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    Cap11 Daune

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    João Victor

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    of 14
    Part Ill Hydration of biopolymers Introduction Water is major constituent in all known forms of lf, It forms $5 per cent by weight of an adult human body. although, as indicated in the table below. i is unevenly distributed. The water content ofthe body is kept constant by an average daily intake of 2.5 litres for an adult as par of solid or liquid food. With increasing ‘age, the proportion decreases: from $5 per cent at age 40 10 47 percent at 70. Fronortons f water invarous 8eues ane tse his ot muscle 79 roin hea 6-81 epidermis fer a Pacenia ws 8 ‘ood pasa oO 9 pour uid an n-0 corbrospinalii 0 56 salva ‘Until the advent of NMR, there was no way ofearrying out an i viv study ofthe water present in tissues. Even with NMR, the water detected is essentially ‘Tree’ \water, in which the movements ofthe water moleculesare sufficiently rapid to allow proton resonance lines (o be observed. The broadening of these lines when pro- uced by strongly bound water makes it undetectable Inimaging by NMR, parameters such as nuclear spin density ot relaxation time T, are measured. As with most such techniques involving instrumentation, the imageis not obtained directly but only after processing the collected data. Here, the production ofa two-orthree-dimensional image requiresa magnetic field gradient to be superimposed on the steady magnetic induction By, As result of tis, each point in the sample has a different precession frequency from its neighbours and spatial displacements are then translated into changes of resonant frequency. By ‘combining pulse sequences of both the radiofrequency fold and the gradient field, and by using three-dimensional Fourier analysis, the nuclear spin density can be reconstructed over a plane or ina volume clement ofthe sample, Images of the ree water in given tssueenable normal and pathological samples tobe compared. Moreover, with the computational speeds now available, it seven possible to monitor the movement of fluids such as the blood flow in the heart in realtime, MOLECULAR BIOPHYSICS [As regards the bound water, there are few experimental methods at present available enabling us o specify its role in biological processes and to propose any ‘models for phenomena such as fTeetor—receptor binding, the integrity of mem- brane structures, the Folding of macromolecular chains, charge transfer (proton ot cleetron) between donor-acceptor pairs, ete. Difficulties arise simply because no ‘comparison can be mace with the sume process in the absence of water: we cannot, for example, examine the spatial folding ofa completely dehydrated peptide chain, In dealing with hydration inthis Part, therefore, we can only offer a saies of experimental and often complementary approaches, coupled with static and «dynamic models. This willenable uso gaint Teast some insightinto the role played Dy water in the structure and functioning of biological systems, The macromolecules occurring in the cellular medium of in Various body fluids (blood. lymph, cerebrospinal fluid, et.) must be solublein water. Ths implies that there are many mechanisms governing interactions between water molecules and the atoms located at the solvent—macromolecule interface. The term fidration coversall such phenomena, which are not only involved in solubility but also pla role in the morphogenesis, the stability andthe dynamics of biopolymers. When hydration is defined inthis way, it corresponds to non-covalent associa tions of water molecules, Moreover, such molecules are not site-bound but are continually changing places with external water molecules, The residence time, i. the average time spent by a water molecule in a hydration site, might atleast ‘qualitatively give us some idea ofthe hydration energy. The residence time isnever Jonger than a nanosevond, It should now beclear that hydration as defined above differs considerably from chemical hydration, which isthe covalent addition ofa water molecule to another molecule, This process generally involves both H* and OH ions and leads to ‘another compound with different chemical properties (the hydration of ethylene, CH=CH. for example, gives ethanol, CHyCH:OH). 11 Properties of water Itisparadoxical that we should sill know so little about a liquid that is universally indaily useand of such importance biologically. Thischapter discusses what wedo ‘know of the physical properties of water, using the classical distinction between its three phases (vapour, liquid and solid) with well-defined transition temperatures when a change of phase occurs, 11.1. The water molecule Weshould expect an sp bond between thes orbital of Hand the two 2porbitalsofO in 0. The angle HOH should be 90° but is actually 104.5°. Hybridization oveurs between the 2s and 2p orbitals of O to form sp° bonds iu the molecule, Two O orbitals remain available, corresponding to the lone-pair electrons and thus bringing about the almost tetrahedral symmetry ofthe water molecule (Fig. 11.1) with the following parameters: OH distance = 0.957 A, HOH angle = 104.5°, angle between free orbitals of O~ 109 There are two important consequences of the geometrical and electronic prop- extos of H30. |The charge distribution can be represented diagrammatically by two positive point charges atthe positions of the H atoms and two negative point charges ina plane perpendicular to that of the molecule. Water therefore has & high dipole moment p= 1,834D (debys) = 6.11 x 10-Cm, and a quadrupole moment of about 1.87x10-"Cm*, The mean polarizability is 1.444 x 10° ms the distribution of polarizability is not known but is very neatly isotropic (see Box I1.1) ‘The presence of two free orbitals on the O atom and the polarity of the OH bonds account for the ease with which the tetrahedral coordination of HO ‘occurs, especially with other water molecules, The energy of the association ‘of water molecules into dimers, trimers, etc., can be calculated, and out ofall the possible associations, che trimer Geeta 1 OH OHH is the most stable. Next come the tetramer with Sq symmetry, the asymmetric ppentamer and the hexamer with S, symmetry. MOLECULAR BIOPHYSICS Figure 114 Synmaty seers ant geometry the water melecule Box 11.1 Charge distribution in H,0 ‘The water molecule can be represented diagrammatically by 49 +9 4 ‘The distance d between the centroids of the positive and negative charges is aiven by 0.957 e0s(52.25*) =0.586A 0 that to obtain a dipole moment of 6.11 x 10-'Cm the charge 2y must be equal to 1.043. 101°C, ie, 0665, where ¢ is the elementary charge. The charge q is thus 0.325. The three vibrational normal modes of H:O, D:O and HOD are shown in Fig. 11.2: the fundamental and harmonic frequencies of these yield the infrared spectrum ofadilute water vapour. When se compare the Frequencies for H:0, D0 and HOD, we see that the HOD molecule appears to have almost uncoupled OH and OD stretching bands, since their frequencies are very close to the corres: ponding onesin HO and D.0. Asa result, HODis often used as an infrared probe of the structure of water in its different states 11.2 Kee 11.21 Structure ‘Water does not have one unique crystalline soli state, but a large number existing ‘vera wide range of temperatures and pressures. In tis chapter, we shall only look atice Land ice I, since the conditions under which they form and are stable are the closestto those ofiquid water. Ice exists at °C undernormal pressures Iteonssts, Ho D,0 | HOD To 2 symmoticar | 9656.65 | 2671.48 | 2726.78 HN: Biting As nC ye Bending | ° SSH Asymmetrical \, Biting 1504.59 | 1178.98 | 1402.20 9785.78 2788.05 | 3707.47 Figure 1.2 vorsional guns states of D-0ana HOD winner Vibrational states Wavenumber (om) Figure. SrucurecticeThe oxygen ‘sore ate in svecessne layers iormed by ‘networkotohar-snaped hexagons Enon ayer the mor erageoltha adjacent one. Each oxygen atom is Surrounded by ourhydogen atoms: the twobebongng othe molecule ae 101A frm tneonygen atom he ote two belonging‘ we neighbouring ‘molecule are a1 75 Each oxy900| Stem at he cane oa eaneern which means hat a eurangleshave he sama ae tcos"(9) = 109" 47 of chair shaped hexagonal layers (Fig. 11.3) having opposite puckering in wo superimposed rings. Each oxygen atom liesat the centre ofa tetrahedron formed by four other oxygen atoms separated by 2.76 A. The hydrogen bonds between OH and O stretch the OH distance slightly o 1,01 A. Le caxis FX 0.242 mm 112 Basal plane caxis 363M Figure 11. Schematcclagram ofice cyeialstormad in antowze peti solitons wth incrasng concerstins. {Fromyang ea, 1968. Reprted ty petmiasion fom Nate, ol 333 252-7 and wh the authors pernision © 1988 Warman Magazines Lis) MOLECULAR BIOPHYSICS The most important structural feature isthe presence of “cavities” Formed by six water molecules at a distance of 294A from the centre of the cavity. The space- filling factor is therefore very small (0.34) and the resultant density of ie (0.924) ‘means that water is anomalous in tat is liquid phase has higher density than the solid. The ability of water to form what may be called ‘cages’ in which foreign molecules can be accommodated will be looked at again later. 11.2.2. Antifreeze peptides Fish living in cold sea-water with a freezing point elose to ~2°C must avoid the freezing of their serum, whose saline concentration is much lower than that of the sea. Their problem is solved by the presence of antifeze polypeptides or lycoproteins, the most common of which isa polymer ofa slycotripeptide Ala- AlacThr with a threonine-bound disaccharide (3-galactosyl{1—3)-a-N-acetyl- galactosamine). I is interesting to note that the depression of freezing point induced by such proteins doesnot depend on the number of molecules, asitdoes inthe classical laws «of eryoscapy: antifreeze polypeptides appear tobe only asefficient as common salt their concentration is expressed in g1~' but are 300500 times more efficient if itisexpressedin moll Another noteworthy pointsthat the melting point of the glycoprotein solutions ‘sclose 100°C. In far, hysteresisis occurring... a delay in the freezing of aqueous solutions containing the polymers Oneof these polypeptides (M ~ 1650)hasbeen crystallized and studied by X-ray dilfractionat a resolution of2.5 A, thus providing new data. Ibwas already known, from measurements of eicular dichroism, thatthe polypeptides form a-helices. ‘Thecrystalhas been shown toconsist ofa unique type of packing of thehelices, each containing four repeated sequences ofthe motif TAAXAAAAAAA (where X isa polar amino acd), ic. a total of 36 Ala residues, The antifreze properties ofthe polypeptide are related to the large dipole moment of the a-helx directed along its axis (se section 15.1. In ie I, the resultant dipole moment ofall the water mol- cculesisprecisely oriented with respect to theerystallographic axes: 508° from thec axis and 60° from the a axis (Fig. 14). The antiparallel alignment of the dipole ‘moment ofthe a-helix (and hence of its axis) and of a crystal of ice 1 enables the peptide to latch on to the surface of the ice erysta Ina dilute solution ofthe polypeptide, the a-helices ie parallel to each other on the prismatic aces ofthe seeds ofice and slow down the rystal growth in the plane ofits basal surface, but allow it to grow ina direction perpendicular to the ¢ axis, For high enough concentrations of the polypeptide, all the a-helices lie parallel to the caxisand cause needle-like growth, In addition, hydrogen bonds are formed between the hydrophilic polypeptide side chains and water molecules on the surface ofthe ie. 11.3 Liquid water Liquid water is well known for its unusual physical properties given its low molecular weight. Unlike its direct homologue, HS, and unlike molecules with analogous structures sueh as CHy, water is liquid from 0°C to 100°C under atmospheric pressure. To maintain itself ina liquid state inthis way. there must be 11.3 UQUID WATER strong interactions between its molecules, and this leads to speculation as to ‘wheter Some form of order might be present. ‘Table 11.1 lists some of the properties of both HO and DO. 11.3.1 Structural study Bernal and Fowler (1933) were the first to provide evidence ofa local structure in liquid water from their classical X-ray studies. From the pattern yielded by small- angle X-ray scattering, its possible to obtain the distribution fiction g(r) of dis- tances r between one water molecule and another (Fig. 1.5) at 4°C. The quantity 4zr'e(r)dr is the number of molecules at distances between r and r+d from the ‘origin. A perfecly isotropic distribution has g(r) = | for any r The fact that e(°) _20¢s through several maxima shows that some short-range order exists defined by ‘neighbours’ at distances of 2.8, 3.7, 4.5 and 7A, but with none beyond 8A, A sphere of radius 8A contains about 70 water molecules, giving some idea of the Table 1 Comparison between HO and 0:0 Propeny ‘mating po tle point ating pec) temperature of maximum deny crea temoeratue (°C) Adimeting heal") ‘AH vapoteaton feaimo) ASmeting calk-' ‘ASveporzation (alk) mol volume oflqudatmeting pat Exclusion opts Partial order Figure 1.5 Dstibuton uncton efor epic wateratec. Figure 116 Faman specva of OD: (al asymmetric band at 25°C: (o)varations ih terperture The pont (Fam Wate, 1968 Reprinted tom Joumalot Ghemcat Physics wth tne permission ofthe Amaiean sue cf Prysis and te author) MOLECULAR BIOPHYSICS size of the ordered domains. Thefist peak, at 2.8 at4°C, shifts 02.94 A at 200°C under pressure Ifa graph of $27°¢(r) against ris plotted, the area under the peak at 2.8 A shows that each molecules surrounded on average by 4~S molecules. Ths igureis 12 for close-packing of spheres (with a space-iling factor of 0.74), while its Found to be 8-10 formany simple liguids. Liquid water therefore hasa very open structure and ‘analmost tetrahedral arrangement of molecules, However, the first peak cannot be identified witha single Gaussian distribution, which seems to indicate the existence ofamixture of structuresevenat short range. Inadition, while the peaksat28, 4.5 and 7A are also found in ice, the peak at 3.7 A is unique to liquid water. Another method of studying the structure of water isto analyse its infrared and Raman spectra. To avoid the coupling between the vibrational modes of two molecules, measurements are made either of the frequency of the OD band (by dissolving HOD in H,0) or of that ofthe OH band (by dissolving in DO). In the later case the wavenumber (3439 em") les between that of ee I(3277em~) and water vapour (3707em~, Although the bandwidth is smaller than that of an isolated water molecule, it is much larger (160em™") than in ie (Sem), Such @ width can only be explained by a structural disorder with a wide variety of local ‘environments. A variation of the position of the Raman band with temperature cccurs between 20 and 90°C (Fig. 11.6) together with the existence of an isosbestie point characterizingan equilibrium between two species clearly visiblein the figure Nevertheless, each of the species retains a bandsvidth that is stil very large. By studying HOD in water at 400°C up to a pressure of 5000 bar, any density between 0.016 and 0.9 can be obtained, thus decoupling the variations in tem: perature and density. Lis then found tha the characteristic frequency of OD tends toa limit of 2620cm~ and noc2720em~' asin the dilute vapour, The bandwidth must therefore be due mainly o rotational interactions between a given molecule ‘and its neighbours, and does not involve hydrogen bonding (s2¢ Box 11.2), ‘The above two methods of investigating the structure of water are com- plementary and cover different structural ranges. In Ft, liquid waters examined Intensity + +t — t + 2700 2600-2500 24002400 Wavenumiber (orn) 11.3 LIQUID waTER Box 11.2. Relationship between the heat capacity C, and energy | fluctuations | Another way of representing the instantaneous local disorder in water isto | éslimateheampiitudesofenereyMuctations.Inacosed system constant, V, Dinthermal equilibrium with theesternal medium (a canonical ensemble), the energy Muetation canbe defined as usual by ob =(E)-(E) By definition: 4B) = So Bexp(— Ek T)/ J exp|-Ej/keT) (2) Lexi By/koT) = Y Bjexp(—Ei/ka) Differentiating both sides with respect to Tat constant Nand F gives (HEVOT y.4 ) exp(—Ej/kaT) + (E) kT?) % DL Brexp(-F,/kuT) = (1/hkaT") > BP exp(—E;/ksT) so that kT (EY/OT) = > Ef exp(-E,/boT 1) expl-£/ba) [LF eww 07 Sexpl-A key] EB) =o ensemble depends only on the temperature and the partial derivative (0E/ a= PC, For water C.is lal g ‘and the molarspecific heatis 8 eal, Since Risabout ‘Teal, iving an amplitude of fluctuations of 1.64 keal mol! at0°Cand 2.24 keal mol~ at 100°C. Thelatterisalmosta quarterof the molar latent heat of vaporization a this temperature, which is about 9.7 keal mol” ‘on very differen timescales depending on the physical method used (Fisenbergand Kauzmann, 1969) and because of this the actual structures detected will be very different. Thus (Fig. 11.7), we ean distinguish throe types of structure: instant neous () vibrational (V) and diffusional (D), The first isa ‘snapshot of water taken over a very short time of less than 10 "8s. Molecules are caught in a given oscillatory and vibrational state, which generally differs from their average state. If the observation time is extended to between 10""" and 10-"s, che image willbe “fuzzy” because of the oscillations and vibrations, but if aay order is present in the liquid it must be detected from the instantaneous positions of the molecules Inelastic seatterng of neutrons allows the molecular motion to be studied in more detail. Iftheinteraction time 7 ofthe neutrons with water moleculesisongenough MOLECULAR BIOPHYSICS D struct Votructure, structure gate ‘to(iee OC) ep (water 0°0)| (ce) a ae) -2 -4'-6 -8 -10%-12"%-i4 -16 | 18 X-ray diffraction “Thermodynamic NMR (chemical shit) roper Epiepeeeee Dielectric relaxation Light Inelastic neutron scattering scattering Uttrasonic absorption IR and Raman spectroscopy oof water structures: rg = perodofelacton ina Bohr afb ron = ‘verage etm between no successive eran. {From esenberg and Kauanann, 968 Ferinod ten The Scture are Properties Wate, 1968 by permission of xo Univers Press) (slow neutrons), a process of diffusion by abrupt jumpsis observed, the mean time between two successive jumps being of the order of 2x 10~'*s, No long-range order can be detected, but at short range there isa tetrahedrallike coordination diferent from that ofive I. There areno free water moleculespresent,astherearein water vapour: this is the V structure finaly, the observation time exceeds 10-"’s, the image is completely blurred by the various molecular movements (vibration, rotation, translation) In thisease, therefore, itis preferable to define a new structure (D) by considering the spatial istribution of molecules with respect to one of them taken as origin. This is ‘equivalent to puttinga camera ona given water molecule and takingasnapshotasit ‘moves. The photograph would not be completely blurred because some local order persists inthe mutual arrangement of molecules, and it will correspond to the information provided by the distribution funetion g(7), There are many experi- meatal techniques for obtaining information about the V and D structures, but there is at present no way of discovering anything about the I structure, which remains purely theoretical. ‘Another point should be made about the lifetime of the V structure. As the temperature is gradually lowered, 7p (see Fig. 11.7) inereases and the V structure persists overlonger times, Ata very low temperature (~80°C), 7pis ofthe order of Several days or weeks, and this new structure is glass. By rapidly freecinga thin pellet of water, it might be possible to fix a structure similar to the V structure, ‘though it will of course differ from one point to another in the glass. When a macromolecule i present, the observed structure will bean instantaneous picture 11.3 LIQUID WATER of the dynamic equilibrium between the macromolecule and its aqueous enviran- ‘ment. This forms the basis for new methods of prepazing specimens for deposition on the grid of an electron microscope. 113.2 Models of liquid water “Many models of liquid water have been proposed asa result of all these observa tions. However, these must nat only have an explanatory value buta predictive one as wel. They must beable to provide calculated values for physical properties such as phase change temperatures, latent heat capacities and specific heat eapacites that agree with experimental values. The models put forward can be divided into two groups, depending on whether ‘the water molecule is assumed to havea single typeof environment or tohave more ‘than one. Inthe atterease, wate is viewed asa mixture or an interstitial compound. The mixture model In the mixturemodel of Frank and Wen (1957), liquid waters assumned to bein the fotm of molecular clusters arranged asin the ie I lati, witha very short lifetime (10-"'s) and with disordered regions. A large number of water molecules would be present in the clusters because of the cooperative nature of hydrogen bond Formation, ‘The advantage of this model is that it lends itself to a quantitative treatment (Nemethy and Scheraga, 1962). Several energy levels accessible to the water ‘molecule are defined and a partition function Z ealeulated from the population in the various levels. The other thermodynamic funetions can be calculated from Z with as ew adjustable parameters as possible. The values for these parameters are ‘chosen to give the best fit between calculated and observed values ofthe physical Properties, The agreement for some quantities is quite good, but the e(7) function and the Raman and infrared bandwidths are more dificult to predict. A crucial factor, however, is thatthe model depends on the assumption that the hydrogen bonds between water molecules ure highly directional, whereas quan- tum-mechanical calculations show that such bonds are deformable and that rotation about them is easy. One model (Grunwald, 1986) assumes a mixture of a tetra-coordinated structure A and a penta-coordinated structure B and is able to explain many of the properties. The relative amount of states A and B depends on the type of physieal property (see Box 11.3). The interstitial model Itisassumed inthe interstitial model that non-coordinated water molecules can be ‘accommodated in the cavities ofa limited ice I lattice, There are, however, serious criticisms ofthis: [Its impossible to see how molecules of liquid water could exist in these “eavities’ without interacting strongly with the lattice molecules and thus destroying the lattice ‘The accommodation of water molecules would necessarily increase the fattice volume and such a process is improbable at normal temperatures and pressures, The interstitial model is to0 close to a solid-state model to be capable of interpreting the thermodynamic properties of liquid water MOLECULAR BIOPHYS! Box 11.3 A two-state model ‘Thisis the model of Grunwald (1986), Water can be considered as a mixture of ‘wostates und B, the irsthavings low energy. low entropy and largevolume, and the second having high energy, high entropy and small volume, Such a ‘model has always been regarded with reservations by theoreticians, butit does pprovidea correct interpretation of spectral daa, hoth Raman and infrared, by ‘using an equilibrium between the wo states ccording toa law of mass ation Tor which 4a T[n(B)/(A)] = (1/ RT) AH ‘Thisisan internal isomerization ofthe lattice and nota dissociation of water ‘polymers’. We cannot assume that the A state is an ‘iceberg’ structure with slitectional hydrogen bonds and the B states onein which hydrogen bondsare ‘broken, for calculation shows that hydrogen bonds can be bent and twisted. and hence suffer considerable deformation but cannot be broken, Iti pre= {erable to view A and B as two types of organization around a molecule. State ‘A has tetrahedral coordination (Bernal snd Fowler model) and state B has five-fold coordination with bifureated hydrogen bonds Tavis the fraction in state A, then K'=(1-a)javand AGan = -RT ln K = AHan~TASh9 Measurements of heat capacity give AHap=23kealmol-' and ASqy = 6.Tkealmol"!K-!. This gives the following variation of a with temperature: 10) 0 25 oa a 0.76 0.694 0.587 0.532 ‘The tetrahedral model predominates at low temperatures. Coordination number Each of the two states is characterized by-a coordination number m defined as the number of hydrogen bonds between a water molecule and its nearest neighbours. The mean coordination number (m) at any temperaturecan then Dedefined by (m) = ama + (-0)ing Using the values in the above ‘table’ gives (m) = 44 ati’Cand4.9at 83°C, However, these values can also be derived fromtheurea under the 2.9 A peak in g(r). Theagreementis satisfactory, with themost recent Polarizability In Kirkwood’s theory of the dielectric constant of water, a coordination parameter gis introduced to take into account the field produced by thedipole ‘moments of neighbouring water molecules, If isthe intrinsie dipole moment, ‘ofa water molecule and the dipole moment due to nesrest neighbours, we have 11.3 LIQUID WATER Similarly, we can attribute values x4 and gy respectively to the two states to | account for the variation in dielectric eonstant with temperature, In this way, | we find gx =3.0-£0.1 and gy = 1.9402, indicating that dhe B state is about | half as polar as the A state. | Volume _ Themolar volumes of tates A and Barein the ratio 6: 5, which corresponds to © theratio ofthe numbersof water moleulescontaine ina given otal vlune ‘If we adopt for the A state the value ys =20mlmo! ' (corresponding to the density of ice), then we deduce that ry ~ 16.7 ml mot” ',givinga volumechange | of -33mimol when pong from A 0 B Tis is wih the considerable | tango ales (rom 310 ~7) obtained fom several diferent experimental, methods iterpreted by a twostate mode. ‘An analysis the phys proper of water ean be taken Tater within theffamework of thismodel However, shouldbe pointed out that itreleson two confgurallons A and B ba arene averages, which wold no longer be Belge gi Seer arte meio ‘hul-state or almost continuous model would be beter. The comments made Previously about the timescale for observation of the stroture of water (Fig 11.7) shold be kept mind. ‘There is at present no entirely satisfactory model, but what is certain s that liquid water is indeed a mixture of several types of molecule depending on the degree of hhydrogen bonding. For each type, the physical properties vary because of local ‘changes in molecular interactions. The rapid interconversion ofthe different types ‘means that liquid water appears to be a homogeneous pure liquid at sufficiently long time scales. In any case itisclearly impossible to attribute to water a so-called [As we shal se later, problems related to the hydration of macromolecules are paradoxically simpler than those related to water alone, insofaras thestructureand

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