Introducing a Fast Method to Determine the Solubility and Metastable Zone Width for Proteins: Case Study Lysozyme

Abstract

The phase diagrams of tetragonal hen egg white lysozyme containing besides thermodynamic (solubility) also kinetic (nucleation) data were determined for pH values of 4.4, 5.0, and 6.0, 3 to 7 wt % sodium chloride concentrations and lysozyme concentrations from 5 to 70 mg/mL by the use of a turbidity technique. This new technique offers a rapid, precise and reliable determination of nucleation and solubility points. These points can be obtained simultaneously within 6 h. The errors of measurements are less than ±0.9 °C for the solubility temperature and less than ±1.0 °C for the nucleation temperature. The solubility data obtained could be extended and described by a good correlation with literature data. The solubility of lysozyme was found to decrease with increasing salt concentration while the nucleation points were observed more early with respect to salt addition; as a consequence the metastable zone is more narrow. The solubility of lysozyme is slightly reduced at higher values of the pH, and the nucleation point is observed later in time. The result is an increase of the metastable zone width.

Determination of the phase diagram for the crystallization of L‐asparaginase II by a turbidity technique ‐ part II. ‐MPD and crystallography studies‐

“…As shown in Figure , it is obvious that the increasing addition of MPD would not change the shape of crystals. This phenomenon of shifting is in a good agreement with that phase diagram of Lysozyme with varied addition of NaCl .…”

“…Compared with the conventional molecules, in protein crystallization there remain many surprising difficulties what might stem from the complicated structure of proteins and enzymes. To date, Lysozyme, the most common subject of protein crystallization studies, has possessed a set of established phase diagrams . By contrast, phase diagram data for the crystallization of L‐asparaginase II in a given solvent are not to be found except for part I of this paper .…”

Determination of the phase diagram for the crystallization of L‐asparaginase II by a turbidity technique

“…Compared with the phase diagram of Lysozyme including MZW supplemented with NaCl published by Maosoongnern et al , good agreement is obtained between L‐asparaginase II and Lysozyme of the nucleation and solubility curves increase with an increase in temperature. However, Lysozyme possesses a wider MZW of 24 – 25 °C in the presence of NaCl at pH 5.0, while L‐asparaginase II only has a maximum MZW of 8–10 °C in the presence of PEG 6000 or ethanol with the same pH.…”

“…However, Lysozyme possesses a wider MZW of 24 – 25 °C in the presence of NaCl at pH 5.0, while L‐asparaginase II only has a maximum MZW of 8–10 °C in the presence of PEG 6000 or ethanol with the same pH. But it is notable that Maosoongnern et al applied a lower agitate speed of 350 rpm for determination the phase diagram of Lysozyme, while the agitating speed of 500 rpm – 1000 rpm was used in the present work. The increasing of agitating speed would result in narrowing of MZW .…”

“…This device has been successfully previously operated to measure the solubility of model compounds such as paracetamol, mannitol, adipic acid and glycine and the maximum deviation was only 5 wt% compared to the literature data . Furthermore, it has already been applied to supplement the phase diagram of Lysozyme at higher temperature areas by Maosoongnern et al . But it is the first time to utilize this technology to investigate the crystallization behavior of L‐asparaginase II.…”