Immobilized pH gradients for isoelectric focusing: Interaction between histones and histone‐like proteins with the charged polyacrylamide matrix

It has been found that polyacrylamide matrices containing immobilized pH gradients interact strongly with at least two classes of proteins, histones and the histone‐like, ‘high‐mobility group’ (HMG) chromatin proteins, forming insoluble complexes. The nature of these interactions has been demonstrated to be purely ionical: the complexes are split by high ionic strength (0.5 M NaCl) and by altering the pH (full disaggregation being obtained at pH 5.5 and at pH 11.5). On the basis of these observations, soluble Immobiline polymers have been made, by polymerizing them in the absence of cross‐linker (N,N′‐methylenebisacrylamide). Several classes of Immobiline polymers can be produced: a) mixed‐type polymers, containing a blend of basic and acidic Immobilines, in such ratios as to obtain any desired pH in solution; b) homopolymers with a pure carboxyl surface; and c) homopolymers with a pure amino surface. These polymers can be synthesized as ‘mono‐pK species’ or as ‘multiple‐pKcoils’, the latter providing a full range of charges over an ample pH‐interval. It has been found that ‘carboxyl’ Immobiline homopolymers preferentially interact with the HMGl component among the HMG class, thus allowing the partial purification from a lysate by a simple precipitation step.