title: Affinity capillary electrophoresis study of the linkage existing between proton and zinc ion binding to bacitracin A1.
abstract: 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.
authors.affiliation: Institute of Biochemistry and Clinical Biochemistry, Catholic University of Rome, Rome, Italy. m.castagnola@uniserv.ccr.rm.cn.it
authors.forename: Massimo
authors.initials: M
authors.lastname: Castagnola
authors.forename: Diana Valeria
authors.initials: DV
authors.lastname: Rossetti
authors.forename: Rosanna
authors.initials: R
authors.lastname: Inzitari
authors.forename: Alberto
authors.initials: A
authors.lastname: Vitali
authors.forename: Alessandro
authors.initials: A
authors.lastname: Lupi
authors.forename: Cecilia
authors.initials: C
authors.lastname: Zuppi
authors.forename: Tiziana
authors.initials: T
authors.lastname: Cabras
authors.forename: Maria Benedetta
authors.initials: MB
authors.lastname: Fadda
authors.forename: Raffaele
authors.initials: R
authors.lastname: Petruzzelli
authors.forename: Bruno
authors.initials: B
authors.lastname: Giardina
authors.forename: Irene
authors.initials: I
authors.lastname: Messana
mesh_terms: D001414:Bacitracin / Q000032:analysis / Q000737:chemistry*; D002021:Buffers; D002413:Cations, Divalent; D019075:Electrophoresis, Capillary / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D008433:Mathematics; D015394:Molecular Structure; D009994:Osmolar Concentration; D011485:Protein Binding; D011522:Protons; D013997:Time Factors; D015032:Zinc / Q000737:chemistry*
chemical_list: D002021:Buffers; D002413:Cations, Divalent; D011522:Protons; D001414:Bacitracin; C026461:bacitracin A; D015032:Zinc