Affinity capillary electrophoresis study of the linkage existing between proton and zinc ion binding to bacitracin A1.
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.
Castagnola
Massimo
M
Institute of Biochemistry and Clinical Biochemistry, Catholic University of Rome, Rome, Italy. m.castagnola@uniserv.ccr.rm.cn.it
Rossetti
Diana Valeria
DV
Inzitari
Rosanna
R
Vitali
Alberto
A
Lupi
Alessandro
A
Zuppi
Cecilia
C
Cabras
Tiziana
T
Fadda
Maria Benedetta
MB
Petruzzelli
Raffaele
R
Giardina
Bruno
B
Messana
Irene
I
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*
D002021:Buffers; D002413:Cations, Divalent; D011522:Protons; D001414:Bacitracin; C026461:bacitracin A; D015032:Zinc