Hydrophobic interaction between alkaline immobilines and ferritin during isoelectric focusing in immobilized pH gradients.

It has been found that three alkaline Immobilines (out of seven weak acids and bases used to generate immobilized pH gradients), having pK values of 6.2, 7.0 and 9.3, act as cross-linking agents, aggregating and precipitating out of solution ferritin and other large macromolecules (e.g., from serum and tissue extracts) present in body fluids and human biopsies. All the acidic Immobilines (pK 3.6, 4.4 and 4.6) and the basic species of pK 8.5 appear to be unreactive. The three precipitin Immobilines mimic cationic detergents, acting on the basis of two different principles at the opposite extremes, by ionic interaction at one end and by hydrophobic bonding at the other end of the molecule. The ionic type of interaction was clearly demonstrated, owing to its sensitivity to pH extremes and to progressively increasing ionic strength. The hydrophobic interaction in the region of the double bond (Immobilines are N-substituted acrylamido acids and bases) was deduced on the basis of the following observations: (a) oxidation of the double bond with introduction of a vicinal diol totally inhibited ferritin aggregation; (b) addition of SH groups to the double bond increased protein precipitation and (c) the protein-Immobiline aggregates were found to be sensitive to alkyl-substituted ureas (especially ethyl- and propylurea), which are known to bind to hydrophobic regions of proteins, and insensitive to urea, which is known to split only hydrogen bonds. Interestingly, neutral and zwitterionic detergents were unable to split the Immobiline-ferritin complexes, suggesting that their large micelles could not have access to the tightly packed Immobiline cross-linking region.