Lysozyme heat capacity

Hen egg-white lysozyme, lyophilized from aqueous solutions of different pH from pH 2.5 to 10.0 and then dissolved in water and in anhydrous glycerol, exhibits a cooperative conformational transition in both solvents occurring between 10 and 100°C (Burova, 2000). The thermal transition in glycerol is reversible and equilibrium follows the classical two-state mechanism. The transition enthalpies AHm in glycerol are substantially lower than in water, while transition temperatures Tm are similar to values in water, but follow similar pH dependences. The transition heat capacity increment ACp in glycerol does not depend on the pH and is 1.25 0.31 kj (mol K) 1 compared to 6.72 0.23 kj (mol K)-1 in water. Thermodynamic analysis of the calorimetric data reveals that the stability of the folded conformation of lysozyme in glycerol is similar to that in water at 20-80°C but exceeds it at lower and higher temperatures. 

Fig. 2. Temperature dependence of the partial specific heat capacity for pancreatic ribonuclease A (RNase), hen egg-white lysozyme (Lys), sperm whale myoglobin (Mb), and catalase from Thermus thermophilus (CTT). The flattened curves are for RNase and Lys with disrupted disulfide cross-links and for apomyoglobin, when polypeptide chains have a random coil conformation without noticeable residual structure (Privalov et al., 1988).

Figure 8. A DSC record (a thermogram) from a thermally induced transition of lysozyme in dilute aqueous solution. Tt = transition temperature ACp = change in heat capacity accompanying the unfolding process. The hatched area is proportional to the enthalpy of transition. Adapted from Privalov (1980).

Fig. 4. Profile of a differential scanning calorimetry experiment done on a synthetic lysozyme. The heat capacity (kilocalories per degree per mole) of the unfolding process was monitored as a function of temperature on a Micro-Cal MC2 instrument. The transition midpoint of protein unfolding corresponds to the temperature at the peak of the curve, and the thermodynamic parameters A H and A Cp are evaluated by the procedure of Privalov.33...

As an example, we will consider the molecular dynamical behavior of egg white lysozyme. The temperature dependence of mobility of fluorescence, spin and Mossbauer labels attached to lysozyme was found to be similar to other investigated proteins the monotonic increase typical for rigid polymers in dry states and in samples with water content (wt) was less than the critical value (wtcr) and drastically burst when wt > wtcr at T > 200 K took place (Frolov et al., 1978 Likhtenshtein, 1979). At similar conditions, experiments on the temperature dependence of heat capacity indicated only a monotonic steady increase for rigid organic material. Recently, in the fully dried lysozyme crystal, similar monotonic behavior of heat capacity was observed in temperatures between 8 and 30°C. At D20 content more than 24 wt %, a slight deviation from the monotony was observed at temperatures above approximately 185 K, which most probably is due to the eutectic melting of NaCl/2H20 present in the samples to prevent water crystallization (Miyazaki et al., 2000). 

Experimental results from studies of Arrhenius dependence of different characteristics of lysozyme are presented in Fig 4.1. (Alfimova and Likhtenshtein, 1979 Likhtenshtein, 1993 Likhtenshtein et al., 2000). The discontinuities on the curves indicate local conformational transitions and are apparently due to the appearance of a more open conformation of the protein. As can be seen from Fig. 4.1., these methods reveal conformational transitions at a temperature of about 30°C, whereas the temperature dependence of the partial heat capacity decreases monotonically in this temperature region. Recently, the presence of the conformational transition in lysozyme was confirmed independently. It was shown that the segmental motion of Trp 108 is hindered by the local cage structure at T < 30°C, although relieved from restricted motion by thermal agitation or by the formation of a ligand complex. 

Miyzaki, Y., Matsua, T., and Suga, H. (2000) Low-temperature heat capacity and glassy behavior of lysozyme crystal J. Phys. Chem. B 104, 8044-8052. 

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. 3. Specific heat of the lysozyme-water system from 0 to 1.0 weight fraction of protein. Least-squares analysis of the linear portion of the heat capacity function from 0 to 0.73 weight fraction of water gives = 1.483 0.009 J K g ...

Figure 4 gives the apparent specific heat capacity [< >(Cpj)] of lysozyme... 

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).

The heat capacity measurements for lysozyme are consistent with data obtained for other globular protein systems, for example, ovalbumin (Bull and Breese, 1968b Suurkuusk, 1974), chymotrypsinogen (Hutchens et ai, 1969 Suurkuusk, 1974), and insulin (Hutchens et al, 1969). For a discussion see Yang and Rupley (1979). 

Blake et al. (1983) refined the structures of human lysozyme (HL) and tortoise egg white lysozyme (TEWL) to 1.5 and 1.6 A resolution, respectively. The diffraction was modeled as arising from three components the protein, ordered water, and disordered water. Most of the water in the crystals (i.e., 60—80%) is disordered. The analysis located 143 molecules of ordered water out of about 350 per HL molecule, and 122 molecules out of 650 per TEWL molecule. The ordered water covers 75% of the available surface of the the protein. One-third (TEWL) to one-half (HL) of the total surface is unavailable for analysis of the adjacent water, owing to crystal contacts or disorder in the protein region. Thus, the estimate of surface coverage is in good agreement with the 300 molecules of water estimated by heat capacity measurements as full hydration (0.38 h). The area covered per water molecule is estimated as 18.9... 

The process of protein hydration is the stepwise addition of water to dry protein, until the hydration end point is reached. Heat capacity measurements (Yang and Rupley, 1979) serve as a framework on which to develop a picture. Figure 38 gives the dependence on hydration level of various time-average properties of lysozyme, over the hydration range 0-0.4 h, from the dry protein to slightly beyond the end point of the process. Curve d shows the dependence of the apparent specific heat on hydration level. It is directly related to the extent to which the thermal response of the lysozyme-water system deviates from ideal behavior. The nonideality of the system shows three discontinuities at 0.07, 0.25, and 0.38 h. 

Fig. 38. Comparison of heat capacity and spectroscopic properties. Effect of hydration on lysozyme time-average properties. (Curve a) Carboxylate absorbance (1580 cm" ) (curve b) amide I shift ( 1660 cm" ) (curve c) OD stretching frequency (—2570 cm ) (curve d) apparent specific heat capacity (curve e) diamagnetic susceptibility. From Rupley elal. (1983).

Pfeil (1981) concluded that a-lactalbumin is less stable than lysozyme, with a lower thermal transition temperature, lower denaturational enthalpy, lower heat capacity change, and lower Gibbs free-energy change. 

Since the values of the thermal diffusivity for myoglobin and water are close, the discrepancy between their thermal conductivities is largely due to differences in their respective heat capacities. At 300 K the specific heat of water is 4.2 J K 1 g-1, whereas we calculate the specific heat of myoglobin to be 1.0 J K 1 g 1 at 300 K. This latter value is reasonably close to measured specific heats for proteins. For example, the specific heat of lysozyme in dilute aqueous solution is 1.5 J K-1 g , and it is 1.3 J K-1 g-1 for the dry protein [154,155]. Still, we should bear in mind that at 300 K the partial specific heat of a hydrated protein may be some 50% larger than the specific heat of a dry protein [156], so that the coefficient of thermal conductivity may be correspondingly larger. 

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. 

The dependence of the heat of sorption on the extent of coverage has been observed to be Irregular, with an extremum in the knee region of the isotherm (, 7). A calorimetric study W has demonstrated a similar Irregularity in the hydration of polysaccharides. The extremum in the heat of sorption for lysozyme ( ) corresponds with one in the heat capacity (see below) that reflects proton redistribution. 

Figure 1 shows the heat capacity of the lysozyme-water... 

Figure 2. Apparent specific heat capacity of lysozyme from 0 to 0.45 g of water...

As noted already, the hydration level above which the protein heat capacity is constant defines completion of the hydration process. The value estimated for lysozyme is 0.38 g of water/g of protein, equivalent to 300 molecules of water/molecule of lysozyme. With regard to other thermodynamic measurements, the sorption Isotherm is not able to define completion of the hydration process, and there can be difficulty in Interpreting scanning calorimetric experiments in terms of completion of hydration, because different states of the system are being compared (frozen and solution, or native and denatured) and during a scanning calorimetric measurement the system is not at equilibrium, allowing reaction rates to influence the response. 

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... 

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. 

---