Abstract The characteristics of manganese and iron binding to human apotransferrin (apo-tf) have been investigated and compared in this study. Both metal ions were taken up by human apo-tf and formed complexes, with the maximum absorbances observed at 410 and 340 nm for manganese-transferrin (Mn-tf) and 465 nm for iron-transferrin (Fe-tf). Addition of manganese (1.5 µg/ml) to the reaction mixture containing iron and apo-tf, reduced Fe binding to apo-tf by 20 percent, in comparison to the control sample. The binding of both metals to apo-tf appears to be time and pH dependent processes. Using the equilibrium dialysis technique, the binding constant of manganese to apo-tf was also determined. The binding constatnt of Mn to apo-tf was calculated, using the Scatchard plot analysis. The calculated Ka was 3.1 × 10 9 M -1 . The binding of manganese and iron to human apo-tf has been discussed and compared in this work, using different biochemical techniques

Akbar Badii

Ali Asghar Moshtaghie

Hadi Kharazi

Mohammad Fazilati

Taghi Hassanzadeh

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Original Article
Manganese and Iron Binding to Human Transferrin 
Ali Asghar Moshtaghie a*, Akbar Badii a, Mohammad Fazilati b, 
Taghi Hassanzadeh a and  Hadi Kharazi a
aDepartment of Clinical Biochemistry, School of Medicine, Razi University, Kermanshah, 
Iran. bDepartment of Food Sciences, School of Agricalture, Isfahan University of Technology, 
Isfahan, Iran.
Abstract
The characteristics of manganese and iron binding to human apotransferrin (apo-tf) have 
been investigated and compared in this study. Both metal ions were taken up by human           
apo-tf and formed complexes, with the maximum absorbances observed at 410 and 340 nm for 
manganese-transferrin (Mn-tf) and 465 nm for iron-transferrin (Fe-tf). Addition of manganese 
(1.5 µg/ml) to the reaction mixture containing iron and apo-tf, reduced Fe binding to apo-tf by 
20 percent, in comparison to the control sample. The binding of both metals to apo-tf appears 
to be time and pH dependent processes. 
Using the equilibrium dialysis technique, the binding constant of manganese to apo-tf was 
also determined. The binding constatnt of Mn to apo-tf was calculated, using the Scatchard plot 
analysis. The calculated Ka was 3.1×10 9 M-1. 
The binding of manganese and iron to human apo-tf has been discussed and compared in 
this work, using different biochemical techniques. 
Keywords: Manganese; Iron; Transferrin; Binding activity. 
Introduction
The trace element manganese (Mn) is 
essential for normal development and body 
functions of animal life (1). Mn is a required co-
factor for many enzymes throughout the body. 
Mn is a required co-factor for arginase (2), 
which is responsible for urea production in the 
liver, superoxide desmutase, which is critical in 
the prevention of cellular oxidative stress, and 
pyruvate carboxylase, an essential enzyme in 
gluconeogenesis (3). 
Thermodynamic modeling of Mn (II) in 
serum suggests that Mn exists in several forms, 
including an albumin-bound form, as a hydrated 
ion, and in complex with bicarbonate, citrate 
and other small molecular weight ligands (4). 
Similar modeling of Mn (III) in serum suggests 
that it is almost 100% bound to transferrein (5, 
6). Mn (II) may be oxidized to Mn (III), which is 
rather reactive and more toxic than Mn (II). Mn 
(III) rapidly associates with transferrin to from a 
stable complex (7, 8). 
Manganese toxicities have been reported 
through occupational exposure, which could 
lead to the occurrence of pathophysiological 
disorders. Neurotoxicity due to the inhalation 
of airborne Mn, has been reported in miners 
working in manganese oxide mines, workers in 
dry cell battery factories and welders (9). 
The exact mechanism by which manganese 
produces neurotoxicity is still a matter of 
speculation. It has been reported that manganese 
* Corresponding author:
   E-mail:  Moshtaghie@pharm.Mul.ac.ir 
Copyright © 2007 by School of Pharmacy
Shaheed Beheshti University of Medical Sciences and Health Services
Iranian Journal of Pharmaceutical Research (2007), 6 (2): 101-106
Received: December 2005
Accepted: April 2006
Moshtaghie AA, Badii A, Fazilati M, Hassanzadeh T and Kharazi H / IJPR (2007), 6 (2): 101-106
neurotoxicity may be associated with its interaction 
with other essential trace elements including iron 
(10), zinc and copper (11). Studies have indicated 
that chronic exposure to manganese appears to 
be associated with an altered blood iron (Fe) 
concentration, due to Mn-Fe interaction at certain 
iron containing proteins (12). 
As manganese binds to plasma transferrin 
(tf), transport of manganese-transferrin (Mn-tf) 
complex into brain has been suggested to rely 
on a transferrin receptor (TFR), which competes 
with iron-transferrin (13) to enter the cells.
Transferrin, the metal-combining globulin of 
blood plasma, possesses two metal binding sites 
(14) and binds to a variety of metals including 
chromium (15), cobalt (16), cadmium (17), zinc 
(18), indium (19, 20) etc. 
With regards to the interference of 
manganese with iron binding to trasnferrin in 
those with manganese overload, the present 
study was established in order to conduct a 
series of methods, to investigate and compare 
binding characterstics of manganese and iron to 
transferrin. 
Experimental
Materials 
Human apo-tf iron free was purchased 
from Sigma Chemical Company, and further 
purified. The purification process was as follow 
(21): human apo-tf (5 mg/ml) was dissolved in 
Earle’s medium, placed in prewashed visking 
sacs, and dialyzed twice against 100 volume 
of 50 mM acetate buffer, pH 5.2, first for 6 h 
and then for a further16 h. Protein solutions 
were then successively dialyzed against 100 
volume of 0.15 mM NaCl, 0.02 M NaHCO3 in 
0.15 M NaCl, and finally Earle’s medium (pH 
7.4). The homogeneity of the prepared apo-
tf was checked by SDS-polyacrylamide gel 
electrophoresis, as reported elsewhere (21). 
No aggregation was noted in the solution, as 
checked at 280 nm.
 
Methods
Preparation of iron and manganese citrate 
complex 
Separate stock standard solutions of FeCl3. 
6H2O (3.0 mM) and/or MnCl2. 4H2O (3.0 mM), 
were prepared in deionized water and mixed 
with an equal volume of 60.0 mM citric acid. 
The solutions were adjusted to pH 7.4 with 1 M 
NaOH and made up to a final concentration of 
1.5 mM iron and manganese. 
Freshly, 50 ml of each stock standard solution 
was prepared in the beginning of each series of 
experiments. 
Spectrophotometeric titration technique of 
metal binding to apo-tf
The binding of iron and/or manganese to 
apo-tf was carried out using a Perkin-Elmer 
UV/visible spectrophotometer (Model 5515), at 
room temperature (22±1°C).
Approximately, 1.0 ml (5 mg) of the prepared 
tf in Earle’s medium was added to a standard 
1-Cm Pre-acid washed glass cuvet. Aliquots 
(1-100 µl) of 1.5 mM metal ion as the citrate 
complex, were added to separate cuvets. The 
cuvets were covered with parafilm, mixed 
thoroughly by vortexing, and left for 2 h at room 
temperature. 
The absorbance of samples prepared were 
individually measured at wavelengths of 465 nm 
for Fe-tf and 410 nm for Mn-tf, respectively. The 
same conentration of apo-tf was used as a blank. 
Throughout this study, all the glasswares were 
soaked overnight in 10% nitric acid and then 
thoroughly rinsed with distilled and deionized 
water, in order to minimize iron and manganese 
contamination. Plasticwares were individually 
perwashed with 10 mM EDTA, followed by 
three washes of distilled and deionized water. 
The equilibrium dialysis technique 
The binding of manganese and iron to human 
serum apo-tf was also investigated, using the 
equilibrium dialysis technique (22) at room 
temperature (20-22°C) in a chamber gassed 
with a 95% O2 / 5% CO2 mixture. A solution of 
apo-tf (5 mg/ml) was placed in a dialysis sac, 
open to the atmosphere, which was immersed 
in a plastic vessel containing 1600 ml of a pH 
7.4 Earle’s medium. 300 µl aliquots of (1.5 mM) 
iron as ferric-citrate or (1.5 mM) manganese as 
manganese chloride solutions were added at set 
time intervals to the buffer solution (1600 ml) 
surrounding the dialysis sac, with the aid of a 
magnetic stirrer and kept at the required pH by 
102
Manganese and Iron Binding to Human Transferrin
bubbling through the O2/CO2 mixture. After 24 h, 
a 100 µl of sample was taken from the inside and 
another 100 µl of sample from the outside of the 
dialysis sac and analysed for iron and manganese 
concentration. Iron concentration was determined 
using phenanthrolin as chromogen (23). 
Manganese level was determined, using a 
flameless Perkin-Elmer model 3030 atomic 
absorption spectrophotometer. The binding 
constant of manganese to apo-tf was calculated, 
using the Scatchard plot analysis (24). 
Results
Spectrophotomeric titration studies 
Binding of iron and manganese to the 
prepared purified apo-tf was studied by 
spectrophotometric and equilibrium dialysis 
techniques. 
The first experiment carried out was the 
measurement of the absorption spectrum of 
iron-trasnferrin complex. This was performed 
at room temperature, as mentioned in the 
experimental section. The absorption spectrum of 
the iron-transferrin complex shows a broad peak 
in the 465 nm region. The same experiment was 
conducted for the determination of the absorption 
spectrum of manganese-transferrin complex. 
The absorption spectrum of the manganese-
transferrin complex shows two peaks at 410 and 
340 nm (Figure 1 and 2). 
The next series of experiments were undertaken 
to study the binding of manganese and/or iron 
to apo-tf. Initially, increasing amounts of iron 
(0.84-8.4 µg/ml) were added to apo-tf solutions 
(5 mg/ml), present with the Earle’s medium (pH 
7.4) and incubated at room temperature for 2 h. 
The absorbance at 465 nm was indicating the 
binding of iron to apo-tf (Figure 3). 
When the apo-tf was titrated with iron in 
the presence of 1.5 µg/ml of manganese as the 
complex with citric acid, Fe binding to apo-tf 
was reduced by 20 percent (Figure 3). 
In order to investigate the effect of pH on 
iron and/or manganese binding activities of ap-
trasnferrin, to a series of plastic tubes containing 
buffer (2 ml), at different pH values within the 
range of 3-10 and apo-tf (5 mg/ml), aliquots of 
Fe-citrate and Mn-citrate were added. The tubes 
were treated as mentioned above. The maxiumum 
binding of Fe to apo-tf was observed at pH 7.0, 
whereas the binding of Mn to apo-tf took place 
at pH 7.5 (Figure 4). 
Equilibrium-dialysis studies 
In order to confirm the binding of iron and/
or Mn to apo-tf, equilibrium dialysis techniques 
were used next. 
Figure  1. Absorption spectrum of Mn-transferrin (Apo-tf, 
Mn-tf). APO-tf was titrated with manganese as a complax with      
citric acid. The maximum wavelength was obtained as men-
tioned in the experimental section. 
Figure  2. Absorption spectrum of Fe-transferrin (Apo-tf, Fe-
tf). APO-tf was titrated with iron as a complex with citric acid. 
The maximum wavelength was obtained as mentioned in the 
experimental section. 
103
Moshtaghie AA, Badii A, Fazilati M, Hassanzadeh T and Kharazi H / IJPR (2007), 6 (2): 101-106
To achieve this goal, a dialysis sac containing 
20 ml of 5 mg/ml apo-trasnferrin was placed 
in the dialysis chamber, as described in the 
experimental section. Aliquots of ferric cirate 
and/or manganes-cirate were added and the 
binding of iron and/or manganese to apo-tf 
studied as desricbed earlier. Results obtained are 
presented in Figures 5 and 6. Figure 5 shows the 
binding of iron to apo-tf and the effect of 200 µg/
l of manganese on the binding activity, whereas 
Figure 6 shows the binding of manganese to 
apo-tf. 
Reduction in iron uptake by apo-tf was seen 
when manganese was added to the outside of 
dialysis sac. 
Calculation of binding constant
In order to find out the association constant 
of manganese to apo-tf, the Scatchard plot 
analysis technique was used. For this purpose, 
this data shown in figure 6 was used and the 
binding constant for manganese binding to 
apo-tf calculated as follows: free manganese 
ion concentration from outside the sac was 
subtracted from the total Mn ion concentration 
within the sac. This was considered as the bound 
fraction. The approximate calculated binding 
constant for manganese apo-tf complex was 
3.1×109 M-1 (Figure 7). The calculated binding 
constant values of Mn to apo-tf are presented in 
Table 1 and the Scatchard plot was constructed 
using the bound/free values versus the bound 
values. 
Finally, the effect of varying concentrations 
of citric acid on manganese binding to apo-tf 
was investigated and the results obtained are 
presented in Figure 8. 
It was shown that the binding of manganese 
Figure 3. Spectrophotometric titration of human apo-tf with 
iron in Earle’s medium at pH 7.4 and the effect of Manganese. 
Each point is the mean±SD of three separate experiments. 
Figure 4. Effect of pH on iron and manganese binding to apo-
tf. Each point is the mean±SD of three separate experiments.
Figure  5. The equilibrium dialysis study, showing the binding 
of iron to human apo-tf in Earle’s medium at pH 7.4 and the 
effect of manganese. Each point is the mean±SD of three 
separate experiments. 
Figure  6. The equilibrium dialysis study, showing the binding 
of manganese to human apo-tf in Earle’s medium at pH 7.4. 
Each point is the mean±SD of three separate experiments. 
104
Manganese and Iron Binding to Human Transferrin
to apo-tf decreased with increasing the 
concentration of citric acid. This confirmed 
that citric acid is not necessary for binding of 
manganese to apo-tf. 
Discussion
Transferrin has been proposed as the major 
manganese binding ligand in the plasma. 
Earlier reports from this laboratory showed 
that cerroluplasmine (Ferroxidase) may play an 
important role in the binding of manganese to 
apo-tf (25). 
Absorption spectra obtained in this study, 
indicated the presence of two peaks at 410 and 
340 nm for the iron-transferrin complex (Figures 
1 and 2), whereas earlier reports from other 
laboratories suggested two peaks at 430 and 330 
nm for the manganese-trasferrin complex and the 
same spectrum for the iron-transferrin complex 
(25). The discrepancies in these findings could 
be due to the buffer composition and other 
experimental conditions. 
Mn (III) can effectively compete with ferric 
ion for binding to transferrin in the blood 
circulation, and thus it is generally accepted 
that Mn (III) is transported extracellulary as a 
transferrin complex. Fe-transferrin complex 
binds to transferrin-receptors at the cell 
membranes and iron releases into the cells 
for further biochemical pathways (27). It has 
been reported that manganese-transferrin 
complex may also follow the same procedure, 
undergoing internalization within endosomal 
vesicles (28). The endosomal vesicle undergoes 
acidification, presumably releasing Mn (III) 
from the transferrin-complex (13), resulting in 
the reduction in iron uptake by the cell.
Although citric acid was necessary for iron 
binding to apo-tf, the binding of manganese 
to apo-tf dose not require citric acid. These 
discrepancies might be due to the chemical 
structure of manganese. The effect of different 
pH values on the binding activity of manganese 
to apo-tf is in agreement with this suggestion. 
Data presented here show that the maximum 
binding occurs at an alkaline pH of 7.5 (Figure 
4), rather than an acidic pH. In fact, this binding 
activity of manganese seems to be dependent 
on the pH of the solutions. Data obtained 
from both the spectrophotomeric titration and 
equilibrium dialysis studies conducted confirm 
the binding of manganese to human apo-tf, with 
a subsequent reduction in iron uptake. This might 
Figure 7.  A typical Scatchard plot for the binding of manganese 
to apo-tf. 
Figure 8. The effect of citrate on the manganese binding to 
apo-tf. 
Table 1. The calculated values for the binding constant of 
manganese to apo-tf 
105
Moshtaghie AA, Badii A, Fazilati M, Hassanzadeh T and Kharaz H / IJPR (2007), 6 (2): 101-106
lead to the accumulation of manganese in those 
exposed to this metal and could be linked to the 
development of severe neurological disorders or 
the appearance of anemia. 
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Manganese and Iron Binding to Human Transferrin

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Manganese and Iron Binding to Human Transferrin

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