**1. Introduction**

Water controls and affects in large extent structure and function of biological macromolecules [1]. This unique molecule has had an essential role in the evolution of living systems, and its functions are manifold. At macromolecular level, very small water amount or small aggre‐ gates are fundamental both in the determination and maintenance of the biologically active

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

structure of proteins, nucleotides, carbohydrates and other biopolymers. The solvent orga‐ nization around the solute macromolecule structures allows folding to the native and func‐ tional conformation and the development of many biological functions such as for example, substrate recognition and binding to an enzyme, protein subunits assembling and to origi‐ nate and stabilize higher order structures such as membranes. A lot of biochemical reactions fundamental in metabolism and synthesis are involving water as universal reagent, such as hydrolysis, condensation, reduction and oxidation. Despite the great interest in the problem, the relationship between the biopolymer conformation and the structure of the water net‐ work cannot still be described with confidence. In the case of proteins, it is well‐known that polypeptide molecules are surrounded in the cell by a hydration shell which can be described as formed by differently interacting water molecules organized in layers surrounding macro‐ molecule. The simplified description of the hydration shells formed by a uniform layering of water molecules around the macromolecule is not realistic: most proteins, for example, offer a surface where binding sites, cleft or crevices provide favorable environments for solvent mol‐ ecules. Such water molecules are organized in clusters or patches decorating the macromol‐ ecule surface and are called "bound" water due to the restricted mobility with respect to the water molecules in bulk. Orientation changes may be related to structural or conformational differences among macromolecules. The bond strength variety of bound water affects mobil‐ ity, reorientation and vibrational properties [2–7].

A variety of experimental techniques were introduced and applied to study the hydration properties of biological macromolecules: DSC (differential scanning calorimetry), NMR (nuclear magnetic resonance), neutron and X‐ray diffraction, gravimetric techniques, as well as UV‐vis and CD spectroscopies have been employed to characterize the extent of water‐ biomolecules interaction. In addition, the problem of water interactions was object of theoreti‐ cal analysis and structure prediction as well as molecular dynamics simulations.

Fourier transform IR absorption spectroscopy represents a powerful method to gain struc‐ tural information on hydrated biological macromolecules, alternative to other well‐estab‐ lished techniques whose application presents severe limitation to dry compounds or in the first hydration events. It requires a minimal sample amount and preparation, and it can be used in a wide variety of conditions and geometries. It enables to study (1) the amplitude and position changes of the main absorption bands associated with characteristic functional macromolecule groups, as a function of water content; (2) the properties of water structured around the biomolecules, using H2 O molecules as probes to detect conformational changes in the macromolecule structure induced by water interactions [8].

H2 O displays a strong IR spectrum with three main bands corresponding to the OH stretch‐ ing, bending and libration modes. Their contribution can be identified in the spectrum of macromolecules and changes in the hydration conditions significantly influence the infra‐ red spectral pattern. The spectral changes observed as a function of water removal may be monitored, correlated to the changes in macromolecule conformation and used to identify the sites of water sorption. The OH stretching band, in particular, by appropriate mathematic manipulation, can be used to build the water adsorption‐desorption isotherms describing the hydration processes governing each water population.

The technique and the related experimental procedures were successfully applied in the past on two macromolecular systems very different in chemical composition and structure. Melanin and lipid array have been object of past publications [9, 10]. The present work is con‐ cerning two proteins, collagen and lysozyme, and their interaction with water.
