Protein precipitation induced by alkaline Immobilines for isoelectric focusing in immobilized pH gradients: Causes and remedies

Thierry Rabilloud, Cecilia Gelfi, Maria Luisa Bossi, Pier Giorgio Righetti

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The mechanism by which three basic Immobilines (pK's 6.2, 7.0 and 9.3) precipitate proteins in solution seems to be due to the presence in these three monomers of variable amounts of oligomers, spontaneously formed during storage by polymerization. These polymerization products range in size from short oligomers (dimers and trimers) to polymers with a molecular mass more than 2000 Da. Oligomers are present in all batches of precipitating Immobilines in substantial amounts, usually at least 15–20%. Only the pK 8.5 Immobiline, in general unable to precipitate proteins from solution, has been found to contain low levèls of oligomers, typically less than 3%, possibly due to intrinsic stability against polymerization, or to its ability to spontaneously hydrolyze in water solutions. None of the batches of pK 6.2, 7.0 and 9.3 Immobilines, unable to precipitate proteins in solution, was found to contain polymers but exhibited a bimodal elution peak from a Bio‐Gel P‐2 sieve gel chromatography, probably corresponding to dimers and trimers. Upon mild polymerization (highly diluted persulfate in presence of oxygen) all these batches were found to precipitate proteins and to contain higher oligomers and polymers. By testing column eluates, it was found that the precipitation power begins at the level of polymers with 10–12 residues, suggesting that these relatively short chains already possess a strong cooperativity in their chemical behavior in solution. Work is in progress to find a suitable solution for completely inhibiting two of the most noxious reactions occurring to Immobilines: hydrolysis and polymerization. There still remains a third problem, which will require a longer‐term investigation: the intrinsic hydrophobicity of the four basic Immobilines. The basic Immobiline homopolymers present two unlike groups for interaction: ionic on one side, hydrophobic on the opposite end. Acidic Immobilines, even when reacted to form much larger polymers, are unable to precipitate ferritin from solution, suggesting that their strongly hydrophilic surface suppresses one of the two binding regions. In a similar manner, when batches of pK 7.0 Immobiline, able to precipitate ferritin, where rendered more hydrophilic by methylolation, their protein precipitation power was abolished, even, though the polymers had not been eliminated. While waiting for the synthesis of more ‘hydrophilic’ Immobilines, a partial remedy is to admix carrier ampholytes (CA) to Immobiline gels. The CA's act as shielding ions, minimizing hydrophobic interactions among proteins and the Immobiline matrix. Mixed CA‐IEF gels should never be prefocused, as the shielding action of CA is maximal in the unfocused state.

Original languageEnglish
Pages (from-to)305-312
Number of pages8
Issue number7
Publication statusPublished - 1987

ASJC Scopus subject areas

  • Biochemistry
  • Clinical Biochemistry


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