Capillary isoelectric focusing: the problem of protein solubility.

A whole family of protein solubilizers, compatible with native structure and maintenance of enzyme activity, is reported for preventing protein precipitation and aggregation at the pI value under conditions of very low ionic strength, as typical of isoelectric focusing methodologies. In addition to mild solubilizers proposed in the past, such as glycols (glycerol, ethylene and propylene glycols) non-detergent sulphobetaines, in concentrations up to 1 M, are found to be quite effective in a number of cases. Other common zwitterions, such as taurine and a few of the Good's buffers (e.g., Bicine, CAPS) are also quite useful in acidic pH gradients and up to pH 8. Addition of sugars, notably saccharose, sorbitol and, to a lesser extent, sorbose (20% in capillary IEF and in the 30 to 40% concentration range in gel-slab IPGs), greatly improved protein solubility in the proximity of the pI. The improvement was dramatic if these sugars were mixed with 0.2 M taurine. In the case of hydrophobic peptide antibiotics, mixtures of 6 M urea and 25% trifluoroethanol were found to markedly improve solubility. All these additives, unlike non-ionic or zwitterionic surfactant, have the advantage of remaining monomeric, i.e., of being unable to form micelles, even at concentrations >1 M. Thus, their elimination from the protein zone can be easily accomplished by gel filtration or by centrifugation through dialysis membranes. Using these additives, capillary IEF of proteins should be now applicable to a number of difficult cases, such as the separation of mildly hydrophobic macroions.