188 J. A. Asenjo and J B Chaudhuri The fact that electrostatic interactions play an important role in the distribution of oteins over reverse micellar and aqueous phase is shown by the dependence of the aqueous phase pH and ionic strength The ph of the solution will affect the solubilisation characteristics of a protein prima rily in the way in which it modifies the charge distribution over the protein surface. With creasing pH the protein becomes less positively charged until it reaches its isoelectric point (pl). At pHs above the pI the protein will take on a net negative charge. If electro- static interactions play a significant role in the solubilisation process, partition with anionic surfactants should be possible only at pHs below the pl of the protein, where the protein is positively charged and electrostatic attractions between the protein and the surfactant head groups are favourable. At pHs above the pl, electrostatic repulsions ould inhibit protein solubilisation Goklen and Hatton(1987) have presented results on the effect of ph on solubilisation tochrome-c, lysozyme, and ribonuclease-A, in AOT/isooctane reverse micelle solu tions. The results were presented as the percentage of the protein transferred from a I mg/ml aqueous protein solution to an equal volume of isooctane containing 50 mM of the anionic surfactant AOT. A summary of their results is presented in Table 7.3 Table 73. Effect of pH on solubilisation Protel pH range of maximum solubilisation 10 5-10 l1,.1 6-11 As anticipated, only at pHs lower than the pI was there any appreciable solubilisation of a given protein, while above the pl the solubilisation appears to be totally suppressed However, at extremes of pH there is a drop in the degree of solubilisation of the proteins due to protein denaturation, observed as precipitate formation at the interface( Chaudhuri eral.,1993) Luisi et al.( 1979)used the quaternary ammonium salt methyl-trioctylammonium chloride(TOMAC) for the transfer of a-chymotrypsin from water to cyclohexane. It was found that the pH had to be reduced to values significantly below the pI (pI =8.)for there to be any appreciable solubilisation. The solubilisation occurred only over a very narrow ph range before decreasing rapidly again with further decreases in the ph of the aqueous feed phase, accompanied by precipitation at the interface Similar results have been obtained by Dekker et al. (1986) for the enzyme a-amylase Significant solubilisation of the enzyme was observed over a narrow pH range in the vicinity of 10-10.5(pI=5.1). In this pH range, all basic residues will be deprotonated d the only charged residues being the carboxyl groups bearing a negative charge188 J. A. Asenjo and J. B. Chaudhuri The fact that electrostatic interactions play an important role in the distribution of proteins over reverse micellar and aqueous phase is shown by the dependence of the aqueous phase pH and ionic strength. The pH of the solution will affect the solubilisation characteristics of a protein prima￾rily in the way in which it modifies the charge distribution over the protein surface. With increasing pH the protein becomes less positively charged until it reaches its isoelectric point (PI). At pHs above the PI the protein will take on a net negative charge. If electro￾static interactions play a significant role in the solubilisation process, partition with anionic surfactants should be possible only at pHs below the PI of the protein, where the protein is positively charged and electrostatic attractions between the protein and the surfactant head groups are favourable. At pHs above the PI, electrostatic repulsions would inhibit protein solubilisation. Goklen and Hatton (1987) have presented results on the effect of pH on solubilisation of cytochrome-c, lysozyme, and ribonuclease-A, in AOT/isooctane reverse micelle solu￾tions. The results were presented as the percentage of the protein transferred from a 1 mg/ml aqueous protein solution to an equal volume of isooctane containing 50 mM of the anionic surfactant AOT. A summary of their results is presented in Table 7.3. Table 7.3. Effect of pH on solubilisation Protein PI pH range of maximum solubilisation cy tochrome-c 10.6 5-10 ribonuclease-A 7.8 1-7 lysozyme 11.1 6-1 1 As anticipated, only at pHs lower than the pl was there any appreciable solubilisation of a given protein, while above the PI the solubilisation appears to be totally suppressed. However, at extremes of pH there is a drop in the degree of solubilisation of the proteins due to protein denaturation, observed as precipitate formation at the interface (Chaudhuri et al., 1993). Luisi et al. (1979) used the quaternary ammonium salt methyl-trioctylammonium chloride (TOMAC) for the transfer of a-chymotrypsin from water to cyclohexane. It was found that the pH had to be reduced to values significantly below the PI (PI = 8.3) for there to be any appreciable solubilisation. The solubilisation occurred only over a very narrow pH range before decreasing rapidly again with further decreases in the pH of the aqueous feed phase, accompanied by precipitation at the interface. Similar results have been obtained by Dekker et al. (1986) for the enzyme a-amylase. Significant solubilisation of the enzyme was observed over a narrow pH range in the vicinity of 10-10.5 (PI = 5.1). In this pH range, all basic residues will be deprotonated and the only charged residues being the carboxyl groups bearing a negative charge