Proton 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

Abstract

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)

Toward a property/function relationship for metallothioneins: Histidine coordination and unusual cluster composition in a zinc‐metallothionein from plants

“…The pH‐dependence of the resonances of histidine imidazole ring protons has been used previously to probe histidine metal coordination 52, 53. Even in the absence of a complete sequential assignment for E C , resonances corresponding to the H(δ2) and H(ε1) imidazole ring protons of His32 and His40 may be readily observed as sharp signals in the low‐field region of 1D 1 H NMR spectra [Fig.…”

The pH-Induced Release of Iron from Transferrin Investigated with a Continuum Electrostatic Model

“…The conventional view has been that, in vivo, iron-loaded transferrin binds to its cellular receptor, is internalized, releases its iron at the lower intravesicular pH (5.5), and is then recycled to the outside of the cell as apoprotein (for recent reviews see Qian and Tang, 1995;De Silva et al, 1996). The only experimentally determined pK a values that have been reported for transferrins, however, are for some of the histidine residues in half-molecules of ovotransferrin (Woodworth et al, 1987) and for some of the histidine residues in human serum apo-transferrin (Kubal et al, 1994). No similar results are available for human lactoferrin, which also has a somewhat different distribution of histidine residues.…”

Role for an Essential Tyrosine in Peptide Amidation

“…Complexes of Fe(III) and Ga(III) show similar ligand exchange rates and pK a values for coordinated water. Gallium has been used as a structural analog of Fe(III) in ironbinding proteins such as transferrin (65)(66)(67), lactoferrin (68), and ovotransferrin (48,69), and the invariant ferric site iron in bovine spleen PAP can be functionally substituted with Ga(III) (59). Because the Fe(III)-tyrosinate site of PALcc is only partially filled, we expected the addition of trivalent metals such as Ga(III) to increase activity.…”