Lysozyme powder

Harvey and Hoekstra (1972) determined the dielectric constant and loss for lysozyme powders as a function of hydration level in the frequency range 10 —10 Hz. At water contents less than 0.3 h, they found a dispersion at 170 MHz, which increased somewhat with increasing hydration, and a new dispersion at about 10 Hz that develops at high hydration. These dispersions, detected by time-domain techniques, remain measurable down to the lowest temperature studied, — 60°C. Water mobility in the hydration shell below 0 C is in line with other observations of nonfreezing water. Above 0.3 h, in the stage of the hydration process at which condensation completes the surface monolayer, water motion increased strongly with increased hydration (Fig. 11). 

Fig. 11. Hydration dependence of dielectric response at 25 GHz. Dielectric constant (e ) and loss (s") of packed lysozyme powder as a function of water content. Frequency, 25 GHz temperature, 25°C. (From Harvey and Hoekstra, 1972.)... 

Fig. 13. Hydration dependence of protonic conduction. The dielectric relaxation time, Ts, is shown versus hydration, h, for lysozyme powders. The relaxation time is proportional to the reciprocal of the conductivity. (A) H20-hydrated samples solid curve, lysozyme without substrate , lysozyme with equimolar (GlcNAc)< at pH 7.0 , with 3x molar (G1cNAc)4 at pH 6.5. The relaxation time is nearly constant between pH 5.0 and 7.0. (B) HjO-hydrated samples solid curve, lysozyme without substrate 9, lysozyme with equimolar (GlcNAcb at pH 7.0. From Careri etal. (1985). 

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).

The use of the percolation model to analyze the d.c. conductivity in hydrated lysozyme powders (Careri et al., 1986, 1988) and in purple membrane (Rupley et al, 1988) introduces a viewpoint from statistical physics that is relevant to a wide range of problems originating in disordered systems. Percolation theory is described in the appendix to this article, for readers unfamiliar with it. Here, we discuss the significance of percolation specihcally for protein hydration and function. 

Fig. 17. Longitudinal H NMR relaxation parameters at 30 MHz for water adsorbed on lysozyme powders derived from the cross-relaxation model after setting the protein relaxation rate equal to 0. Tis the water proton relaxation time and 7", is the time constant characterizing spin transfer between the protein protons and the water protons. From Hilton etal. (1977).

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... 

Lioutas et al. (1986) measured the 0 and resonances of lysozyme powders and solutions, in experiments like those carried out for H by Fullerton et al. (1986). They similarly interpreted discontinuities in the NMR response in terms of three populations of water 20 mol of water per mol of protein (corresponding to 0.025 h) with a correlation time of 41 psec, 140 mol of water (0.17 h) with a correlation time 27 psec, and 1400 mol of water (1.7 h) with a correlation time 17 psec. The differences between these results and those of Fullerton et al. (1986) indicate the difficulty of estimating water correlation times. Lioutas et al. (1987) extended these results by analyzing H resonance data through comparison with the sorption isotherm. D Arcy-Watt analysis of the sorption isotherm gave 19 mol of tightly bound water per mol of lysozyme, 148 mol of weakly bound water, and 2000 mol of multilayer water. These classes plus two more types, corresponding to water in solutions... 

Fig. 22. Hydration dependence of amide hydrogen exchange in lysozyme powder at pH 5. Individual samples of pH 5 fully labeled (with H O) lysozyme were equilibrated at 25°C for 24 hr at various water contents obtained by isopiestic equilibration ( ) or by the addition and mixing of solvent (A). The samples were then dissolved to a concentration of 20 mg/ml and 100-/U.1 aliquots were analyzed by gel filtration. The arrow indicates the 24-hr solution H . end point. H, represents the number of hydrogens remaining unexchanged. From Schinkel el al. (1985).

The diamagnetic susceptibility is a measure of the averaged electronic distribution in bulk matter. Careri et al. (1977, 1980) showed that the differential diamagnetic susceptibility per gram of water adsorbed on lysozyme powders reached the bulk water value at 0.2 h. Lysozyme behaved as a normal diamagnetic substance. The diamagnetic susceptibility and the enthalpy of sorption for lysozyme change similarly at low hydration. 

We start from the experimental observation that lysozyme powders exhibit a single critical hydration level for the onset of enzyme activity and the onset of surface motions, displayed in the dynamics of a spin... 

Careri G, Giansanti A, Rupley lA. Proton percolation on hydrated lysozyme powders. Proc. Nat. Acad. Sci. U.S.A. 1986 83 6810-6814. 

The effect of "residual water" on either protein stability or enzyme activity continues to be a topic of great interest. For example, several properties of lysozyme (e.g., heat capacity, diamagnetic susceptibility (Hageman, 1988), and dielectric behavior (Bone and Pethig, 1985 Bone, 1996)) show an inflection point at the hydration limit. Detailed studies on the direct current protonic conductivity of lysozyme powders at various levels of hydration have suggested that the onset of hydration-induced protonic conduction (and quite possibly for the onset of enzymatic activity) occurs at the hydration limit. It was hypothesized that this threshold corresponds to the formation of a percolation network of absorbed water molecules on the surface of the protein (Careri et al., 1988). More recently. Smith et al., (2002) have shown that, beyond the hydration limit, the heat of interaction of water with the amorphous solid approaches the heat of condensation of water, as we have shown to be the case for amorphous sugars. 

Careri, G., Giansanti, A., and Rupley, J.A. Critical exponents of protonic percolation in hydrated lysozyme powders, Phys. Rev. A, 37, 2703, 1988. 

Relaxation Measurements. NMR measurements have been used to examine water-protein interactions in solution and in hydrated powders (2). Hilton et al (24) have shown that the motional properties of water in partially hydrated powders of lysozyme are best characterized as those of a viscous liquid. Dielectric relaxation spectra of water in lysozyme powders (28) distinguish... 

Carerl, G.C. Gratton, E. Yang, P.-H. Rupley, J.A. "Protein-Water Interactions. Correlation of Infrared Spectroscopic, Heat Capacity, Diamagnetic Susceptibility and Enzymatic Measurements on Lysozyme Powders," submitted for publication, 1979. 

Figure 2. Water proton relaxation in hydrated lysozyme powder (0.17 g HjO/g lysozyme) at 57.5 MHz at 253 K. Amplitudes were measured after the second pulse of a 180°-7-90° sequence with the 180° pulse width either (%) 8.6 /tsec or (Q) 55...

Three times crystallized, dialyzed, and lyophilized hen egg white lysozyme powder from Sigma was used after a subsequent dialysis and lyophilization. Hydration of the dry powder was accomplished through the vapor phase by exposing the sample to a constant relative humidity for 5 days. In preparing the lysozyme-D20 sample, the lysozyme was twice dissolved in D2O and held at 40 deg C for 24 h both times before lyophilization. 

Figure 3. Temperature dependence of various proton relaxation times in lysozyme powder samples at 57.5 MHz. ( ) indicates for the lysozyme-DgO sample other symbols refer to the lysozyme-H,0 powder (0) protein R (O) water Ri," fAJ wa/er (%) water Rr. ...

Scfainkel, J.E., Downer, N.W., Rupley, J.A. (1985) Hydrogen exchange of lysozyme powders. Hydration dependence of internal motions. Biochemistry, 24 (2), 352—366. 

Figure 96 DC conductivity, calculated from capacitance data measurements, vs hydration level h of the lysozyme powder. Left panel dc conductivity a normalized by the dc conductivity co of the dry sample. Right panel dc conductivity vs hydration level in a double-logarithmic scale. Solid lines show the slopes, corresponding to the critical exponents 1.3 and 2.0 for 2D and 3D percolation, respectively. Reprinted, with permission, from [592].

Below we show how the appearance of spanning water networks may be detected in computer simulations. In particular, a percolation transition of water upon hydration was studied by simulations in model lysozyme powders and on the surface of a single lysozyme molecule. In protein crystals, increase in hydration of a biomolecular surface may be achieved by applying pressure. In some hydration range, pressurization leads to the formation of spanning water networks enveloping the surface of each biomolecule. Finally, the formation of the spanning water network is shown for the DNA molecule at various conformations and for different forms of DNA. 

The percolation threshold of water found in simulations should be compared with experiments performed in the lysozyme powder [591]. At ambient temperature, it was observed at the hydration level h = 0.152 0.016 g of water per gram of dry lysozyme, which corresponds to the water mass fraction C 0.132 [591]. The percolation threshold seen in simulations thus occurs ath 0.183, i.e., at slightly higher hydration level than in experiment. This should be considered as rather good... 

Figure 99 Distributions ns of clusters with S water molecules in densely packed lysozyme powder at T = 400 K. Mass fraction of water increases from C = 0.128 (top) to 0.201 (bottom). Circles represent at C = 0.151, when the spanning cluster exists with probability of about 50%, while squares correspond to C = 0.173, when the fractal dimension of the largest cluster is close to the 2D percolation threshold value. The distributions are shifted consecutively by one order of magnitude each, starting from the bottom. Reprinted, with permission, from [401].

Figure 108 Average number h of water-water hydrogen bonds on the surface of a flexible lysozyme molecule and in the rigid lysozyme powder shown as functions of JV (number of water molecules per lysozyme) and hydration level h (data from [630]).

Figure 109 Fractal dimension of the largest cluster df as a function of the average number h of H-bonds between water molecules at the surface of rigid (open squares) and flexible (solid squares) lysozymes and in the hydrated lysozymes powder (open circles). Reprinted, with permission, from [631].

P. Pissis, A. Anagnostopoulou-Konsta, Protonic percolation on hydrated lysozyme powders studied by the method of thermally stimulated depolarization currents, J. Phys. D Appl. Phys. 23... 

G. Careri, M. Geraci, A. Giansanti, J. A. Rupley, Protonic conductivity of hydrated lysozyme powders at megahertz frequencies, Proc. Natl. Acad. Sci. U.S.A. 82 (1985) 5342-5346. 

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