M.G. Bridelli

Self-assembly of melanin studied by laser light scattering

The unknown molecular weight and chemical structure of melanin place the study of these pigments outside the range of the classical biochemical techniques; thus in this paper the problem of characterizing these heterogeneous biopolymers was approached by means of light scattering techniques, static and dynamic. The static technique allowed us to identify the macromolecular properties (MW and R(g)(2)(1/2)) of melanin extracted from sepia inksac and of two synthetic analogues: L-Dopa melanin obtained by autooxidation and by enzymatic oxidation by Tyrosinase. By dynamic light scattering (DLS), the hydrodynamic radius R(h) was measured to monitor the temporal behaviour of the polymerization and aggregation processes and R(h) variation by changing the chemical constraints of the polymerization medium, such as pH and ionic strength. The fractal dimension d of the aggregates of melanin, both natural and synthetic, in the past only recognized during the aggregation of the synthetic one by lowering the pH of the medium, was a useful parameter to further investigate and compare the structure of melanin granules of differing origins, revealing for the natural sample, a structure with clusters that are spherical, not largely hydrated and self-assembled, following a reaction limited aggregation kinetics (d=2.38).

Sequential hydration-dehydration studies of lysozyme by the thermally stimulated depolarization currents (TSDC) technique

To investigate the physical state of water in hydrating biological macro-molecules, the dielectric properties of water in hen egg lysozyme pellets with various moisture contents were studied using the thermally stimulated depolarisation currents technique. The water dipoles appeared to be directly involved in the relaxation processes, such that, by increasing the content of water of sorption from ho = 0.075 to ho = 0.29, the current density recorded increased abruptly at moisture content above 0.075. At a fixed starting hydration level, the time evolution of water content was also studied by isothermal sample aging in dynamic vacuum: the TSDC spectra changed in both intensity and position of their main peaks (TM = 245 K, 190 K, 150 K) with moisture content and showed hysteresis. The complex behaviour of the TSDC response can be compared with the results obtained with the same technique on other biological macromolecules and suggests possible models for water configurations and rearrangements.

The hydration structure studied by TSDC. Myoglobin and haemoglobin

The coordination scheme of water in myoglobin and haemoglobin, the two oxygen carrier proteins in vertebrates, is still far from being fully understood. The TSDC (thermally stimulated depolarization currents) technique is a sensitive tool to study water molecules bound to biological macromolecules and characterized by mobility and order degree, quite different from those of bulk water. TSDC spectra for both proteins were recorded in the temperature range 100 to 300 K, at very low hydration levels (h=0.01 to 0.62) obtained with different procedures. Different classes of water molecules were found: the preferred hydration sites and the water dipole activation energies are discussed. In particular, the existence of a critical hydration level which induces a partially irreversible structural transition was monitored in myoglobin. The technique allowed estimation of the average number of hydrogen bonds established by the water molecules in the inner cages of haemoglobin at different hydration levels.

TSDC as a tool to monitor the electret state induced by water in biomolecules

TSDC (thermally stimulated depolarisation currents) technique, applied to a variety of natural and synthetic biopolymers has provided a valuable contribution to the understanding of the complex behaviour of the structured water, thanks to its high sensitivity and selectivity. Mechanisms such as noninteracting dipole reorientation, Maxwell-Wagner relaxation, protonic percolation, are envisaged as responsible for the peaks associated with water in a variety of configurations. The plastifying role played by water in macromolecular phase transitions, induced by the water network rearrangement, have been monitored through the careful analysis of the TSDC spectra. In the present paper the results obtained by different authors are reviewed.

Water induced bioelectret state in lysozyme

The role of water in a bioelectret such as lysozyme at different hydration levels is studied by coupling TSDC and i.r. absorption measurements. In addition to the 312 K-band reported by Mascarenhas, our TSDC spectra exhibit new, weaker but anyway relevant bands related to water in different coordinations. At fixed starting hydration levels, the time evolution of water contents was studied by isothermal sample aging in dynamic vacuum: 1) the whole TSDC spectrum was reduced, the decrease being more relevant on the high temperature side and 2) the water related i.r. bands decreased as well.

Hydration structure analysis of lysozyme amyloid fibrils by thermally stimulated depolarization currents (TSDC) technique

Thermally stimulated depolarization currents technique has been employed to investigate the conformation of hen egg white lysozyme in native and amyloid form, in the state of powder at very low hydration level. The technique, able to detect the current generated by thermally activated reorientation of water dipoles previously oriented by an electric field, exploits H2O dipoles, belonging to the solvation shell, as a probe to gain information on the protein conformation.

Hydration effects on myoglobin and hemoglobin monitored by TSDC spectroscopy

TSDC spectra of myoglobin (Mb) and human hemoglobin (Hb) have provided a great deal of information about the properties and role played by water molecules organized in the structure of both proteins at very low hydration degree /spl delta/ (/spl delta/=0.01+0.62). In particular, complementary TSDC and optical absorption spectroscopies applied to Mb have highlighted a critical hydration level which induces a partially irreversible structural change. TSDC analysis of Hb has supplied information about the hydration network, particularly with regard to the average number of hydrogen bonds established by the water molecules in the inner cages of the polymer at different hydration degrees.

Water induced TSDC in hemoglobin and myoglobin

The hydration of hemoglobin (Hb) and myoglobin (Mb) has been investigated by the Thermally Stimulated Depolarization Currents (TSDC) technique. The TSDC spectra as a function of the hydration degree (/spl delta/=0.01+0.3 grams of water/grams of protein) and of the annealing time in vacuum were recorded in the temperature range 100-300 K on pellet samples. The distribution of water dipoles for both heme-proteins shows four main peaks in the range 170-300 K, indicating the presence of at least four different classes of bound water. A fifth peak at very low temperatures (T/sub m//spl sim/150 K) appears only at low hydration levels and probably it is not related to water dipole relaxations. Possible environments of water dipoles responsible for the TSDC peaks are discussed.

I.V.V. Dipoles in LiF:Ti

ITC technique is exploited to search dipolar defects, induced in LiF by Ti, whose valence state is not well known. ITC spectra (78–350 K) exhibit at least one peak at 235 K: 1) its amplitude increases with nominal Ti-concentration and annealing temperature; 2) its shape does not depend on thermal treatment nor on Ti-concentration nor on the dopant type (TİO2 or Li2TiF6);3) it is not a single peak; 4) it is very weak in air grown crystals. Critical analysis of ITC data compared with optical and EPR spectra suggest that Ti3+ compensated by two cation vacancies is responsible for 235 K peak.

Thermally Stimulated Depolarization Currents (TSDC): A sensitive technique for analyzing protein structure

The water molecules surrounding proteins as a thin layer and those packaged in pockets and cavities shape and control their structure. Thermally Stimulated Depolarization Currents (TSDC) technique has been applied to investigate the hydration structure of six proteins with different structural motifs: pepsin, β-lactoglobulin, α-chymotrypsin, bovine serum albumin, human serum albumin and myoglobin, at very low hydration level (water vapor activity aw≈0.80) both in the native state and after treatment in trifluoroethanol/water mixture 80% (v/v). A combined approach based on the use of the TSDC technique, able to distinguish H2O dipoles belonging to the solvation shell in terms of their order degree and mobility, and of FTIR and CD spectroscopies has allowed us to reexamine the problem of conformational stability of macromolecules as a function of their hydration.