Lysozyme, solubilization

Figure 14.4 Gel image of proteins extracted from a mixed carbonic anhydrase lysozyme tissue surrogate. Lane M, molecular weight marker lane 1, a 1 2 mol ratio mixture of native, non-formalin-treated carbonic anhydrase and lysozyme lane 2, mixed surrogate with 1 2 mol ratio carbonic anhydrase lysozyme, solubilized and retrieved in 20mM Tris-HCl, pH 4.0, with 2% SDS lane 3, mixed surrogate with 1 2 mol ratio carbonic anhydrase lysozyme, solubilized and retrieved in 20mM Tris-HCl, pH 6.0, with 2% SDS. Protein bands corresponding to lysozyme monomer (a), carbonic anhydrase monomer (b), and the putative lysozyme-carbonic anhydrase heterodimer (c) are indicated. For more detail, see Reference 25.

Enzymatic catalysis Phase behavior Phase behavior, UV-visible, lysozyme solubilization Phase behavior, UV-visible, SAXS, lysozyme solubilization Phase behavior, SANS, UV-visible SANS, simulation... 

Another approach developed to increase the solubility of proteins in a bulk aqueous phase is the use of reverse microemulsions. Zhang et al. (84) reported the GAS-based precipitation of lysozyme solubilized in AOT reverse micelles in iso-octane using pressurized CO2. Comparing the on-line UV-vis (ultraviolet-visible) spectra of processed and unprocessed lysozyme, the authors concluded that the lysozyme was not denatured. The use of reverse micelles to dissolve proteins in a bulk organic phase is a promising variation of the GAS technique. The use of reverse micelles could potentially increase the stability of proteins because they would be in a primarily aqueous local environment until precipitation. 

FIGURE S Effect of pH on protein solubilization in AOT-iso-octane system with 0.1 M KCI (O) cytochrome c, ( ) lysozyme, (a) ribonudease-a (from Goklen and Hatton49 by courtesy of Marcel Dekker, Inc.). 

It would be expected that the trend described above should also be verified for proteins. Goklen and Hatton confirmed this hypothesis (38, 39), at least for low molecular weight proteins. They studied the solubilization of three proteins, cytochrome C, ribonuclease and lysozyme of similar size but with distinct pi, from a 1 mg/ml aqueous protein solution into a 50 mM AOT/isooctane organic phase (figure 2) and showed that no extraction occurs for pH > pi, i.e. for pH values where the net charge of the protein is negative. As soon as the pH decreases below the pi value, there is an abrupt enhancement of the protein solubilization the surfactant and the protein bear opposite charges and electrostatic interactions become attractive. The decrease of protein solubilization at low pH values is interpreted by the authors in terms of protein precipitation at the interface due to denaturation. 

Figure 2. Effect of pH on solubilization of three proteins in AOT system (o) cytochrome c (pl=10.6) ( ) lysozyme (pl=ll.l) (A) ribonuclease (pl=7.8). (Reproduced with permission from Ref. 38. Copyright 1987 M. Dekker.)...

A more complete analysis must also include the nature of the ions (45). Thus, as an example, Goklen (46) noted that calcium ions permitted to solubilize cytochrome c beyond its pi although it was not possible with potassium. At a given pH, Leser et al. (47) reported that at 0.1 M salt concentration, solubilization of ribonuclease, lysozyme and tr)q)sin were more efficient with CaCl2 than with MgCl2. Taking these results into account, it is not surprising to note that the choice of the buffer influences solubilization as well. Results on the influence of the pH are thus undissociable from the type of buffer used in the experiments. 

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