J. Phys. Chem. B 2002, 106, 899-901
899
Relation of Hydrophobic Effect with Salt Effect: On the Viewpoint of Cluster Structure Akihiro Wakisaka*,† and Yutaka Watanabe‡ National Institute of AdVanced Industrial Science and Technology, Onogawa 16-1, Tsukuba, Ibaraki 305-8569, Japan, and Gifu Prefectural Institute of Health and EnVironmental Sciences, Naka-Fudogaoka 1-1, Kakamigahara, Gifu 504-0838, Japan ReceiVed: August 8, 2001; In Final Form: NoVember 12, 2001
We have studied the salt effect on the clustering of a nucleoside (cytidine) in water by means of an electrospray mass spectrometer, which is specially designed for the analysis of clusters isolated from solution. In comparing the formation of cytidine clusters in the presence of NaCl, KCl, or MgCl2, Mg2+ promoted the formation of cytidine clusters until octamer, whereas the effects of Na+ and K+ on the cytidine clustering was not as efficient as that of Mg2+. It was also demonstrated that the observed salt effect on the cytidine clustering is related to the structures of observed hydrated ions, that is, Mg2+ formed its hydrated clusters with less than 12 water molecules, whereas Na+ and K+ with less than 6 water molecules.
The ionic environment in water is indispensable for making the ideal structure or conformation of biological macromolecules including ionic groups, e.g., DNA, RNA and proteins; accordingly, salt effect has been studied extensively.1 As for the origin of such salt effect, the electrostatic interaction of the macromolecules including ionic groups with coexisting counterions is generally pointed out; however, it must be also pointed out that the salt effect is strongly related with the hydrophobic effect. Organic molecules in aqueous solution are easy to form selfaggregation to minimize the interface with water molecules. This is called hydrophobic effect.2 In this hydrophobic self-aggregation, the mutual separation between the organic molecules and water molecules plays important role. Because this mutual separation is originated from strong water-water interaction, the hydrophobic interaction should be markedly influenced by the presence of ions. This is because the ion-water interaction will affect the microscopic water structure. In fact, the solubility of neutral organic molecules in water is controlled by the presence of salts, namely salting-out or salting-in,3 which indicates that the hydrophobic effect is influenced by the coexisting ions. Also, on the viewpoint of the biological importance, the relation of the hydrophobic effect with the salt effect must be explained on the basis of the microscopic structure. Here, we report the experimental evidence to show how the coexisting ions affect the aggregation of a nucleoside (cytidine) in water on the basis of the molecular clustering observed through the mass spectrometry. In comparing the formation of cytidine clusters in the presence of NaCl, KCl, or MgCl2, it is suggested that the cytidine clustering is related to the structures of hydrated ion (Na+, K+, and Mg2+) clusters. These observations showed a kind of microscopic origin of the relation between the hydrophobic effect and the salt effect. To have information on the microscopic structures in solutions, we have used mass spectrometry specially designed for the measurement of clusters isolated through fragmentation of * To whom correspondence should be addressed. E-mail:
[email protected]. † National Institute of Advanced Industrial Science and Technology. ‡ Gifu Prefectural Institute of Health and Environmental Sciences.
liquid droplets.4,5 Especially, for the analysis of electrolyte solution, an electrospray interface was used to form the liquid droplets. As shown in Figure 1 schematically, the experimental setup is composed of a five-stage differentially pumped vacuum system, a homemade electrospray interface and a quadrupole mass filter (Extrel, C50). For the electrospray, electric voltages were supplied to the nozzle and three skimmers (E1-E4), and nitrogen gas was flowing to maintain an appropriate pressure balance (P1-P5) to the formation of clusters through the fragmentation of liquid droplets. The electric voltages (E1-E4) and the pressures (P1-P5) were kept as follows: (E1, E2, E3, E4: +4530, +298, +296, +240 V), (P1, P2, P3, P4, P5: 741, 10.6, 7 × 10-3, 1.7 × 10-5, 1.0 × 10-6 Torr). When an ionic solution is injected into high electric field between the nozzle and the first skimmer through a fused silica capillary tube (i.d., 0.1 mm) at a flow rate of 0.01 ml/min, positively multi-charged liquid droplets including excess cations are generated according to the polarity of the electric field through the electrospray principle. The resulting multi-charged liquid droplets fly to the second, third, and fourth chamber through the electric potential and the pressure slopes. The multicharged liquid droplets are fragmented into clusters via adiabatic expansion and electrostatic repulsion during the flight, as shown in Figure 1. The clusters including ions were analyzed by the quadrupole mass filter without using any external ionization. To see the relation of the salt effect with the hydrophobic effect, we measured mass spectra for aqueous solutions of cytidine (0.002 mol/l) in the presence of a salt (0.001 mol/l; NaCl, KCl, or MgCl2). The cytidine as shown below is a nucleoside, which has the same hydrophobic moiety with a DNA-component nucleotide. Therefore, the salt effect on the cytidine clustering will provide an insight into the biological self-aggregation, too. Figure 2a-c show mass spectra of clusters observed for aqueous cytidine solutions in the presence of NaCl, KCl, and MgCl2, respectively. The salt effect on the cytidine clustering and its relation to the hydrated-ion structure are obviously demonstrated in Figure 2. In each spectrum, the cytidine cluster (XCn: X ) Na+, K+, or Mg2+, C ) cytidine, n ) 1, 2, 3,‚‚‚) and the hydrated-ion cluster (X(H2O)m: X ) Na+, K+ or Mg2+,
10.1021/jp0131018 CCC: $22.00 © 2002 American Chemical Society Published on Web 01/10/2002
900 J. Phys. Chem. B, Vol. 106, No. 5, 2002
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Figure 1. Schematic picture of mass spectrometer specially designed for the analysis of clusters in electrolyte solutions. E and P denote electric potential and pressure, respectively. Pt represents a platinum electrode to supply the electric potential E1. RP and TMP correspond to rotary pump and turbo molecular pump for vacuum. Electrospray to form positively multi-charged liquid droplets is performed between the nozzle and the first skimmer, that is, in the electric field between E1 and E2.
m ) 0, 1, 2, 3,‚‚‚) are clearly observed. One must be careful that XCn and X(H2O)m clusters of X ) Mg2+ are observed as a half of real mass number, because the peak position is determined by (mass of cluster, M )/(charge of cluster, Z ). The mass distribution of cytidine self-aggregation clusters in the presence of NaCl (Figure 2a), Na+Cn: n ) 1, 2, 3, ‚‚‚, shows a maximum at n ) 2. The Na+Cn clusters with n g 2 decrease monotonically with increase of the number of cytidine, and Na+C5 is only a little amount of formation. In the presence of KCl (Figure 2b), the mass distribution of cytidine clusters, K+Cn, shows similar to that of the Na+Cn. The difference between Na+Cn and K+Cn is so small, but the distribution for the large size is a little bit favorable for Na+Cn. In contrast, in the presence of MgCl2 (Figure 2c), the clustering of cytidine is promoted remarkably. The mass distribution of Mg2+Cn clusters shows a maximum around n ) 3 or 4, and even n ) 7 and 8 are observed clearly. The effect of Mg2+ on the promotion of cytidine clustering is outstanding. In consequence, the effect of ions on the promotion of the cytidine clustering is in the order of Mg2+ . Na+ g K+. The structures of the hydrated-ion clusters observed in Figure 2 are closely related to the cytidine clustering described above. To show the mass distribution of the hydrated-ion clusters more clearly, 0-200 amu regions of Figure 2 are expanded in Figure 3. For the aqueous solution including NaCl, nonhydrated Na+ and hydrated Na+, Na+(H2O)m with 0 e m e 6, are observed as a series of clusters (Figure 3a). The peaks of Na+(H2O)m with m ) 5 and 6 are so small but detectable. Similarly, nonhydrated K+ and hydrated K+, K+(H2O)m with 0 e m e 6, are recognized as a series of clusters (Figure 3b). The mass distributions of the hydrated clusters for Na+ and K+ look alike, but the ratio of the nonhydrated K+ to the hydrated K+ clusters
is slightly higher than the case of Na+. Because Na+ have higher charge density than K+, the interaction with water will be stronger. In contrast with the hydration for Na+ and K+, Mg2+ forms much larger hydration structure. As shown in Figure 3c, Mg2+(H2O)m clusters with 1 e m e 12 are formed remarkably, and nonhydrated Mg2+ is not observed. The hydration number of Mg2+ greatly exceeds that of Na+ and K+. It is also a good contrast that the Mg2+C1 is strongly interacting with water molecules as observed for Mg2+C1(H2O)m with 0 e m e 6, but the Na+C1 and K+C1 are not so much. When the ions interact with the cytidine, the hydration for the ions will become weaker. The strongly hydrated Mg2+C1 observed in Figure 2c or 3c indicates that Mg2+-water interaction overwhelms Na+-water and K+-water interactions. Furthermore, the observed Mg2+(H2O)m clusters are thought to be composed of the first and the second hydration shell, because twelve water molecules are difficult to coordinate in the first hydration shell. The large intensity drop between m ) 6 and 7 might suggest the fact that six water molecules are coordinated to form stable first hydration shell and the other six water molecules are in the second hydration shell. The hydration numbers for ions have been extensively studied through the experimental and theoretical methods such as X-ray and neutron diffraction experiments, molecular dynamic and Monte Carlo simulations, etc.6 As for the Mg2+ hydration, it was reported that the first and the second hydration shell are composed of 6 and 12 water molecules, respectively.6 On the other hand, as for the Na+ and K+ hydrates, the second hydration shell was not confirmed experimentally; moreover, the hydration number corresponding to the first hydration shell was 3∼8, which was dependent on the experimental method.6 The mass spectra of hydrated ion clusters observed here give an experimental evidence to show the difference in the hydrations for Na+, K+, and Mg2+ clearly. However, it must be careful that much more water molecules are surrounding ions in solution than the observed hydration numbers. The weakly interacting molecules are vaporized during the fragmentation of liquid droplets into the clusters. Accordingly, the observed clusters do not correspond to ones existing in real solution exactly, but the observed difference reflects the ion-molecular and intermolecular interactions in solution directly.4,5 The microscopic structure in the aqueous solutions (NaCl, KCl, and MgCl2) can be discussed on the basis of the observed hydrated ion cluster structures. The largest hydration shell is constructed around Mg2+, and the second hydration shell is
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J. Phys. Chem. B, Vol. 106, No. 5, 2002 901
Figure 3. Mass spectra expanded for 0-200 amu regions of Figure 2. (a), (b), and (c) correspond to the expanded spectra of Figure 2a, b, and c, respectively. The numbers of water molecules, m, in the hydrated clusters are shown on the peaks. This clearly indicates that hydration for Mg2+ is firmer than hydrations for Na+ and K+. Figure 2. Mass spectra of clusters isolated from aqueous solutions including cytidine (0.002 mol/l) and a salt (0.001 mol/l: a, NaCl; b, KCl; and c, MgCl2). (a) In the presence of NaCl, cytidine self-aggregation clusters, Na+Cn: n ) 1,2,3,4,5, and hydrated Na+, Na+(H2O)m, are observed as main peaks shown by bold. Na+C1 is weakly hydrated to form Na+C1(H2O) and Na+C1(H2O)2. The other Na+Cn clusters were hardly hydrated. Double charged clusters will be formed through the inter-cluster interaction, e.g., Na+C2 + Na+C3 f (Na+)2C5. (b) In the presence of KCl, the resulting clusters are similar to those observed in the presence of NaCl. (c) In the presence of MgCl2, the cytidine selfaggregation, Mg2+Cn: n ) 1,2,3,‚‚‚8, hydration of Mg2+, Mg2+(H2O)m, and hydration of Mg2+C1, Mg2+C1(H2O)m, are promoted more abundantly. Mg2+Cl-Cn with n ) 1,2,3,4, Mg2+C2(H2O) and some other clusters are identified.
observed only for Mg2+. This indicates that the most ordered water structure is formed around Mg2+. On the other hand, the considerable ion-water interactions reach mainly within the first hydration shell of Na+ and K+. In consequence, the effect of ions on the promotion of the hydration structure is in the order of Mg2+ . Na+ g K+. The distribution of the hydrated ion clusters has the same tendency as that of the cytidine clusters. If the cytidine clustering is attributed to the electrostatic interaction with the ions, the observed effect of ion on the cytidine clustering will be reasonably explained by the charge density of the ions, which is the same as the observed hydrated ion clusters. This is a direct effect of ion. However, another possibility can be pointed out, that is, the hydrophobic effect may be promoted by the presence of ions with higher charge density. The hydrophobic molecules are excluded from the hydrogen-bonding network of water. The
formation of ordered hydrated ion like hydrated Mg2+ may increase such hydrophobic effect. This is a kind of indirect effect of ion. References and Notes (1) (a) Chen, S. W.; Honig, B. J. Phys. Chem. B. 1997, 101, 91139118. (b) Khan, M. O.; Mel’nikov, M. S.; Jo¨nsson, B. Mocromolecules 1999, 32, 8836-8840. (c) Begg, G. E.; Morris, M. B.; Ralston, G. B. Biochemistry 1997, 36, 6977-6985. (d) Smith, J. S.; Scholtz, J. M. Biochemistry 1998, 37, 33-40. (e) Smith, J. S.; Scholtz, J. M. Biochemistry 1996, 35, 7292-7297. (f) Jelesarov, I.; Du¨rr, E.; Thomas, R. M.; Bosshard, R. Biochemistry 1998, 37, 7539-7550. (2) Blokzijl, W.; Engberts, J. B. F. N. Angew. Chem. Int. Ed. Engl. 1993, 32, 1545-1579. (3) (a) Tu, E. B.; Ji, G.; Jiang, X. Langmuir 1997, 13, 4234-4238. (b) McDevit, W. F.; Long, F. A. J. Am. Chem. Soc. 1952, 74, 1773-1777. (4) (a) Nishi, N.; Yamamoto, K. J. Am. Chem. Soc. 1987, 109, 73527261. (b) Nishi, N.; Koga, K.; Ohshima, C.; Yamamoto, K.; Nagashima, U.; Nagami, K. J. Am. Chem. Soc. 1988, 110, 5246-5255. (5) (a) Wakisaka, A.; Yamamoto, Y. Chem. Commun. 1994, 21052106. (b) Yamamoto, Y.; Sato, Y.; Wakisaka, A. Chem. Commun. 1994, 2727-2728. (c) Wakisaka, A.; Yamamoto, Y.; Akiyama, Y.; Takeo, H.; Mizukami, F.; Sakaguchi, K.; J. Chem. Soc., Faraday Trans. 1996, 92, 3339-3346. (d) Wakisaka, A.; Akiyama, Y.; Yamamoto, Y., Engst, T.; Takeo, H.; Mizukami, F.; Sakaguchi, K.; Jones, H. J. Chem. Soc., Faraday Trans. 1996, 92, 3539-3544. (e) Wakisaka, A.; Carime, H. A.; Yamamoto, Y.; Kiyozumi, Y. J. Chem. Soc., Faraday Trans. 1998, 94, 369-374. (f) Wakisaka, A.; Kobara, H. J. Mol. Liq. 2000, 88, 121-127. (g) Wakisaka, A.; Komatsu, S.; Usui, Y. J. Mol. Liq. 2001, 90, 175-184. (6) Ohtaki, H.; Radnai, T. Chem. ReV. 1993, 93, 1157-1204.
