Protein adsorption to the bare silica wall in capillary electrophoresis quantitative study on the chemical composition of the background electrolyte for minimising the phenomenon.
A novel method is reported for quantifying protein adsorption to naked silica tubings and for assessing the efficacy of amino quenchers added to the background electrolyte. It consists of flushing a fluorescently-labelled protein (myoglobin) into a capillary equilibrated in Tris-acetate buffer, pH 5.0, until full saturation of the potential adsorbing sites. Desorption is then affected by driving electrophoretically sodium dodecyl sulphate (SDS) micelles into the capillary from the cathodic reservoir: the peak of eluted material is quantified fluorometrically by using a dual laser beam instrument able to read the fluorescein-isothiocyanate-labelled myoglobin at 520 nm and the internal standard (sulphorodamine) at 630 nm. As potential quenchers, a series of monoamines have been investigated (triethylamine, triethanolamine, ethylamine), followed by diamines (putrescine, cadaverine and hexamethonium bromide) and finally by oligoamines [spermidine, spermine and TEPA (tetraethylenepentamine), i.e., a tri- a tetra- and a pentamine, respectively]. Two values of molarities have been derived: a value at 50% (a kind of a dissociation constant) and a value at 90% inhibition of binding of macromolecules to the silica surface. According to these figures of merit, mono- and diamines are rather poor quenchers of interaction with the wall, since the 50% values are of the order of 50-100 mM and the 90% values reach as high as 560 mM. On the contrary, oligoamines, especially spermine and TEPA, are most effective, since the 50% molarities are in the sub-millimolar range and the 90% values are of the order of ca. 1 mM. Figures of merit have also been derived for different washing procedures. Those most commonly adopted in routine practice, i.e., of washing with either 1 M NaOH or with 1 M HCl, or with both, leave behind traces of proteins still bound to the wall, whereas the SDS micelle electrophoretic desorption seems to be 100% effective.
Verzola
B
B
University of Verona, Department of Agricultural and Industrial Biotechnology, Italy.
Gelfi
C
C
Righetti
P G
PG
D000327:Adsorption; D000588:Amines / Q000737:chemistry; D004573:Electrolytes / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000379:methods; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D008823:Micelles; D009211:Myoglobin / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D011786:Quality Control; D012822:Silicon Dioxide / Q000737:chemistry*; D012967:Sodium Dodecyl Sulfate
D000588:Amines; D004573:Electrolytes; D005456:Fluorescent Dyes; D008823:Micelles; D009211:Myoglobin; D011506:Proteins; D012967:Sodium Dodecyl Sulfate; D012822:Silicon Dioxide; D016650:Fluorescein-5-isothiocyanate