Hen egg-white proteins

Hofmeister series — The Hofmeister series (HS) originates from the ranking of anions and cations toward their ability to precipitate a mixture of hen egg white proteins [i]. This protein precipitation can be explained simply in terms of the extent of ions binding to water (i.e., as a salting-out effect). The HS has been shown to have a much more general utility with a broad range of biophysical phenomena, which include the stability and crystallization of biological macromolecules, enzyme activity, DNA-protein interactions, etc. The traditional and extended HS [ii] is shown as... 

Based on fractionation data for hen egg white proteins including hen egg white lysozyme, ovalbumin, and avidin. 

Analysis of the Debye—Waller B factors suggests that 33—35 waters are strongly bound. These are located mostly at positions that are equivalent in the HL and TEWL structures. Hagler and Moult (1978) noted the similarity in water positions determined for two crystal forms, triclinic and tetragonal, of hen egg white lysozyme. The waters found for the hen egg white proteins are also largely equivalent to ones found for HL and TEWL. These observations suggest that essential features of the water structure in the crystal are intrinsic properties of the hydrated protein and would be found also in the solution state. 

In addition to the studies of various crystalline forms of domestic hen egg-white lysozyme, the structures of human and tortoise egg-white lysozymes have been determined (for crystal data see Table III). Artymiuk and Blake (1981) refined the structure of the human enzyme to 1.5 A resolution. The main objectives of this study were to determine the extent of differences in structure from that of the hen egg-white protein, to discover the location of water molecules, and to test the validity of the method of restrained refinement. The particular restrained least-squares approach to refinement described in their paper appears to have been validated. The two proteins were found to be closely homologous, but there were small differences (e.g., in a helices), details of which can be obtained from consulting their paper. 

In the 13 a-lactalbumins listed in Table VI, seven have His contents of three residues per molecule, four have four residues, and two have two residues. Four of the mammalian lysozymes have two His residues, the remainder varying from one to five residues. Four of the hen egg-white proteins have no His residues, the remainder varying from one to five residues. 

Salts are known to influence several properties of aqueous solutions in a systematic way (122,123). The effect of different aiuons and cations seems to be ordered in a sequence this theory was already proposed by Hofmeister in 1888 (124) from a series of experiments on the salts ability to precipitate hen-egg white protein. Numerous other properties of aqueous salt solutions are also found to be systematically salt dependent, such as the surface tension or the surface potential (122). However, the exact reason for the observed specific cation and anion sequences is still not fully understood (125). Model calculations (126), as well as nuclear magnetic relaxation experiments (127), propose a delicate balance between ion adsorption and exclusion at the solute interface. This balance is tuned by the solvent (water) stmcture modification according to the ion hydration (128, 129) and hence is possibly subject to molecular details. 

Figure 3. Comparison of lysozyme adsorption on hydrophobic (DDS), negatively-charged (silica), and positively-charged (APS) surfaces. Step adsorption isotherms for the hen egg-white protein are plotted in Panel A, and for the human milk protein in Panel B.

Phosphoamino acids that are part of proteins known to bind metal ions are posttranslational modifications introduced by specific protein kinases (Meggio et al, 1981 Vogel and Biidger, 1982c). The bovine milk protein casein and the hen egg-white protein ovalbumin, as well as possibly the human saliva acidic proline-iich proteins share sequence homology of their phosphorylated sites. Dephosphorylation of such sites by enzymatic phosphatase treatment usually reduces the affinity of such proteins for metal-ion binding (Bennick et al., 1981). Hence it is likely that dianionic phosphoryl moieties are directly involved in the complexation of metal ions. This seems particularly important for the two polyelectrolyte proteins that contain large amounts of phosphoserine residues, phosvitin purified from egg yolk (Ta-borsky, 1974), and the phosphoprotein purified from dentine (Linde et al, 1980). 

Fig. 6-3. Column chromatography of hen egg-white proteins on DE52 using 0.025 M-Tris/HCl buffer, pH 7.5 at (a) laboratory scale (1.5 em i.d. x 15.5 cm) at a flow rate of 2 mL min and (b) process scale (45 cm i.d.xl6cm) at a flow rate of lOOOmLmin. ...

Fig. 6-6. Rechromatography using DE52 of individual fractions eluting during the saturation loading of a laboratory column (1.5 cm i.d.xl5.5 cm) of DE52 with lOmgmL hen egg-white proteins. Samples of Fraction 1 (a), Fraction 2 (b) and Fraction 8 (c) were chromatographed at laboratory scale (1.5 cm i.d.x 15.5 cm) using 0.025 M-TVis/HCl buffer at a flow rate of 1 mL min . ...

Both attractive forces and repulsive forces are included in van der Waals interactions. The attractive forces are due primarily to instantaneous dipole-induced dipole interactions that arise because of fluctuations in the electron charge distributions of adjacent nonbonded atoms. Individual van der Waals interactions are weak ones (with stabilization energies of 4.0 to 1.2 kj/mol), but many such interactions occur in a typical protein, and, by sheer force of numbers, they can represent a significant contribution to the stability of a protein. Peter Privalov and George Makhatadze have shown that, for pancreatic ribonuclease A, hen egg white lysozyme, horse heart cytochrome c, and sperm whale myoglobin, van der Waals interactions between tightly packed groups in the interior of the protein are a major contribution to protein stability. 

Privalov et al (1989) studied the unfolded forms of several globular proteins [ribonuclease A, hen egg white lysozyme, apomyoglobin (apoMb), cytochrome c, and staphylococcal nuclease]. Unfolding was induced by 6 M Gdm-HCl at 10°C, heating to 80°C, or by low pH at 10°C with cross-links cleaved (reduction and carboxamidomethylation or removal of heme). The unfolded forms showed CD spectra (Fig. 27)... 

Squaraine dyes 10b, 39a, 39b, 41a, 41c, 41d, and 41e were used to measure different proteins such as BSA, HSA, ovalbumin, avidin from hen egg white, lysozyme, and trypsin (Fig. 12) [58]. It is difficult to predict correlations between the dyes structures and the affinity or sensitivity of the dyes for different proteins. All squaraine probes exhibit considerable fluorescence increases in the presence of BSA. Dicyanomethylene-squaraine 41c is the brightest fluorescent probe and demonstrates the most pronounced intensity increase (up to 190 times) in presence of BSA. At the same time, the fluorescent response of the dyes 10b, 39a, 39b, 41a, 41c, 41d, and 41e in presence of other albumins (HSA and ovalbumin) is, in general, significantly lower (intensity increases up to 24 times). Dicyanomethylene-squaraine 41a and amino-squaraines 39a and 39b are the most sensitive probes for ovalbumin. Dyes 41d, 10b, and 41e containing an A-carboxyalky I -group demonstrate sufficient enhancement (up to 16 times) in the presence of avidin. Nevertheless, the presence of hydrolases like lysozyme or trypsin has only minor effects on the fluorescence intensity of squaraine dyes. 

We found that solutions of hen egg white lysozyme, bovine ribonuclease A (RNase A), or a 1 2 mol ratio of bovine carbonic anhydrase lysozyme formed opaque gels within 2 min when mixed with an equal volume of 20% NBF.25,26 Multi-protein tissue surrogates comprised of 50% w/v lysozyme and up to four additional proteins have also been formed (Fowler et al., unpublished results). After overnight fixation, the surrogates were firm and sliced easily with a razor blade for sampling. To determine the optimal... 

Fig. 11.14. Positive-ion ESI spectra of (a) hen egg white lysozyme and (b) the protein after ad(htion of 1,4-dithiothreitol. Reproduced from Ref. [88] by permission. American Chemical Society, 1990.

Beau has applied the DCC concept to a number of carbohydrate systems [13-16]. Carbohydrate-binding proteins often exhibit weak ligand interactions with millimolar dissociation constants, a quite different scenario to the enzyme-small molecule DCLs discussed thus far. The protein target initially chosen for study was hen egg-white lysozyme (HEWL), a glycosidase known to bind A-acetyl-o-glucosamine (d-G1cNAc) with... 

Material. 3 X crystallized, dialyzed and lyophilized Grade 1 Hen Egg White Lysoz3mie was obtained from Sigma Chemical Company, and used without further purification. Purity of the protein was verified by both gel electrophoresis on acrylamide and chromatography on a column packed with anion exchange resin (Cl form) Sephadex DEAE. The sample showed a single peak in these analyses. 

Besler M, Steinhart H, Paschke A (1997) Allergenicity of hen s egg-white proteins IgE binding of native and deglycosylated ovomucoid. Food Agric Immunol 9 277-288... 

It has been suggested that the exons could correspond to functional units of proteins and that new proteins could have evolved by the recombination of different exons.27 Early circumstantial evidence was consistent with this idea. For example, hen egg white lysozyme DNA contains four exons. Exon 2 has the... 

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