Drakenberg, Torbjörn

Gustavsson, Hans

Lindman, Björn

Parello, Joseph

Reimarsson, Pétur

Elsevier - Publisher Connector 

Counterion NMR in polyelectrolyte solutions 25Mg2+ and 43Ca2+ interaction with DNA

Volume 108, number 2 FEBS LETTERS December 1979 
COUNTERION NMR IN POLYELECTROLYTE SOLUTIONS 
25Mg2+ and 43Ca2+ interaction with DNA 
PCtur REIMARSSON, JosephPARELLO+, Torbjam DRAKENBERG, Hans GUSTAVSSON and Bjijrn LINDMAN 
Physical Chemistry I and 2, Chemical Center, POB 740, S-220 07 Lund, Sweden and +Equipe de Recherche de Biophysique 
no. 140, CNRS USTL, 340 60Montpellier, France 
Received 25 October 1979 
1. Introduction 
The importance of electrostatic effects on the 
physicochemical and biological properties of nucleic 
acids has long been recognised. It has been found that 
ion binding to nucleic acids cannot be described in a 
simple way in terms of equilibrium constants; there- 
fore there has been a rapidly increasing interest in 
polyelectrolyte systems, concerning mainly the 
development of models amenable to theoretical 
analysis and the implications of polyelectrolyte 
phenomena in biological situations [l-S]. The con- 
cept of counterion condensation advocated by 
Manning (reviewed [ 11) has been found to give a 
good first-order description of the interaction of ions 
with polyelectrolytes. According to this model, the 
condensation occurs above a certain critical linear 
charge density (1 elementary charge/7.1 i% for mono- 
valent counterions and 1 charge/l4.2 8, for divalent 
counterions), and in a solution containing only one 
type of counterion, the charge fraction of the poly- 
electrolyte remains constant over a broad concentra- 
tion range. 
For the study of counterion-polyion interactions 
at a molecular level, NMR should be a most useful 
method, and it has been shown, using 2 3 Na+ and 
35C1- as examples, that quadrupolar effects in NMR 
give significant information on the mode and 
dynamics of ion binding [6-91. In particular the 
motional characteristics of Na’ bound to DNA and 
tRNA have been elucidated [9]. From a biological 
point of view, the study of Mg” and Ca2+ is especially 
intriguing. However, as discussed in [lo], available 
EIsevier/North-HoIland Biomedical Press 
isotopes have unfavourable NMR characteristics and 
Mg and Ca are therefore among the least studied ele- 
ments. The use of isotopeenriched 2sMg and 43Ca 
considerably improves the situation; it was found 
that the interactions of Mg2+ [ 1 l] and Ca2* [ 121 
with a muscular protein, parvalbumin, produce 
marked effects in 2sMg and 43Ca quadrupole relaxa- 
tion. However, one problem was encountered, that 
is, for high affinity binding to proteins, chemical 
exchange is often too slow to allow NMR studies. 
Also 2sMg2+ NMR has been used to study binding 
properties of bovine serum albumin and nitrogenase 
Fe protein [ 133. The quadrupole relaxation for 2sMg2+ 
has been shown sufficiently effective to give large line 
broadening effects also when the metal is bound to 
smaller ligands, such as small peptides, whereas for 
43Ca2+ the quadrupole relaxation in not so effective 
and in this case chemical shifts due to complexation 
could be observed [14,15]. 
It was anticipated in [ 121 that the Ca2+ and Mg” 
ion exchange should be faster when bound to biolog- 
ical polyelectrolytes than to specific sites in proteins, 
and as shown here, with DNA as an example, the 
metal ion binding may indeed be profitably explored. 
2. Experimental 
Calf thymus DNA of low molecular weight 
(“3.5 X 1 OS ; Szo = 7.7) was prepared as in [ 161. 
The DNA-phosphate concentration was determined 
by the A260 assuming that 1 .O A,,, is equivalent to 
1.5 X lo4 M DNA-P. 43Ca and 2sMg were purchased 
439 
Volume 108, number 2 FEBS LETTERS December 1979 
from Oak Ridge, TN, as 43CaC0i (61% enrichment) 
and “MgO (98% enrichment), respectively. The 
2sMg and 43Ca linewidth studies were made at 
6.095 MHz and 6.73 MHz, respectively, on a modi- 
fied Varian XL-1 00-l 5 spectrometer operating in the 
FT mode. External proton lock was employed, the 
acquisition times were 0.1 s for 2sMg and 0.2 s for43Ca. 
The number of transients varied from 500-10 000, 
depending on linewidth and solution. Each reported 
linewidth is the average of at least 2 different measure- 
ments and the resulting errors are < 5%. Temperature 
was maintained accurate to O.S”C by a stream of dry 
thermostatted gas. 
3. Results and discussion 
In fig.l,2 we report the temperature dependence 
of 2sMg2+ and 43Ca2+ relaxation, respectively, in the 
presence of DNA. (In the absence of DNA, relaxation 
is much slower and decreases monotonically with 
increasing temperature, cf. [ 111.) The experimental 
30 
20 
2 
L 
10 
0 
6 
5 
2.4 
23 
2 
pIi= 
pH=71 
1 i:rrl 
0 2 4 6 6 
[DNA-P], m 
43Ca2’ -DNA II/___ 
- 
1 
40 60 60 
t ,“C 
Fig.1. Study of the Ca2’-DNA system by 43Ca NMR. The 
dependence of the linewidth on temperature for a solution 
containing 7 .l mM DNA-P, 3.5 mM Ca*‘, 57 mM Na’ in 
13 mM Tris-HCl, at pH 5.2. Insert: Effect of DNA concen- 
tration on the 43Ca linewidth. Successive volumes of a stock 
DNA solution (4.25 X 10m2 M DNA-P in 75 mM Tris-HCl. 
at pH 7 .l) added to a 1.5 3 ml solution containing 40 mM 
Ca2’, 65 mM NaC in 15 mM Tris-HCl, at pH 7.1. Tempera- 
ture 27°C. Total volume variation 13%. 
104 
0 20 40 
t ,“C 
60 80 
Fig.2. Dependence of the 25Mg linewidth on temperature for 
a solution containing 7.1 mM DNA-P, 88 mM Mg”, 21 mM 
Na’ in 50 mM Tris-HCl, at pH 7.4. 
results are given as linewidths which are related to the 
transverse relaxation time, T2, as T2-’ = rrAvl . The 
important features of fig.1 are the marked in&ease in 
relaxation rate with increasing temperature, and the 
low relaxation rate above the DNA melting point: 
this is considered to correspond to a transition from 
a double-stranded helical DNA to a single-stranded 
random coil state. For 2sMg2+ (fig.2) there is likewise 
a marked decrease in relaxation at DNA melting while 
below this temperature there is a slow variation with 
a wide maximum. A similar curve is obtained for 
transfer RNA [ 171. Parallel optical studies of the 
denaturation of double-stranded DNA in the presence 
of Ca2+ and Mg2+ show that the melting transition 
occurs at 80-85°C under conditions similar to those 
used for the NMR experiments. 
The principles of ion quadrupole relaxation studies 
of macromolecular systems have been reviewed [ 18,191. 
(In [ 181 a detailed account of the theory is given.) 
For both 2sMg and 43Ca, quadrupole interactions 
give the dominating relaxation mechanism. The 
observed relaxation can be written as a sum of con- 
tributions from different sites. That is: 
~Av+ = T2-’ = Ep/(T,i + vex ,i) 
Here pi is the fraction of ions in site i, rex i is the 
life-time of the ion in site i and T2i is the intrinsic 
440 
Volume 108, number 2 FEBS LETTERS December 1979 
relaxation time of site i. T-j: = Ki2~, where K = 0.947 
for 2sMg (spin 5/2) and 0.403 for 43Ca (spin 7/2), 
x is the quadrupole coupling constant describing the 
strength of the interaction between the ion and the 
site and 7c the correlation time. Here we have assumed 
that the extreme-narrowing condition applies, i.e., 
w~~ << 1 (o is the resonance Larmor frequency in 
rad/s); this is not established for these ions but does 
not affect the general conclusions of this work. 
Current polyelectrolyte theories [l-.5] relate 
counterion binding to the charge density of the 
cylinder or to a linear charge density; the Manning 
ion condensation model [l] is in qualitative agree- 
ment with solutions of the Poisson-Boltzmann equa- 
tion for different geometries [20]. The linear charge 
density is extensively decreased at the melting of 
DNA, and the dramatic counterion relaxation changes 
at high temperatures are in qualitative agreement with 
the transition from a double- to a single-stranded DNA. 
According to the Manning ion condensation model 
[ 11, considering only the linear charge density, the 
Mg2+ and Ca2+ binding should change from -88% 
polyion charge neutralization to a much lower level 
(-50%). From the temperature dependence of counter- 
ion relaxation it can be deduced, at least for Ca2+, 
that 7ex i dominates over Tzi [ 181. With the aid of the 
Manning’ condensation model (88% charge neutralisa- 
tion) and the measured line broadenings, the average 
lifetime of Mg2+ bound to DNA was estimated to be 
-0.4 ms at 4°C while Ca2+ has an average lifetime of 
-9 ms at 25°C and -3 ms at 50°C. It is clear that 
analysis of the data of fig.l,2 in terms of activation 
parameters must be preceded by rather detailed stu- 
dies of the binding equilibria. 
If the ion condensation model were applicable 
under the conditions of this work, one would expect 
that the difference between the observed linewidth, 
and that of a solution without DNA, should be pro- 
portional to the DNA concentration at a constant ion 
concentration. As shown in fig.3, there is a marked non- 
linearity in the plot of linewidth of 25Mg versus DNA 
concentration which is in contrast to many other 
macromolecular systems [ 181. It has also been 
observed that the excess linewidth of 23Na+ in transfer 
RNA solutions is not linear with respect to tRNA 
concentration [ 171. However, it is generally not 
expected that the charge fraction of a polyelectrolyte is 
independent of concentration. 
25Mg2*- DNA 
lo- 
; 
Z 
Z 5- 
0 0.5 
[DNA-:, mM 
1.5 2.0 
Fig.3. Dependence of the “Mg excess linewidth on DNA 
concentration at 28°C. Successive volumes of a stock DNA 
solution (1.54 X lo-’ M in 75 mM Tris-HCl, at 7.1) added 
to a 1 SO ml solution containing 96 mM Mg”+, 17 mM Na’ in 
15 mM Tris-HCl,at pH 7.1. Total volume variation 13%. 
This report clearly demonstrates the feasibility of 
NMR studies of Ca2+ and Mg2’ interaction with DNA 
and also shows that direct access to both kinetics and 
thermodynamics of ion binding can be obtained. The 
kinetic information is particularly difficult to obtain 
by other techniques, and the marked difference in 
exchange kinetics between Mg” and Ca2+, which 
probably reflects a difference in hydration state of the 
bound ions, will be examined in detail elsewhere. 
Acknowledgements 
This work is contribution no. 14 from Equipe de 
Recherche de Biophysique and was partly supported 
by the DClegation B la Recherche Scientifique et 
Technique (MCcanismes de reconnaissance B 1’6chelle 
molCculaire). The Swedish Natural Science Research 
Council provided project and travel grants to Bjijrn 
Lindman. We are grateful to Professor Sture ForsBn 
for valuable discussions. 
References 
[1] Manning,G.S.(l978)Quart.Rev.Biophys. 11,179-246. 
[2] Manning,G. S. (1977) Biophys. Chem. 7,141-145. 
[3] Eisenberg, H. (1977) Biophys. Chem. 7,3-14. 
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Volume 108, number 2 FEBS LETTERS December 1979 
141 Eisenberg, H. (1976) Biological Macromolecules and 
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[S] Oosawa, F. (1971) Pol~elec~oIytes, Dekker, New York 
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[7] Gustavsson, H., Lindman, B. and Bull, T. E. (1978) 
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[P] Burton, D., Forsin, S. and Reimarsson, P. (1980) sub- 
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[lo] Lindman, B. and For&, S. (1979) in: NMR and the 
Periodic Table (Harris, R. and Mann, B. eds) ch. 7, 
Academic Press, New York. 
[ 1 I ] Cave’, A., Parello, Drakenberg, T., Thuhn, E. and 
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[12] Parello, J., Lilja, H., Cave, A. and Lindman, B. (1978) 
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[16] Gour~vitcll,M.,Pui~donlenech,P.,Cav~,A., Etienne,G., 
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[18] Lindman,B.andFors&n,S.(1976)in: Chlorine, 
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[JP] Fors~n,S.andL~dman,B.(l97S)Chem.Brit.l4,29-35, 
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442 


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Counterion NMR in polyelectrolyte solutions 25Mg2+ and 43Ca2+ interaction with DNA

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