Lysozyme, isoelectric point

Just as individual amino acids have isoelectric points, proteins have an overall p/ because of the acidic or basic amino acids they may contain. The enzyme lysozyme, for instance, has a preponderance of basic amino acids and thus has a high isoelectric point (p/= 11.0). Pepsin, however, has a preponderance of acidic amino acids and a low- isoelectric point pi 1.0). Not surprisingly, the solubilities and properties of proteins with different pi s are strongly affected by the pH of the medium. Solubility- is usually lowest at the isoelectric point, where the protein has no net charge, and is higher both above and below the pi, where the protein is charged. 

The effect of pH on the protein adsorption on CMK-3 was also investigated [152], The monolayer adsorption capacities obtained under various pH conditions are plotted in Figure 4.12, where the maximum adsorption was observed in the pH region near the isoelectric point of lysozyme (pi of about 11). Near the isoelectric point, the net charges of the lysozyme molecule are minimized and would form the most compact assembly. A similar pH effect was also seen in the adsorption of cytochrome c on CM K-3. Although the nature of the surface of mesoporous silica and... 

The following polar amino acid compositions with their internal pK values have been determined for two small proteins, lysozyme and calmodulin. Estimate their isoelectric points. 

The study of the VF IVg = f(C) dependence and the determination of cmin has been performed also for lysozyme solutions at different pH values and for NaDoBS solution with 0.1 mo dm 3 NaCl added [84,95]. Fig. 10.10 depicts the dependence of the probability for observation of black spots AN/N (curve 1) for films from lysozyme solutions with different concentrations at pH = 11.45 (isoelectric point). The techniques is described in Chapter 2. Izmailova and Yampolskaya [96] have investigated foam films from lysozyme solutions and found the concentration of black films formation c = 2.35-10"6 mol dm 3. As reported in [84], this concentration corresponds to a 100% probability for formation of stable black films. To the beginning of black spot formation in films from lysozyme solution with probability 5% corresponds the concentration of 1.82-1 O 6 mol dm 3. The expansion of black spots and formation of a black film occurs, as a rule, from the periphery to the centre, analogous to that for films from BSA solutions and its mixtures with lysozyme [97]. 

The lowest residual concentration of lysozyme found by extrapolation of the linear segment of the VFIVg = f(C) dependence gives the value of 2.1-10 6 mol dm 3 which is close to the concentration of the onset of black spot formation (Fig. 10.10, curve 2). At pH values less than the isoelectric point that corresponds to the formation of thick films from solutions of such surfactants [96], the VF/Vg = f(C) dependence has an S-shaped course. 

If thin films are formed in a system (NaDoS and NaDoBS in the absence of electrolyte, or lysozyme, that is not in its isoelectric point) the lower concentration limit cannot be distinguished unambiguously. In these systems the minimum concentration is found from the particular conditions under which the process is conducted (rate of gas feed, dispersity, foam height in the column, etc.) and the limit is no clearly expressed. 

Lysozyme shows a high buffer capacity in the acidic region around pH 5 and has almost no buffer function above pH 10. It appears that this lowest point is around the isoelectric point of lysozyme, which is reported to be about pH 11 (22], Ribonuclease has a good buffer function in the acidic region around pH 5... 

Because of the high isoelectric point and limited molecular size, it is not surprising that isolation of the lysozymes has proceeded generally along fairly similar paths. In the isolation from milk and egg white from a variety of species, the lysozyme has been adsorbed on an ion exchanger, such as Amberlite IRC-50 (Rohm and Haas, Philadelphia, PA), CM-cellulose (Whatman Inc., Clifton, NJ), CM-Sephadex (Pharmacia, Uppsala, Sweden), and BioGel-CM 30 (Bio-Rad, Richmond, CA), from an ammonium sulfate fraction of the original egg white, or skim milk. After elution it is often purified further by gel filtration on Sephadex G-50 or another suitable medium. Whitaker (1963) noted abnormal retention on Sephadex gel under certain conditions. He attributed this to interaction with the dextran, possibly related to the enzymatic function. 

Kronman et al. (1965) and Kronman and Holmes (1965) appear to be the first to have studied the effects of acid on a-lactalbumin and report that this protein, adjusted to pH values below its isoelectric point, exhibits a hypsochromic shift in its absorption spectrum between 270 and 300 nm. Spectral shifts in this region usually reflect changes in the environment of Trp and Tyr residues. The conformational change is a complex one, involving a series of steps. Because of the nature of the shift, the numbers of Trp and Tyr residues present, and the relative magnitudes of e for Trp and Tyr, Kronman and co-workers concluded that the shift results from environmental alterations for more than one of the buried Trp residues. (At the time of this study, three Trp residues were considered to be buried in bovine a-lactalbumin.) There appears to be no corresponding effect for hen egg-white lysozyme. [However, note the effect of acetic acid studied by Kato et al. (1984).]... 

One feature of the ruminant and Old World monkey stomach lysozymes is their low isoelectric points (pH —7.0-9.0) compared with the high pH value (10.0-12 0 for many lysozymes. Arg appears to have been selected against, and L vi selected for, during the recent evolut> a-ary history of the ruminant lysozymes (Stewart and Wilson, 1987). Lys and Arg are considered generally to be the epitome of conservative, neutral replacements (Zuckerkandl and Pauling, 1965 Jukes, 1978). 

Rezwan, K., Meier, L.P, and Gauckler, L.J., A prediction method for the isoelectric point of binary protein mixtures of bovine serum albumin and lysozyme adsorbed on colloidal titania and alumina particles, Langmuir, 21, 3493, 2005. 

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