Lysozymes

Lysozyme is a 14.4-kDa cationic protein (pi 10) with the ability to kill a wide range of Gram-positive bacteria. It is present in both azurophilic and specific granules of neutrophils and is also found in the granules of monocytes and macrophages, in blood plasma, tears, saliva and airway secretions. In human neutrophils it is present at 1.5-3 jug/106 cells. Since its discovery in 1922 by Fleming, it has been widely studied by protein biochemists, and its three-dimensional structure has been precisely defined. It exerts its ef- 

Lysozyme has often been chosen as a simple example of how an enzyme works. It is said that Alexander Fleming (who later discovered penicillin) when he had a cold, at one time let the drips from his nose fall onto a bacterial colony on a Petri dish. Rather than throw it away, he kept it to see what would happen. He discovered that his nasal discharge inhibited the growth of the bacterium and this led to the discovery of the mildly antibiotic substance lysozyme in tears. He gave it this name because it is 

Because lysozyme is a protein, the presence of phenolics as well as the degree of clarification will affect activity. Plant phenolics are well-known to react with enzymes, thereby decreasing activity (Rohn et al., 2002). Reflecting this, lysozyme is more active in white wines than reds, most likely due to the differences in polyphenolic content (Daeschel et al., 2002  

Delfini et al., 2004). In support, Daeschel et al. (2002) reported that activity of lysozyme was greatly reduced in a Cabernet Sauvignon wine after 28 days but not in Riesling. Delbni et al. (2004) reported that lysozyme binds to suspended solids thus decreasing activity after clarification. 

Lysozyme may also influence protein stability in white wines. Thus, fining trials should be conducted prior to bottling wines treated with the enzyme. Although evidence is lacking, utilization of lysozyme may have an indirect sensory impact on palate structure similar to that of proteinaceous fining agents used in red wines. 

As discussed in section 8.2.5, lysozyme has been isolated from the milk of a number of species human and equine milks are especially rich sources. In view of its antibacterial activity, the large difference in the lysozyme content of human and bovine milks may have significance in infant nutrition. It is claimed that supplementation of baby food formulae based on cows milk with egg-white lysozyme gives beneficial results, especially with premature babies, but views on this are not unanimous. 

Aggregates obtained by heating lysozyme (from Sino-American Biotechnology Co.) solution at 80°C for 30 min. 

The cell walls of gram-positive bacteria are formed from peptidoglycan (synonymous with murein). Peptidoglycan consists of repeating units of the disaccharide N-acetylglucosamine 

The objective of a study of Van Nostrum group (Verheyen et al., 2010) was to develop a method to functionalize LZM with methacrylamide functions, for subsequent immobilization and controlled release from a hydrogel network. LZM has six amine groups in the side chains of the lysine residues and one amine at its fV-terminus. The reactivity of these seven groups is different and well documented. Three of them, localized on the surface of the macromolecule, are most reactive and readily available to 

The modification reaction is well controlled and the number of linkers introduced per protein molecule can be tailored by changing the reaction conditions. LZM can be modified with up to three methacrylamide moieties without major conformational changes. The lytic activity was still 60% after introducing an average of 2.3 methacrylamide units. The modified LZM was successfully immobilized into a hydrogel network and subsequently released by reduction of the degradable linker. 

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Hayward, S., Kitao, A., Berendsen, H.J.C. Model-free methods to analyze domain motions in proteins from simulation A comparison of normal mode analysis and molecular dynamics simulation of lysozyme. Proteins 27 (1997) 425-437. 

In an early study of lysozyme ([McCammon et al. 1976]), the two domains of this protein were assumed to be rigid, and the hinge-bending motion in the presence of solvent was described by the Langevin equation for a damped harmonic oscillator. The angular displacement 0 from the equilibrium position is thus governed by... 

Simulation of Small Ligands Bound in T4-lysozyme L99A... 

Extraction of Bound Xenon from Mutant T4-Lysozyme... 

Fig. 2. Work performed to extract xenon from T4 lysozyme L99A in 27 independent simulations of 100 ps each.

Hermans, J., Wang, L. Inclusion of loss of translational and rotational freedom in theoretical estimates of free energies of binding. Application to a complex of benzene and mutant T4-lysozyme. J. Am. Chem. Soc. 119 (1997) 2707-2714... 

Mann, G., Prins, J., Hermans, J. Energetics of forced extraction of ligand Simulation studies of Xe in mutant T4 lysozyme as a simple test system. Bioohys. J., in preparation (1998)... 

Morton, A., Baase, W. A., Matthews, B. W. Energetic origins of specificity of ligand binding in an interior nonpolar cavity of T4 lysozyme. Biochemistry 34 (1995) 8564-8575. 

The critical factor for any method involving an approximation or an extrapolation is its range of application. Liu et al. [15] demonstrated that the approach performed well for mutations involving the creation or deletion of single atoms. The method has also been successfully applied to the prediction of the relative binding affinities of benzene, toluene and o-, p-, and m-xylene to a mutant of T4-lysozyme [16]. In both cases, however, the perturbation to the system was small. To investigate range over which the extrapolation may... 

The presented algorithm was applied to 4 proteins (lysozyme, ribonuclease A, ovomucid and bovine pancreatic trypsin inhibitor) containing 51 titratable residues with experimentally known pKaS [32, 33]. Fig. 2 shows the correlation between the experimental and calculated pKaS. The linear correlation coefficient is r = 0.952 the slope of the line is A = 1.028 and the intercept is B = -0.104. This shows that the overall agreement between the experimental and predicted pKaS is good. 

Tanford, C., Roxby, R. Interpretation of protein titration curves Application to lysozyme. Biochem. 11 (1972) 2192-2198. 

A detailed examination of LN behavior is available [88] for the blocked alanine model, the proteins BPTI and lysozyme, and a large water system, compared to reference Langevin trajectories, in terms of energetic, geometric, and dynamic behavior. The middle timestep in LN can be considered an adjustable quantity (when force splitting is used), whose value does not significantly affect performance but does affect accuracy with respect to the reference trajectories. For example, we have used Atm = 3 fs for the proteins in vacuum, but 1 fs for the water system, where librational motions are rapid. 

Table 3. Percentage error for LN compared to reference Langevin trajectories (at 0.5 fs) for energy means and associated variances for lysozyme over 60 ps at 7 = 20 ps At = 0.5 fs, Atm = 3 fs, and At = k Atm, where k2 ranges from 1 for LN 1 to 96 for LN 96.

Another view of this theme was our analysis of spectral densities. A comparison of LN spectral densities, as computed for BPTI and lysozyme from cosine Fourier transforms of the velocity autocorrelation functions, revealed excellent agreement between LN and the explicit Langevin trajectories (see Fig, 5 in [88]). Here we only compare the spectral densities for different 7 Fig. 8 shows that the Langevin patterns become closer to the Verlet densities (7 = 0) as 7 in the Langevin integrator (be it BBK or LN) is decreased. 

Fig. 11. The Speedup of LN at increasing outer timesteps for BPTI (2712 variables), lysozyme (6090 variables), and a large water system (without nonbonded cutoffs 37179 variables). For lysozyme, the CPU distribution among the fast, medium, and slow forces is shown for LN 3, 24, and 48.

B. R. Brooks and M. Karplus. Normal modes for specific motions of macromolecules Application to the hinge-bending mode of lysozyme. Proc. Natl. Acad. Sci. USA, 82 4995-4999, 1985. 

The visuahzation of hundreds or thousands of connected atoms, which are found in biological macromolecules, is no longer reasonable with the molecular models described above because too much detail would be shown. First of aU the models become vague if there are more than a few himdied atoms. This problem can be solved with some simplified models, which serve primarily to represent the secondary structure of the protein or nucleic acid backbone [201]. (Compare the balls and sticks model (Figure 2-124a) and the backbone representation (Figure 2-124b) of lysozyme.)... 

Figure 2-124. The most common molecular graphic representations of biological molecules (lysozyme) a) balls and sticks b) backbone c) cartoon (including the cylinder, ribbon, and tube model) and of inorganic molecules (YBajCujO , d) polyhedral (left) and the same molecule with balls and sticks (right),...

Weber, P. L. Buck, D. R. Capillary Electrophoresis A Past and Simple Method for the Determination of the Amino Acid Composition of Proteins, /. Chem. Educ. 1994, 71, 609-612. This experiment describes a method for determining the amino acid composition of cyctochrome c and lysozyme. The proteins are hydrolyzed in acid, and an internal standard of a-aminoadipic acid is added. Derivatization with naphthalene-2,3-dicarboxaldehyde gives derivatives that absorb at 420 nm. Separation is by MEKC using a buffer solution of 50 mM SDS in 20 mM sodium borate. 

Fig. 13. Preferential iateraction parameter vs lyotropic number for lysozyme on (° ) bovine semm albumin and ( ) Toyopead.

Many enzymes have been the subject of protein engineering studies, including several that are important in medicine and industry, eg, lysozyme, trypsin, and cytochrome P450. SubtiHsin, a bacterial serine protease used in detergents, foods, and the manufacture of leather goods, has been particularly well studied (68). This emphasis is in part owing to the wealth of stmctural and mechanistic information that is available for this enzyme. 

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