Hans Gustavsson

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

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; therefore 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 concept 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 monovalent 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 polyelectrolyte remains constant over a broad concentration 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 35C1as 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

23Na+-NMR studies of cation binding to multi-chain and single-chain glycosaminoglycan peptides

Abstract 1. 1. 23 Na + nuclear magnetic relaxation studies of cation binding to single-chain chondroitin 4-sulphate and dermatan sulphate and a multi-chain chondroitin 4-sulphate-polypeptide complex have been performed. Excess relaxation rates (longitudinal and transverse) for the bound sodium have been measured during titration experiments. The data were used to calculate the correlation time and the fraction of bound ions. This approach was also used to analyze the relative affinities of Na + , K + and Ca 2+ . 2. 2. The excess transverse relaxation rates for 23 Na + (obtained from line-width measurements or the decay of transverse magnetization) in solutions of single-chain and multi-chain chondroitin sulphate were recorded as a function of the degree of neutralization. There was a steep increase in transverse relaxation rate for the multi-chain preparation around pH 7 which was not seen with single chains. This could be ascribed to an increase in the correlation time for bound sodium. The alteration in binding properties observed with multi-chained material around pH 7 suggested a change in the quadropolar coupling constant for 23 Na in this range. The behaviour of a multi-chained specimen (as expressed in excess line-width) during titration gradually changed to that of single chains upon prolonged exposure to alkali. 3. 3. Competition experiments using KCl additions to multi-chained sodium chondroitin sulphate at pH 12.3 and 3.3 indicated that Na + and K + have very similar affinities at high pH. However, at low pH there was an increased affinity for K + (expressed as a faster decrease in excess relaxation rate when KCl was added). 4. 4. Competition experiments using multi-chain chondroitin sulphate and Ca 2+ at pH 11.4 showed increasing excess relaxation rates during the initial phase. Above 4 mM Ca 2+ simple exchange of bound Na + with Ca 2+ was observed. The former behaviour was not seen at pH 3.3 nor with single-chain chondroitin sul[hate at pH 11.4. It is suggested that endogeneous Ca 2+ present in the multi-chain preparation is responsible for the pH dependence. The increased excess transverse relaxation rate seen with the multi-chained specimen upon Ca 2+ additions was referred to an increase in the correlation time for bound Na + . 5. 5. The most likely explanation for the abrupt increase in correlation time for Na + bound to multi-chain chondroitin sulphate around pH 7 is an aggregation between the side-chains. In the presence of Ca 2+ , cross-linking of chains (clustered together on the protein core) was promoted and the local mobility of the Na + ions was further reduced. In addition to the formation of Ca 2+ bridges between chains, the results also imply increased binding of counter ions owing to a higher local charge density.

23Na+NMR in solutions of mucopolysaccharides

Polyelectrolytes have a wide occurrence in biological systems and their function is greatly influenced by abundant small ions, for example, Na+, K’, Mg2* and Ca2+. The quadrupole relaxation method for studying ion binding to macromolecules, the principles of which are in [l] , should constitute a very general experimental approach to the problem of ion binding in biological systems. While the method has won wide-spread use in the field of protein chemistry [l-3] , ion binding to biological polyanions like nucleic acids [4,5], humic acids [6-91 and others [lo] has not been penetrated to the same depth. One reason for this is that in the case of proteins, it is much easier to isolate relaxation effects (e.g., using competition experiments) directly related to the biological function while this is not so for the polyanions. To be of any significant value, quadrupole relaxation studies of ion binding to polyelectrolytes must include studies of both longitudinal (T,) and transverse (TZ) relaxation times as a function of the degree of ionization. Even so the analysis is not without difficulties because several factors may influence the measured relaxation rates. However, a recent analysis [ 1 l] of rather extensive 23Na* relaxation data for a synthetic polyanion, polymethacrylic acid (PMA), gave consistent results and provides a suitable basis for the discussion of polyelectrolyte systems in general. Mucopolysaccharides, occurring in the extracellular matrix of connective tissues, form a group of biological polyanions for which the interactions with small cations is of great significance [12,13] ; e.g., there is certainly a critical interplay between ion binding phenomena and functionally important con-

113Cd NMR study of cadmium ion interaction with polyelectrolytes

113Cd NMR is examined as a tool for the study of ion-polyelectrolyte interactions. Binding of Cd2+ to polystyrenesulfonate gives chemical shift changes of up to 35 ppm, thus demonstrating that the method is very sensitive. Information on the Cd2+ exchange rate, on the fraction of bound ions, and on the competition of the ion with Ca2+ and Mg2+ (partly from 25Mg NMR) is obtained. The applicability of the method is compared with that of the quadrupole relaxation method. Both electrostatic theories and site-binding models have been used to describe the interaction between Cd2+ and polyanion. Only the site-binding model agrees with the experimental results.

NUCLEAR MAGNETIC RESONANCE STUDIES ON HYDRATION AND INTERACTIONS OF COUNTERIONS IN SOME COLLOID SYSTEMS

Nuclear magnetic resonance, NMR, of alkali and halide ions has been shown to be sensitive to interactions and microdynamics of surfactant systems. In this work the shielding of 23 Na + and 133 Cs + nuclei has been studied, as well as the quadrupole relaxation of 23 Na + and 37 Cl − . By the introduction of Fourier transform techniques much lower concentrations could be studied than previously feasible; for example with 23 Na + , measurements were made down to 0.5 · 10 −3 m. This makes many more problems accessible to study. Systematic studies of especially the sodium ion binding was performed as a function of surfactant concentration, alkyl chain length and polar end-group. The mechanism of counterion binding was inferred to change markedly with polar end-group, while alterations in the other factors have smaller effects on the ionic interactions. The water isotope effect in shielding was found to provide interesting novel information on counterion hydration. As a complement to the study of surfactant aggregates, the 23 Na + and 37 CI − relaxation and shielding were also studied in the presence of different polyanions, polyampholytes and polycations at different acid dissociation degrees. An anomalous variation of counterion binding with the polyion acid dissociation degree is observed for certain systems.

Molecular and Ionic Behaviour at Water-Amphiphile Interfaces

In systems containing aggregates of amphiphilic compounds there is a spatial separation between the hydrocarbon chains and the water molecules. The decisive feature is the interface betwen the two distinctly different regions and to examine, for example, the energetic and kinetic conditions for the formation of various amphiphilic aggregates a prerequisite is information on molecular interactions and dynamic events in the interface region. While the hydrophobic interaction, although not understood in detail, can be accounted for rather well semiempirically, our understanding of the interactions in the polar part is deficient. A factor of almost 100 higher c.m.c. of ionic than of non-ionic surfactants may be given as a single example of the importance of the interactions in the polar part of the amphiphile.

Counterion NMR in Some Polyelectrolyte Systems

NMR quadrupole relaxation rates of 23Na+ were used to study counterionpolyion interactions for both synthetic and biological macromolecules. The non-exponential relaxation and the non-equality of T1 and T2 permitted an analysis in terms of correlation times, quadrupole coupling constants and amount of counterion binding.

Counter-ion nuclear magnetic resonance chemical shifts in micellar solutions

It is demonstrated that the binding of alkali and halide ions to micellar aggregates may be followed by studying n.m.r. chemical shifts of alkali and halide nuclei.