1H NMR study of effects of synergistic anion and metal ion binding on pH titration of the histidinyl side-chain residues of the half-molecules of ovotransferrin.
Separation of ovotransferrin into C-terminal (OTf/2C) and N-terminal (OTf/2N) half-molecules has made possible the resolution of all expected histidinyl C(2)H resonances by proton nuclear magnetic resonance at 250 MHz. The chemical shift of many of the resonances decreases with increasing pH, allowing construction of titration curves, whereas a few resonances fail to titrate. On formation of the GaIIIOTf/2(C2O4) ternary complexes, two of the low-field C(2)H resonances in each half-molecule fail to titrate. This behavior implicates the imidazole groups giving rise to these resonances as ligands to the bound metal ion. A third C(2)H resonance in each half-molecule undergoes a marked reduction in pK'a on formation of the ternary complex. The imidazole group displaying this resonance is implicated in a proton-relay scheme involved in binding the synergistic anion, oxalate, and a water of hydration on the bound metal ion. The titration curves for the various imidazole resonances have been fit to a four-parameter equation involving estimation of the pK'a, the limiting chemical shift values, and a Hill constant n. Hill constants of less than 1 can be rationalized by correcting the titration curve for the charge Z on the protein as a function of pH and the work function w. The titration curve for the imidazole group in OTf/2C involved in the proton-relay scheme shows a value for n greater than 1, which suggests positive cooperativity in the titration of this residue. The basis for this behavior cannot be rationalized at this time.(ABSTRACT TRUNCATED AT 250 WORDS)
D000838:Anions; D001665:Binding Sites; D003207:Conalbumin / Q000378:metabolism*; D004527:Egg Proteins / Q000378:metabolism*; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008670:Metals / Q000378:metabolism; D010070:Oxalates / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D014508:Urea / Q000378:metabolism
D000838:Anions; D004527:Egg Proteins; D046911:Macromolecular Substances; D008670:Metals; D010070:Oxalates; D003207:Conalbumin; D006639:Histidine; D014508:Urea