Lysozyme film hydration events: An IR and gravimetric study

Abstract

By a combined gravimetric and ir technique, spectra of protein films are recorded during sorption isotherms at constant water content h (mg D2O/mg dry protein) in the range 0 les; h ⩽ 0.35 at 27 and 38°C. Computer-aided differential analysis shows the effect of progressive hydration on some significant sites of the protein such as the ionizable acidic side chains and the backbone amide carbonyls, as well as the spectrum of the adsorbed water itself. In order to derive thermodynamic properties of these sites, the measured sorption isotherm is decomposed in terms of a model which postulates the existence of two classes of primary sorption sites only, and these two contributions are independently checked by the ir data. The free energy of binding of the strong and weak binding sites is found to be 2.0 ± 0.2 and 0.40 ± 0.1 kcal/mol, respectively. A water-induced transition region is clearly detected in all the observed properties at 0.06 < h < 0.10 at 38°C and is shown to be due to changes involving both the structure of the absorbed water and the coverage of the absorption sites. A detailed picture of the hydration events is offered, and the relevance of these findings to protein dynamics is discussed

Water Binding in Legume Seeds

“…4) (3,6). Calculated values for those parameters are listed in Table II for the five different species and tissue types at different temperatures.…”

“…In making this assumption, we follow the scheme of RUPLEY et al (21) and CARERI et al (3). This may be an unfair assumption, and if it is in error, it would be reflected in the estimates of weakly binding sites (Table III).…”

“…The number of sorption sites and strength of water binding were determined from the D'Arcy/Watt parameters using equations and constants derived earlier (3,6). The strength of water binding onto strong sorption sites (AHint (s)) the number of strong sorption sites, the number of weak sorption sites, and the number of multimolecular sites are given by Eqs.…”

“…sites (w) = c x N/(M x f(AHin,(w)) (7) No. sites (m) = k' x NIM (8) where Q is the molecular partition coefficient for primary sorption sites ranging from 0.8 to 1.25 depending on temperature (3), R is the ideal gas constant, Tis the temperature, fJAHin, (w)) is a temperature related function of the strength of weakly binding sites (ranging from 0.997 to 1.001 (dimensionless) for temperatures between 5 and 35°C [3]), N is Avogadro's number, and M is the gram mol wt of water (3,6), and s, w, and m identify strong, weak, and multimolecular binding. (Table I).…”

Adiabatic compressibility of globular proteins.

“…In particular, if one existing equilibrium is pressure dependent-i.e., AV, 7 0 in the corresponding free energy term-it contributes to the solution adiabatic compressibility. (18) an upper limit for the water associated with charged groups to be k = 0.10. This value is obtained by considering the total number ofcharged groups and assuming that 30 ml of water is associated with each mole of charged groups (21).…”