Isoelectric focusing and non‐isoelectric precipitation of ferritin in immobilized pH gradients: An improved protocol overcoming protein‐matrix interactions

Abstract

It has been impossible, up to now, to focus ferritin (a large, ca. 450 000 Da, ironstorage protein ubiquitous in mammalian systems) in immobilized pH gradients (IPG). This was not a sieving problem, as ferritin focuses readily in a 4%T polyacrylamide (or polytrisacryl) gel in conventional carrier ampholyte isoelectric focusing. A strong interaction with the IPG matrix was suspected, but an auto-buffered (pH 6.0) soluble IPG polymer failed to precipitate ferritin from solution, although it altered its visible spectrum, suggesting the formation of soluble complexes. By a Takeo plot, the dissociation constant (Kd)of the ferritin-IPG matrix complex was estimated to be 0.1 mM. However, even a 100-fold dilution of Immobilines into the IPG matrix with a concomitant addition of competing ions (up to 3 % carrier ampholytes) failed to produce good focusing patterns. An unsuspected, hitherto unreported, precipitating power of free Immobilines in solution on the ferritin macroion was found to be the key to the riddle. The precipitating ions are (in order of aggregation power): pK 9.3 > pK 7.0 > pK 6.2. None of the acidic Immobilines (pKs 3.6,4.4 and 4.6) and, among the basic, only the pK 8.5, exhibit any precipitation power on ferritin and other complex protein mixtures. In a new IPG pH 4-5 interval, containing only the pK 4.6 as buffering and the pK 8.5 Immobilines as titrant ions, excellent focusing patterns of ferritin were obtained. It is hypothesized that the precipitating Immobilines act as cationic detergents, and are able to cross-link macroions with a mixed-type, hydrophobic-ionic interaction at the two extremities of the molecule. Guidelines are given for handling this major problem on a short-term basis, the longterm goal being the replacement of the 'unfavorable' Immobilines (pKs 9.3,7.0 and 6.2) by new chemical structures of the desirable type (the non-precipitating pK 8.5 species). P. G. Righetti el a1Electrophoresis 1987,8,62--70

Two-Dimensional Electrophoresis of Membrane Proteins Using Immobilized pH Gradients

“…It was noted quite early that resolution of some hydrophobic proteins (membrane proteins) was poor and others were lost when separated by IPGs. These observations were attributed to hydrophobic interactions between the proteins and the basic acrylamido derivatives of the IPG matrix (9,10). More recently the protein patterns were compared after running 2-D gels with either CA-IEF or IPG-IEF for some membrane preparations (11).…”

Isoelectric Focusing with Immobilized pH Gradients

“…It is a fact that, in the absence of CAs, these same proteins would essentislly fail to enter the gel and mostly precipitate or g i v e elongated smears around the application site (in general cathodic sanple loading). As the same pnenomenon recently happened even with ferritin, a cytoplasmic, freely soluble protein, we were forced to explore in m r e aecail the effect of CAs on iPG matrices ana to broaden the meaning of our first explanation [30]. It was found that, on a relative hydrophobicity scale, the four basic imbilines (pKs 6.2.…”

Recent Advances in Capillary Electrophoresis of DNA Fragments and PCR Products in Poly(N-substituted Acrylamides)

“…In 1992, Zewert and Harrington (40,41), proposed a poly(ethylene glycol) producing more porous gels. Despite its advantages, its use in isoelectric focusing (29,30) and DNA separations methacrylate-acrylamide copolymer for electrophoresis, as an alternative to agarose and polyacrylamides. (31) was not satisfactory, due to its inherent instability in alkaline solutions (it degrades with zero-order kinet-Although these authors additionally reported its use for separating hydrophobic proteins (42), their results ics!)…”