Modeling the migration behavior of rabbit liver apothioneins in capillary electrophoresis

Abstract

Metallothioneins (MTs) are a group of low-molecular-mass proteins (6-7 kDa) characterized by their great affinity for heavy metal ions. At acidic pH, the bound metal ions are released from the amino acidic structure and MTs lead to apothioneins. In this study, a general equation is used to model the electrophoretic mobility of rabbit liver apothioneins as a function of the pH of the separation electrolyte. The ability of these relationships to explain the migration behavior of these relatively complex polyprotic proteins in the pH range between 2 and 6 has been investigated. Their relevant ionization constant values in the studied pH range were estimated and employed for molecular charge calculations. The classical semiempirical relationships between electrophoretic mobility and charge-to-mass ratio (me vs. q/Malpha) were tested for prediction of their electrophoretic separations. The accuracy of the separations predicted at acidic pH was confirmed by CE-ESI-MS.

Modeling the electrophoretic behavior of quinolones in aqueous and hydroorganic media

“…In a previous study, we found that the classical polymer model (a 5 1/2) yielded excellent correlations when it was employed to study the m e of several peptide hormones and neuropeptides over the pH range between 2 and 12 in aqueous media [10,16]. Several other authors have drawn similar conclusions for different sets of peptides using the Stoke's law, the Offord's surface area law or minor modifications derived from any of the three [1,2,[9][10][11][12][13][14][15][16][17][18]. Several modifications have been suggested to these classical relationships, but in our opinion the classical formulations still providing the best results in the whole pH range [9,10,16].…”

“…The classical semiempirical relationships, which relate electrophoretic mobility and charge-to-mass ratio (m e versus q/M a ), have been extensively used to explain migration behavior of peptides and proteins [1,2,[9][10][11][12][13][14][15][16][17][18]. In general, the m e of a peptide is proportional to its q and inversely proportional to its Stoke's radius (r).…”

“…3). The q/M 2/3 values for each quinolone were obtained at the optimum pH values in the different media (Equation 2 and Table 1) and an electropherogram for the separation of the mixture was easily simulated, by showing a pure Gaussian peak for each quinolone [9,16,35]: where q/M 2/3 are the charge-to-mass ratio values that define the x-scale, h 0 and q i /M i 2/3 are the height and charge-to-mass ratio at the peak maximum for each quinolone, respectively, and s is the SD of the Gaussian peak which is half the value of the width at half height (w 1/2 ) of the simulated electrophoretic peak. A value of 90% was arbitrarily selected as h 0 for all the electrophoretic peaks.…”

“…peptides, quinolones or flavonoids) [3][4][5][6][7][8] until relatively large polyprotic proteins like rabbit liver apothioneins, which bear up to 34 ionizable groups, most of them with similar acidity constant values [9]. The classical semiempirical relationships relating electrophoretic mobility to structural parameters, such as charge and mass or number of amino acid residues have been also extensively investigated for the physicochemical characterization mostly of peptides and proteins [1,2,[9][10][11][12][13][14][15][16][17][18]. It has been demonstrated that if accurate acidity constant values are available, the electrophoretic separations can be easily predicted taking into account the classical semiempirical relationships between electrophoretic mobility and charge-to-mass ratio (m e versus q/M a ) [9,10,16].…”

“…The classical semiempirical relationships relating electrophoretic mobility to structural parameters, such as charge and mass or number of amino acid residues have been also extensively investigated for the physicochemical characterization mostly of peptides and proteins [1,2,[9][10][11][12][13][14][15][16][17][18]. It has been demonstrated that if accurate acidity constant values are available, the electrophoretic separations can be easily predicted taking into account the classical semiempirical relationships between electrophoretic mobility and charge-to-mass ratio (m e versus q/M a ) [9,10,16]. The literature about this topic for analytes different from peptides and proteins is scarce and to the best of our knowledge no papers are reporting the study of these relationships in hydroorganic media [19][20][21].…”