The role of water in biological systems

Journal of Molecular Structure, 291(1993) 425-437 0022-2860/93/$06.00 0 1993 - Elsevier Science Publishers B.V. All rights reserved

425

The role of water in biological systems’ A. Bertoluzzaa’ *, C. Fagnanoa, M.A. Morellib, A. Tintia, M.R. TosiC aDipartimento

di Biochimica “G. Moruzzi”, Sezione di Chimica e Propedeutica Biochimica. Universitci di Bologna, via Belmeloro 812, Bologna, Italy ‘Dipartimento di Chimica “G. Ciamician”, Universitd di Bologna, via Selmi 2, Bologna, Italy ‘Istituto di Citomorfologia Normale e Patologica de1 CNR, via di Barbiano I/10, Bologna, Italy; Centro di Studio Interfacoltci sulla Spettroscopia Raman, Universitd di Bologna, Bologna, Italy (Received

19 April 1993)

Abstract A general model of the liquid water structure consistent with its physical, chemical, structural, and also biological properties has not been described in the literature so far. Therefore, different structure models of liquid water have been taken into account in relation to its OH stretching Raman modes. Some factors (structure factor, H-bond cooperativity) typical of liquid and biological water have been considered. Also, the different roles played by water in biological systems are elucidated and discussed by means of a few general structure models, suitable for explaining the perturbations induced on liquid water by solutes (ionic, acids and bases, apolar) and surfaces. In particular, the possibility of strong acid-base H-bond interactions between different groups has been explored together with the biological role of water. Furthermore, the modulating effect of water on the H-bond strength and the consequent properties have been hypothesized. Finally, the behaviour of water structure in tissues and its possible correlation with normal and pathological conditions are reported.

Introduction The study of the structure and properties of liquid water has always received much interest, especially because of the discrepancy between experience and theory. The impossibility of explaining the structure and properties of liquid water by a unique general model has encouraged the enunciation of new structural models suitable for explaining some of, but not all, the properties of liquid water. The same problem exists for aqueous solutions where, in addition to the effect of the * Corresponding author. I This work is devoted to Professor Camille Sandorfy who contributed, with many meaningful studies, to the charao terization of the H-bond, and so to that of water which is one of the most important H-bonded systems.

solute on the water structure, the consequences of the direct interaction between solute and water molecules are superimposed. A particular case is the water in biological systems, where the problems of structure and properties of liquid water and solutions are present, and also new situations due to water being in contact with a solid surface. Moreover, the water in tissues is frequently subjected to different effects according to normal or pathological situations and its behaviour can sometimes be a pathological marker. In this work, all the above-mentioned problems will be evaluated by considering the structural elements of the water molecule common to some of the proposed models for liquid water, the “structure factor” of liquid water which depends on the temperature, the “cooperativity” of the

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H-bond of the biological water, the “perturbation” of water molecules due to solutes, biological molecules and surfaces, and finally the “disorganization” of water structure in tissues as a pathological consequence. In all cases we shall make reference to vibrational spectroscopic Raman and infrared measurements, the main investigation techniques used in this work. Structural models of liquid water

Several models have been proposed to explain the structure of liquid water. The main ones are the following. (i) The model of Bernal and Fowler [l] who proposed the presence of ice H-bonds to a certain extent in liquid water, although the extensive rigid three-dimensional network of the solid has disappeared. Pople [2] developed a model for liquid water in which the majority of H-bonds are regarded as distorted rather than broken, and Bernal [3] extended the distorted H-bond model. (ii) The model of Samoilov [4] who considered liquid water to be similar to ice, but with molecules in the interstitial cavities. Danford and Levy [5] and Narten et al. [6] proposed an interstitial model similar in some respects to Samoilov’s model. In addition, Pauling [7] presented a “water hydrated model” hypothesis consisting of H-bonded water molecules in pentagonal dodecahedra, bonded together to form an open structure of clathrates, where guest water molecules can be included. (iii) The model of Frank and Wen [8] in which a continuum of molecular configurations for liquid water is considered. (iv) The Nemethy and Sheraga [9] or “flickering clusters” model, in which differently structured clusters and non-structured unbonded molecules rapidly form and break.

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Also, the interpretation of the vibrational spectra of liquid water is referred to these or similar models generally subdivided into “continuum”, “interstitial” and “mixture”. In particular, the O-H stretching modes more easily detectable in the Raman spectra have been the topics of much discussion [lo-181 and have been interpreted not only by mixture [19] and continuum [19,20] models, but also by a fluctuating H-bond model [21,22], which is a hypothesis very similar to the continuum model. A new contribution to the interpretation of Raman spectra has been provided by the use of derivative spectroscopy for resolving spectral bands [23-251. One of the most important studies of the O-H stretching region in the Raman spectrum of liquid water is that of Walrafen [lo, 1l] who split the O-H stretching band into four gaussian components, assigning two bands (about 3535cm-’ and about 3622cm-‘) to the stretching modes of non-Hbonded water and two dilfercnt bands (about 3247 and about 3435cm-‘) to water lattice modes. This interpretation results from the trend of the relative intensities of the different O-H components as the temperature rises and from the detection in the spectra of an isosbestic point at about 346Ocm-‘, even though the existence of an isosbestic point has subsequently been considered as fortuitous [17]. Murphy and Bernstein [13] split the broad O-H Raman band into five components and suggested an equilibrium between tricoordinated (C,) and tetracoordinated (C,,) water species. In this framework, Scherer [17] reviewed the Raman O-H stretching bands of water and suggested that liquid water can be formed by symmetrical (with two strong H-bonds) and asymmetrical (with one strong and one weak H-bond) water species. More recently, Sastry and Singh [26] explained the five components obtained from the second derivative plots of the Raman spectrum of liquid water as being due to the presence of three types of associated water species: one involved in two moderately strong H-bonds, another involved in two weak H-bonds, and the third involved in one strong and one weak H-bond.

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Beyond the different interpretations of the O-H stretching region of liquid water, the hypothesis of the presence of water molecules interacting with one another, with different “numbers” and “strengths” of H-bonds, arises. This hypothesis is supported by considering the modifications undergone by water molecules on going from the solid to the liquid state, using a more general approach than that based on the different structure models. It is important to notice that the directions of the O-H bonds and of the electron pairs for the molecules in the liquid state are not tetrahedral as in ice. Consequently, the directions of the O-H bonds and of the H-bonds formed in liquid water can be considered as fluctuating within certain values. In fact, the relation between the 13’~~ angle of the O-H’ and O-H2 bonds and the A polarity of the respective i’ and i2 hybridized oxygen orbitals, the relation between the @I1 angle of the oxygen 1’ and 1” electron lone pairs and the ~1polarity of the respective hybridized orbitals, and also the relation between X and ~1, are shown by the following formulae [27,28]:

can be considered, and for water molecules different H-bond conditions can coexist (completely or partially bonded and also free) that are not necessarily of coincident strength. Therefore, in the liquid state the presence of water molecules interacting with each other via a different number of H-bonds with variable strengths, depending on) temperature, can be supposed. “Structure factor” of liquid water and its biological properties

The following experiment is meaningful with regard to the title of this section [29]. A sample of distilled water is boiled and then subdivided in two equal parts; one of these is left to cool slowly to, for example, 10°C; in contrast, the other one is frozen and then melted to bring the sample temperature up to 10°C. In this way the two samples are alike with respect to composition, volume, temperature and pressure. They show equal chemical, physical and structural properties; there are no measure-

(1)

1+x2cosB’~2=o 1 + /.&2 cos elJ1 = 0 2 1+X2+]=

Only in the case when X and p are equal to fi do 61,2and 13’~”assume the tetrahedral values 109”28’. As X decreases, h increases and the corresponding bond angles behave in a proportionally irreversible manner. It follows that in the liquid state it is impossible to assign such a well-defined structure to water as that of ice, with specific positions and interactions of water molecules. Moreover, unlike the solid, in the liquid only short-range structure

2

1

(2) (3)

ments that can differentiate between them. Contrary to all expectations, however, the two water samples show a different biological activity [3034], e.g. the growth and the photosynthesis of a spirogira are accelerated in the cultures containing water arising from the melting of ice. Also, protozoa reproduction is faster in cultures containing water arising from ice melting than in those containing water arising from vapour condensation.

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Moreover, these differences are notable enough for scientists [33] to have related the phenomenon to the increase in plankton biological activity following glacier melting and microorganism abundance in Arctic waters. This difference in biological activity could be explained by considering that liquid water has a “structure factor” at every temperature (and pressure), and that there is some “hysteresis” on reaching this structure factor [29]. In fact, if liquid water immediately reaches the structure factor values at every temperature, the two samples would be exactly alike. Vice versa, if some hysteresis exists in reaching the structure factor at that temperature, it can be supposed that at 10°C the water arising from the melting of ice holds an excess of molecules interacting in an “ice like” structure, unlike the water molecules of the other sample which reaches the same temperature, but from a higher one, where there is less association between water molecules. This is a very important problem and the hysteresis hypothesis could explain not only the properties of pure water but also of mineral waters which, in the bowels of the Earth, are subjected to varying temperatures and pressures, and hence to a structure factor different from those of the same mineral water when bottled. In fact, since ancient times, it has been well-known that mineral waters have different pharmacological and clinical properties if they are drunk at the source compared with away from their origin. As regards “biological water”, that is the water that constantly regenerates in living organisms, besides the above-mentioned structure factor, a new structural property must be introduced in order to explain some biological properties of these neo-formed liquids. An example is the cerebrospinal fluid that is developed daily in living organisms by a process of secretion or dialysis of the ephithelium which covers the coroid plexus. Pauling [7] attributed the effect of anaesthetic agents to the formation of hydrate microcrystals in the encephalonic fluid, in which the trapping of various protein side chains and ions increases the impedance of the network. On the basis of this kind

A. Bertoluzza et al./J.‘Mol. Strut.. 297 (1993) 425-437

Fig. 1. Cooperative H-bonding of water molecules. of conception, Pauling also predicted that merely lowering the temperature would also create narcosis, and this seems to be true. Klotz [35], as an alternative explanation of the effect of narcotic agents, proposed a change in H+ transport owing to the creation of local short circuits. In every case the effect of the general anaesthetic agents has been attributed to a structural modification of water in the encephalonic fluid. Even if no real information about the following hypothesis is available, it can be suggested that the neo-formed biological liquids, such as encephalonic fluid forming more times in 24 h, could be characterized by water molecules interacting with each other via a “cooperative H-bond” (Fig. l), which is able to transfer a chemical (e.g. H+ ion) or electrical perturbation from one end to the other by means of a mechanism proposed by Klotz [35], and more recently reviewed in a book on the proton transfer in H-bonded systems [36]. The formation of hydrate microcrystals by anaesthetics would cause the loss of the cooperative character of the H-bonds of biological water, and consequently the transfer of the chemical or electrical stimulus would be modified. The essential part of this hypothesis, which is to be verified, consists of assigning an H-bond cooperativity to biological water, which is needed for biological. and physiological functions. Perturbations of liquid water structure induced by solutes and studied by Raman spectroscopy: their applications to systems of biological interest The water in biological systems is subjected to the following different interactions. (i) Water interactions with acid-base groups bonded by means of H-bonds of different strengths. This type of interaction plays an important role in living systems. (ii) Water interactions with dissolved electro-

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lytes. A simple example is a solution of ionic salts, such as NaCl, but a more complex interaction can be formed between water and strong acids and bases, even if this case is less important at a biological level. (iii) Water-water interactions due to van der Waals associations of apolar solutes. Consequently, the water molecules are reorganized in a structure which tends to avoid interactions with solute molecules. Also, this type of association is important in the biological field. (iv) Surface-modified water, that is the water in contact with a solid surface. Synonymous of this situation are the terms “vicinal”, “surface-modified” and “interfacial” water. Water interactions with acid-base groups bonded by means of H-bon& of different strengths In biological systems, acidic and basic centres can interact not only directly by means of Hbonds (Fig. 2(a)), but also indirectly via water molecule “bridges” [35] (Fig. 2(b)) which mediate the acid-base interaction. Water molecules can realize their mediating action by means of two main orders of phenomena: the “cooperativity” and the “strength” of OH0 H-bonds. The cooperativity of water H-bonds has been previously considered; next, the case of the strength of the H-bonds of two

(a)

W

Fig. 2. Interaction of acidic and basic centres in biological systems via H-bonds “bridges”.

(a) directly and (b) by water molecule

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groups very common in biological molecules, PO; organic phosphate (hereafter simplified to PO; and -NH2 amine, will be discussed. As regards the PO; organic phosphate, Raman and infrared spectra of the 1 : 1 adducts between this group of phosphatydylcoline PC and HA acids with different strengths have shown the formation of a (P02HA) H-bond of varying strength depending on ]ApK,] = ]pKF* - PK,“>‘~) [371. The spectra of the same HA acids adsorbed in the vapour phase onto a thin layer of PC behave similarly [37]. When pK,P02H (M 1) is approximately the same acid where as pKF* (as for dichloroacetic pK, = 1.3), that is when ]ApK,] x 0, the infrared spectrum shows the formation of strong . ..H...O)H-bonds with the appearance (PO2 of two bands at about 2600 and 1950 cm-‘, classified as B and C type bands [37,38]. By the addition of acids of increasing strength, that is when pK:* < PK,P~~~, the IR spectra show a gradual decrease of the intensity of these new components and the bands are no longer detectable in the case of the hydrochloric acid adduct (pK,” = -7.4); in addition a new band at about 1020 cm-’ increases. This has been attributed to the v P-OH stretching mode of a (P02-H.. . Cl-) weak H-bond. Also, when the strength of acid HA decreases, that is when pKF* > pKaM2”, the bands at 2600 and 1950 cm-’ show an intensity decrease and in the case of methylamine hydrochloride (pKF* = 10.7) can no longer be detected. On the contrary, a band at about 1250 cm-i, attributed to a weak (PO; . . . H-A) H-bond, is present. A similar phenomenon has been observed for the -NH;! group of propylamine (pKfH+ = 10.61) in the 1 : 1 adducts with HA acids of varying strength (going from trifluoroacetic acid, pK, = -0.26, to dimethylamine hydrochloride, pK, = 10.71) [39]. From the spectra we observed: (i) an H transfer from the acid to the amino group and the formation of a weak @I+--H. . . A-) H-bond when the amino group interacts with the strongest acid; (ii) an increase in the H-bond strength as the acid strength decreases and pK, increases; (iii) the formation of a strong (N. . . He . . N)+ H-bond in

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the case of dimethylamine hydrochloride, that is when pKaW % pKfH’ or JApK,] M 0. In the biological field there are many examples of H-bonds of variable strength going from weak (where the H is asymmetrically placed on one or the other of the terminal atoms) to strong (where the H is asymmetrical or almost symmetrical) [37]. Moreover, particular types of H-bonds of biological interest are: the amidic (-CONH&H+ [40], and peptidic (-CONH-)rHf [41], (-COOHOOC-)and (-NH2HH2N-)+ hemiprotonated groups [42], and other hemiprotonated groups involving the imidazolic ring [43]. As Zundel showed [43], a strong H-bond is easily polarizable and Grotthus proton conductivity is possible [44]. It has also been supposed that protons might flow by such a mechanism through the lipophile part of a biological membrane via imidazole residues of the hystidines of the membrane proteins. In fact, the IR sprectrum of poly-L-hystidine shows a continuum absorption due to the formation of a strong easily polarizable (N . a. H . . . N)+ H-bond

shows a large continuum absorption in the 3600700 cm-’ region, centered at about 1500 cm-‘, which decreases when water molecules are added to the system, and the spectrum reverts to n-propylamine + n-propylamine - HCl, showing a progressive weakening of the H-bond. Table 1 shows the NH stretching Raman bands of n-propylamine : n-propylamine - HCl : water addition compounds, with the water molecule numbered from 0 to 10. The experimental results show, in agreement with expectations, that the H-bond strength (NHN in the examined case) is strongly affected by the presence of water molecules. Consequently, the biological functions (e.g. proton conductivity) associated with strong H-bonds are also modified. Therefore, the biological properties due to a strong H-bond between structural centres of biological molecules can be modulated by water molecules in the neighbourhood of these centres, according to the model of Fig. 2(b). In this way water can play a mediating role, retaining the strong character of the interaction and the hydrogen polarizability when the number of water molecules is low and so keeping a cooperative H-bond character. As the number of water molecules rises, the H-bond strength decreases progressively. This phenomenon is due to a wider dispersion of the interaction by a higher number of water molecules not all necessarily arranged in a cooperative way.

(431. A question now arises: what is the effect of water on the strength of the H-bonds and consequently on their polarizability and proton conductivity? Regarding this, we have studied the effect of water on the strong (N . . . H . . . N)+ H-bond in the n-propylamine : n-propylamine. HCl 1 : 1 adduct with ]ApK,] = 0 upon addition of water molecules to the system [45]. The IR spectrum of the adduct Table 1 N-H stretching modes in the Raman spectra of n-propylamine, the presence of water molecules

n-propylamine

hydrochloride

Compound

N-H stretching modes (cm-‘)

Propylamine Propylamine.HCl Propylamine : propylamine Propylamine : propylamine Propylamine : propylamine Propylamine : propylamine Propylamine : propylamine Propylamine : propylamine Propylamine : propylamine

3368 m

and their addition compound in

3313s 3050 sh

HCl HCl HCl HCl HCl HCl HCl

1:1 : water : water : water : water : water : water

1: 1: 1: 1: 1: 1:

1:1 1:2 1:3 1:4 1:5 1 : 10

3365 sh 3361 sh 3360 w 3360 w 3360w 3365 w

3267 m 3280 m 3288 m 3288 m 3297 m 3297 m 3306m

3175w 3173w 3177w

3055 sh 3060 sh (3060) (3065) (3065) (3065)

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Water interactions with dissolved electrolytes The addition of solute molecules to water alters the observed vibrational Raman spectrum, and solutes have been classified as “structure makers” or “structure breakers” [ 17,461. Examples of “structure maker” solutes are constituted by large organic ions as tetra-alkylammonium halides, while examples of “structure breaker” solutes are ions such as BFT, PF;, ClO,, I-. This approach has been criticized by a number of authors who prefer to interpret the spectral changes in terms of specific ion-water (or molecule-water) interactions. Nevertheless, it can be justified by considering that some complex cations, such as organic ions, act on the structure of water in a similar way to a temperature decrease. Vice versa, complex anions act on the water structure in a similar way to a temperature increase. Moreover, it must be observed that the concentrations of the examined solutions are generally rather high, with a low water : solute ratio [47]. Therefore, in these cases a model where solute ions or molecules interact by different bonds (H-bonds, van der Waals forces, etc.) with water molecule aggregates prevails with regard to the model of liquid water structure proposed by Frank and Wen [8] characterized by three distinct zones. Likewise, the behaviour observed by Luck [47] on the Raman spectra of hydrate water in crystalline hydrates, where band maxima lie mainly between water vapour and ice spectra, can be explained. An interesting example of solute-water interactions is found in aqueous solutions of strong inorganic acids and bases. Usually, these measurements are interpreted assuming the presence of H30+ ions in the acid solutions and of OH- ions in the basic ones. The Raman spectra show a different OH stretching trend as the acid or base concentration rises [48]. In particular, we have observed two different trends, one corresponding to acid solutions with a concentration <6M HCl and the other to acid solutions with a concentration between 6 and 12 M. In the first case, a progressive strengthening of the 3460 cm-’ band of the liquid water occurs, and

this band moves to 343Ocm-’ in the spectrum of the 6M HCl aqueous solution. At the same time a progressive decrease of the 3225 and 3635cni’ bands takes place. The appearance in the spectra of two isosbestic points at 3105 and 3350 cm-’ could indicate the presence of two main types of water at equilibrium in these conditions. Moreover, the Raman spectra of the 6 M HCl and 5.5 M NaCl aqueous solution at about the same concentration have a very similar trend consisting of the intensification of the band at 3460 cm-* (at 3430 cm-’ for HC16 M and at 3450crn-’ for NaC15.5 M) and in the weakening of the 3225 cm-’ band. This trend can be interpreted on the grounds of two phenomena. The first, of a structural type, regards the formation of cationic (H20),H+ or (HzO),Na+ and anionic (H20),$1- hydrated species, which involve a demolition of the H-bond structure of liquid water, modifying the frequency and the intensity of bending and stretching OH bands. The second, of a mechanical type, concerns a different coupling of Fermi resonance between the vl OH stretching and the 29 bending overtones derived from the modifications due to the above-mentioned interactions, either on the O-H stretching modes or on the bending modes of water molecules. The similar trend of the Raman spectra of the 6 M HCl and 5.5 M NaCl solutions suggests that the interaction of H+ and Na+ cations with water is of a comparable magnitude. The Raman spectra of more concentrated aqueous HCl solutions (from 6 to 12M) show a progressive decrease of the 343Ocm-’ band of 6M HCl and, at the same time, in the spectral region of the lower wavenumber, a broad band appears [48]. This trend can be assigned to (HzO),H + ions with a low coordination number (3.4 in 12 M HCl) characterized by very strong Hbonds. One of these bonds can be represented by the resonance between these two forms: );i-H-0,

/

c,

\

/O-H-h<

showing strong H-bond interactions between a proton of HsO+ and a water molecule, and conse-

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A. Bertoluzza

quently the facility of proton migration into the (H20),H+ ion. In addition, the spectra of NaOH aqueous solutions show a similar trend with increasing concentration: a progressive intensity decrease of the 3460 and 3225 cn-’ bands of liquid water, a progressive increase of the 3635cm-’ shoulder of the liquid water shifting at 3605cm-’ in the 13.4M NaOH aqueous solution, and the appearance of a broad continuous band increasing in size from the O-H stretching region towards lower wavenumbers [48]. Also, the trend of concentrated aqueous solutions of NaOH can be ascribed to the presence of (H20),0Hions characterized by strong (0. . . H . . . O)- hydrogen bonds of the type

H\-

,-J...H-_O

/

-

H\O- Ho.0

and with a coordination number n decreasing as the solution molarity increases. Here, the band at about 3600 cm-‘, intensifying as NaOH concentration increases, can be assigned to the OHstretching mode in (H20),0Hwhich is not involved in 0 . ..H.+.O strong H-bonding, in agreement with ab initio calculations on (H20),0Hionic structure [49]. The analogy of the Raman spectra of concentrated aqueous solutions of strong inorganic bases and acids is also confirmed by their IR spectra. In fact, Ackermann [50] has shown that the IR spectra of 10 M HCl and 15 M NaOH aqueous solutions are comparable, and characterized by a corresponding continuum absorption in which some components at about 2900, 1700 and 1200 cm-’ appear. The above-mentioned different aqueous solutions can be used as reference models to characterize the role of water at a biological level. An example is the denaturant effect of ionic solutes, e.g. alkaline halogenides, guanidinium salts, etc., on proteic aqueous solutions when solutes are added at high concentrations. A general interpretation of the denaturant effect considers the weakening of the interactions stabilizing the secondary protein structure and is generally ascribed to liquid water structure perturbation by denaturant substances.

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The Raman spectra of the concentrated NaCl solutions ilustrated above, and those of guanidine hydrochloride [51] agree, showing, as the main cause of the denaturant action, the lack of liquid water structure instead of a perturbation of this structure due to the denaturant. The formation of hydrated cations and anions with a comparatively low coordination number involves a breakdown of the liquid water structure and the absence of phenomena (H-bond interaction, cooperativity of the same bonds, etc.) which mediate between interand intramolecular interactions of biological molecules. Analogously, the conformational transition of nucleic acids, polynucleotide pairs and the sequences of nucleotides are regulated by the water included in these biological systems. In the case of poly (dG-dC).poly (dG-dC) aqueous solutions, the presence of NaCl modifies dramatically the conformation. At low salt concentrations (e.g. 1 M NaCl) the B form is present; at high concentrations (e.g. 4 M NaCl) the Z form is present [52]. We observed a similar conformational transition for the IR spectrum of a DNA film obtained from a 3M NaCl solution [53]. Therefore, it can be supposed that as the salt concentration rises in the solution, the liquid water structure is modified by the coordination both of the cation and the anion. Hence, the interactions, mediated by water molecules amongst the centres of biological molecules must also undergo a modification. The case of strong concentrated aqueous basic and acidic solutions is not usually found in biological systems because, as a rule, these are buffered at physiological pH. In spite of this, there can exist anomalous situations for which the pH changes remarkably, e.g. flogosis, and in this case the above-mentioned models can help to understand the modifications undergone by water molecules. Water-water interaction due to van der Waals associations of apolar solutes

It is known that the heat capacity of a solution increases on addition of apolar solutes and that

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this rise has been in part assigned to an organizing ability of these groups on the structure of liquid water [54]. Moreover, crystallizable hydrates - clathrates of known structure [55] are formed, e.g. C12.H20, Kr.6H20, CH4.6Hz0, CHCls - 18H20. Unlike ionic solutions, the non-dependence of the hydration energy of apolar solutes on the type of molecule shows that these hydrates do not originate from a specific interaction of the apolar molecule with water, but rather from a permanent interaction between water molecules due to the rearranging effect of the apolar molecule on the water structure. The basis of many studies on protein-water interaction [54] is the behaviour of apolar solutes on liquid water structure, as groups or side chains with apolar character can be observed in a protein molecule. Two main theories have been proposed: by Klotz [56] and by Kauzmann [57]. The first theory considers that the stabilization of proteins in aqueous solutions is due to “hydrotactoids” - crystalline hydrates - formed around the side chains of proteins; this is analogous to what happens in aqueous solutions of apolar solutes. As a consequence of this, the effect of foreign substances (e.g. urea in protein denaturation) can be understood by considering the disorganization of the “hydrotactoid” structure. The second theory, based on thermodynamics, considers the formation of hydrophobic bonds between apolar side groups of proteic molecules, since these bonds are established due to the tendency of apolar groups to avoid water. The role of water structure on the stabilization of the proteic molecule is, surely, much more complicated and important than the two proposed models which can, therefore, be considered only as parts of a more general theory. Moreover, it can be observed that the presence of apolar groups also perturbs those effects (H-bond cooperativity, mediation of the H-bond strength, etc.) that are at the basis of the role of water at a biological level.

Surface-modljied water (water in contact with a solid surface)

Water in contact with a solid surface is also generally described as “vicinal”, “surface modified” and “interfacial” water. This water has properties which can be significantly different from bulk water. These properties arise from the fact that surfaces in general, and therefore those of biological interest, are characterized by electronic surface states (described by Tamm [58] and Shockley [59] for metallic surfaces, by Levine and Mark [60] for ionic salt surfaces, etc.) which interact specifically with the surrounding adsorbate molecules to modify their structure and reactivity. An example is aluminium chloride which as a thin layer reacts with acyl halogenides and the corresponding anhydrides, forming (RCO)+ oxycarbonium ions which are not always found in the adduct compounds. This property, therefore, is assigned to particular electronic states on aluminium chloride film surfaces [61]. Another more pertinent example of this work is the “anomalous water” or “polywater” studied by Deryagin and Churaev [62], and Fedyakin [63] in the early sixties. This particular type of water forms by condensation of a liquid from an atmosphere of unsaturated water vapour in narrow silica capillaries: on cooling, the liquid separates into two phases, one removed by distillation which appears to be ordinary water, and another which shows a complex freeze-melt behaviour, as well as high density and viscosity. Controversy arose in the seventies with claims that this argument did not solve the problem. In fact, some researchers assigned the properties of polywater to a new polymorphic type of water; in contrast, others ascribed them merely to the presence of impurities. For a brief history see Franks [64]. We have also studied this topic and, on the basis of some vibrational spectroscopic measurements, believe that a strong H-bond perturbation on water molecules due to the reactive surface centres exists, and that this perturbation takes place by forming water molecule associations characterized

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by OH0 H-bonds stronger than those present in liquid water [65]. From this point of view, the anomalous water could also belong to a wider category which considers the perturbation on water molecules near solid surfaces, interfaces, membranes, gels and also on biological systems. In this last case, according to the structure of biological surfaces (hydrophilic, hydrophobic, characterized by cations and anions, and also by acidic and basic groups) perturbations on the surrounding water similar to those described in the previous paragraphs of this work can be foreseen. These perturbations originate in particular from the reactivity of the electronic states of biological surfaces and from the extension of the surface interaction through the successive layers of water molecules surrounding the surface. From this point of view, some surface situations due to crystalline defects, vacancies, interface cracks, etc., can produce on surface water perturbations more marked than those produced by the same groups isolated or in solution. Moreover, in these situations, the above illustrated structural models (acid-base concentrated salts, concentrated interactions, acidic or basic solutions, apolar van der Waals associations) find new and interesting applications. For instance, the model of concentrated aqueous solution is more comparable with water surrounding biological surfaces than the more common one of dilute aqueous solution. In the literature there are few spectroscopic studies on surface-modified water, but some meaningful works of Drost-Hansen and others on the properties of water near solid interfaces including the biological one and on the role of “vicinal” water at a biological level, are reported [66-691.

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An important point could be the cooperative effect of H-bonds in the continuous formation of water in biological fluids, which is fundamental to specific biological and physiological functions, such as proton and electric stimulus transfer. A perturbation of the H-bond cooperativity in biological water, due to apolar or ionic solutes or to surface interactions of biological molecules, involves a modification of its biological functions. An example, discussed above, is that of local anaesthetics. Another point involves conduction by H+ ions, which is an important phenomenon in the transfer of biological processes. Figure 2(b) shows an acidbase interaction in side-chains of biological molecules mediated by a water chain mechanism. The proton transfer mechanism depends, as already seen, not only on the cooperativity of the water H-bonds, but in particular on the strength of the H-bonds between the water molecules, that is on [Ap&] of the two interacting centres, and on the number of water molecules of the chain. With an analogous mechanism suggested by Klotz [35], an active site X can perform its enzymatic activity through the mediation of the chain of water molecules without. the need of contact between interacting centres (Fig. 3). As a consequence, an induced perturbation on the organization of the water chain (e.g. the effect

Some speculations on the contributions of water to the understanding of phenomena at a chemical, physiological and pathological level In order to characterize the molecular basis of some fundamental biochemical and physiological phenomena, we start from the results obtained in the previous sections.

Fig. 3. Mediation of enzymatic activity via a chain of water molecules.

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of polar or apolar solutes) causes a modification of proton conduction and, hence, of biochemical and physiological properties related to this conduction. A third important point concerns the possible formation of strong H-bonds at a biological level and the effect exerted on the H-bond strength by interaction of a limited number of water molecules. Some types of strong H-bonds in biological molecules have been previously described, and these strong H-bonds involve the greatest molecular modification of the coinvolved species. In principle, if we suppose that through this perturbation a specific biological or physiological activity can arise, this activity could be reversibly modulated by water molecules interacting with this strong H-bond. When water is present, the H-bond strength decreases and hence the biological property weakens. However, by eliminating water, the H-bond can regain its strength and consequently also regain the biological property. This model could explain biological functions due to strong H-bond formation and the modulation of water on the reversibility of these functions (an example could be the primary transmitter-receptor interaction). Finally, as regards the pathological aspect, water characterization could be a useful marker for this situation. Water, in fact, is arranged in normal tissues in a “homogeneous” way among biological molecules being subjected to: (i) cooperative H-bond interactions between acid and base centres interacting via water molecule chains; (ii) “structure maker” and “structure breaker” actions by dissolved electrolytes; (iii) structural organization induced by the associations of hydrophobic parts of biological molecules; (iv) surface perturbations. Therefore, pathological modifications of structural groups in biological molecules which have an interaction with water involve, as a consequence, modifications of water homogeneity. An example is the cataract where the interaction among amino acid residues of protein molecules

produces “fluorphors”; these are molecular aggregates giving rise to lens opacity. This phenomenon happens with a loss of water homogeneity as shown by Raman spectra [70]. Another, more general, example concerns the changes in water proton relaxation times in pathological tissues. NMR data for tumours show greater values of magnetic resonance longitudinal relaxation time Ti for water protons compared with normal tissues [71]. The increasing Ti for water in tumours has been associated with a decrease in the content of bonded water, compared with normal tissue. A possible explanation could be the break down in tumours of structural biological groups which homogeneously interact with water, leading to greater mobility of water which rearranges dishomogeneously in tissues according to the above-mentioned general hypothesis. Once again, it is shown that water is not an “inactive witness” in the behaviour of biological molecules, but an important component that acts in different ways, one of which is to modulate the strength and the cooperativity of the H-bond. References

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