Hydration of lysozyme

Table 23.2. Steps in the hydration of lysozyme. The hydration level is given in g water g-1 protein, and numbers in parentheses refer to number of water ... 

Poole and Finney (1983b), using direct difference IR and laser Raman spectroscopy, studied the hydration of lysozyme. On the basis of these measurements, they suggested that there are conformational changes just below the hydration level for the onset of enzyme activity (i.e., 0.2-0.25 h). This conclusion conflicts with that of Careri et al. (1979b). Poole and Finney (1984) extended these measurements to lactalbumin. 

Neutron diffraction crystallographic studies of the dynamics and hydration of lysozyme are discussed in Section XI. 

Study by Careri et al. (1980), who used IR to study the events that occur during the hydration of lysozyme. [For later developments in this work, see the article by Rupley and Careri, this volume.]... 

Carerl et al ( ) have carried out a careful infrared spectroscopic examination of the hydration of lysozyme. Figure 3 compares the spectroscopic results with heat capacity measurements. The principal conclusions are the following 1) the first two steps in the hydration process. Regions IV and HI, are seen in the Infrared measurements. The discontinuity at 0.07 li observed in the dependence on hydration of the carboxylate, amide, and water bands (Figure 3), corresponds to the juncture of Regions IV and III. 2) The Increase in carboxylate intensity within Region IV means that proton redistribution follows... 

Remmele, R. L., Stushoff, Carpenter, J. F. Real-time spectroscopy analysis of lysozyme during Lyophilization structure-hydration behavior and influence of sucrose. American Chemical Society Symposium, Ser. 567 (Formulation and delivery of proteins and peptides) 1994. 1994 American Chemical Society... 

Disposable cuvettes are used only once to eliminate the possibility of lysozyme carryover. All microcentrifuge tubes and pipette tips are autoclaved, and buffers and BSA solutions are not used beyond 7 days. The M. lysodeikticus suspension is prepared 18-24 hr before use and set at 37° with shaking, to hydrate fully. It was found that substrate prepared just before use settled much faster and interfered with the signal at the low concentrations of enzyme used. 

It is worthwhile to present some examples of the types of results available from Monte Carlo simulations of peptide solvent systems. In Figure 6 we present the convergence of the energy of hydration of the lysozyme crystal over a million configurations. 

Figure 6. The average energy of the water of hydration of the triclinic lysozyme crystal as a function of the number of configurations generated in the Monte Carlo simulation. The upper curve (A) corresponds to the cumulative statistical average, and the lower curve fU gives the statistical average over sequential sets of 5,000...

Fig. 10.3. Water molecules in a cavity of lysozyme. Only the surrounding residues are displayed. The isosurfaces of water oxygen (green) and hydrogen (pink) for the 3D distributions larger than 8 (left), the most probable model of the hydration structure reconstructed from the isosurface plots (center), and the crystallographic...

The hydration of a protein can be described in several steps [622, 810, 827-829]. If the molar ratio water/lysozyme is gradually increased, a number of discrete levels of hydration are observed [810, 828], as established by a variety of methods summarized in Fig. 23.1 and in Ihble 23.2 ... 

Hydration of an alanine side-chain. In human lysozyme, the methyl group of Ala92 is surrounded by four water molecules located on a semi-circle ([625], Fig. 23.6). These hydration waters are buried in the protein which might be the reason why they are so well ordered. We can also assume that such hydration schemes occur at the protein periphery but they are not seen in the X-ray analyses due to larger-thermal motion and/or disorder of the water molecules. 

The point of full hydration determined by the heat capacity response corresponds to 0.38 h, or 300 mol of water per mol of lysozyme. The... 

Fig. 4. The apparent specific heat capacity of lysozyme from 0 to 0.45 g of water per gram of protein. The curve is calculated. The heat capacity measurements were made with lyophilized powders of lysozyme, appropriately hydrated, except for the four measurements indicated by the square symbols, for which the sample was a film formed by slowly drying a concentrated solution of lysozyme. From Yang and Rupley (1979).

Fig. 14. Hydration dependence of capacitance [9 C, in picofarads (pF)] of the composite capacitor containing a sample of lysozyme powder of pH 3.11 as a function of hydration level of the protein. The capacitance data are given for three frequencies. The hydration level was decreased from the high-hydration limit of more than 0.35 h to the low-hydration limit of near 0.07 h by passage of a stream of dry air through the apparatus. The evaporation rate E (O grams of water evaporated per minute) decreases to 0 at the low-hydration limit. From Careri et al. (1986).

Tredgold et al. (1976) measured the polarization of hydrated hlms of lysozyme at 0.1 Hz and concluded that the apparent high dielectric constant was attributable to protonic conduction in the water of crystallization. 

Fullerton et al. (1986) measured H spin-lattice relaxation during dehydration of lysozyme solutions to a nearly dry state, and during rehydration of lyophilized lysozyme powder by isopiestic equilibration and, for high hydration levels, by titration with water. Breaks in the NMR response were found at 0.055, 0.22-0.27, and 1.22-1.62 h (Fig. 19 shows the two higher hydration discontinuities in slope). Estimates of the water correlation times are 10 , 2 x 10 , and 5 X 10 sec, respectively, for the three classes of water defined by the breaks. The 0.055... 

Kakalis and Baianu (1988) obtained similar results for lysozyme. They estimated 180 mol of hydration water per mol of lysozyme in the absence of salt. In 0.1 M NaCl solution the hydration was 290 mol/mol. 

Ruggiero et al. (1986) measured the ESR spectra of samples of lysozyme, myoglobin, and hemoglobin with covalently bound spin labels and noncovalently bound spin probes, in solution and in the partially hydrated powder, over the temperature range 120—260 K. The several proteins behaved similarly. The solution samples differed from the powders in showing a change in spectrum shape at 210 K, understood to represent freezing of water in the hydration shell. 

Fucaloro and Forster (1985) found substantially different behavior for the hydration dependence of the tryptophan lifetime of chymotrypsino-gen A (Fig. 24). Below 0.15 h the lifetime was constant above 0.15 h the lifetime decreased from the dry protein value of about 3 nsec to the dilute solution value of near 2 nsec at a hydration level above 0.4. The change in lifetime near 0.15 h is sharp, as for a phase change. The per-colative phase transition of lysozyme is at this hydration level. 

Comparison of neutron scattering of lysozyme at 0.07 and 0.20 h (Smith et al., 1987) showed that hydration decreased elastic scattering and increased inelastic scattering between 0.8 and 4.0 cm". This observation is consistent with an increase in the number of low-frequency modes. Normal mode analysis indicates that the lowest frequency mode of lysozyme and the hinge-bending mode fall in this frequency range (Brooks and Karplus, 1985 Bruccoleri et al., 1986 Levitt et al., 1985). Hydration of a protein has little effect on the scattering spectrum, outside of that noted above (Cusack, 1989). 

Fig. 29. Enzymatic activity of lysozyme as a function of water content (grams of water per gram of sample), at pH 8, 9, and 10. , O, A, Measurements on powders hydrated by isopiestic equilibration ,, A, solvent added to powder. Powder samples were the 1 1 (GlcNAc)6-lysozyme complex, obtained by lyophilization. The reaction rate (no sec ) was determined by product analysis. From Rupley etcd. (1980).

Fig. 30. Comparison of ESR and enzyme activity changes with hydration. Effect of hydration on lysozyme dynamic properties. (Curve f) Log rate of peptide hydrogen exchange. (Curve g) , Enzyme activity (log uo) O, rotational relaxation time (log t ) of the ESR probe TEMPONE. From Rupley et al. (1983).

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