PubMed · 13652775
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D KASSAY, Z LABAS. 1959. A study on intersegmental septa.. https://doi.org/10.1378/chest.35.6.625
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A new, simple and accurate micellar electrokinetic chromatography (MEKC) method is established for quantification of hydrocortisone, polymyxin B and Zn-bacitracin in local pharmaceutical preparations. The separation was carried out at 25 degrees C and 25 kV, using a 15 mmol L(-1) phosphate-15 mmol L(-1) borate buffer (pH 8.2), 60 mmol L(-1) sodium dodecylsulfate (SDS), and 10% methanol-water (v/v) as background electrolyte. Under these conditions the analysis takes about 23 min. The method has been applied for quantifying these compounds in two different commercial pharmaceutical products and the method gave good results when compared with a reference spectrophotometric multivariate calibration method.
Binding of Zn(2+) to bacitracin A(1) was studied by capillary electrophoresis in water/2,2,2-trifluoroethanol (70/30 v/v) at different apparent pH values in order to estimate the association constant of metal, the acidic dissociation constants and the Stokes radii of both free and bounded peptide in apolar environment. The Stokes radii of the free peptide species were compared with those in aqueous solution, as obtained in a recent study performed by our group, indicating that apolar environment stabilizes bacitracin A(1) in a conformational structure with the lateral chain of apolar amino acids exposed on the external surface. This conformation of the macrocyclic dodecapeptide is ready to interact with Zn(2+) ion, as pointed out by the strong increase of the association constant measured in water/2,2,2-trifluoroethanol with respect to the value obtained in aqueous solution. In addition, whereas Zn(2+) ion binding in aqueous solution provides a sensible reduction of peptide Stokes radius, no sensible variations following to ion binding were observed in hydro-organic solution. The present results suggest that the apolar environment, rather than the metal ion binding, could be responsible for the conformational transition that brings bacitracin A(1) towards its biologically active structure.*
Measurements by capillary electrophoresis (CE) of bacitracin A(1) effective mobility at different pH values permitted to estimate the five acidic dissociation constants and the Stokes radii at different protonation stages of the macrocyclic dodecapeptide. The pK(a) values were 3.6 and 4.4 for the two carboxylic groups of the lateral chains of D-Asp-11 and D-Glu-4, respectively, 6.4 for the aza-atom of the imidazole ring of His-10, 7.6 for the amino group of N-terminal Ile-1 and 9.7 for the delta-amino group of D-Orn-7, very close to the values obtained by other researchers by titration experiments. In agreement with a rigid macrocyclic structure the Stokes radii of different protonated forms ranged only between 14.3 and 14.8 A. Best fitting procedures performed on experimental mobility measured at two different pH values (5.50 and 6.72) in the presence of increasing Zn(+2) concentration allowed confirming the model that assumes the binding of Zn(+2) to P(0) peptide form with a 1.5 x 10(3) M(-1) intrinsic association constant. Following to Zn(+2) binding, the pK(a) of the amino group of N-terminal Ile-1 is shifted from 7.6 to 5.9 and the Stokes radius is reduced of about 3 A. The mean charge of the bacitracin A(1)-Zn(+2) complex resulted +1.67 and +1.12 at pH 5.50 and 6.72, respectively. These results suggest that the amino group of N-terminal Ile-1 is not essential for Zn(+2) binding.