Study of the Binding of Iron and Indium to Human Serum Apo ...

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Moshtaghie & Ghaffaritrichloride (3 mM) were prepared in deionizedwater and mixed with the equal volume of 60 mMcitric acid solution. The solutions were adjusted topH 7.4 with 1 N NaOH and made up to a finalvolume of 50 ml by deionized water [10].Spectrophotometric titration technique for metalbinding to apo-tf. The binding of iron and/orindium to apo-tf was carried out usingspectrophotometric titration technique. Thistechnique was performed at room temperature(25°C). To achieve spectrophotometric titration forbinding of either iron or indium to apo-tf, 200 µlapo-tf (107.5 µM) in 50 mM Tris-HCL buffer, pH7.4, 0.02M NaHCO 3 , was added to a standard 1 cmpre-acid washed glass tubes. Aliquots of 1.2 mMiron-citrate (1:20) or 1.2 mM indium-citrate (1:20)complexes were then added to the tubes and thevolumes made up to 2 ml with the same buffer, theymixed vigorously by vortexing. The tubes werecapped and left for up to 2 h at room temperature.The absorbance of the test tubes was taken usingPerkin-Elmer UV/Vis spectrophotometer model551-S [8, 10].Equilibrium dialysis technique for metal bindingto apo–tf. The binding of iron to human apo-tf andthe effect of indium on iron binding activity werealso investigated using equilibrium dialysis at 2-4°C. Two ml solution of apo-tf (21.5 µM) wasplaced in a dialysis sac opened to the atmosphere,which was immersed in a glass vessel contain 160ml 50 mM Tris-HCl buffer, pH 7.4, 0.02 MNaHCO 3 [10]. Fifty µl of iron (1.2 mM) as ferriccitrateor indium r o indium (1.2 mM) as indiumcitratesolutions were added at time intervals to thebuffer surrounding the dialysis sac with the aid of amagnetic stirrer. After 24 h, 100 µl of sample frominside and 100 µl from outside of the dialysis sacwas removed and analyzed for iron. Ironconcentration was determined using Brittenhammethod [11]. The binding constant of iron to apo-tfwithout indium was calculated using Scatchardrelationship (i.e. [B]/[F] = -1/K[B] + n[E] T /K S ) andplot of [B]/[F] against [B], [12]. Where [B] isconcentration of bound ion to protein, [F] isconcentration of free ion, n is number of bindingsite, [E] T is concentration of total protein and K S isthe dissociation constant.The effect of pH on the binding of iron andindium to apo-tf. To a series of pre-acid washedtest tubes containing 200 µl apo-tf (107.5 µM), 45µl of iron solution (1.2 mM) or indium (1.2 mM) as74complex with citric acid were added and thevolumes made up to 2 ml by Tris-HCl buffer withvarying pH within the range of 5.8 to 8. Thesolutions were vortexed and incubated for 2h atroom temperature. The absorbance of the test tubeswas measured by spectrophotometer.Metal ions interactions with each other forbinding to apo-tf. The binding of iron to apo-tf andthe effect of indium on iron binding activity andvice versa, were also investigated by spectrophotometrictitration. Initially, the effect of indiumon iron binding to apo-tf was studied. Prepared apotf(107.5 µM) in 200 µl, in 50 mM Tris-HCl buffer,pH 7.4, 0.02M NaHCO 3 was added to pre-acidwashed test tubes first. Then 55 µl of indium (1.2mM), as complex with citrate (1:20) and 0-55 µl ofiron (1.2 mM) as complex with citric acid wereadded to the series of tubes and the volumes weremade up to 2 ml with the same buffer. The solutionswere then mixed vigorously by vortexing and leftfor up to 2 h at room temperature. The absorbancesof the test tubes were measured at 465 nm. Thesame experiment was also repeated without indium.Next, the effect of iron on binding of indium toapo-tf was studied. As mentioned above 200 µl ofapo-tf was added to a series of test tubes. Ironsolutions (55 µl) and 0-55 µl of indium solutionwere added and the volumes were made up to 2 mlwith Tris-HCl buffer, pH 7.4. The absorbance of thetest tubes was measured at 255 nm. This study wasalso repeated in the absence of iron.Absorbance0.60.50.40.30.20.10Tf-FeTf-InAp o -Tf200 300 400 500Wavelength (nm)Fig. 1. Absorption spectrum (200-550 nm) of apo-tf, irontransferrinand indium-transferrin. ([Apo-tf] = 10.75 µM, [Fe +3 ]= [In +3 ] = 27 µM, pH 7.4, t= 25°C).
Iranian Biomedical Journal 7 (2): 73-77 (April 2003)A (465 nm)0.120.10.080.060.040.020ARESULTSThe absorption of apo-tf, iron-transferrin andindium-transferrin complexes was measured first.The results in Figure 1 show that the absorptionspectrum of the indium-transferrin complex has twopeaks at 280 and 255 nm. Iron-transferrin showed abroad peak at 465 and 280 nm and apo-tf at 280 nm.The effect of different pH within the range of 5.8to 8 on the binding of iron and indium to apo-tfwere studied. It showed that both iron and indiumbind to apo–tf mostly at pH 7.4. The lower the pH,the less binding of these metals to apo-tf (Fig. 2).0.55.6 6.1 6.6 7.1 7.6 8.1BpHThe effect of different amounts of iron (0 to 33µM) on binding of apo-tf and the effect of indium(33 µM) on iron up-take by apo-tf wereinvestigated. Initially, it was found that eachmolecule of apo-tf was saturated withapproximately 2 atoms of iron, corresponding to 1.4µg iron per mg transferrin (Fig. 3A, curve Tf-Fe),when indium (33 µM) was added, the absorbance oftransferrin-iron at 465 nm was reduced byapproximately 16% (Fig. 3A, curve Tf-Fe+In). Thebinding effect of different amounts of indium (o to33 µM) as a complex with citric acid and the effectof iron on indium binding to apo-tf were alsoinvestigated. The results show that each molecule ofapo-tf was also saturated with approximately 2atoms of indium, corresponding to 2.85 µg indiumper mg transferrin (Fig. 3B, curve Tf-In). Thepresence of iron (33 µM) in the reaction mixturesreduced the absorbance of transferrin-indiumcomplex at 255 nm by approximately 48% (Fig. 3B,curve Tf-In+Fe).A (465 nm)0.140.120.10.080.060.040.020A0 0.5 1 1.5 2 2.5 3 3.5Molar ratio of Fe+3 / TfTf-FeTf-Fe+In0.40.60.5BA (255 nm)0.30.2A (255 nm)0.40.30.20.105.6 6.1 6.6 7.1 7.6 8.1pHFig. 2. Effect of different pH (5.8 to 8) on iron binding (A)and ( 8 . 5 to 8) noig nidnib nor(A) and indium binding (B) toapo-tf. Each point is the mean of three separate experiments.([Apo-tf] = 10.75 µM, [Fe= +3 ] = [In +3 ] = 27 µM, t = 25°C).0.10Tf-InTf-In+Fe0 0.5 1 1.5 2 2.5 3 3.5Molar ratio of In+3 / TfFig. 3. Spectrophotometric titration of human apo-tf withdifferent concentration of i n or(0 to 33 µM) in the presence andabsence of indium (33 µM) (A) and also spectrophotometrictitration of human apo-tf with different concentrations ofindium (0–33 µM) in the presences and the absence of iron (33µM ) (B). Each point is the mean of three experiments. ([Apotf]= 10.75 µM, pH 7.4, t = 25°C).75
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