Studies On The Properties Of Gallium(Ⅲ) And Chromium(Ⅲ)-Transferrins And The Mechanism Of Their Transport

The interactions of gallium(â…¢) or chromium(â…¢) with transferrins and the transport mechanism of transferrin-receptor meditated metal-transferrin by cells have been investigated at a molecular level and cell level in order to reveal rules of the compounds in anticancer and treatment in diabetes. The results are shown as follows:Properties of gallium(â…¢) or chromium(LLI) binding to transferrins at a molecular level1. Ovotransferrin is an important member of transferrin family and has a dual role in both the transport of iron and the antibacterial function. However, the releasing and binding of a metal ion are different in the N-terminal and C-terminal binding site of ovotansferrin. It maybe be ascribed to the different structure of the both binding sites. To explain the case we have studied the interactions of Ga³⁺ and these phenolic hydroxl compounds such as 8-hydyoxyl quinoline, salicylic acid, EHPG and HBED and ovotransferrin. We try to research the disparity of hydrogen-bonds of those compounds by fluorescence spectra technique. The founding implies that the O-H…O type intramolecular hydrogen bonds for the phenolic groups of Tyr 92 and Tyr 191 residues at the N-terminal binding site and there are O-H…N ype intramolecular hydrogen bonds for Tyr431 and Tyr524 residues at the C-terminal binding site.2. Detailed studies on characterization of Cr³⁺ binding to apoovotransferrin were conducted by means of the fluorescence and difference UV spectra. Thework below was carried out:(1). The conditional stability constants and site selectivitywhen HCO₃^- or C₂O₄²- as a synergistic anion , respectively, the interaction of Cr³⁺ with ovotransferrin has been studied at pH 7.4 and 0.1M Hepes, room temperature and air-saturated by the fluorescence spectra. It indicates Cr³⁺ prefers the C-terminal site to the N-terminal site. The conditional stability constants for lgK_N =5.65±0.12 and lgK₂= 13.08±0.24 were determined by using Modkf by difference UV spectra.(2). The conformational change experimentation(a) The alternation of the surrounding of tryptophan residues buried inside the protein was free from interference from Cr3+ by the synchronous fluorescent approach, (b) The binding of Cr3+ with apoovotransferrin was conducted by means of a resonance light scattering technique. The finding showed the fluorescent intensity was gradually increased in Xmax- The volume increase for Cr3+ binding to N-terminal ovotransferrin was larger than that for its corresponding partner. The result implied the conformational change of ovotransferrin rose upon Cr3+ binding to apoovotransferrin. (c) It has no effect on the shape of globe of the total protein molecule involved in Cr3+ binding to apoovotransferrin by fluorescent polarization method, (d) The results shows TNS may bind to the hydrophobic region of helix 5 in domain N2 due to pivoting on helix 11 in the conformational change with the addition of a metal ion. TNS and Tb3+ are located in the same place of Tf. (e)The binding of Cr3+ to apoOTf leads to stable structure of OTf.(3). The rates at which Cr3+ was removed from the C-terminal monochromium ovotransferrins by citrate were evaluated by UV difference spectra in 0.1 M Hepes, pH 7.4 and at 37 °C. The results indicate that the pathway of Cr3+removal from the C-terminal binding site is to the combination of saturation and first-order kinetics, but letter is main way.3. The preparation and research of half-molecule transferrins are an important bridge to understand the relation between the structure and function. Iron was added to apoOTf to 30% saturation or Tb3+ was added toapoOTf to 40% saturation and protein was digested with trypsin at 37°C and pH 8.1. The N-terminal half-molecule of OTf was obtained by means of gel electrophoresis, UV-Visible and fluorescence spectra technique. By comparing the interaction of apoOTf, N-terminal half-molecule and TNS, a conclusion can be drawn that their structure are different. The result suggested that cooperative interactions between the lobes exit.The mechanism of transferrin-receptor meditated metal-transferrin transport at a cell level1. The mechanism of Gac-Tf-FeN transporting into and out of HL60 cells has been investigated by comparing transports between Gac-Tf-FeN and apoTf by means of 125I-labeled transferrin. An association constant for Gac-Tf-FeN was two times that for apoTf. Gac-Tf-FeN and apoTf of cell surface-bound displayed similar kinetics during the uptake, but the release rates of internalized Gac-Tf-FeN and apoTf from cells were different which showed characteristic disparate. The release continued to occur during the incubation of Gac-Tf-FeN in the presence of nonradioactive apoTf. Neither NaN3 nor NH4CI could completely block internalization of Gac-Tf-FeN, but they prevented the release of Gac-Tf-FeN from the cells. Excess cold unlabeled apoTf could overcome the block in the release due to NH4CI but not NaN3. The binding and internalization of Gac-Tf-FeN could be competitively inhibited by nonradioactive apoTf. It implies that both bind to the same receptor on the membrane and the localization of Gac-Tf-FeN resembles that of apoTf inside cells. Pretreated cells with pronase abolished the binding of Gac-Tf-FeN significantly. On the basis of these findings, the "transferrin receptor" for the mechanism of Gac-Tf-FeN transport is proposed by HL60 cells.2. Cell-associated counts (CPM) studies on the uptake of 125I labled transferrin contain Cr3+ species by K562 cells showed that like apoTf, Cr-Tf and Cr2-Tf can bind to the cell receptors. The sequence of three species binding to transferrin is apoTf