COOPERATIVE INTERACTION OF POLYSACCHARIDE MOLECULES IN WATER : A ROLE OF CONNECTIVITY PROPERTIES OF H-BONDS WITHIN THE SOLVENT ?

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COOPERATIVE INTERACTION OF

POLYSACCHARIDE MOLECULES IN WATER : A ROLE OF CONNECTIVITY PROPERTIES OF

H-BONDS WITHIN THE SOLVENT ?

P. San Biagio, F. Madonia, P. Sciortino, M. Palma-Vittorelli, M. Palma

To cite this version:

P. San Biagio, F. Madonia, P. Sciortino, M. Palma-Vittorelli, M. Palma. COOPERATIVE INTER- ACTION OF POLYSACCHARIDE MOLECULES IN WATER : A ROLE OF CONNECTIVITY PROPERTIES OF H-BONDS WITHIN THE SOLVENT ?. Journal de Physique Colloques, 1984, 45 (C7), pp.C7-225-C7-233. �10.1051/jphyscol:1984725�. �jpa-00224290�

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J O U R N A L D E PHYSIQUE

Colloque C7, supplkment au n09, Tome 45, septembre 1984 page C7-225

COOPERATIVE INTERACTION OF POLYSACCHARIDE MOLECULES IN WATER : A ROLE OF CONNECTIVITY PROPERTIES OF H-BONDS WITHIN THE SOLVENT ?

P.L. San Biagio, F. Madonia, F. Sciortino, M.B. Palma-Vittorelli and M.U. Palma

Istituto di Fisica deZZrUniversita', Istituto AppZicazioni InterdiscipZinari deZZa Pisica, Cons. Naz. Ric. via Arckirafi 36, 1-90123 Palemno, Italy

~ 6 s u m 6 - Nous avons Qtudi4 la transition vers un ordre surmolkculaire d'un simple polysaccharide structure1 en solution aqueuse et les propri6tds struc- turelles de la phase ordonne'e. Nous avons observ6 des instabilitks des propri.6t6.s d'agrsgation et de transport 2 travers la transition. Nous discu- tons le possible r8le de la st6r60dynamique du syst&me eau-biomol6cule la lumiere du concept de parcours de connexion des liaisons hydrog'ene.

Abstract - We study the transition to supramolecular ordering of a simple biostructural polysaccharide in water, and the structural properties of the ordered phase. Instabilities in aggregation and transport properties are observed across the transition. The possible role of the stereodynamics of the biomolecule-water system. as a whole is discussed in terms of the notion of connectivity pathways of hydrogen bonds.

I - INTRODUCTION

Modelling the interplay between the rearrangement of solvent molecules in the presence of solutes and the functional conformation of a biomolecule in water is a very important step towards a deeper understanding of biomolecular stability. To this purpose, an experimental study spanning across a conformational transition of solute biomolecules in a simple two-components aqueous system may prove fruitful. Particu- larly so if pattern recognition between solute molecules at relatively low zoncentra- tion is the initial step of the transition.

As it was suggested long ago 111 this modelling must include the dynamic domain, in order to account for the second half of the phase space available to the interacting solvent and solute molecules. There has been a very early indication of the interest of taking jointly into account biomolecular dynamics and conformation 12-41, as a source for the understanding or the prediction of novel phenomena 13/41. This has been followed by an increasing amount of interest concerning dynamic aspects of biomolecular physics, their computer simulation, their conjectured or established relevance to functional efficiency, and more in general the role of the time variable, that is dynamics, fluxes and fluctuations 1113-101.

Our modelling could not ignore recent progresses in the understanding of the structure of liquid water in terms of the topologic notion of percolation, as proposed theoretically /11/ and visualized by Molecular Dynamics simulation 1121. The ex- istence of flickering connectivity pathways of H-bonds in water surrounding solutes has been visualized by Monte Carlo /13/ and Molecular Dynamics /12/ simulation. On plain statistical thermodynamic grounds, we have proposed a model wich visualizes at a microscopic level how these topologic structures, as pinned at or modified by different sites at biomolecular surfaces, or as rearranged upon a conformational transition of a solute biomolecule could have a significant role in biomolecular configura- tional stability and ordering/14/15/.

In this connection, a revisitation of thermoreversible sol-gel transitions in aqueous biomolecular systems appears promising. Difficulties encountered in formulating a u- Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:1984725

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'27-226 JOURNAL DE PHYSIQUE

niversal description of these transitions may possibly reflect their sensitivity to molecular details 1161. Similar difficulties and interest concern the dynamic aspects of transitions /17/18/.

In the present paper we report preliminary results of non-equilibrium studies of correlation spectroscopy across the thermoreversible sol-gel transition of Agarose- water systems. Agarose (M.w.-100,000 - 120,000) is an unbranched and essentially un- charged biostructural polysaccharide which in aqueous solutions forms thermoreversible gels, down to concentrations of 0.3% w/v or less /19/20/. Gelation is caused by molecular ordering in double helices, followed by partial supramolecular ordering 1201, both occurring cooperatively as evidenced by a large thermal hysteresis 1211. A Monte Carlo simulation of the interaction at 300 OK of the Agarose double helix and solvent water has evidenced the occurrence of H-bond connectivity pathways around the double helix 1221. The thermal hysteretical cycle is altered by substitution of H 0 with D20, and the effect appears to be entropic /14/15/21/. Gelation kinetics at dig- ferent temperatures and up to thousands of hours of observation have also been studied 1231. Solvent deuteration causes non-trivial effects. The nature of the transition 1241, the time required for it to occur, and the structural characteristics of gels so obtained, depend strongly upon the selected temperature and polymer concentration 1231241. A narrow temperature interval separates two markedly different gelation behaviours /15/23/. In the same interval, or close to it, ample transient fluctuations of the scattered light have been reported to occur in the course of cooling, but they have not been followed up 1251. Light scattering experiments at temperatures below the sol-gel transition have revealed the occurrence of long term, long range fluctuations 1241261.

In view of the interest of dynamic studies and also because a sol-gel transition temperature is hardly defined in these systems 1231, the present experiments were done in non-equilibrium conditions. We have used solvent perturbations for studying the role of solvent in pattern recognition and in molecular organization and dynamics. To this purpose, we have studied across the transition the fluctuations of scattered light intensity in different solvents and compared them to similar fluctuations that we have observed in transport properties as measured non-perturbatively, using minute quantities of polystyrene latex spheres as probes 1271. Further information on the role of the solvent was obtained from data on turbidity of gels formed in different conditions, analyzed so as to allow sorting out the effects on the mean square fluctuations of the polymer molar partial volume, and the spatial correlation function of the static fluctuations. We shall discuss our results in terms of the men- tioned microscopic model which makes use of the notion of connectivity pathways of H-bonds

.

I1 - EXPERIMENTAL

Photon scattering and correlation measurements were done with a Malvern RR 102 spec- trometer, a Malvern Log-Lin 7027 correlator, and a Coherent 52 Argon Laser operated at 514.5 nm. Data collected with a logarithmic channel spacing were analyzed with the Malvern software which uses the exponential sampling method 1281. The same software was used for analysis of data collected in the linear 60-channel mode, but in this case we also used the procedure described in Ref.27, modified so as to allow fittings with two exponential plus a constant whenever necessary. The two different analyses gave results which coincided within a very narrow margin. This added to reliability.

In most experimental conditions, diffusion coefficients to be measured ranged within a not so large interval, and advance information was available on the number and dispersivity of the mobile species involved. This, and the presence of a flat back- ground component in the normalized correlogram, made the use of linearly spaced chan- nels and our method of fitting more convenient in the present case. The method of CU-

mulants 1291301 was also used occasionally, that is when high polydispersity was expected or possible. Data were taken at a constant 90' scattering angle, an extension of experiments to various angles being now under way. Photon correlation spectrometry was also used for studying viscosity at a microscopic scale and non-perturbatively.

To this purpose we measured the translational diffusion coefficient of monodisperse

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p o l y s t i r e n e m i c r o s p h e r e s of 0.04 micrometer nominal r a d i u s , added i n minute q u a n t i - t i e s d u r i n g p r e p a r a t i o n of specimens 1271.

T u r b i d i t y was measured w i t h a J a s c o 505 Uvidec s p e c t r o m e t e r . I n e v e r y e x p e r i m e n t , t e m p e r a t u r e was c o n t r o l l e d and scanned w i t h a Haake programmable t h e r m o s t a t and a c t u - a l l y measured w i t h a Fluke thermometer u s i n g a p l a t i n u m s e n s o r .

Agarose was Seakem HGT ( P ) , l o t no.62933, w i t h s u l f a t e c o n t e n t s l e s s t h a n 0.15%. The powder was d i s s o l v e d i n t h e s o l v e n t by keeping t h e s e a l e d samples i n b o i l i n g w a t e r f o r a b o u t 20 min. Samples were t h e n f i l t e r e d through 0.2 micron f i l t e r s , a t 70-90 OC.

I f n o t d i f f e r e n t l y s t a t e d , t e m p e r a t u r e was t h e n d e c r e a s e d from 70 O C a t t h e r a t e of 1.8 OC/hr. Water was M i l l i p o r e Super Q and D 0 was from Norsk Hydro ( O s l o , Norway).

E t h a n o l was s t a n d a r d r e a g e n t g r a d e and i t was added t o s o l v e n t w a t e r d u r i n g p r e p a r a - 2 t i o n of samples. A molar f r a c t i o n of 0.03 E t h a n o l i n w a t e r was used a s a c o n v e n i e n t s o l v e n t p e r t u r b a t i o n / 3 1 / . E x t e n s i o n t o d i f f e r e n t a l c o h o l s and c o n c e n t r a t i o n s i s now under way.

111 - RESULTS

Experiments w i t h s o l s a t 70 O C .

An Agarose c o n c e n t r a t i o n of 0.5% w/v was chosen f o r t h e p r e s e n t s t u d y , o n t h e b a s i s of p r e l i m i n a r y photon c o r r e l a t i o n s p e c t r o m e t r y (PCS) e x p e r i m e n t s . S o l s a t d i f f e r e n t Agarose c o n c e n t r a t i o n s i n H 0 and a t 70 O C were observed. Two e x p o n e n t i a l s p l u s a c o n s t a n t ( s e e f o r e g o i n g sec2tion) g a v e v e r y s a t i s f a c t o r y f i t t i n g s of c o r r e l o g r a m s , showing t h e p r e s e n c e o f : i ) a p a u c i d i s p e r s e s p e c i e s , w i t h hydrodynamic r a d i u s of t h e o r d e r of 1,500-2,000 Angstrom, and i i ) a s i m i l a r l y p a u c i d i s p e r s e s p e c i e s w i t h hydrodynamic r a d i u s of t h e o r d e r of 150 Angstrom. T h i s l a t t e r v a l u e compares f a v o u r a b l y w i t h t h e e x p e c t e d a v e r a g e r a d i u s of a random c o i l e d Agarose polymer and d o e s n o t depend upon polymer c o n c e n t r a t i o n , i n t h e range of o u r measurements. The p r e s e n c e of s p e c i e s h a v i n g r a d i i s i m i l a r t o t y p e i ) h a s a l r e a d y been r e p o r t e d and d i s c u s s e d i n t h e l i t e r a t u r e / 2 4 / , b u t t o t h e p r e s e n t p u r p o s e we a r e o n l y concerned w i t h t y p e i i ) . Non-perturbative measurements of v i s c o s i t y were a l s o done by PCS a s d e s c r i b e d i n t h e p r e v i o u s s e c t i o n . R e s u l t s r e l a t i v e t o t h r e e s o l v e n t s and d i f f e r e n t Agarose c o n c e n t r a - t i o n s a r e shown i n Fig.1, where i s t h e measured v i s c o s i t y , i s t h e v i s c o s i t y of t h e s o l v e n t ( a s measured by t h e same method), (q-%)/% i s t

%

e s o c a l l e d s p e c i f i c v i s c o s i t y , d i s t h e c e n t e r - t o - c e n t e r a v e r a g e d i s t a n c e between Agarose m o l e c u l e s , and

F i g . 1 S p e c i f i c v i s c o s i t y vs. d/2R i n Agarose s o l s a t 70 O C i n H20 ( o ) , D20 (+) and 0.03 Mol. f r a c t . Et-OH s o l u t i o n (2). Arrow c o r r e s p o n d s t o 0.5% w/v Agarose concen- t r a t i o n .

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C7-228 JOURNAL DE PHYSIQUE

R their measured hydrodynamic radius. Note the marked rise in the viscosity at value one in the abscissa, corresponding to the overlap threshold concentration 1161, at which the polymer random coils are just about in contact.The concentration of 0.5%

W/V was chosen for the present experiments for its being close to this threshold value. To be noted that points relative to different solvents fall on one and the same curve when plotted vs. the scaled variable d/2R. This adds reliability to results of our PCS experiments and data analyses.

Instabilities and fluctuations across the sol-gel transition.

Viscosity and light scattered at 90' were measured across the sol-gel transition, for a decreasing-temperature scanning rate of 1.8 Oc/hr, starting from 70 OC. Results of a typical run are shown in Fig. 2, where specific viscosity and the normalized number of scattered photons averaged over a 100 sec. time basis are plotted against tempera-

Fig. 2 - Specific viscosity (-o-o-) as measured by 0.04 radius micrometer spheres, and 100sec.-average, normalized counts of photons scattered at 90'. Typical run with a sample of 0.5% w/v Agarose in H 0, and with temperature decreasing at 1.8 OC/hr.

The arrow indicates the temperature of occurrence of the sol-gel transition, as moni- 2 tored turbidometrically at the same scanning rate.

2' ^Q

ture. An aliquote of the same specimen, identically treated, was used for recording the occurrence of the sol-gel transition, as revealed by the steep increase of opti- cal turbidity 1211. At our scanning rate, the width (between 25% and 75% of the increase in turbidity) of the transition so measured is about 6 OC and its 50% point is indicated on the temperature scale in the Figure. The observed viscosity fluctuations could in principle be due to an artefact caused by the appearance and disap- pearance of large species, capable of "entraining" the photon correlation function.

As it will be discussed elsewhere, all our efforts for finding an explanation of this kind were unsuccessful. This was essentially due to the fact that in our conditions the contribution of polystyrene spheres dominates the correlation function. Results must therefore be ascribed to viscosity fluctuations. Sequences of events in repeated runs at decreasing temperatures in H 0 systems have in common the following features:

i) microscopic viscosity is observed to fluctuate well before the start of the turbi- 2 dometric gelation curve. Measurements at constant temperature in this range were not attempted, but it is known from other experiments that here an exceedingly slow gelation process occurs, originating a new type of gel structure 1231; ii) average value and fluctuation amplitude of specific viscosity start both decreasing when the turbidometrically monitored transition is about at its midpoint; iii) intensity and fluctuations of light scattered at 90' start instead increasing at about this temperature; iv) the final value of specific viscosity measured by our probes is smaller

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than that relative to sols at 70 OC, indicating that empty spaces in the gel are sizeably larger than our probes /26/27/.

In D20, viscosity fluctuations, turbidometric gelation curve, and scattered light fluctuations all appear at temperatures about 2 OC higher than in H20. However, the temperature at which large viscosity fluctuations decrease is about 4 OC higher, so that their interval of occurrence is narrowed.

In H 0 with 0.03 molar fraction Ethanol, the turbidometric gelation curve occurs at a temperature some 2 or 3 2 OC lower than in H20. The start of viscosity fluctuations is also similarly shifted but they start decreasing at the same temperature as for H 0 ,

with a resulting narrowing of their interval of occurrence. The temperature at wiich large fluctuations of scattered light intensity appear is not much affected.

Qualitative features of the observed effects of solvent perturbations are such as to make impossible a unified description in terms of the variable T/x relevant to mass fluxes, or in terms of a shift in temperature, to compensate for the slightly altered strength of hydrogen bond. A combined use of both changes of temperature scale does not appear more viable. As it has already been discussed /15/ and as we shall recall in our conclusions, this is particularly meaningful.

Turbidity spectra of formed gels

A preliminary study of the role of temperature, Agarose concentration, and solvent in the kinetics of isothermal gelation, containing also partial information on the structural characteristics of the already formed gel, has been published elsewhere /23/. We have prepared a series of gels at 0.5% w/v Agarose, by isothermal gelation at several temperatures and measured their turbidity spectra in the range 350 - ⁸⁰⁰

nm , up to when no appreciable further variation with time was obse'rved. This required waiting times ranging from several hours to several weeks. For a better comprehension of information contained in the present results, we recall that if 8 ^is

the partial molar volume of Agarose, turbidity can be expressed in our case as the product of < A m j , which somehow measures the degree of supramolecular aggregation of polymer strands, by a function of the wavenumber of the light propagating in the gel /24/32/. This function is determined by the spatial correlation function of the static fluctuations /32/. As we shall discuss in detail elsewhere, it can be shown that its average slope when represented on a log-log scale is determined by the correlation length.

In Fig.3,a we show spectra of gels obtained in H 0 at several temperatures. From Fig.3,b again it appears that spectral differences cannot be accounted for by a mere 2 temperature shift. In Fig.3,b turbidity at a given wavenumber is plotted against the temperature at which gels were formed. In account of the constancy of wavenumber, we conclude that turbidity maxima evidence corresponding maxima in mean square fluctuations <A$>. The maximum shifts towards lower temperatures and becomes less pro- nounced by going from D 0 to H20 to 0.03 molar fraction Ethanol in water. Further, it appears that spectral differences cannot be accounted for by a mere temperature 2 shift. The log-log representation in Fig.3,~ shows that at 20 and 38 OC the mean square static fluctuations of the polymer partial molar volume in the formed gel are measurably affected, in the order D O>H O>EtOH, while their correlation functions are

2 2

less so; and that at 44 OC, both these mean square fluctuations and their correlation functions are markedly affected.

IV - COMMENTS AND CONCLUSIONS

We have reported for a simple aqueous system of a biostructural polysaccharide, the onset and the evolution of instabilities and fluctuations, across the thermoreversible phase transition that the system undergoes upon cooling. .Reference to better known critical phenomena is not straightforward, because the transition exhibits a large thermal hysteresis, the origin of cooperativity is scarcely understood, and the definition of a critical temperature and of an order parameter across the sol-gel transition is not trivial / 1 6 / . The reported instabilities and fluctuations, however,

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JOURNAL DE PHYSIQUE

1 ambda Inm) ^*IB

El I ambda (nml

Pi

1

Temperature [ CI _log_(n/I ambdal

Fig. 3 - Turbidity of 0.5% w/v Agarose gels isothermally formed at selected temperatures.

A ) Spectra of H 0 gels

B) Turbidity at fixed wavelength (400 nm) of gels vs. temperature at which they 2 were formed: H20 (01, D 0 (+), 0.03 Mol. fract. Et-OH solution ( 2 )

C) Turbidity of gels forme2 at three selected temperatures and in three different solvents, vs. wavenumber in the solvent. Symbols as in B).

Here n is the index of refraction of the solvent.

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evidence and concern a role of stereodynamics, mass fluxes and hydrodynamics of the entire biomolecule-water system. The behaviour reported here may more generally be typical of transitions to the gel state 1271.

In our specific system, transition to the gel state implies a coil-to-helix transfor- mation (with no intrahelical hydrogen bonds) followed by aggregation of helices in bundles 1201. Results that we have presented suggest that the onset of hydrodynamic/viscometric instabilities be mainly related to the former, and that similar fluctuations in scattered light be instead mainly related to the latter. If so, the present approach possibly offers for the first time a decoupled study of the two processes.

The two solvent perturbations used here are expected to act in complementary ways, in our model based on the possible role of H-bond connectivity pathways. The mass difference between H and D reduces significantly the librational and vibrational am- plitudes in D20. It has been suggested that the reduced orientational disorder will facilitate propagation of order along connectivity pathways of hydrogen bonds /14/15/. This agrees with results of molecular dynamics simulation 1331. On the other hand, addition of monohydric alcohols to the aqueous solvent, in addition to trivial effects due to the altered dielectric constant, is known to alter e.g. the difference between the standard free energies of the two possible conformations of Hemoglobin by an overall, non bulk-electrostatic effect of the solvent as a whole

/ 3 1 / . This effect can be synthetically thought to be due to a decreased hydrophobic

interaction 1311. It might be accounted for in terms of an altered free energy contribution of the H-bonds connectivity pathways, as perturbed by alcohol molecules and their hydrophilic/hydrophobic hydration and interaction /15/. In short, molecular organization and aggregation can be expected to be more efficient in D20 and less efficient in H 0-Ethanol solutions, on the basis of this model.

2

Solvent perturbation results are summarized as it follows. In D 0: i) viscometric instabilities occur in a narrower temperature interval, as if helices aggregate more 2 efficiently as soon as they form; ii) amplitude of scattered light fluctuation is larger, as if potential aggregates could be larger in D20, and iii) <A*'> values are larger, again speaking for a more efficient aggregation. (All this possibly implies some recognition process). At variance, in Ethanol solutions, although fluctuations of scattered light are not delayed in this case with respect to viscosity fluctuations, aggregation as measured by < A 4 ' > appears to be less efficient.

Predictions from the model appear thus confirmed by present data. The model itself, however, needs to undergo much more critical tests and scrutiny. If confirmed, it would in essence amount to saying that an increased stability or a disturbance of the H-bonds connectivity pathways in water may interfere with the coming together of solute biomolecules. Hydrophobic surfaces, inasmuch that they alter the connectivity pattern, would appear capable of interfering with such processes just as well as hydrogen bonding sites.

We express our sincere thanks to Prof. L. Cordone, Prof. S. Fornili and Dr. M. Mi- gliore for several discussions and access to unpublished results; to Mr. F. Ficarra, Mr. F. Nicotra, Mr. M. Lapis and Mr. A. La Gattuta for valuable technical help, and to Ms. M. Genova-Baiamonte and Ms. M. Giannola for producing the processed typescript. The present work was supported by Consiglio Nazionale delle Ricerche (In- /

stitute for Interdisciplinary Applications of Physics), by CRRNSM (Palermo) and by M.P.I. local fundings.

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