicating a complete displacement of protein from the binding sites.
INTRODUCTION In the development of knowledge about the structure and function of DNA and RNA, a considerable amount of attention has been paid to their nature as polyelectrolytes. This is especially true with respect to their amphoteric behavior, for their acid-base titration curves have been carefully studied b y several investigators 1-e. The study of the interaction of other small ions with nucleic acids has also yielded important information with respect to the conformational and other aspects of nucleic acid structure 7-13. Early results in this area showed that small cations interact with DNA at neutral p H to form stable complexes. More recently, it has been shown that certain di- and multi-valent cations stabilize the double helix of DNA against heat denaturation 14-17. Both the complex formation and the stabilizing effect * Present address: Department of Pharmacology, McGill University, Montreal, Canada. Biochim, Biophys. Acta, 157 (I968) I27-139
128
K . Y. C H A N G , C. W . C A R R
have been considered to be most likely the result of ion-pair formation between the small cation and the multiple phosphate groups of the DNA backbone. Recent studies with several physicochemical techniques have confirmed this idea 18-*°. The present report is concerned with a study of the binding of calcium with DNA and how it is affected by other cations. The effect of p H on calcium binding is first established and then the competitive relationships between calcium and several types of cations is investigated. In addition some studies have been made with both artificial and natural DNA-protein complexes. The competitive effects between calcium and cationic proteins is clearly shown.
EXPERIMENTAL
Procedure The binding measurements were made b y the technique of equilibrium dialysis, the procedure being as follows: 0.20 % DNA solutions were prepared b y dissolving the highly polymerized material in I mM NaC1. The p H was adjusted to the desired value b y the addition of dilute HC1, acetic acid, N H i O H or N a O H as the case might be. The strong acid or base was used to obtain the extreme p H values, and the weak acid or base was used to obtain the intermediate values. I0 ml of DNA solution was placed inside a cellophane dialyzing membrane, and an equal volume of CaC1, or other chloride salt of known concentration was placed outside. The resulting system was then shaken for 8 h at room temperature, preliminary experiments showing that equilibrium was always attained in that time. The two compartments were then analyzed for their various constituents. As a check, control systems were run from time to time in which the inside compartment contained water and the outside compartment contained the salt solution under investigation. Calcium was determined b y titration with ethylene diamine tetraacetic acid according to the method of SOBEL AND HANOK~1. In the determination of calcium in the presence of lanthanum or thorium, I ml of I M ammonium buffer at p H Io.o was added to 5 ml of solution being analyzed and allowed to stand overnight. This resulted in the quantitative precipitation of La (OH)3 or Th (OH)4 leaving the calcium in solution. An aliquot of the supernatant fluid was then titrated in the usual way. The p H was measured with a Beckman Zeromatic p H meter. The phosphorus content of each of the DNA samples was determined by the method of FISKE AND SUBBARow z2. To determine the Donnan distribution factor for the counterions, a measurement of the membrane potential was made with two saturated calomel electrodes which were connected to the two solutions inside and outside the membrane through saturated KC1 bridges. There was an appreciable Donnan correction only in experiments in which the calcium binding was measured at low concentration ( < 2 raM), i.e., in those experiments in which the effect of free Ca ~+ concentration on the degree of binding was measured. In nearly all of the experiments reported here, the conditions were such t h a t the negative binding sites were saturated with bound calcium or other bound cations so there was no detectable Donnan effect. From the data collected in these experiments, the binding of Ca 2+ was calcuBiochim. Biophys. Acta, 157 (1968) lZ7-139
C a 2+ BINDING TO D N A
129
lated to be the difference between the total concentration of calcium in the inside compartment and the free Ca 2+ concentration, which, in the absence of an appreciable Donnan effect, was assumed to be the same as the concentration measured in the outside compartment. The results are expressed in terms of tile ratio of the moles of calcium bound to the moles of phosphorus in the sample (Ca/P).
Materials Three samples of highly polymerized DNA were studied. These were salmon sperm DNA from Mann Research Laboratories, calf thymus DNA from Nutritional Biochemicals Corp., and calf thymus DNA prepared in our laboratory b y the method of SCHWANDER AND SIGNER23. On the basis of measurements of the extinction coefficient at 260 m# (ref. 24, 25) and the fact that all of the samples dissolved very slowly in I mM NaC1 forming very viscous solutions, it was concluded that all the samples were highly polymerized. The diamines, 1,2-diaminoethane, 1,4-diaminobutane, and 1,6-diaminohexane were E a s t m a n Organic Chemicals; the spermidine and spermine were obtained from the Sigma Chemical Co. The artificial DNA-protein complexes were prepared by blowing a jet of 5.0 ml of protein solution from a syringe pipet into 5.0 ml of 0.2 % DNA solution in 1.0 mM NaC1 contained in a cellophane dialyzing bag, generally causing flocculation of DNA-proteinate. The resulting complex was dialyzed against I0.0 ml of CaC12 in the external compartment. Highly polymerized fish sperm DNA was used that was obtained from Mann Laboratories, Inc. The proteins, protamine sulfate and histone, were obtained from Nutritional Biochemicals Corp. The protamine sulfate was changed to protamine chloride b y anion exchange with Dowex-2 resin to avoid the interference of sulfate in the calcium determination. Chicken erythrocyte nuclei were isolated b y the method of DOUNCE AND LAN26. The isolated nuclei were stained with methylene blue and examined microscopically as a morphological criterion for the integrity. The very viscous nature of the nuclear suspensions was observed as another criterion of their integrity. Fish sperm nuclei were obtained from Nutritional Biochemicals Corp. as a preparation of protamine nucleinate.
RESULTS AND DISCUSSION
A. Binding studies with D N A The first experiments were carried out to determine the effect of free Ca 2+ concentration on the degree of binding. I t was found that as the concentration of free Ca z+ increases, the binding increases until a m a x i m u m is reached at 4-5 mM. This value is such that Ca/P is 0.48, and it remains constant up to a concentratior of 50 mM Ca 2+. Effect o/ p H on Ca 2+ and Mg ~+ binding Three different samples of DNA were studied over a wide range of pH, the Ca 2+ concentration being kept at 4.5-5.0 mM to insure maximal binding (Fig. i). Biochim. Biophys. Acta, 157 (1968) 127-139
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Within the pH range of 2-12 the three samples yielded identical curves for Ca *+ binding, and in addition, with one sample, the Mg~+ binding was the same. The nature of the curve is such that it is almost completely in quantitative agreement with what might be predicted on the basis of the acid-base titration curves of highly polymerized DNA. In the first place, Ca/P is constant and equal to o.475:~o.oI3 over the pH range of 5.0-9. 5. This is just slightly less than equivalent to the primary phosphate groups which are completely ionized in this pH range. The constancy of this value over this wide pH range shows that there are no detectable secondary phosphate groups; however, the fact that it is consistently slightly less than 0.50 is somewhat puzzling. If the value of 0.475 is not due to some unrecognized error, then it may be surmised that about 95 % of the phosphate groups in these DNA samples are flee and the remaining 5 % are unavailable for binding Ca ~+ for some unexplained reason. Below pH 5.0 the amount of binding decreases, most probably because of the protonization of the amino groups on the nitrogen bases. From the published acidbase titration data for DNA from pH 7.0 to pH 3.0, it is evident that at p H 3.0, 2 protons have been taken up per 4 phosphorus atoms. This would decrease the net phosphate charge so that the predicted Ca *+ binding would result in Ca/P being equal to 0.25. The value we obtained at pH 3.0 is 0.26. The base compositions of calf thymus DNA and fish sperm DNA are similar enough so that these calculated contributions to charge are essentially the same for both types *~. Apparently the positively charged groups form an internal bond with adjacent phosphate groups stoichiometrically, and calcium cannot bind with the phosphate groups unless the positively charged groups are deprotonized. These intramolecular non-covalent bonds are apparently strong enough so that calcium cannot compete with them to any significant extent. As the pH drops below 3.o, the decrease in Ca *+ binding is greater tkan the increase in positive charges such that the binding reaches zero at pH 2.0. At this pH the quantity of protonized amino groups is about 65 °/o of the phosphate groups which would leave free phosphate groups equivalent to o.17 in our Ca/P expression. This discrepancy in stoichiometry suggests that when less than half of the original phosphate groups are free, the formation constant with calcium becomes less so that at the concentration of 5 mM Ca ~+, less than maximal binding is obtained. Biochim. Biophys. Acta, I57 (I968) I 2 7 - I 3 9
Ca 2+ BINDING TO D N A
131
At p H greater than 9.0, the Ca 2+ binding increases above its equivalence with the phosphate groups and reaches a value of about 0.75 at p H 11. 5. The binding curve in this region is again correlated with the acid-base titration curve of highly polymerized DNA. This increase in the binding curve is most likely due to the ionizable enol groups of guanine and thymine 4. Thus a total of 2.0 equiv/4 P of enol groups are available for binding at high pH, which would mean that the upper limit for Ca/P would be about 0.75. These results on the binding of divalent cations with DNA are in general agreement with the various reports in the literature. SHACK, JENKINS AND THOMPSET7 have reported the binding of Mg *+ with DNA to be a coulombic interaction with the phosphate groups and that there was close to equivalence between the Mg z+ binding and the phosphorus content. WIBERG AND NEUMAN9 have shown that the saturation binding of Ca 2+, Mg ~+, and Mn ~+ with DNA is the same and is equivalent to the phosphorus content. In addition their results also show that the binding reaches its m a x i m u m at about I mM free Ca 2+. FELSENFELD AND HUANG ~7, in conductimetric studies with synthetic polynucleotides and DNA, have also demonstrated the equivalence between Mg ~+ binding and phosphorus content. In addition to confirming these earlier findings, our results also show the effect of low and high p H on the binding of these ions, which has not been previously demonstrated, an effect which is quite consistent with the H + titration curve for highly polymerized DNA 2,4.
Ca2+-Na + competition
Experiments were carried out to determine the effect of the presence of Na + on the binding of Ca 2+ b y DNA at p H 6.5 (Fig. 2). The total Ca 2+ concentration was kept constant at 5.0 mM, and the Na + concentration was varied b y the addition of NaCI. It is seen that the binding of Ca ~+ is decreased in a regular manner until it approaches zero at 25o mM Na +. This is strongly indicative once more that the binding of the small divalent cations is largely coulombic in nature. In view of these results and the results of others, it is quite likely that there is competition between Na + and Ca ~+. From the data in Fig. 4, at the half suppression of Ca ~+ binding, the concentration of flee Ca ~+ is about 4.5 mR[ and that of free Na + about 70 mM, the ratio of these concentrations being 16. WIBERG AND NEUMAN9 have also shown that in the presence of 15o mM NaC1, the binding of calcium is markedly depressed, a behavior which they suggest might be due to competition. According to their data, the binding of Ca ~+ in 15o mM NaC1 is one-half its m a x i m u m value when the free Ca 2+ is about IO mM. The ratio of these concentrations is 15, just about the same as obtained from our data. SHACK and coworkers 7,28 and FELSENFELD AND HUANG ~7,29 have also suggested that Na + competes with the divalent cations for binding with polynucleotides. Earlier statements in the literature suggest that the binding site for divalent cations, especially Mg a+, with polynucleotides is the nitrogen bases 8°,81. However, it has already been pointed out b y STEINER AND BEERS 82, especially in the light of the data of FELSENFELD AND HUANG, that this conclusion was not the most likely. The recent more direct evidence of LYoNs AND KOTINTM, FELDMAN AND KEILtg, and HAPPE AND MORALES20, and the consequences of our binding data also strongly support the concept of phosphate binding sites instead of the nitrogen bases. Biochim. Biophys. dcta, 157 (1968) 127-I39
132
K . Y . CHANG, C. W. CARR
C a2+-Diamine competition The effect of organic divalent cations on the binding of Ca 2÷ with DNA was determined b y measuring Ca 2+ binding at p H 6.0 in the presence of three different aliphatic diamines of increasing chain length, 1,2-diaminoethane, 1,4-diaminobutane, and 1,6-diaminohexane (Fig. 3). The total Ca 2+ concentration was kept constant at 5.0 raM, and the diamine concentration was varied b y the addition of the chloride salts. I c is seen that the binding of Ca 2+ decreases markedly in the presence of these diamines, with the value approaching zero at about 60-70 mM diamine. I t will also be observed that there appears to be slight differences among these compounds, the order of their ability to suppress Ca 2+ binding being 2C > 4 C > 6C. When "competition constants" are calculated from the free concentrations of the two competing species at the half point of maximal Ca 2+ binding, values are obtained which range from o.6 for diaminohexane to o. 9 for diaminoethane*. This would indicate that Ca 2+ has only a slightly greater affinity for DNA than the small, divalent organic cations. The results are of special interest because it reinforces the idea that the binding of these small cations is primarily a coulombic interaction with phosphate groups. o5 I 0.5 ~ 0,4
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FELSENFELD AND HUANG~9have also shown that the binding of certain diamines with synthetic polynucleotides is very much like that of Mg *+, but that it is slightly weaker. Similarly, other investigators have shown that diamines such as those studied here stabilize undenatured DNA b y increasing the "melting" temperature, Tm (refs. 14-17). I t is suggested t h a t complex formation of the amines with the DNA phosphate groups is the cause of the increased stability.
Ca ~+ competition with tri- and tetravalent cations I n these experiments the binding of tri- and tetravalent cations with DNA is studied b y the determination of the binding of Ca *+ in the presence of the polyvalent cations. I n this indirect method we have considered that the negative charges on * Kdiamine/Ca =
(Diaznine-- D N A ) (Ca--DNA)
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Biochim. B i o p h y s . Acta, 157 (1968) I 2 7 - 1 3 9
CAm+ BINDING TO D N A
133
DNA are neutralized stoichiometrically b y the various mixtures of Ca 2+ and polyvalent cation and that the fraction of DNA which is not bound b y Ca 2+ is bound by the other cation. It has been reported by FELSENFELD AND HUANGs3 that spermine 4+ can displace Ba m+from poly(U) stoichiometrically. In the first experiment with La 8+, the concentration of La ++ was varied (Fig. 4). It is seen that the binding of Ca m+ is decreased markedly by the addition of low concentrations of La 3+, it reaching zero when the free La 3+ concentration is 2.o raM. In a second experiment it was desired to determine the effect of Ca m+concentration on its binding to DNA in the presence of several different polyvalent cations, the inorganic La 3+ and Th 4+ and the organic spermidine 8+ and spermine 4+. The polyvalent cation concentration was maintained constant at 2.5 mM, and the Cam+ concentration was varied from 5.0 mM to ioo mM. The results are shown in Fig. 5. 05 I
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Fig. 5. The binding of Ca 2+ by DNA in the presence of trivalent and tetravalent cations. Salmon sperm DNA, p H 4.5. Fig. 6. The binding of Ca ~+ by DNA-protamine complexes at various pH's. Salmon sperm DNA, [Ca 2+] 5.0 mM.
Once more it is seen that the charge effects appear to be the decisive factor in determining the degree of interaction that occurs. The displacement of trivalent cations b y Ca2+ is about the same for either La ~+ or spermidine 3+. The concentration of Ca2+ which causes one-half displacement of the trivalent cation is about 15-2o raM. The curve for the tetravalent cations is similar to the one for the trivalent cations except that it is somewhat displaced to the right. The concentration of Ca ~+which causes one-half displacement of the tetravalent cations is about 40-60 mM. Thus, a fourth charge is apparently somewhat effective in adding to the binding strength of the cation. The data which have been presented appear to be quite consistent and in conformity with the idea that the binding of small cations by DNA is primarily an electrostatic interaction with phosphate groups. Thus, for undenatured DNA, the monovalent cation, Na +, is bound very weakly, but strongly enough so that it competes to an appreciable extent with divalent cations. The divalent cations are bound nearly stoichiometfically and with considerably more affinity than monovalent cations. At saturation each divalent cation combines with two phosphate groups in such a manner that just about all of these groups are occupied. In many, or perhaps Biochim. Biophys. Acta, 157 (1968) 127-139
134
I~. Y. CHANG, C. W. CARR
all, instances such a cation combines with one phosphate group in one strand and the nearest neighbor phosphate group in the other strand in order to account for the effectiveness of these cations in increasing the "melting" temperature of DNA. The La 3+ and spermidine are also apparently bound stoichiometrically and are bound much more strongly than divalent cations. This would indicate that there are three phosphate groups close enough together to produce a much stronger electrostatic interaction. Th 4+ and spermine are bound more tightly than the trivalent cations; however, the increase in binding affinity appears to be somewhat less than that observed between the divalent and trivalent cationic interactions. This would indicate that a fourth anionic charge of the DNA is always located at some greater distance from each cluster of three charges. Thus, these results are strongly in concurrence with the accepted double helical model for DNA in which there is no detectable breaking or branching of the structure and in which the phosphate groups are 7 3. apart in symmetrical distribution over the surface of the double helix in a nearly endless rhombic array.
B. Binding studies with DNA-protein complexes Artifical DNA-protein complexes The way in which nucleic acids and proteins interact in the nucleus has for a long time been attributed primarily to an electrostatic interaction between positively charged proteins and negatively charged nucleic acids. Many data now seem qualitatively, at least, to substantiate this idea ~,35. In view of our results in the previous section, it became of interest to us to study the interaction of DNA with proteins by means of Ca2+-binding measurements. The first experiments were carried out with DNA and protamine, and the results are shown in Fig. 6. From the pronounced decrease in Ca/P, it is easily seen that protamine masks the sites for binding of Ca 2+ with DNA. A quantitative evaluation of the decrease in Ca/P in the p H range 6- 9 shows that the Ca 2+ displaced is equivalent to the amount of positive charge added in the form of protamine when 0.05 % protamine is used se. When higher concentrations are used, there is a further decrease in Ca/P, but it is less than equivalent to the amount of protamine added. This is most likely caused b y a steric effect such that after 50-60 ~o of the DNA-phosphate groups are covered b y protamine, the remaining areas for protamine binding are too small to accommodate whole molecules. Some of the positive groups of the protamine combine with phosphate groups leaving the remainder of the protamine molecule unreacted with nucleic acid. Thus, it requires a considerable excess of protamine to reduce Ca/P to o.02-0.03. rn a similar series of experiments with the positively charged protein, calf thymus histone, a similar behavior is observed (Fig. 7). With a o.o5 % solution of protein, the decrease in Ca/P at pH 6.0 is just about equivalent to the calculated total positive charges of the protein sT. However, as the amount of protein is increased, the further decrease in Ca/P becomes less than is equivalent to the charged groups of the proteins. This effect is greater with histone than with protamine, presumably because these molecules are larger, titus resulting in a greater steric effect when many of them compete for the charged groups of DNA. When the same experiment was carried out using lysozyme in place of histone, Biochim. Biophys. Acta, i 5 7 (i968) i 2 7 - 1 3 9
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the results were quite similar. With o.I % lysozyme in the presence of o.i % DNA, the decrease in Ca/P in the p H range 5-6.5 was 0.22, which is equivalent to the total positive charge of the added lysozyme. Above p H 6.5 there was a further increase in Ca/P which was considered to be additional binding to the lysozyme. When serum albumin was added to DNA at p H ' s below the isoelectric point of the albumin, there was also a lowering of Ca/P. In this case, it was found to be equivalent to the net positive charge of the albumin. These experiments with DNA and various proteins were carried out at a Ca ~+ concentration which is sufficient to produce maximal binding with the free D N A phosphate groups. I t has been presumed that this binding is a quantitative measure of those groups that are not involved in the DN'A-protein bonding. This is based primarily on the fact that there is essentially a stoichiometric agreement between Ca ~+ displacement and protein cationic groups at low protein concentration. Thus, for these different proteins, they apparently bind strongly enough to prevent any significant competition with Ca 2+ at the level of 5.0 mM free Ca 2+. To determine the competitive effect of Ca ~+ on the protein binding of DNA, experiments were carried out in which p r o t a m i n e - D N A and histone-DNA mixtures were exposed to increasing Ca ~+ concentrations at p H 7.0 (Fig. 8). The amount of histone added was somewhat less than would be expected to be stoichiometfic with respect to the DNA. I t is seen that at low concentrations of Ca 2+ there is leveling off at Ca/P -----o.18, when the free Ca 2+ reaches 5.0 mM. I t is assumed that this represents the free DNA, unreacted with protein. As the Ca ~+ concentration is increased further, a second rise begins at 6-7 mM Ca 2+ and approaches a second plateau at 15 mM Ca *+ with the value of Ca/P slightly greater than 0.5. Our interpretation of this behavior is that the increased Ca 2+ concentration displaces the protein b y a competitive effect, very similar to that observed in our earlier experiments with spermidine- and spermine-DN'A complexes. For the experiment with protamine, enough protamine was added to react with all of the nucleic acid so that Ca/P remained essentially zero at 5 mM flee Ca 2+. The displacement of protamine then begins at a somewhat higher concentration of Ca *+ than for histone, which would be expected for the greater cationic charge density in protamine than in histones. Biochim. Biophys. Acta, I57 (1968) 127-139
136
K. Y. CHANG, C. W. CARR
Natural DNA-protein complexes Most of our data obtained for natural DNA-protein complexes has been obtained from chicken erythrocyte nuclei. They have been isolated in two different media, physiological saline and 0.25 M sucrose-o.oo 3 M CaC12 (ref. 26), In our first experiments with these nuclei, we studied the effect of p H on the binding of Ca 2+ at a free Ca ~+ concentration of 4.5-5,0 mM (Fig. 9)- The curve which was obtained is quite reproducible from one preparation to the next using the same method of isolation, and, in addition, it is seen that the two different methods of isolation resulted in no significant difference. I t will also be noted t h a t there is a strong similarity between this curve and the curves obtained previously for artificial complexes. In the p H range 3.0-6.5 there is a lowering in Ca/P which seems to be almost certainly due to the blocking of sites b y protein. Although the amino acid composition of the histone fraction from chicken erythrocyte nuclei has been reported aT, there is uncertainty in the amount of histone that comprises the total nuclear protein. Thus, a comparison of the positive charge of the histones with the decrease in Ca/P is not feasible. Nevertheless, we would like to propose that the value for Ca/P at p H 6.0 is a quantitative indication of the DNA that is not bound b y protein. i
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I n the p H range 6.5-7.5, the further increase in binding which occurs is similar to that observed with the histone and lysozyme complexes with DNA. I t can be surmised here as it was in the previous instances that this increased binding is caused b y interactions with anionic binding sites in the nuclear protein. In another experiment with chicken erythrocyte nuclei, protamine was added to the nuclei (Fig. 9). I t will be seen that the remaining free DNA can be "covered" with protamine just as in the previous experiments with DNA-protamine complexes. Lysozyme also can block some of the free DNA from binding with Ca 2+. On the other hand, a larger protein such as serum albumin is unable to block any sites in the p H range where it is positively charged (pH 4.5). I t can be surmised t h a t the albumin molecule is too large to occupy any of the DNA sites that are accessible to protamine, especially, and to lysozyme to some extent. In the binding measurements with fish sperm nuclei (Fig. IO), the general shape of the curves is the same as for chicken erythrocyte nuclei; however, there is considerBiochim. Biophys. Acta, 157 (1968) 127-139
C a 2+ BINDING TO D N A
137
ably less uncombined DNA as indicated b y the low values for Ca/P at pH 6.o. This agrees with the data of VENDRELY, KNOBLAUCH-MAZENAND VENDRELY88 that there is equivalence between the arginine content and phosphate content of fish sperm nuclei. As with the other nuclei, there is also a rise in binding in the pH range 6.5-7.5, the magnitude of which varies for the different samples. Thus, for the two types of nuclei studied, the value for Ca/P at pH 5.5-6.5 appears to represent those phosphate groups of DNA that are not combined with strongly binding substances such as cationic proteins and polyamines. The variation in the values for Ca/P above pH 6.5 apparently reflects differences in types of proteins and other substances that are present in the nucleus'. With erythrocyte nuclei a single rise is obtained above pH 6.5 indicating that a single general type of protein is the principle non-nucleic acid component. For the fish sperm nuclei, two characteristic breaks are obtained in the curve suggesting that there are two different types of binding substances present. In a final experiment the effect of Ca ~+ concentration on the binding at constant p H was determined (Fig. II). It is seen that the data obtained with the nuclei are very similar to the data obtained with the artificial complexes, especially the DNAhistone complex. In the range of 6-15 mM Ca 2+, the protein is apparently displaced allowing the Ca/P to increase and eventually approach 0.5. ,
0.8
F
r
i
i
i
~
,
i
DNA - HEstone, Artificiof, Complex DNA - Protamine, Artlficia~ Cornp!e: Nuclei, Chicken RBC
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0.0
i
0
i
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I
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I 16
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I, 20
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Fig. II. The binding of Ca 2+ b y chicken erythrocyte (RBC) nuclei a t c o n s t a n t p H a n d increasing Ca 2+ concentration.
The displacement of proteins from their binding with DNA as revealed by Ca ~+ binding measurements is quite similar to our data obtained with polyamineDNA complexes. The range of free Ca ~+ concentration which produced the displacement of one-half of the histone is lO-12 mM while for spermidine 3+ and La 3+ it is 20 mM. Similarly, for protamine it is 15-2o mM and for spermine 4+ and Th*+ it is 40--60 mM. These data confirm the previous proposals for an electrostatic bonding of positively charged proteins to DNA in the nucleus. We would go further and suggest that the interaction of histones and protamines with DNA is one in which each point of attachment involves the interaction of a two or three positive charge cluster of the protein with a three negative charge cluster of the DNA. For the protamines this may be extended to four changes, but the contribution of the fourth charge appears * Some p r e l i m i n a r y e x p e r i m e n t s with nuclei from certain m a m m a l i a n cells indicate t h a t in addition to proteins, phospholipids m a y contribute to the Ca ~+ binding at the higher p H range.
Biochim. Biophys. Acta, 157 (1968) I 2 7 - I 3 9
138
K . Y . CHANG, C. W. CARR
to be relatively small. Amino acid analyses of histones indicate the occurrence of clusters of 2-3 positively charged amino acids 3", and for protamine it is quite evident that the arginine residues in groups of four3t As was pointed out earlier in this paper, the rhombic array of the phosphate groups in DNA provides a continuous succession of 7/~ triangles as clusters of three negative charge to serve as binding sites foc cations. On the basis of the data reported here, it appears to be possible to probe the nature of isolated cellular particulates with a relatively non-disruptive technique. The usefulness of this approach has also been demonstrated recently with ion-binding studies on another cellular particulate, the ribosome4°. It is anticipated that further studies with purified biological macromolecules and with artificial and natural complexes of these substances will yield further information about the nature of the forces which hold such complexes together. ACKNOWLEDGEMENT This
investigation
08412 from the National
was
supported
Institutes
by
of Health,
research
grants
Public Health
HE 01618
and
GM
Service.
REFERENCES I R. A. Cox, A. S. JONES, G. E. MARSH AND A. R. PEACOCKE, Bioehim. Biophys. Acta, 21
(1956 ) 576.
2 3 4 5 6 7 8 9 io ii 12 13 14 15 16 17 18 19 20 21 22 23 24
25 26 27 28 29 3° 31 32 33
R. A. C o x AND A. R, PEACOCKE, J. Chem. Soc., (1956) 2499. R. A. Cox, Biochim. Biophys. Acta, 68 (1963) 4Ol. J. M. GULLAND, D. O. JORDAN AND H. F. W. TAYLOR, J . Chem. Soc., (1947) 1132. A. R. MATHIIESON AND S. MATTY, J. Polymer Sci., 23 (1957) 747. R. F. STEINER AND R. F. BEERS, Nature, 179 (1957) lO76. J. SHACK, R. J. JENKINS AND J. M. THHOMPSETT, J. Biol. Chem., 203 (1953) 373. P. D. LAWLEY, Biochim. Biophys. Acta, 21 (1956) 481. J. S. WIBERG AND W. F. NEUMAN, Arch. Bioehem. Biophys., 72 (1957) 66. G. ZUBAY AND P. DOTY, Bioehim. Biophys. Acta, 29 (1958) 47. G. FELSENFELD, "The Molecular Basis o/ Neoplasia", U n i v . of T e x a s Press, A u s t i n , 1962, p. lO4. K. C. BANERJEE AND D. J. PERKINS, Bioehim. Biophys. Acta, 61 (1962) I. J. W . LYoNs AND L. KOTIN, J. Am. Chem. Soc., 86 (1964) 3634. H. TABOR, Biochemistry, i (1962) 496. G. L. EICHHORN, Nature, 194 (1962) 474. H. R. MAHLER AND B. D. MEHROTA, Biochim. Biophys. Aeta, 55 (1962) 252. M. MANDEL, J. Mol. Biol., 5 (1962) 435. J. w . LYoNs AND L. KOTIN, J. Am. Chem. Soc., 87 (1965) 178I.. I. FELDMAN AND E. KEIL, J. Am. Chem. Soc., 87 (1965) 3281. J. A. HAPPE AND M. MORALES, J. Am. Chem. Soc., 88 (1966) 2o77. A. E. SOBEL AND A. HANOK, Proc. Soc. Exptl. Biol. Med., 79 (1951) 737. C. H. FISKE AND Y. SUBBARow, J. Biol. Chem., 81 (1929) 629. H. SCHWANDER AND R. SIGNER, Helv. Chim. Aeta, 38 (195o) 1521. E. CHIARGAFFAND J. N. DAVlDSON, The Nucleic Acids, Vol. I, A c a d e m i c Press, N e w York, 1955. D. O. JORDAN, The Chemistry o~ Nucleic Acids, B u t t e r w o r t h , W a s h i n g t o n , 196o. A. L. DOUNCE AND T. H. LAN, Science, 97 (1943) 584 • G. FELSENFELD AND S. HUANG, Biochim. Biophys. Acta, 37 (196o) 425 . H. KAHLER AND J. SHACK, J. Phys. Chem., 61 (1957) 649. G. FELSENFELD AND S. HUANG, Biochim. Biophys. Aeta, 34 (1959) 234. G. ZUBAY, Bioehim. Biophys. Aeta, 32 (1959) 233. L. CAVALIERI, J. Am. Chem. Soe., 74 (1952) 1242. R. F. STEINER AND R. F. BEERS, Poly~ucleotides, Elsevier, A m s t e r d a m , 1961. G. FELSENFELD AND S. HUANG, Biochim. Biophys. Aeta, 51 (1961) 19.
Biochim. Biophys. Acta, 157 (1968) 127-139
Ca ~+ BINDING TO D N A
139
J- S. MITCHELL, The Cell Nucleus, B u t t e r w o r t h s , London, 196o. J. BONNER AND P. O. P. Ts'o, Nucleohistones, Holden-Day, San Francisco, Calif., 1964. K. FELIX, H. FISCHER AND A. KREKELS, Progr. Biochem. Biophys. Chem., 6 (1956) 2. J. M, NEELIN, P. X. CALLAHAN, D. C. LAMB AND I~. MURRAY, Can. J. Biochem., 42 (1964) 1743. 38 R. VENDRELY,A. KNOBLAUCH-MAZEN AND C. VENDREL¥, ill J. S. MITCHELL, The Cell Nucleus, B u t t e r w o r t h s , London, 196o, p. 2oo. 39 H. BUSCH, Histones and Other Nuclear Proteins, Academic Press, New York, 1965. 4 ° Y. S. CHOI AND C. W. CARR, J. Mol. Biol., 25 (1967) 331.
34 35 36 37
Biochim. Biophys. Acta, 157 (I968) 127-139
127
BBA 95861
T H E B I N D I N G OF CALCIUM W I T H D E O X Y R I B O N U C L E I C ACID AND D E O X Y R I B O N U C L E I C A C I D - P R O T E I N COMPLEXES
KIM YONG CHANG* AND CHARLES W. CARR Department o/ Biochemistry, University of Minnesota Medical School, Minneapolis, Minn. (U.S.A.)
(Received November I7th, 1967)
SUMMARY
The binding of Ca ~+ to highly polymerized DNA in the presence of several different small cations has been studied b y the technique of equilibrium dialysis. In the neutral range of pH, the cations Na +, diamine *+, La 2+, Th 4+, spelmidine 3+ and sperimine 4+ compete with Ca ~+ for the phosphate binding sites. The competitive effect for a given ion is almost entirely a function of the magnitude of the cationic charge. The effect of p H on the binding of Ca z+ also shows the electrostatic nature of the binding process because the binding curve is closely correlated with the H + titration curve in the p H range 2.0-11.0. In studies with DNA-protein complexes, competition between Ca 2÷ and cationic proteins is also shown. When the Ca ~+ concentration is below 5 raM, cationic proteins such as protamine and histones will block DNA binding sites for Ca 2+ in direct proportion to the concentration of cationic groups present. When the concentration of Ca ~÷ is greater than 20 mM, Ca 2+ is bound to its full-capacity to D N A , indicating a complete displacement of protein from the binding sites.
INTRODUCTION In the development of knowledge about the structure and function of DNA and RNA, a considerable amount of attention has been paid to their nature as polyelectrolytes. This is especially true with respect to their amphoteric behavior, for their acid-base titration curves have been carefully studied b y several investigators 1-e. The study of the interaction of other small ions with nucleic acids has also yielded important information with respect to the conformational and other aspects of nucleic acid structure 7-13. Early results in this area showed that small cations interact with DNA at neutral p H to form stable complexes. More recently, it has been shown that certain di- and multi-valent cations stabilize the double helix of DNA against heat denaturation 14-17. Both the complex formation and the stabilizing effect * Present address: Department of Pharmacology, McGill University, Montreal, Canada. Biochim, Biophys. Acta, 157 (I968) I27-139
128
K . Y. C H A N G , C. W . C A R R
have been considered to be most likely the result of ion-pair formation between the small cation and the multiple phosphate groups of the DNA backbone. Recent studies with several physicochemical techniques have confirmed this idea 18-*°. The present report is concerned with a study of the binding of calcium with DNA and how it is affected by other cations. The effect of p H on calcium binding is first established and then the competitive relationships between calcium and several types of cations is investigated. In addition some studies have been made with both artificial and natural DNA-protein complexes. The competitive effects between calcium and cationic proteins is clearly shown.
EXPERIMENTAL
Procedure The binding measurements were made b y the technique of equilibrium dialysis, the procedure being as follows: 0.20 % DNA solutions were prepared b y dissolving the highly polymerized material in I mM NaC1. The p H was adjusted to the desired value b y the addition of dilute HC1, acetic acid, N H i O H or N a O H as the case might be. The strong acid or base was used to obtain the extreme p H values, and the weak acid or base was used to obtain the intermediate values. I0 ml of DNA solution was placed inside a cellophane dialyzing membrane, and an equal volume of CaC1, or other chloride salt of known concentration was placed outside. The resulting system was then shaken for 8 h at room temperature, preliminary experiments showing that equilibrium was always attained in that time. The two compartments were then analyzed for their various constituents. As a check, control systems were run from time to time in which the inside compartment contained water and the outside compartment contained the salt solution under investigation. Calcium was determined b y titration with ethylene diamine tetraacetic acid according to the method of SOBEL AND HANOK~1. In the determination of calcium in the presence of lanthanum or thorium, I ml of I M ammonium buffer at p H Io.o was added to 5 ml of solution being analyzed and allowed to stand overnight. This resulted in the quantitative precipitation of La (OH)3 or Th (OH)4 leaving the calcium in solution. An aliquot of the supernatant fluid was then titrated in the usual way. The p H was measured with a Beckman Zeromatic p H meter. The phosphorus content of each of the DNA samples was determined by the method of FISKE AND SUBBARow z2. To determine the Donnan distribution factor for the counterions, a measurement of the membrane potential was made with two saturated calomel electrodes which were connected to the two solutions inside and outside the membrane through saturated KC1 bridges. There was an appreciable Donnan correction only in experiments in which the calcium binding was measured at low concentration ( < 2 raM), i.e., in those experiments in which the effect of free Ca ~+ concentration on the degree of binding was measured. In nearly all of the experiments reported here, the conditions were such t h a t the negative binding sites were saturated with bound calcium or other bound cations so there was no detectable Donnan effect. From the data collected in these experiments, the binding of Ca 2+ was calcuBiochim. Biophys. Acta, 157 (1968) lZ7-139
C a 2+ BINDING TO D N A
129
lated to be the difference between the total concentration of calcium in the inside compartment and the free Ca 2+ concentration, which, in the absence of an appreciable Donnan effect, was assumed to be the same as the concentration measured in the outside compartment. The results are expressed in terms of tile ratio of the moles of calcium bound to the moles of phosphorus in the sample (Ca/P).
Materials Three samples of highly polymerized DNA were studied. These were salmon sperm DNA from Mann Research Laboratories, calf thymus DNA from Nutritional Biochemicals Corp., and calf thymus DNA prepared in our laboratory b y the method of SCHWANDER AND SIGNER23. On the basis of measurements of the extinction coefficient at 260 m# (ref. 24, 25) and the fact that all of the samples dissolved very slowly in I mM NaC1 forming very viscous solutions, it was concluded that all the samples were highly polymerized. The diamines, 1,2-diaminoethane, 1,4-diaminobutane, and 1,6-diaminohexane were E a s t m a n Organic Chemicals; the spermidine and spermine were obtained from the Sigma Chemical Co. The artificial DNA-protein complexes were prepared by blowing a jet of 5.0 ml of protein solution from a syringe pipet into 5.0 ml of 0.2 % DNA solution in 1.0 mM NaC1 contained in a cellophane dialyzing bag, generally causing flocculation of DNA-proteinate. The resulting complex was dialyzed against I0.0 ml of CaC12 in the external compartment. Highly polymerized fish sperm DNA was used that was obtained from Mann Laboratories, Inc. The proteins, protamine sulfate and histone, were obtained from Nutritional Biochemicals Corp. The protamine sulfate was changed to protamine chloride b y anion exchange with Dowex-2 resin to avoid the interference of sulfate in the calcium determination. Chicken erythrocyte nuclei were isolated b y the method of DOUNCE AND LAN26. The isolated nuclei were stained with methylene blue and examined microscopically as a morphological criterion for the integrity. The very viscous nature of the nuclear suspensions was observed as another criterion of their integrity. Fish sperm nuclei were obtained from Nutritional Biochemicals Corp. as a preparation of protamine nucleinate.
RESULTS AND DISCUSSION
A. Binding studies with D N A The first experiments were carried out to determine the effect of free Ca 2+ concentration on the degree of binding. I t was found that as the concentration of free Ca z+ increases, the binding increases until a m a x i m u m is reached at 4-5 mM. This value is such that Ca/P is 0.48, and it remains constant up to a concentratior of 50 mM Ca 2+. Effect o/ p H on Ca 2+ and Mg ~+ binding Three different samples of DNA were studied over a wide range of pH, the Ca 2+ concentration being kept at 4.5-5.0 mM to insure maximal binding (Fig. i). Biochim. Biophys. Acta, 157 (1968) 127-139
13o
K. Y. CHANG, C. W. CARR 0.5
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Fig. I. T h e m a x i m u m b i n d i n g of Ca *+ a n d Mg *+ b y v a r i o u s D N A s a m p l e s as a f u n c t i o n of p H . Fig. 2. T h e b i n d i n g of Ca *+ b y D N A in t h e p r e s e n c e of NaC1. Calf t h y m u s D N A , p H 6.5, [Ca *+] 5.o raM.
Within the pH range of 2-12 the three samples yielded identical curves for Ca *+ binding, and in addition, with one sample, the Mg~+ binding was the same. The nature of the curve is such that it is almost completely in quantitative agreement with what might be predicted on the basis of the acid-base titration curves of highly polymerized DNA. In the first place, Ca/P is constant and equal to o.475:~o.oI3 over the pH range of 5.0-9. 5. This is just slightly less than equivalent to the primary phosphate groups which are completely ionized in this pH range. The constancy of this value over this wide pH range shows that there are no detectable secondary phosphate groups; however, the fact that it is consistently slightly less than 0.50 is somewhat puzzling. If the value of 0.475 is not due to some unrecognized error, then it may be surmised that about 95 % of the phosphate groups in these DNA samples are flee and the remaining 5 % are unavailable for binding Ca ~+ for some unexplained reason. Below pH 5.0 the amount of binding decreases, most probably because of the protonization of the amino groups on the nitrogen bases. From the published acidbase titration data for DNA from pH 7.0 to pH 3.0, it is evident that at p H 3.0, 2 protons have been taken up per 4 phosphorus atoms. This would decrease the net phosphate charge so that the predicted Ca *+ binding would result in Ca/P being equal to 0.25. The value we obtained at pH 3.0 is 0.26. The base compositions of calf thymus DNA and fish sperm DNA are similar enough so that these calculated contributions to charge are essentially the same for both types *~. Apparently the positively charged groups form an internal bond with adjacent phosphate groups stoichiometrically, and calcium cannot bind with the phosphate groups unless the positively charged groups are deprotonized. These intramolecular non-covalent bonds are apparently strong enough so that calcium cannot compete with them to any significant extent. As the pH drops below 3.o, the decrease in Ca *+ binding is greater tkan the increase in positive charges such that the binding reaches zero at pH 2.0. At this pH the quantity of protonized amino groups is about 65 °/o of the phosphate groups which would leave free phosphate groups equivalent to o.17 in our Ca/P expression. This discrepancy in stoichiometry suggests that when less than half of the original phosphate groups are free, the formation constant with calcium becomes less so that at the concentration of 5 mM Ca ~+, less than maximal binding is obtained. Biochim. Biophys. Acta, I57 (I968) I 2 7 - I 3 9
Ca 2+ BINDING TO D N A
131
At p H greater than 9.0, the Ca 2+ binding increases above its equivalence with the phosphate groups and reaches a value of about 0.75 at p H 11. 5. The binding curve in this region is again correlated with the acid-base titration curve of highly polymerized DNA. This increase in the binding curve is most likely due to the ionizable enol groups of guanine and thymine 4. Thus a total of 2.0 equiv/4 P of enol groups are available for binding at high pH, which would mean that the upper limit for Ca/P would be about 0.75. These results on the binding of divalent cations with DNA are in general agreement with the various reports in the literature. SHACK, JENKINS AND THOMPSET7 have reported the binding of Mg *+ with DNA to be a coulombic interaction with the phosphate groups and that there was close to equivalence between the Mg z+ binding and the phosphorus content. WIBERG AND NEUMAN9 have shown that the saturation binding of Ca 2+, Mg ~+, and Mn ~+ with DNA is the same and is equivalent to the phosphorus content. In addition their results also show that the binding reaches its m a x i m u m at about I mM free Ca 2+. FELSENFELD AND HUANG ~7, in conductimetric studies with synthetic polynucleotides and DNA, have also demonstrated the equivalence between Mg ~+ binding and phosphorus content. In addition to confirming these earlier findings, our results also show the effect of low and high p H on the binding of these ions, which has not been previously demonstrated, an effect which is quite consistent with the H + titration curve for highly polymerized DNA 2,4.
Ca2+-Na + competition
Experiments were carried out to determine the effect of the presence of Na + on the binding of Ca 2+ b y DNA at p H 6.5 (Fig. 2). The total Ca 2+ concentration was kept constant at 5.0 mM, and the Na + concentration was varied b y the addition of NaCI. It is seen that the binding of Ca ~+ is decreased in a regular manner until it approaches zero at 25o mM Na +. This is strongly indicative once more that the binding of the small divalent cations is largely coulombic in nature. In view of these results and the results of others, it is quite likely that there is competition between Na + and Ca ~+. From the data in Fig. 4, at the half suppression of Ca ~+ binding, the concentration of flee Ca ~+ is about 4.5 mR[ and that of free Na + about 70 mM, the ratio of these concentrations being 16. WIBERG AND NEUMAN9 have also shown that in the presence of 15o mM NaC1, the binding of calcium is markedly depressed, a behavior which they suggest might be due to competition. According to their data, the binding of Ca ~+ in 15o mM NaC1 is one-half its m a x i m u m value when the free Ca 2+ is about IO mM. The ratio of these concentrations is 15, just about the same as obtained from our data. SHACK and coworkers 7,28 and FELSENFELD AND HUANG ~7,29 have also suggested that Na + competes with the divalent cations for binding with polynucleotides. Earlier statements in the literature suggest that the binding site for divalent cations, especially Mg a+, with polynucleotides is the nitrogen bases 8°,81. However, it has already been pointed out b y STEINER AND BEERS 82, especially in the light of the data of FELSENFELD AND HUANG, that this conclusion was not the most likely. The recent more direct evidence of LYoNs AND KOTINTM, FELDMAN AND KEILtg, and HAPPE AND MORALES20, and the consequences of our binding data also strongly support the concept of phosphate binding sites instead of the nitrogen bases. Biochim. Biophys. dcta, 157 (1968) 127-I39
132
K . Y . CHANG, C. W. CARR
C a2+-Diamine competition The effect of organic divalent cations on the binding of Ca 2÷ with DNA was determined b y measuring Ca 2+ binding at p H 6.0 in the presence of three different aliphatic diamines of increasing chain length, 1,2-diaminoethane, 1,4-diaminobutane, and 1,6-diaminohexane (Fig. 3). The total Ca 2+ concentration was kept constant at 5.0 raM, and the diamine concentration was varied b y the addition of the chloride salts. I c is seen that the binding of Ca 2+ decreases markedly in the presence of these diamines, with the value approaching zero at about 60-70 mM diamine. I t will also be observed that there appears to be slight differences among these compounds, the order of their ability to suppress Ca 2+ binding being 2C > 4 C > 6C. When "competition constants" are calculated from the free concentrations of the two competing species at the half point of maximal Ca 2+ binding, values are obtained which range from o.6 for diaminohexane to o. 9 for diaminoethane*. This would indicate that Ca 2+ has only a slightly greater affinity for DNA than the small, divalent organic cations. The results are of special interest because it reinforces the idea that the binding of these small cations is primarily a coulombic interaction with phosphate groups. o5 I 0.5 ~ 0,4
,
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Fig. 3. T h e b i n d i n g of Ca 2+ b y D N A in t h e p r e s e n c e of d i a m i n e s . S a l m o n s p e r m D N A , p H 6.0, [Ca 2+] 5.0 raM. Fig. 4. T h e b i n d i n g of Ca z+ b y D N A in t h e p r e s e n c e of L a 8+. S a l m o n s p e r m DIq'A, p H 4.5-
FELSENFELD AND HUANG~9have also shown that the binding of certain diamines with synthetic polynucleotides is very much like that of Mg *+, but that it is slightly weaker. Similarly, other investigators have shown that diamines such as those studied here stabilize undenatured DNA b y increasing the "melting" temperature, Tm (refs. 14-17). I t is suggested t h a t complex formation of the amines with the DNA phosphate groups is the cause of the increased stability.
Ca ~+ competition with tri- and tetravalent cations I n these experiments the binding of tri- and tetravalent cations with DNA is studied b y the determination of the binding of Ca *+ in the presence of the polyvalent cations. I n this indirect method we have considered that the negative charges on * Kdiamine/Ca =
(Diaznine-- D N A ) (Ca--DNA)
(Ca *+) ( D i a m i n e *+)
Biochim. B i o p h y s . Acta, 157 (1968) I 2 7 - 1 3 9
CAm+ BINDING TO D N A
133
DNA are neutralized stoichiometrically b y the various mixtures of Ca 2+ and polyvalent cation and that the fraction of DNA which is not bound b y Ca 2+ is bound by the other cation. It has been reported by FELSENFELD AND HUANGs3 that spermine 4+ can displace Ba m+from poly(U) stoichiometrically. In the first experiment with La 8+, the concentration of La ++ was varied (Fig. 4). It is seen that the binding of Ca m+ is decreased markedly by the addition of low concentrations of La 3+, it reaching zero when the free La 3+ concentration is 2.o raM. In a second experiment it was desired to determine the effect of Ca m+concentration on its binding to DNA in the presence of several different polyvalent cations, the inorganic La 3+ and Th 4+ and the organic spermidine 8+ and spermine 4+. The polyvalent cation concentration was maintained constant at 2.5 mM, and the Cam+ concentration was varied from 5.0 mM to ioo mM. The results are shown in Fig. 5. 05 I
I
I
!i ,
04
Ca/p
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]
I
I
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:'
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Fig. 5. The binding of Ca 2+ by DNA in the presence of trivalent and tetravalent cations. Salmon sperm DNA, p H 4.5. Fig. 6. The binding of Ca ~+ by DNA-protamine complexes at various pH's. Salmon sperm DNA, [Ca 2+] 5.0 mM.
Once more it is seen that the charge effects appear to be the decisive factor in determining the degree of interaction that occurs. The displacement of trivalent cations b y Ca2+ is about the same for either La ~+ or spermidine 3+. The concentration of Ca2+ which causes one-half displacement of the trivalent cation is about 15-2o raM. The curve for the tetravalent cations is similar to the one for the trivalent cations except that it is somewhat displaced to the right. The concentration of Ca ~+which causes one-half displacement of the tetravalent cations is about 40-60 mM. Thus, a fourth charge is apparently somewhat effective in adding to the binding strength of the cation. The data which have been presented appear to be quite consistent and in conformity with the idea that the binding of small cations by DNA is primarily an electrostatic interaction with phosphate groups. Thus, for undenatured DNA, the monovalent cation, Na +, is bound very weakly, but strongly enough so that it competes to an appreciable extent with divalent cations. The divalent cations are bound nearly stoichiometfically and with considerably more affinity than monovalent cations. At saturation each divalent cation combines with two phosphate groups in such a manner that just about all of these groups are occupied. In many, or perhaps Biochim. Biophys. Acta, 157 (1968) 127-139
134
I~. Y. CHANG, C. W. CARR
all, instances such a cation combines with one phosphate group in one strand and the nearest neighbor phosphate group in the other strand in order to account for the effectiveness of these cations in increasing the "melting" temperature of DNA. The La 3+ and spermidine are also apparently bound stoichiometrically and are bound much more strongly than divalent cations. This would indicate that there are three phosphate groups close enough together to produce a much stronger electrostatic interaction. Th 4+ and spermine are bound more tightly than the trivalent cations; however, the increase in binding affinity appears to be somewhat less than that observed between the divalent and trivalent cationic interactions. This would indicate that a fourth anionic charge of the DNA is always located at some greater distance from each cluster of three charges. Thus, these results are strongly in concurrence with the accepted double helical model for DNA in which there is no detectable breaking or branching of the structure and in which the phosphate groups are 7 3. apart in symmetrical distribution over the surface of the double helix in a nearly endless rhombic array.
B. Binding studies with DNA-protein complexes Artifical DNA-protein complexes The way in which nucleic acids and proteins interact in the nucleus has for a long time been attributed primarily to an electrostatic interaction between positively charged proteins and negatively charged nucleic acids. Many data now seem qualitatively, at least, to substantiate this idea ~,35. In view of our results in the previous section, it became of interest to us to study the interaction of DNA with proteins by means of Ca2+-binding measurements. The first experiments were carried out with DNA and protamine, and the results are shown in Fig. 6. From the pronounced decrease in Ca/P, it is easily seen that protamine masks the sites for binding of Ca 2+ with DNA. A quantitative evaluation of the decrease in Ca/P in the p H range 6- 9 shows that the Ca 2+ displaced is equivalent to the amount of positive charge added in the form of protamine when 0.05 % protamine is used se. When higher concentrations are used, there is a further decrease in Ca/P, but it is less than equivalent to the amount of protamine added. This is most likely caused b y a steric effect such that after 50-60 ~o of the DNA-phosphate groups are covered b y protamine, the remaining areas for protamine binding are too small to accommodate whole molecules. Some of the positive groups of the protamine combine with phosphate groups leaving the remainder of the protamine molecule unreacted with nucleic acid. Thus, it requires a considerable excess of protamine to reduce Ca/P to o.02-0.03. rn a similar series of experiments with the positively charged protein, calf thymus histone, a similar behavior is observed (Fig. 7). With a o.o5 % solution of protein, the decrease in Ca/P at pH 6.0 is just about equivalent to the calculated total positive charges of the protein sT. However, as the amount of protein is increased, the further decrease in Ca/P becomes less than is equivalent to the charged groups of the proteins. This effect is greater with histone than with protamine, presumably because these molecules are larger, titus resulting in a greater steric effect when many of them compete for the charged groups of DNA. When the same experiment was carried out using lysozyme in place of histone, Biochim. Biophys. Acta, i 5 7 (i968) i 2 7 - 1 3 9
C a *+ BINDING TO D N A 0.8
i
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Fig. 7. The binding of Ca 2+ by DNA-histone complexes at various pH's. Salmon sperm DNA, [Ca*+~ 5.0 mM. Fig. 8. The binding of Ca 2+ by protamine and histone complexes of DNA at constant p H and increasing Ca 2+ concentration. Salmon sperm DNA,
the results were quite similar. With o.I % lysozyme in the presence of o.i % DNA, the decrease in Ca/P in the p H range 5-6.5 was 0.22, which is equivalent to the total positive charge of the added lysozyme. Above p H 6.5 there was a further increase in Ca/P which was considered to be additional binding to the lysozyme. When serum albumin was added to DNA at p H ' s below the isoelectric point of the albumin, there was also a lowering of Ca/P. In this case, it was found to be equivalent to the net positive charge of the albumin. These experiments with DNA and various proteins were carried out at a Ca ~+ concentration which is sufficient to produce maximal binding with the free D N A phosphate groups. I t has been presumed that this binding is a quantitative measure of those groups that are not involved in the DN'A-protein bonding. This is based primarily on the fact that there is essentially a stoichiometric agreement between Ca ~+ displacement and protein cationic groups at low protein concentration. Thus, for these different proteins, they apparently bind strongly enough to prevent any significant competition with Ca 2+ at the level of 5.0 mM free Ca 2+. To determine the competitive effect of Ca ~+ on the protein binding of DNA, experiments were carried out in which p r o t a m i n e - D N A and histone-DNA mixtures were exposed to increasing Ca ~+ concentrations at p H 7.0 (Fig. 8). The amount of histone added was somewhat less than would be expected to be stoichiometfic with respect to the DNA. I t is seen that at low concentrations of Ca 2+ there is leveling off at Ca/P -----o.18, when the free Ca 2+ reaches 5.0 mM. I t is assumed that this represents the free DNA, unreacted with protein. As the Ca ~+ concentration is increased further, a second rise begins at 6-7 mM Ca 2+ and approaches a second plateau at 15 mM Ca *+ with the value of Ca/P slightly greater than 0.5. Our interpretation of this behavior is that the increased Ca 2+ concentration displaces the protein b y a competitive effect, very similar to that observed in our earlier experiments with spermidine- and spermine-DN'A complexes. For the experiment with protamine, enough protamine was added to react with all of the nucleic acid so that Ca/P remained essentially zero at 5 mM flee Ca 2+. The displacement of protamine then begins at a somewhat higher concentration of Ca *+ than for histone, which would be expected for the greater cationic charge density in protamine than in histones. Biochim. Biophys. Acta, I57 (1968) 127-139
136
K. Y. CHANG, C. W. CARR
Natural DNA-protein complexes Most of our data obtained for natural DNA-protein complexes has been obtained from chicken erythrocyte nuclei. They have been isolated in two different media, physiological saline and 0.25 M sucrose-o.oo 3 M CaC12 (ref. 26), In our first experiments with these nuclei, we studied the effect of p H on the binding of Ca 2+ at a free Ca ~+ concentration of 4.5-5,0 mM (Fig. 9)- The curve which was obtained is quite reproducible from one preparation to the next using the same method of isolation, and, in addition, it is seen that the two different methods of isolation resulted in no significant difference. I t will also be noted t h a t there is a strong similarity between this curve and the curves obtained previously for artificial complexes. In the p H range 3.0-6.5 there is a lowering in Ca/P which seems to be almost certainly due to the blocking of sites b y protein. Although the amino acid composition of the histone fraction from chicken erythrocyte nuclei has been reported aT, there is uncertainty in the amount of histone that comprises the total nuclear protein. Thus, a comparison of the positive charge of the histones with the decrease in Ca/P is not feasible. Nevertheless, we would like to propose that the value for Ca/P at p H 6.0 is a quantitative indication of the DNA that is not bound b y protein. i
--
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Fig. 9. T h e b i n d i n g of Ca 2+ b y c h i c k e n e r y t h r o c y t e (RBC) nuclei a t v a r i o u s p H ' s . [Ca*+ 1 5.0 m M . Fig. io. T h e b i n d i n g of Ca 2+ b y f i s h s p e r m nuclei a t v a r i o u s p H ' s . [Ca ~+] 5.0 raM.
I n the p H range 6.5-7.5, the further increase in binding which occurs is similar to that observed with the histone and lysozyme complexes with DNA. I t can be surmised here as it was in the previous instances that this increased binding is caused b y interactions with anionic binding sites in the nuclear protein. In another experiment with chicken erythrocyte nuclei, protamine was added to the nuclei (Fig. 9). I t will be seen that the remaining free DNA can be "covered" with protamine just as in the previous experiments with DNA-protamine complexes. Lysozyme also can block some of the free DNA from binding with Ca 2+. On the other hand, a larger protein such as serum albumin is unable to block any sites in the p H range where it is positively charged (pH 4.5). I t can be surmised t h a t the albumin molecule is too large to occupy any of the DNA sites that are accessible to protamine, especially, and to lysozyme to some extent. In the binding measurements with fish sperm nuclei (Fig. IO), the general shape of the curves is the same as for chicken erythrocyte nuclei; however, there is considerBiochim. Biophys. Acta, 157 (1968) 127-139
C a 2+ BINDING TO D N A
137
ably less uncombined DNA as indicated b y the low values for Ca/P at pH 6.o. This agrees with the data of VENDRELY, KNOBLAUCH-MAZENAND VENDRELY88 that there is equivalence between the arginine content and phosphate content of fish sperm nuclei. As with the other nuclei, there is also a rise in binding in the pH range 6.5-7.5, the magnitude of which varies for the different samples. Thus, for the two types of nuclei studied, the value for Ca/P at pH 5.5-6.5 appears to represent those phosphate groups of DNA that are not combined with strongly binding substances such as cationic proteins and polyamines. The variation in the values for Ca/P above pH 6.5 apparently reflects differences in types of proteins and other substances that are present in the nucleus'. With erythrocyte nuclei a single rise is obtained above pH 6.5 indicating that a single general type of protein is the principle non-nucleic acid component. For the fish sperm nuclei, two characteristic breaks are obtained in the curve suggesting that there are two different types of binding substances present. In a final experiment the effect of Ca ~+ concentration on the binding at constant p H was determined (Fig. II). It is seen that the data obtained with the nuclei are very similar to the data obtained with the artificial complexes, especially the DNAhistone complex. In the range of 6-15 mM Ca 2+, the protein is apparently displaced allowing the Ca/P to increase and eventually approach 0.5. ,
0.8
F
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,
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The displacement of proteins from their binding with DNA as revealed by Ca ~+ binding measurements is quite similar to our data obtained with polyamineDNA complexes. The range of free Ca ~+ concentration which produced the displacement of one-half of the histone is lO-12 mM while for spermidine 3+ and La 3+ it is 20 mM. Similarly, for protamine it is 15-2o mM and for spermine 4+ and Th*+ it is 40--60 mM. These data confirm the previous proposals for an electrostatic bonding of positively charged proteins to DNA in the nucleus. We would go further and suggest that the interaction of histones and protamines with DNA is one in which each point of attachment involves the interaction of a two or three positive charge cluster of the protein with a three negative charge cluster of the DNA. For the protamines this may be extended to four changes, but the contribution of the fourth charge appears * Some p r e l i m i n a r y e x p e r i m e n t s with nuclei from certain m a m m a l i a n cells indicate t h a t in addition to proteins, phospholipids m a y contribute to the Ca ~+ binding at the higher p H range.
Biochim. Biophys. Acta, 157 (1968) I 2 7 - I 3 9
138
K . Y . CHANG, C. W. CARR
to be relatively small. Amino acid analyses of histones indicate the occurrence of clusters of 2-3 positively charged amino acids 3", and for protamine it is quite evident that the arginine residues in groups of four3t As was pointed out earlier in this paper, the rhombic array of the phosphate groups in DNA provides a continuous succession of 7/~ triangles as clusters of three negative charge to serve as binding sites foc cations. On the basis of the data reported here, it appears to be possible to probe the nature of isolated cellular particulates with a relatively non-disruptive technique. The usefulness of this approach has also been demonstrated recently with ion-binding studies on another cellular particulate, the ribosome4°. It is anticipated that further studies with purified biological macromolecules and with artificial and natural complexes of these substances will yield further information about the nature of the forces which hold such complexes together. ACKNOWLEDGEMENT This
investigation
08412 from the National
was
supported
Institutes
by
of Health,
research
grants
Public Health
HE 01618
and
GM
Service.
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139
J- S. MITCHELL, The Cell Nucleus, B u t t e r w o r t h s , London, 196o. J. BONNER AND P. O. P. Ts'o, Nucleohistones, Holden-Day, San Francisco, Calif., 1964. K. FELIX, H. FISCHER AND A. KREKELS, Progr. Biochem. Biophys. Chem., 6 (1956) 2. J. M, NEELIN, P. X. CALLAHAN, D. C. LAMB AND I~. MURRAY, Can. J. Biochem., 42 (1964) 1743. 38 R. VENDRELY,A. KNOBLAUCH-MAZEN AND C. VENDREL¥, ill J. S. MITCHELL, The Cell Nucleus, B u t t e r w o r t h s , London, 196o, p. 2oo. 39 H. BUSCH, Histones and Other Nuclear Proteins, Academic Press, New York, 1965. 4 ° Y. S. CHOI AND C. W. CARR, J. Mol. Biol., 25 (1967) 331.
34 35 36 37
Biochim. Biophys. Acta, 157 (I968) 127-139