Quantitation of protein binding to the capillary wall in acidic, isoelectric buffers and means for minimizing the phenomenon

Notwithstanding the use of acidic, amphoteric, isoelectric buffers with iso electric points (pl) in the pH 2-3 range, adsorption of proteins to the nak ed silica wall can be non-negligible. Two such buffers have been tested: im inodiacetic acid (IDA; pi 2.23, apparent pH 3.2 in 7 M urea) and aspartic a cid (pl 2.77, apparent pH 3.7 in 7 M urea). Three potential quenchers of su ch interactions have been tested: hydroxyethylcellulose (HEC; number averag e molecular mass, M-r 27 000), TEPA (tetraethylenepentamine) and a novel, q uaternarized piperazine [N(methyl-N-omega-iodobutyl)-N'-methylpiperazine] ( Q-Pip), either alone or in binary and ternary mixtures. Human alpha- and be ta-globin chains have been used as test proteins in capillary electrophores is separations. It has been found that mixtures of these compounds are the worst possible remedy. E.g., a ternary mixture comprising 0.5% HEC, 0.5 mM TEPA and 1 mM Q-Pip still leaves behind 4.5% adsorbed protein onto the sili ca surface in runs in IDA buffer and 7 M urea (pH 3.2). Conversely, 0.5 mM TEPA or 1 mM Q-Pip, when used alone, minimize adsorption down to only 1.8% and 0.5%, respectively. When the same globin chain separations are performe d in Asp and 7 M urea (pH 3.7), the situation is much worse: 44% protein is adsorbed in a ternary mixture of 0.5% HEC, 1 mM Q-Pip and 0.5 mM TEPA. How ever, when used alone, 0.5 mM TEPA and 1 mM Q-Pip reduce globin adsorption to levels of 8% and 5%, respectively. TEPA and Q-Pip are found to be in all cases the best quenchers of protein interaction to naked fused-silica; in addition they exhibit the unique property of smoothing the base-line and gi ving reproducible runs. The best method for desorbing bound protein was fou nd to be an electrophoretic step consisting in driving sodium dodecylsulpha te micelles from the cathodic reservoir. (C) 2000 Elsevier Science B.V. All rights reserved.