An electron paramagnetic resonance study of bovine alpha-lactalbumin-metal ion complexes.
alpha-lactalbumin has at least three distinct cation binding regions: a Ca(II)-Gd(III) site, a Cu(II)-Zn(II) site and a VO2+ site as observed from electron paramagnetic resonance (EPR) studies of complexes with the bovine protein. Gadolinium, which bound to the calcium site of the protein with a subnanomolar dissociation constant, yielded EPR spectra at 9.5 GHz (X-band) that exhibited features from g = 8 to g = 2. At 35 GHz (Q-band) the central fine structure transition (Ms = 1/2----Ms = -1/2) gave a well-defined powder pattern. The zero-field splitting was large, as reflected in the second-order splitting of the central fine structure transition of about 1 kG. There was also evidence for additional, low affinity binding site(s) for Gd(III). Addition of either Zn(II) or Al(III) did not affect the amplitudes or positions of the bound Gd(III) EPR spectrum. The Cu(II)-alpha-lactalbumin complex gave a typical axially symmetric spectrum (g parallel = 2.260, g perpendicular = 2.056, A parallel = 171 G) with a partially resolved superhyperfine interaction attributable to at least one directly coordinated nitrogen ligand. Addition of Cu(II) to Gd(III)-alpha-lactalbumin gave an EPR spectrum that was a superposition of signals from the individual Gd(III)- and Cu(II)-alpha-LA spectra. The absence of any magnetic interactions in the Gd(III)-Cu(II)-alpha-lactalbumin species indicated that the two cation sites were more than 10 A apart. On the other hand, addition of Zn(II) to Cu(II)-alpha-lactalbumin gave a set of EPR lines due to free or loosely bound Cu(II), confirming that the Cu(II) was displaced by zinc.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D003300:Copper / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D005682:Gadolinium / Q000378:metabolism; D007768:Lactalbumin / Q000378:metabolism*; D008670:Metals / Q000378:metabolism*; D011485:Protein Binding; D014638:Vanadates; D014639:Vanadium / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D008670:Metals; D014639:Vanadium; D014638:Vanadates; D003300:Copper; D007768:Lactalbumin; D005682:Gadolinium; D015032:Zinc; D002118:Calcium