Abstract

Abstract

In order to explore the transfer mechanism of chromium(III) in mammals, a novel complex [Cr(ASA)(en)₂]Cl· 2H₂O, bis(ethylenediamine-κ²N,N′)(4-aminosalicylic acid-κ²O,O′) chromium(III) monochloride dihydrate was synthesized (4-aminosalicylic acid=H₂ASA, ethylenediamine=en). The crystal structure belongs to orthorhombic system with the space group P2₁2₁2₁ by means of X-ray diffraction. The characteristic for transfer of Cr³⁺ from the compound to the low-molecular-mass chelator EDTA and the iron-binding protein apoovotransferrin (apoOTf) was followed by UV-visible (UV-Vis) and fluorescence spectra in 0.01 mol·L⁻¹ Hepes at pH 7.4. The second order rate constants were calculated. Those spectra in conjunction were used to obtain more accurate information about the interaction of chromium complex with apoOTf. The experimental results indicate that Cr³⁺ can be transferred from the complex to apoOTf with the retention of the 4-aminosalicylic acid acting as a synergistic anion.

The interaction between mercuric ion and apoCopC in the absence or presence of cupric ion was investigated through difference UV spectra in Hepes buffer (10 mmol·L⁻¹) at pH 7.4. The results suggest that mercuric ion can bind to C- and N-terminal binding sites of apoCopC, and the conditional binding constants were calculated to bek_N=(6.79±1.12)×10⁶ mol⁻¹·L and k_C=(3.06±0.05)×10⁵ mol⁻¹·L. Using urea as a chemical agent, the conformational stabilities of apoCopC and Hg²⁺_N-CopC-Hg²⁺_C were monitored by fluorescence spectrum in Hepes buffer (50 mmol·L⁻¹) at pH 7.4. The free energy of stabilization is (14.69±0.85) and (16.66±0.55) kJ·mol⁻¹, respectively. Hg²⁺_N-CopC-Hg²⁺_C is more stable than apoCopC.

The interaction of gallium(III) with the ligands containing phenolic group(s), such as salicylic acid, 8-hydroxyquinoline, N,N'-bis(2-hydroxybenzyl)ethylenediamine-N,N′-diacetic acid (HBED), N,N′-ethylenebis[2-(o-hydroxyphenyl)glycine (EHPG), and ovotransferrin, was studied, respectively, by means of fluorescence in 0.01 mol/L Hepes at pH 7.4 and room temperature. Fluorescence intensity showed an increase when gallium(III) was bound to 8-hydroxyquinoline and HBED. In contrast, it was decreased with the interaction of gallium(III) with salicylic acid and EHPG. At pH 7.4, there was N···HO type intramolecular hydrogen bond in the former, and the latter existed O···HO type intramolecular hydrogen bond. Fluorescence titration of apoovotransferrin with gallium(III) displayed that the fluorescence intensity was decreased at the N-terminal binding site, while enhanced at the C-terminal binding site. It can account for the O···HO type intramolecular hydrogen bonds for the phenolic groups of Tyr92 and Tyr191 residues at the N-terminal binding site. And there are N···HO type intramolecular hydrogen bonds for Tyr431 and Tyr524 residues at the C-terminal binding site. In addition, under the same conditions, the conditional binding constant of gallium(III) with EHPG or HBED determined by fluorescence method is lg K_Ga-EHPG=19.18 or lg K_Ga-HBED=19.08.

The rates at which aluminum was removed from the N- and C-terminal monoaluminum ovotransferrins by pyrophosphate were evaluated by UV difference spectra in 0.01 mol/L Hepes, pH=7.4 and at 37 °C. Pesudo first-order rate constants as a function of pyrophosphate concentration were measured. The results indicate that the pathways of aluminum removal are different. For the N-terminal binding site, aluminum removal follows simple saturation kinetics, while the removal of aluminum from the C-terminal binding site reverts to the combination of saturation and first-order kinetics. The saturation component is consistent with a rate-limiting conformational change in the protein as has been reported. We propose that the first-order kinetics mechanism is attributed to a pre-equilibrium process. The rate constants of saturation kinetics are accelerated from both terminals with the addition of 0.1 mol/L chloride to the monoaluminum ovotransferrin solutions, whereas the rates of the first-order kinetics are decreased for the C-terminal binding site. The effect of chloride ionic strength causes a continuing increase on k_obs for the N- and C-terminal binding sites. Moreover, the kinetics behavior of the N-terminal is more easily affected by chloride than that of the C-terminal. In the experiment presumably the N-terminal site is apparently kinetically more labile than the C-terminal site.