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Affinity capillary electrophoresis study of the linkage existing between proton and zinc ion binding to bacitracin A 1
Author(s) -
Castagnola Massimo,
Rossetti Diana Valeria,
Inzitari Rosanna,
Vitali Alberto,
Lupi Alessandro,
Zuppi Cecilia,
Cabras Tiziana,
Fadda Maria Benedetta,
Petruzzelli Raffaele,
Giardina Bruno,
Messana Irene
Publication year - 2003
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200390100
Subject(s) - protonation , chemistry , capillary electrophoresis , titration , imidazole , zinc , crystallography , dissociation constant , electrophoresis , peptide , stokes radius , ion , dissociation (chemistry) , ionic radius , stereochemistry , analytical chemistry (journal) , chromatography , inorganic chemistry , organic chemistry , size exclusion chromatography , biochemistry , receptor , enzyme
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 p K 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 δ‐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 Å. 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 1.5×10 3 M ‐1 intrinsic association constant. Following to Zn +2 binding, the p K 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 Å. 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.