Different binding thermodynamics of Ni2+, Cu2+, and Zn2+ to bacitracin A1 determined by capillary electrophoresis.
Thermodynamics of the binding of Ni(2+), Cu(2+) and Zn(2+) to bacitracin A(1) was studied by capillary electrophoresis measuring the peptide effective mobility at different pH in the presence of increasing concentration of the three ligands. The affinity follows the order Ni(2+) > Cu(2+) > Zn(2+), with association constant values of (2.3 +/- 0.1)x10(4), (4.9 +/- 0.2)x10(3), and (1.5 +/- 0.1)x10(3) M(-1), respectively. The only model able to rationalize mobility data implies that metal ion binds to the P(0) peptide form. Moreover, mobility values indicated a change of bacitracin A(1) acidic properties on Ni(2+) and Cu(2+) binding, with a shift of the pK(a) of N-terminal Ile-1 from 7.6 to about 5 and of the pK(a) of the delta-amino group of D-Orn-7 from 9.7 to about 7. Even though on Zn(2+) binding a shift of the N-terminal Ile-1 pK(a) was observed, restrictions in the pH range suitable for investigation, due to precipitation phenomena, did not allow establish if the shift of D-Orn-7 lateral chain pK(a) also occurred. Nonetheless, if present, the shift should be limited to the 7.8-9.7 range. Mobility data indicated that the Stokes radius of the complexes is ca. 3 A lower than that of the free peptide. The present results indicate that metal-ion binding to bacitracin A(1) is more complex than previously assumed.
Castagnola
Massimo
M
Institute of Biochemistry and Clinical Biochemistry, Catholic University of Rome, Largo F. Vito 1, I-00168 Rome, Italy. m.castagnola@icrm.cnr.it
Rossetti
Diana V
DV
Inzitari
Rosanna
R
Lupi
Alessandro
A
Zuppi
Cecilia
C
Cabras
Tiziana
T
Fadda
Maria B
MB
Onnis
Giuseppina
G
Petruzzelli
Raffaele
R
Giardina
Bruno
B
Messana
Irene
I
D001414:Bacitracin / Q000737:chemistry* / Q000378:metabolism; D002413:Cations, Divalent / Q000737:chemistry / Q000378:metabolism; D003300:Copper / Q000737:chemistry* / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D015394:Molecular Structure; D009532:Nickel / Q000737:chemistry* / Q000378:metabolism; D011522:Protons; D013816:Thermodynamics; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism
D002413:Cations, Divalent; D011522:Protons; C492695:bacitracin A1; D001414:Bacitracin; D003300:Copper; D009532:Nickel; C026461:bacitracin A; D015032:Zinc