5602
J . Phys. Chem. 1989, 93, 5602-5611
Anion-Speclflc Salt Effects in Aqueous Agarose Systems. 2. Nuclear Spin Relaxation of Ions in Agarose Gels and Solutions Lennart Piculell* and Svante Nilsson Physical Chemistry 1 , University of Lund, Chemical Center, Box 124, S-221 00 Lund, Sweden (Received: November 2, 1988) Transverse and longitudinal relaxation rates of 35Cl-,*IBr-, I4No3-, SC14N-,23Na+,and 87Rb+in various agarose/salt systems (including systems containing mixed salts) have been measured as functions of salt concentration, agarose concentration, and temperature, in an attempt to (i) obtain molecular information on the origin of “lyotropic” anion effects on agarose gel stability and (ii) elucidate general features of ion spin relaxation in thermoreversible polysaccharide gels. In agarose gels and in dilute systems containing aggregates of ordered agarose, the transverse relaxation rate (and, in a few cases, also the longitudinal relaxation rate) of all investigated ions was found to be enhanced, relative to that of reference salt solutions. However, no such relaxation enhancement was seen in agarose solutions. In addition to this general “structure” effect, the relaxation of all anions (but not the cations) was found to contain a contribution that diminishes on addition of salt, giving direct evidence of anion binding to agarose gels. Anion competition for the binding sites was demonstrated, and the affinity of the anions for the sites, as deduced from the competition studies and independent evaluation of the binding constants (varying in the range 3-12 M-I), was found to vary roughly according to the lyotropic series. The nature of the binding sites is unclear, but from relaxation data they appear to be very few (one site per 300-10000 disaccharide units). The magnitude and the agarose concentration dependence of the “structure” contribution to the anion relaxation enhancement demonstrate that this contribution derives from ions dynamically quite perturbed relative to the bulk state, and it is proposed that the large “structure” effect seen for anions is related to the weak preferential adsorption of anions, to agarose, which was inferred previously (part
I. Introduction Salts as additives are known to modify the properties of aqueous systems containing colloidal particles or macromolecules even in cases when the latter particles or molecules carry no net charge. In two consecutive articles, this and the preceding one1 (henceforth referred to as part l ) , we report studies on one such case, viz., systems containing the linear, gel forming polysaccharide agarose. The gelation of agarose normally involves a conformational transition, induced by a lowering of the temperature, of randomly coiled agarose molecules to ordered double helices which are insoluble in water and hence form larger aggregated structures constituting the gel In part 1 we found, by optical methods, that high concentrations of salt (of the order of 1 mol/dm3) affect these transitions significantly, shifting the gel setting and gel melting temperatures of agarose as well as affecting the structure of the gel as evidenced by the sample turbidity. The direction and the magnitude of the salt-induced changes were found to depend rather sensitively on the anion of the simple salt, in accordance with the well-known lyotropic series. The experimental results of part 1 thus add to an extensive body of experimental result^,^-^ including previous studies on a g a r o ~ e , ~ . ~ documenting so-called lyotropic salt effects in aqueous environments. While part 1 thus deals with the effects of different salts as manifested on macromolecular properties (such as conformational transitions and aggregation/gelation of agarose), this paper will focus on direct experimental studies of the added salt, or the ions themselves, in the systems. Such studies are valuable since they may give direct information relevant to the understanding of the molecular mechanism behind lyotropic salt effects-an issue long debated.6+7In particular, studies of the individual ions may help (1) Piculell, L.; Nilsson, S. J. Phys. Chem., preceding paper in this issue. (2) Dea, I. C. M.; McKinnon, A. A.; Rees, D. A. J. Mol. Biol. 1972,68, 153. (3) Arnott, S.; Fulmer, A,; Scott, W. E.; Dea, I. C. M.; Moorhouse, R.; Rees, D. A. J. Mol. Biol. 1974, 90, 269. (4) Rees, D.A,; Morris, E. R.; Thom, D.; Madden, J. K.In The Polysaccharides; Aspinall, G. O., Ed.; Academic Press: New York, 1982; Vol. 1, p 195. (5) McBain, J. W. Colloid Science; Heath: Boston, 1950; Chapter 9. (6) von Hippel, P. H.; Schleich, T. In Structure and Stability of Biological Macromolecules; Timasheff, S. N., Fasman, G. D.,Eds.; Marcel Dekker: New York, 1969; p 417. (7) Collins, K. D.; Washabaugh, M. W. Q.Reu. Biophys. 1985, 18, 323. (8) Letherby, M. R.; Young, D. A. J. Chem. Soc., Faraday Trans. 1 1981, 77, 1953.
0022-3654/89/2093-5602$01.50/0
to clarify whether lyotropic effects are best understood as bulk (solvent structure) or as interfacial effects. One of the most powerful techniques for obtaining such molecular information is N M R of the ions, and especially spin relaxation rate measurements, which is the method we have used here. The spin relaxation rates of ions are known to be very sensitive to phenomena such as ion binding or weaker perturbations of ion solvation and dynamics in the vicinity of macromolecules or a t interface^.^*'^ Furthermore, by N M R one may selectively monitor each of the different (ionic) species in a complex mixture, a possibility which we will frequently make use of here. Previously, ion N M R has been utilized in the study of aniondependent (lyotropic) effects in macromolecular systems containing, inter alia, proteins,” poly(ethy1ene oxide),12 and carrag e e n a n ~ . ~Measurements ~,~~ of quadrupole splittings of ions have been utilized to extract similar information on preferential interactions of anions with surfactant aggregates in lyotropic liquid crystals.lS Limited data on cationk6and anion” relaxation in agarose gels have been presented previously, but, as far as we are aware, no systematic study of ion relaxation in any agarose/salt system has been performed prior to this one. Recent studies of ion (both cation and anion) spin relaxation in carrageenan gels and SO IS'^,'^*'^-^ have demonstrated large and interesting relaxation effects in gelling and nongelling samples containing carrageenan molecules in the ordered conformation, but the interpretation of these effects has been complicated partly because of the fact that carrageenans are charged, and thus nonspecific electrostatic interactions must influence the ionic distributions. Agarose, being uncharged but otherwise similar to the carrageenans in both (9) Lindman, B.; ForsBn, S. Chlorine, Bromine and Iodine NMR Physico-Chemical and Biological Applications; Springer-Verlag: Berlin, 1976. (10) Lindman, B. In NMR of Newly Accessible Nuclei; Laszlo, P., Ed.; Academic Press: New York, 1983; Vol. 1, Chapters 8 and 9. (1 1 ) Norne, J.-E.; Hjalmarsson, S.-G.; Lindman, B.; Zeppezauer, M. Biochemistry 1975, 14, 3401. (1 2) Florin, E. Macromolecules 1985, 18, 360. (13) Grasdalen, H.; Smidsrad, 0. Macromolecules 1981, 14, 1842. (14) Norton, I. T.; Morris, E. R.; Rees, D.A. Carbohydr. Res. 1984, 134, 89. (1 5) Rendall, K.; Tiddy, G. J.; Trevethan, M. A. J. Colloid Interface Sci. 1984, 98, 565. (16) Andrasko, J. J. Magn. Reson. 1974, 16, 502. (1 7) Morris, V. J.; Belton, P. S . J . Chem. Soc., Chem. Commun. 1980,983. (18) Grasdalen, H.; Smidsrd, 0. Macromolecules 1981, 14, 229. (19) Belton, P. S.; Morris, V. J.; Tanner, S. F. Int. J. Biol. Macromol. 1985, 7, 53. (20) Piculell, L.; Nilsson, S.; Strom, P. Carbohydr. Res., in press.
0 1989 American Chemical Society
The Journal of Physical Chemistry, Vol. 93, No. 14, 1989 5603
Salt Effects in Aqueous Agarose Systems
relaxation of the nuclei of the polysaccharide chain i t ~ e l f 2 ~(cf. 3~~ 5 1 ' eq 7).
0
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TI"C Figure 1. Normalized I4NO> rex,
-
From elementary binding theory we obtain, under the same conditions as in eq 1 ( P B
