F. Cavatorta

Dynamics of hydrated starch saccharides

We report here elastic neutron scattering data on glucose and on two of its polymeric forms: amylose and amylopectin. We have covered the hydration range from the dry state to about 0.6 g water/g dry saccharide. The data indicate, in all the analysed systems, the presence of a dynamic glass-like transition similar to that observed in hydrated proteins. The fact that this feature is observed also in a relatively small molecule like glucose confirms the hypothesis already put forward by other authors, that this transition in biomolecular species is essentially triggered and driven by the interaction of the macromolecule with the network of fluctuating H-bond of the solvent.

Rayleigh scattering of Mössbauer radiation in oriented fibres of hydrated biopolymers

The Rayleigh scattering of Mössbauer radiation (RSMR) has been measured on films of highly oriented hydrated polynucleotides (A-NaDNA) and polysaccharides (Na-hyaluronate). Both DNA and hyaluronate (HA) have helical secondary structures with a similar pitch (28.2 Å for A-DNA, and 32.8 Å for Na-HA), but they differ in the basic elements which make up the helices and in the extent of water-biopolymer interactions. These differences are responsible for the diverse stiffness of the polymer backbone, and also affect the dynamics of the first hydration layers. For both samples the elastic scattering intensity shows a sharp peak at about 2 Å−1 only for samples oriented withQ parallel to the fibre direction. Its position is close to that of the first maximum in the structure factor of bulk water; it is, however, much narrower than in pure H2O and it is similar to a crystalline Bragg peak. It can be attributed to an ordered structure of water along the double helices. From the temperature dependence of the elastic intensity under the peak maximum, the mean square displacement of water oxygens in the direction parallel to the helices has been deduced. The thermal diffuse scattering intensity is also peaked at the sameQ values of the elastic intensity, indicating the presence of coherent vibrational excitations propagating along the ordered water filaments.

Synthesis and characterization of [N(PPh3)2]+ cyanometallates. Crystal structure of [N(PPh3)2]3[Fe(CN)6].2H2O

Abstract The complex salts [N(PPh3)2]n[M(CN)6] (M=Fe, n=3 or 4; M=Ru, n=4; M=Co, n=3) and [N(PPh3)2]2- [Fe(CN)5(NO)] have been prepared by metathetical reactions between [N(PPh3)2]Cl and the corresponding alkaline cyanometallates. The iron(II) and ruthenium(II) hexacyano complexes are rather unstable in the air, the former being completely oxidized in a short time even in the solid state. All compounds have been characterized by spectroscopic techniques. The crystal structure of [N(PPh3)2]3[Fe(CN)6]·2H2O has been determined by X-ray diffraction methods.

The binding of 1,N6-ethenoNAD to bovine liver glutamate dehydrogenase: studies using the time-correlated single photon counting fluorescence technique

The time-correlated single photon counting (TCPC) fluorescence technique has been used as a novel approach to investigate ligand-protein interaction, for the case of the binding of the fluorescent coenzyme analogue 1,N6-ethenoNAD (epsilon NAD) to bovine liver glutamate dehydrogenase in the presence of glutarate, a substrate analogue which stabilizes the complex. System calibration was performed using solutions of epsilon ADP and carefully purified epsilon NAD mixed at variable molar ratios (pH 7.0, 0.05 M sodium phosphate buffer, 20 degrees C). The fluorescence lifetimes obtained after deconvolution were 2.4 ns (for epsilon NAD) and 23 ns (for epsilon ADP), in good agreement with literature values obtained under similar conditions. epsilon NAD binds to glutamate dehydrogenase in the presence of 50 mM glutarate, with a fluorescence quantum yield enhancement factor, Q, of about 17-fold, as previously reported (Favilla, R. and Mazzini, A. (1984) Biochim. Biophys. Acta 48-57). For this system, fluorescence lifetime values were obtained after deconvolution as 2.4 ns for free epsilon NAD and 21 ns for bound epsilon NAD. These values did not vary appreciably with enzyme concentration nor with degree of saturation, thus reflecting the existence of only one spectroscopically relevant type of complex. Addition of either GTP or ADP did not affect the lifetime of epsilon NAD bound to the enzyme, but only its affinity, thus allowing calculations of binding strengths. In the case of a simple binding (i.e., in the absence of GTP) the dissociation constant of the complex could be derived from a simple relationship, in which only the ratio between the pre-exponential factors and the parameter gamma, which represents the molar fraction of epsilon NAD molecules free in solution in the open conformation, are to be taken into account. The results are in good agreement with those reported by some of us (reference above) using a steady-state fluorescence technique, which by itself is, however, unable to resolve the number of relevant species present in the system.

Rayleigh Scattering of Mössbauer Radiation in Hydrated Amylose

The Rayleigh Scattering of Mössbauer Radiation (RSMR) has been used to investigate the temperature dependence of the dynamics of hydrated amylose, one of the saccharide components of starch. The mean square atomic displacements measured from 80 K to 300 K indicate the existence of a “glass-like” dynamic transition at about 220 K, similar to that observed in other biopolymers as for instance globular proteins. The RSMR results have been compared to those obtained by elastic neutron scattering measurements on the same samples.

The distribution of water in highly ordered fibres of hyaluronic acid

Abstract Diffractometer D19 at the ILL is being used to locate water around hyaluronate, a polysaccharide present in the extracellular matrix of mammalian connective tissue and the capsule of some bacteria. Data to 3 A resolution have been collected from hydrated fibres where H 2 O has been replaced by D 2 O.

The diffusive dynamics of water confined in ganglioside micelles

Abstract We have investigated by QENS the dynamics of water associated to gangliosides. The dependence of the QENS line-broadening versus Q indicates that proton diffusion is restricted when investigated over scale lengths of about 6–8 A; at smaller distances the diffusivity parameters are similar to those of pure water at a lower temperature.

QENS investigation of the dynamics of starch saccharides

We present here quasi-elastic neutron scattering results on D20 hydrated samples of amylose, one of the main saccharide components of starch. Two different sample hydrations (h = 0.5 and 1.0 g D2O (g dry amylose)-1 have been investigated in the temperature range 170 to 350 K. Below 260 K only an elastic contribution is present in the spectra, while a quasi-elastic component shows up above this temperature. The elastic incoherent structure factor (EISF) associated with this component changes considerably with increasing temperature. For the sample with hydration h = 0.5 the confinement volume increases by a factor of four in going from 300 to 350 K, and the proportion of hydrogen involved in the confined diffusion motion increases as well from 30 to 55%. Similar effects are observed at the higher hydration investigated. The observed dynamics can be associated with the known plasticising role of water in polysaccharides.

Dynamics of hydrated saccharides of interest in food science

Abstract We have used IN13 at the ILL to investigate the dynamics of amylose and amylopectine performing elastic scans from 20 to 320 K at different hydration levels. In the dry samples, the atomic dynamics is harmonic in the whole temperature range explored. For the hydrated samples the mean square atomic displacement, 〈u2〉, is almost hydration independent and harmonic up to ∼230 K for amylose, and ∼250 K for amylopectine. A ‘glass-like’ kinetic transition then shows up with a marked increase in 〈u2〉: this behaviour is qualitatively similar to that observed in other biopolymers like globular proteins, but here the transition temperature is higher and it markedly varies with hydration.

Large-scale structure of polyacrylamide gels

Abstract We present small-angle neutron scattering data on polyzcrylamide gels obtained in the presence of polyethylene glycol. The microsegregation process caused by the competition between gelation and phase separation produces gels with particularly large pore size.