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© Division of Chemical Education • www.JCE.DivCHED.org • Vol. 86 No. 8 August 2009 • Journal of Chemical Education 969
In the Laboratory
Bioinorganic chemistry is an interdisciplinary field that relies on a solid understanding of both biological and inorganic chemistry and employs wide-ranging synthetic, analytical, and physical techniques from both chemistry and biology. While bioinorganic chemistry lecture courses are becoming more com-mon, available laboratory experiments are usually traditional coordination chemistry experiments with some biological rele-vance (1, 2). With the recent availability of inexpensive, purified metalloproteins in relatively large quantities, it is now possible to add a new dimension to bioinorganic laboratory experiments. For example, Donlin et al. developed a laboratory experiment to spectroscopically examine the rate of iron removal from the iron-storage protein ferritin and to investigate the structural aspects, including metal binding, of ferritin using computer modeling (3). Williams and co-workers and McQuate and co-workers also developed bioinorganic laboratory experiments investigating aspects of metal binding in carbonic anhydrase (4–6).
Because the iron-binding protein transferrin is available in gram quantities and has a shelf life of five years when refriger-ated, it is ideal for use in a student laboratory setting. In this experiment, students gain experience in making biological buffer solutions and handling a protein by loading iron into transferrin and characterizing the Fe(III) protein by UV–vis spectroscopy. They then use the commercially-available iron-chelating drug Deferiprone to measure the rates of iron removal from transferrin, as monitored by the decrease in iron loading of transferrin. Since measuring the ability of a ligand to remove iron from transferrin is a method used to rate the effectiveness of compounds for use in iron chelation therapy, this experiment is particularly relevant in this field (7, 8).
Background
Iron is an essential element required by most organisms, and is involved in a multitude of biological processes including oxygen transport, the synthesis of DNA, and electron transport (9). Within the body, iron homeostasis (the balance of iron uptake, transport, and storage) is tightly regulated due to the toxic nature of non-protein-bound iron, since the formation of oxygen radical species by non-protein-bound Fe(II) has been im-plicated in cellular damage and disease (10–14). Furthermore, the insoluble nature of Fe(III) at neutral pH makes solubility of iron a potential difficulty for organisms (9). Thus, iron must be tightly bound and solubilized, and mammals, including humans, achieve this by utilizing a complex iron transport and storage system.
Serum transferrins are the major iron-transporting proteins in blood and are part of a family of transferrin proteins that includes serum transferrins, ovotransferrin, and lactotransfer-rins (15). Transferrin proteins are small, containing approxi-
mately 700 amino acids (MW ~80 kDa) and are synthesized primarily in the liver (9, 16). Structurally, transferrin consists of a single polypeptide chain with N-terminal and C-terminal lobes. Each lobe contains two domains that are joined by a short peptide to create a hydrophobic metal binding site (9, 15, 17). Transferrin reversibly binds two Fe(III) ions with high affinity (Ka = ~1020 L mol–1); the amino acid residues coordinating iron in each lobe include two tyrosine, an aspartate, and a his-tidine residue, along with a bidentate carbonate anion (CO3
2–) (Figure 1) (18). In this coordinating environment, the positive charge of the Fe(III) is balanced by the negative charges from the tyrosine and aspartate residues; the negative charge of the carbonate anion is balanced by neighboring positive charges on nearby amino acids (19). Transferrin can also bind other metal ions, including bismuth(III), gallium(III), indium(III), aluminum(III), copper(II), manganese(II), zinc(II), nickel(II), and ruthenium(III); however, transferrin binding affinity is highest for iron. Binding of non-iron metal ions by transfer-rin may have a significant role in the transport and delivery of essential metal ions, diagnostic radioisotopes, and toxic metal ions to cells (17).
Conformational changes occur in transferrin that are associated with iron binding or release. When iron is bound, each domain moves to enclose iron in the metal binding site. Similarly, upon release of iron, the metal-binding domains move apart; these “closed” and “open” conformations have been ob-served through crystallography (Figure 2) (15, 20). Once iron is bound to transferrin it is internalized by the cell through a process known as receptor-mediated endocytosis; iron is then released and either used or stored in ferritin (21).
Researchers have studied iron removal from transferrin to gain a better understanding of its mechanism of iron binding, and also how iron is stored and transported throughout the body. Additionally, iron availability has been implicated as a limiting nutrient for bacterial infections since the high affinity
Using Proteins in a Bioinorganic Laboratory Experiment: Iron Loading and Removal from TransferrinErin E. Battin, Ashley Lawhon, and Julia L. Brumaghim*Department of Chemistry, Clemson University, Clemson, SC 29634; *[email protected]
David H. HamiltonDepartment of Chemistry, Rockhurst University, Kansas City, MO 64110
O
O
O
O
O
O P
O
N
N
P P
P
Fe3∙
Figure 1. The amino acid residues that bind iron in the C- and N-terminal lobes of transferrin; the protein backbone is indicated by P.
970 Journal of Chemical Education • Vol. 86 No. 8 August 2009 • www.JCE.DivCHED.org • © Division of Chemical Education
In the Laboratory
of transferrin for iron prevents elevated levels of non-protein-bound iron, thereby limiting bacterial growth (22, 23). Inves-tigating the mechanism of bacterial iron sequestration during infection may have significant implications in the treatment and prevention of serious bacterial infections, including tuberculosis and peptic ulcers (24, 25).
Non-protein-bound iron also can react with naturally oc-curring hydrogen peroxide to generate reactive oxygen species (ROS), including superoxide radical (O2
–) and hydroxyl radical (OH), in the Fenton reaction (11):
Fe(III) + •OH + OH−Fe(II) + H2O2
ROS have been implicated in increased oxidative stress and development of cancer, inflammatory, cardiovascular, and neurodegenerative diseases (26–29). Iron chelating agents may provide a way to prevent oxidative damage by keeping the non-protein-bound iron concentration low, thereby decreasing oxida-tive stress. For example, because long-term transfusion therapy is required to treat β-thalassemia, a genetic blood disorder that causes decreased hemoglobin production, a continual increase
in iron concentration occurs in patients since humans have no mechanism for iron excretion (30, 31). Similarly, hemochro-matosis, a genetic disorder associated with elevated iron levels due to increased intestinal iron absorption, results in fatigue, diabetes, heart disease, and death (32). Chelation drugs must be used to remove this excess iron, and continuing efforts are focused on developing new chelating agents that would have improved properties over currently used drugs (33).
Several chelating agents are used for treatment of iron overload. For example, Desferrioxamine B (Desferral; Figure 3) is available to treat iron overload diseases, such as hemochroma-tosis and β-thalassemia (8, 34). Desferral is a very effective iron chelator with relatively low toxicity; however, it is expensive and can only be administered by continuous intravenous infusion, re-sulting in poor patient compliance (8, 35). Another drug used to treat iron overload is 1,2-dimethyl-3-hydroxy-4(1H)-pyridone, also known as Deferiprone (Figure 3) (33). Deferiprone can be administered orally rather than intravenously; unfortunately, the high toxicity of this compound has led the FDA to ban the use of Deferiprone in the United States (8). Testing chelators for their ability to kinetically remove iron from transferrin is a good method for evaluating their potential usefulness as chelation drugs. This laboratory experiment will consist of monitoring iron loading into transferrin and then studying the kinetics of iron removal by the drug Deferiprone using UV–vis spectroscopy.
Summary of Procedure
This experiment can be conducted in an advanced bio-inorganic, biochemistry, or inorganic laboratory course where students have been previously introduced to both UV–vis spectroscopy and kinetic concepts in lecture. The experiment requires two, three-hour laboratory sessions with a pre-lab lec-ture and assignment, as described in the online material, prior to starting the first three-hour laboratory session.
Figure 2. The crystal structure of ovotransferrin: apo-ovotransferrin (left) and diferric ovotransferrin (right); carbonate anion not shown. Structures were obtained from the Protein Data Bank with identifiers 1AOV and 1DOT, respectively.
NHO O
O
NH
NHO O
O
NH
NHO O
H3N (CH2)5 (CH2)2 (CH2)5 (CH2)5 CH3(CH2)2
∙
N O
OH
Figure 3. Chemical structures of Deferiprone (top) and Desferri-oxamine B (bottom).
© Division of Chemical Education • www.JCE.DivCHED.org • Vol. 86 No. 8 August 2009 • Journal of Chemical Education 971
In the Laboratory
In the first three-hour laboratory session students begin the experiment by preparing an iron nitrilotriacetic acid, Fe(NTA), stock solution (pH 4.0). A solution of human apotransferrin is also prepared, and the initial concentration is determined by measuring the protein absorbance at 280 nm using UV–vis spectroscopy. Because of the low solubility of Fe(III) at pH 7.4, iron is loaded into transferrin by Fe(NTA) and monitored us-ing UV–vis spectroscopy; no changes in the UV–vis spectra are observed when iron loading is complete. The percentage of iron loading in transferrin is estimated by measuring the absorbance at 470 nm and calculating the concentration using a transferrin extinction coefficient of 87,200 L mol–1cm–1 (35). Students perform column chromatography to remove any unbound iron, and the diferric transferrin concentration and quantity of iron loading is re-determined.
In the second laboratory session, students remove iron from diferric transferrin using Deferiprone. After preparing a stock solution of Deferiprone (commercially available from VWR International), the chelating solution is added to the protein. The kinetics of iron removal from transferrin is then monitored using UV–vis by measuring absorbance at 460 nm for 50 minutes (3000 scans); kinetic experiments are performed at either 37 °C or 25 °C. Using a spreadsheet program, students fit the kinetic data to the equation At = A∞(1 − e–kobst) + A0e– kobst to determine the pseudo-first-order rate constant for iron re-moval (kobs) from transferrin by Deferiprone; kobs is defined as –ln[(At − A∞)∙(A0 − A∞)]∙t. Determination of kobs and calculation of the second-order rate constant (k2) from kobs enables students to compare their measured rate constant for iron removal by Deferiprone to published rate constants for iron removal with other chelating agents.
Hazards
NaOH and HCl can cause severe burns. Iron salts, tris (base), and Deferiprone can be harmful if ingested in large quantities and can be irritating to eyes and respiratory system. Nitrilotriacetic acid is a possible human carcinogen (Class B). Because of the possibility of infectious agents being present, human apotransferrin should be handled at the bio-safety level 2 as recommended in the CDC–NIH manual Biosafety in Microbiological and Biomedical Laboratories (36). To avoid these biosafety issues, bovine serum transferrin can be substituted for human serum transferrin for these experiments. If this substitu-tion is made, the extinction coefficients for the apotransferrin and Fe(III) transferrin will be 85,000 L mol–1 cm–1 at 278 nm and 4500 L mol–1 cm–1 at 463 nm, respectively (37). Slight dif-ferences in the rate of iron removal from bovine serum transfer-rin may occur; however, these will not be significant owing to structural similarities with human transferrin.
Results
The concentration of apotransferrin is determined using UV–vis spectroscopy prior to iron loading. The protein con-centration should be 50–100 μM. If concentrations are lower than 50 μM instructors can have students use a larger quantity (>20 mg) of apotransferrin.
To determine the efficacy of Deferiprone at removing iron from transferrin, apotransferrin was first loaded with iron using iron nitrilotriacetic acid and monitored using UV–vis spectros-
copy at 25 °C. UV–vis spectra demonstrating efficient iron load-ing are shown in Figure 4; complete loading is indicated by little or no change in absorbance at 470 nm upon addition of iron. Excess unbound iron is removed using a Sephadex G25-column (PD-10). Once protein purification is complete, the transferrin concentration and the quantity of iron loading is re-determined. The concentration of the iron-loaded transferrin should be simi-lar to the original concentration of apotransferrin.
UV–vis spectra (25 °C) illustrating the decrease in absor-bance as iron is removed from transferrin is shown in Figure 5. As expected, spectra show the opposite behavior observed for iron loading. A plot of ln[(At − A∞)∙(A0 − A∞)] versus time for iron removal by Deferiprone is shown in Figure 6 (at 25 °C and 37 °C). The pseudo-first-order rate constants (kobs) can be determined from the slope of the resulting line. The average kobs for iron removal determined by all students completing this laboratory experiment was approximately 1.2 s–1 at 25 °C and 2.2 s–1 at 37 °C.
Figure 5. UV–vis spectra showing a decrease in absorbance as iron is removed from transferrin at 25 oC.
350
0.4
0.2
0.0
400 450 500 550 600
iron rich
iron depleted
Abs
orba
nce
Wavelength / nm
Figure 4. Typical UV–vis spectra obtained by students, showing an increase in absorbance as apotransferrin is loaded with iron at 25 oC. The legend indicates equivalents of iron nitrilotriacetic acid, Fe(NTA), added to apotransferrin.
400
1.0
0.5
0.0
500 600 700
apotransferrin with:
2.8 equiv iron2.4 equiv iron2.0 equiv iron1.6 equiv iron1.2 equiv iron0.8 equiv iron0.4 equiv iron0.0 equiv iron
Abs
orba
nce
Wavelength / nm
972 Journal of Chemical Education • Vol. 86 No. 8 August 2009 • www.JCE.DivCHED.org • © Division of Chemical Education
In the Laboratory
Conclusions
This laboratory provides students with an excellent hands-on introduction into bioinorganic chemistry by exploring the rate of iron removal from transferrin by Deferiprone, which has real-world applications in drug testing and design. By utilizing this simple experiment, students have a better understanding of the biological role of transferrin, and how UV–vis spectroscopy is used to determine the performance of potential iron-chelating drugs.
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Supporting JCE Online Materialhttp://www.jce.divched.org/Journal/Issues/2009/Aug/abs969.html
Abstract and keywords
Full text (PDF) Links to cited URL and JCE articles Figures 2, 4, and 5 in color
Supplement
Instructions for the students, including prelab assignment and postlab questions and an evaluation form
Notes for the instructor, including a summary of the student evaluations
At
– A
∞
A0
– A
∞
ln1
0
−1
−2
0 500 1000 1500 2000 2500
−3
−4
Time / s
iron removal 37 °C
iron removal 25 °C
Figure 6. A graph of ln[(At – A∞)/(A0 – A∞)] versus time for iron removal from transferrin by Deferiprone versus time at 25 oC and 37 oC. The slope is –kobs. (The data at 37 oC starts below 0 owing to the few second time delay between mixing the sample, loading it into the spectrometer, and then pressing “start”.)
1
Supplemental Material
Using Proteins in a Bioinorganic Laboratory Experiment: Iron Loading and Removal from Transferrin A Bioinorganic Chemistry Experiment
JCE Section: In the Laboratory
Authors: Erin E. Battin,1 Ashley Lawhon,1 David H. Hamilton,2 and Julia L. Brumaghim1
Author Information:
1Department of Chemistry, Clemson University, Clemson, SC 29634-0973. Telephone Number:
864-656-0629; Fax Number: 864-656-6613; E-mail Address: [email protected],
2Department of Chemistry, Rockhurst University, Kansas City, MO 64110; Telephone Number:
816-501-4000; E-mail Address: [email protected].
Corresponding Author: Julia L. Brumaghim1
Corresponding Author Information:
1Address: Department of Chemistry, Clemson University, Clemson, SC 29634-0973, USA;
Telephone Number: 864-656-0481; Fax Number: 864-656-6613; E-mail Address:
[email protected] (preferred method of communication).
Word Count: 7,079
2
Using Proteins in a Bioinorganic Laboratory Experiment:
Iron Loading and Removal from Transferrin
A Bioinorganic Chemistry Experiment
Authors: Erin E. Battin,1 Ashley Lawhon,1 David H. Hamilton,2 and Julia L. Brumaghim1
Author Information: 1Department of Chemistry, Clemson University, Clemson, SC 29634-
0973. Telephone Number: 864-656-0481; Fax Number: 864-656-6613; E-mail Address:
[email protected], [email protected], [email protected].
2Department of Chemistry, Rockhurst University, Kansas City, MO 64110; Telephone Number:
816-501-4000; E-mail Address: [email protected].
Abstract
With the increasing availability of metalloproteins it is now possible to incorporate them
into bioinorganic laboratory experiments. Thus, we have developed a laboratory experiment
where students use UV-vis spectroscopy to determine the rate of iron removal from transferrin
by a well-known, commercially-available iron chelating drug, Deferiprone. Students gain
experience in both chemical and biological laboratory techniques, while assessing the efficacy of
a drug that is physiologically relevant to humans.
Keywords: Upper-division Undergraduate, Inorganic Chemistry, Transferrin, Hands on
Learning/Manipulatives, Laboratory Instruction, Bioinorganic Chemistry, UV-Vis Spectroscopy
3
Lab Summary
Iron is an essential metal, used for many physiological functions including O2 transport in
hemoglobin and incorporated in iron-sulfur clusters used by electron-transfer proteins (1, 2).
Maintaining iron homeostasis (balance) is important for organisms, since increased iron levels
lead to disease and cellular damage (3-5). Patients with β-thalassemia require regular blood
transfusions and consequently suffer from iron overload, leading to heart and liver damage (6).
Similarly, elevated body iron concentrations are associated with increased intestinal iron
absorption in hemochromatosis (7). To treat this iron overload, researchers have focused on
removing iron from transferrin, the iron transport protein in blood (8). We have developed a
laboratory experiment to give students experience with real-world drug testing and bioinorganic
chemistry.
Iron chelating drugs, including desferrioxamine B (Desferral), are available to treat iron overload
disease, including hemochromatosis and β-thalassemia. Desferral is a very effective iron
chelator with relatively low toxicity; however, it is extremely expensive and can only be
administered by daily subcutaneous infusion, resulting in poor patient compliance (9, 10).
Currently, Desferral is one of a very few iron chelating drugs administered for treatment of iron
overload disease, although research continues to develop other iron chelators that can be
administered orally, are inexpensive, and are non-toxic.
Deferiprone is the first chelating agent that is effective when administered orally for the
treatment of iron overload diseases. Unlike Desferral, however, Deferiprone is banned in the
United States because of high toxicity. Nevertheless, Deferiprone is still administered to treat
iron overload diseases in many other countries (9, 10). Thus, our laboratory experiment focuses
on loading iron into the transferrin protein and measuring the kinetics of iron removal from the
4
protein using Deferiprone. Testing the ability of a compound to remove iron from transferrin is
commonly used to screen potential drugs for the treatment of iron overload disease, and will give
students relevant laboratory experience in working with proteins and an understanding of how
compounds are evaluated as possible new drugs.
Time and curriculum level
Because the laboratory experiment combines both biochemistry and inorganic chemistry,
it can be utilized for several topics of discussion in bioinorganic chemistry, biochemistry, or
inorganic chemistry. Completion of this laboratory experiment should occur during lectures that
highlight metal uptake and binding, development and efficacy of metal-chelating drugs, and UV-
vis spectroscopy and kinetics. This experiment requires two, three-hour laboratory sessions with
students working individually or in small groups. Students should perform the experiment in an
advanced laboratory course after they have been introduced to buffer solutions, UV-vis
spectroscopy, and kinetics concepts from a lecture course.
Procedures, techniques, and concepts presented
During this experiment, students will utilize advanced laboratory procedures and
techniques to determine the efficacy of a well-known iron-binding drug, Deferiprone. After
determining the concentration of apotransferrin, students prepare an iron solution (Fe(NTA)2)
that loads iron into apotransferrin, which is monitored through UV-vis spectroscopy; column
chromatography is also used to remove excess unbound iron from the transferrin solution. Once
fully loaded, the kinetics of iron removal by Deferiprone are determined using UV-vis
spectroscopy.
5
Performing this laboratory experiment provides students with an opportunity to utilize
important laboratory techniques such as working with proteins, preparing buffered solutions,
column chromatography, and UV-vis spectroscopy. These experiments enable the student to
conceptualize how a protein, such as transferrin, functions physiologically. Students also
recognize how a simple technique like UV-vis spectroscopy can be efficiently used to monitor
the efficacy of a drug that treats disease in humans. Students will be able to compare the rate of
iron removal from transferrin by Deferiprone to iron-removal rates of other known iron
chelators, giving students an idea of the efficacy of Deferiprone relative to other iron chelators
being developed as potential drugs for the treatment of iron overload. Students are also exposed
to the interface of biology and inorganic chemistry, an important interdisciplinary field not
experienced by most students, particularly in a laboratory setting. Additionally, students become
familiar with a very useful resource known as the Protein Data Bank (PDB) that allows the
exploration of 3-dimensional structural data of biological macromolecules, such as transferrin.
Usefulness of experiment
This laboratory will be very useful for illustrating the principles of bioinorganic
chemistry, particularly the ability of proteins to bind metals, drug development, and analytical
techniques, such as UV-vis spectroscopy and kinetics. Without the hands on experience that this
laboratory experiment provides, students have a difficult time conceptualizing how techniques
like UV-vis spectroscopy and kinetics can be used to determine potential drug efficacy. In
addition, students often have difficulty visualizing the structure of a protein and understanding
the important role metals play in protein function. By performing this laboratory experiment,
students learn how to use a UV-vis spectrophotometer for kinetics experiments, enabling
6
students to physically observe iron loading of a protein found in the body and the rate of iron
removal by a metal-chelating drug. Using human serum transferrin and an iron-chelating drug
for the treatment of iron overload makes this laboratory particularly relevant to their own lives.
Students also gain a much greater understanding of the structure and function of transferrin, iron
overload diseases, and the process for the development of real-world drugs used to treat iron
overload diseases, which would most likely not be discussed in detail in a typical lecture course.
Results
Students completing this laboratory experiment obtained results typical of those shown in
Figures 1 and 2. Students should observe increasing absorbance as iron is loaded into
apotransferrin (Figure 1); once absorbance has stopped increasing upon addition of iron, students
should recognize that this is indicative of transferrin being fully loaded. Students then collect
data on the iron removal from transferrin by the iron-chelator Deferiprone at 25˚C and 37˚C
which is then plotted similarly to the data in Figure 2; from the slope of this graph, the psuedo-
first-order rate constant (kobs) for iron removal can be calculated. The average kobs determined
by students was 1.2 s-1 at 25˚C and 2.2 s-1 at 37˚C. From their data analysis, students should
determine that higher temperatures result in a faster rate of iron removal. In addition, students
must calculate the second-order rate constant (k2) from kobs and compare their measured k2 value
for iron removal from transferrin to similar rate constants reported for other iron chelators.
7
Figure 1. Typical UV-vis spectra obtained by students, showing an increase in absorbance as apotransferrin is loaded with iron at 25˚C. Legend indicates equivalents of iron nitrilotriacetic acid, Fe(NTA)2, added to apotransferrin.
0
0.2
0.4
0.6
0.8
1
350 450 550 650Wavelength (nm)
Abs
orba
nce
Apotransferrin0.4 Equiv. Iron0.8 Equiv. Iron1.2 Equiv. Iron1.6 Equiv. Iron2.0 Equiv. Iron2.4 Equiv. Iron2.8 Equiv. Iron
Figure 1. Typical UV-vis spectra obtained by students, showing an increase in absorbance as apotransferrin is loaded with iron at 25˚C. Legend indicates equivalents of iron nitrilotriacetic acid, Fe(NTA)2, added to apotransferrin.
0
0.2
0.4
0.6
0.8
1
350 450 550 650Wavelength (nm)
Abs
orba
nce
Apotransferrin0.4 Equiv. Iron0.8 Equiv. Iron1.2 Equiv. Iron1.6 Equiv. Iron2.0 Equiv. Iron2.4 Equiv. Iron2.8 Equiv. Iron
0
0.2
0.4
0.6
0.8
1
350 450 550 650Wavelength (nm)
Abs
orba
nce
Apotransferrin0.4 Equiv. Iron0.8 Equiv. Iron1.2 Equiv. Iron1.6 Equiv. Iron2.0 Equiv. Iron2.4 Equiv. Iron2.8 Equiv. Iron
-4
-3
-2
-1
0
1
0 500 1000 1500 2000 2500
Time (s)
Iron Removal 37˚CIron Removal 25˚C
ln[(A
t-A∞)/(
Ao-
A∞)]
Figure 2. A graph of ln[(At-A∞)/(Ao-A∞)] versus time for iron removal from transferrin by Deferiprone versus time at 25˚C and 37˚C. The slope is -kobs.
8
Summary of student evaluations
Student evaluations were completed after performing this experiment as part of a
semester-long advanced chemistry laboratory course with 17 students, and after offering this as
an elective experiment in an independent study course (2 students). From the results of this
evaluation, students typically did not have extensive experience working with proteins nor were
they extremely comfortable with the idea of working with proteins prior to completing this
laboratory experiment (average student ratings for Questions 1 and 2 were 3.4 and 2.1,
respectively, averages are calculated from 16 evaluations). Most felt that after having completed
this experiment they would be more comfortable working with proteins in the future; the average
student rating for Question 7 was 4.0. Students also agreed that this laboratory experiment gave
them insight into the chemistry of iron in biological systems, and a better understanding of how
the efficacy of iron chelating drugs were tested (average student ratings for Question 3 and
Question 4 were 4.2 and 4.4, respectively). Similarly, students were interested in this laboratory
experiment because it had real-world applications in drug design (average student rating for
Question 6 was 4.4). They also felt that the experiments and data analysis were an appropriate
level for the course (average student rating for Question 5 was 3.9); however, some disliked the
time required to prepare the solutions or obtain the spectral data. Several students remarked that
they liked the biological relatedness of the laboratory experiment and found that using the same
techniques researchers used to evaluate iron chelators for therapeutic use made the experiment
more interesting.
This laboratory experiment achieved its goals in that students gained experience in both
biological and chemical laboratory techniques, including working with proteins, buffer solutions,
column chromatography, and UV-vis spectroscopy. Students saw how a relatively simple
9
technique, such as UV-vis spectroscopy, could be used to monitor the efficacy of the drug
Deferiprone, and how iron removal by other iron chelating agents compares to Deferiprone.
They were also exposed to an interface of science that incorporates both biology and chemistry,
which enabled students to conceptualize how a protein like transferrin functions and also how to
use the Protein Data Bank to gain insight into the structure of transferrin and other iron-
containing proteins found in the body.
10
Transferrin Kinetics Laboratory Evaluation
Please remember your answers on this evaluation will be used to improve this laboratory experiment and will not influence your grade in this course. Thank you for taking the time to complete this form. Please answer the following questions based on the following scale: 1: strongly disagree 2: disagree 3: neutral 4: agree 5: strongly agree 1. I felt comfortable with the idea of working with proteins prior to completing this laboratory experiment. 1 2 3 4 5 2. I had experience working with proteins previously to completing this laboratory experiment. 1 2 3 4 5 3. This laboratory gave me insight into the chemistry of iron in biological systems. 1 2 3 4 5 4. This laboratory helped me to better understand how iron chelating molecules are tested for efficacy as drugs. 1 2 3 4 5 5. I thought the experiments and data analysis were at an appropriate level for this course. 1 2 3 4 5 6. I liked being introduced to experiments that are used in real-world drug design. 1 2 3 4 5 7. I would feel more comfortable working with proteins in the future after completing this laboratory experiment. 1 2 3 4 5 8. What did you enjoy most about this transferrin kinetics experiment? 9. What did you dislike most about this transferrin kinetics experiment? 10. Please make any additional comments about this experiment on the back of this page.
11
Related experiments
Raymond et al. have extensively investigated iron binding to and removal from
transferrin with numerous iron-chelating agents (8, 11). Although these experiments provide a
great deal of background information, reference material, and experimental design details for this
laboratory experiments, the experiments described by Raymond et al. were not designed for use
in an undergraduate laboratory course. Use of these references, however, is incorporated into
this experiment in a manner suitable for an undergraduate laboratory course.
In a related laboratory experiment by Donlin et. al., students examine the rate of iron
removal from the iron-storage protein ferritin (12). Similar to this experiment, the concentration
of iron in ferritin was determined using UV-vis, then Fe(III) was reduced to Fe(II) with various
reducing agents, and the rate of iron removal from ferritin was monitored spectroscopically. In
addition to the described kinetics experiments, this laboratory experiment also focuses on
computer modeling of ferritin-iron binding and introduces students to three-dimensional
structures and molecular-structure function relationships. This ferritin experiment, although
similar, is unlike our laboratory experiment in that the iron-transferrin binds two iron(III) ions
and is the transport protein in blood, whereas the cellular iron storage protein ferritin stores up to
~3500 iron(III) ions per protein (11, 12). In addition, a primary focus of our laboratory
experiment is to determine the efficacy of an iron-chelating drug, Deferiprone, to remove iron
from transferrin, an experiment that is relevant to both drug testing and design as well as human
disease. Similarly, removal of iron by iron-chelating drugs target transferrin rather than ferritin
due to accessibility of iron. The ferritin experiment described by Donlin et. al. would
complement this laboratory experiment, particularly to augment bioinorganic lectures that focus
on metal transport and storage, metal-protein interactions, spectroscopy, and kinetics.
12
Williams et al. developed a kinetics laboratory experiment investigating the rate of zinc
removal from carbonic anhydrase, an enzyme that catalyzes the conversion of carbon dioxide to
bicarbonate (13). Similar to this transferrin laboratory experiment, the rate of zinc removal was
determined spectroscopically using various chelating agents. Structurally, carbonic anhydrase is
a very different protein from transferrin with extremely different biological activity, and the
experiment focuses on zinc removal, a process that is not biologically relevant for the carbonic
anhydrase enzyme. The chelating agents used to remove zinc from carbonic anhydrase were
chosen based on ease of use rather than physiological relevance. It is also worth noting that both
bovine and human carbonic anhydrase are commercially available in much smaller quantities and
at significantly higher prices than transferrin. Other laboratory experiments, including “Carbonic
Anhydrase and Metalloderivatives: A Bioninorganic Study” and “Kinetics of Formation of
Cobalt(II)- and Nickel(II) Carbonic Anhydrase”, also investigate metal binding in carbonic
anhydrase using spectroscopic studies (14, 15).
Hazards
1. NaOH is very corrosive and can cause severe burns.
2. HCl is extremely corrosive and can cause serious injury, including severe burns.
3. Skin and eye burns can occur with FeCl3⋅6H2O exposure and it can be harmful if ingested.
4. Nitrilotriacetic acid is a possible carcinogen for humans (Class 2B) and can cause irritation
if skin or eye contact and inhalation occurs.
5. Human apotransferrin should be handled at a bio-safety level 2 for any potential infectious
agents present, including HIV and syphilis. Guidelines for handling bio-safety level 2
materials are located in the CDC/NIH manual "Biosafety in Microbiological and
13
Biomedical Laboratories", 1999, or can be found at the following web address:
http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4s3.htm. To avoid these biosafety issues,
bovine serum transferrin can be substituted for human serum transferrin for these
experiments. If this substitution is made, the extinction coefficients for the apotransferrin
and ferric transferrin will be 85,000 M-1cm-1 at 278 nm and 4500 M-1cm-1 at 463 nm,
respectively (16). Small differences in the rate of iron removal from bovine serum
transferrin may occur; however, these should not be significant due to structural similarities
with human transferrin.
6. NaCl is not believed to present a health hazard.
7. NaHCO3 causes slight skin and eye irritation.
8. Tris (base) is hygroscopic and can result in skin, eye and respiratory irritation.
9. Deferiprone can cause eye and skin irritation and can be toxic if ingested in large quantities.
Troubleshooting
Areas of trouble that might occur while completing this laboratory experiment should be
minimized if students follow the notes provided in the Student Handout. However, one area
where students might require additional assistance is in using the Sephadex column. First, be
sure that students have read and understand the Using the Sephadex G25-column (PD-10)
section of the Student Handout. After reading these instructions, students should understand
that after the tip of the column is cut, the liquid in the column should be discarded. Several
milliliters of Tris buffer should then be added and allowed to enter the column; only after all
of the Tris buffer has entered the column should students add their iron loaded transferrin
sample. Once all of the transferrin sample has entered the column, students then add
14
additional Tris buffer to force the iron loaded transferrin through the column and allow
collection of the orange band.
Problems may also arise with the transferrin concentration in the initial protein solution.
The laboratory instructions state that concentrations of apotransferrin should be between 50-
100 μM. Obtaining initial protein concentrations within this range is critical, since at lower
concentrations sufficient UV-vis absorbance may not be observed in order to carry out the
subsequent kinetics data acquisition. If transferrin concentrations are too high, more of the
protein is used than necessary, and the UV-vis absorbances may exceed the linear absorbance
limit of the spectrophotometer, preventing determination of accurate rates of iron removal.
Adapting this experiment
This laboratory experiment is very easily adapted for use in any undergraduate advanced
laboratory course, and is particularly relevant for bioinorganic, inorganic, or biochemistry
laboratory courses. Transferrin is a relatively inexpensive protein and can be stored for
several years when refrigerated. In addition, the UV-vis spectrophotometer required for this
experiment is a very common instrument found at nearly all educational institutions. This
multidisciplinary laboratory experiment also provides instructors with experimental ideas
that can be adapted to suit laboratories with a biological or chemical focus as described in the
Instructor Notes section.
15
Lab Documentation
STUDENT HANDOUT
Pre-laboratory assignment
Prior to the first three-hour laboratory session, you should receive and read the copy of
the laboratory experiment and complete calculations for the desired solutions in the experimental
procedure. You should also explore the Protein Data Bank (PDB) found at
http://www.rcsb.org/pdb/home/home.do. The PDB allows you to find and study the
three-dimensional structures of biological molecules, including apo- and diferric ovotransferrin.
For this pre-laboratory assignment, you should locate and examine the structures of both apo-
and diferric ovotransferrin from the PDB identifiers given in the caption of Figure 4. You should
also locate one other type of transferrin protein or an iron-containing protein found in humans
and provide a brief synopsis, including PDB identifier, structure, function, and other relevant
material, about the protein.
Introduction
You have recently been diagnosed with hemochromatosis, a genetic disease resulting in
increased absorption of iron in the intestines and excessive iron accumulation in the liver and
other organs. Symptoms of hemochromatosis include extreme fatigue, headaches, joint pain,
diabetes, heart disease, and even death (7). You were told by your physician that donating blood
is a simple and effective way for treating hemochromatosis; however, even with blood donation
your symptoms are persisting. You contact your physician to determine if there is any other
medication that can be prescribed to treat hemochromatosis, and learn that a drug, Deferiprone,
has been used to treat hemochromatosis because it chelates (binds) to the excess iron and
16
removes it from your body. It is also active when taken orally in pill form. Unfortunately, your
physician cannot prescribe Deferiprone because it has been banned in the United States by the
Food and Drug Administration (FDA) (10).
Being an inquisitive scientist, you decide to research Deferiprone. From your research,
you find that although not prescribed in the United States, Deferiprone is still used in other
countries, including Europe and India (6, 10). Uncertain why some countries prescribe
Deferiprone and the United States does not, you want to determine the efficacy of Deferiprone
for yourself. After having investigated how pharmaceutical companies test chelation drugs, you
decide that determining the rate of iron removal from transferrin, an iron transport protein found
in humans (11), would be a simple method for determining the efficacy of Deferiprone.
In your laboratory, you have a UV-vis spectrophotometer available to monitor iron
loading and then the rate of iron removal from transferrin by Deferiprone. Once your
experiments are completed, you can decide whether Deferiprone is effective at iron removal
based on how quickly iron is removed from transferrin.
Background information
Iron is an essential element required by most organisms, and is involved in a multitude of
biological processes including oxygen transport, the synthesis of DNA, and electron transport
(1). Within the body, iron homeostasis (balance) is tightly regulated due to the toxic nature of
non-protein bound iron, since the formation of oxygen radical species by non-protein bound
Fe(II) has been implicated in cellular damage and disease (3-5, 17, 18). Furthermore, the
insoluble nature of Fe(III) at neutral pH makes solubility of iron a potential difficulty for
17
organisms (1). Thus, iron must be tightly sequestered (bound) and solubilized, and mammals,
including humans, achieve this by utilizing a complex iron transport and storage system.
Serum transferrins are the major iron-
transporting proteins in blood and are part of a family
of transferrin proteins that include serum transferrins,
ovotransferrin, and lactotransferrins (11). Transferrin
proteins are small, containing approximately 700
amino acids (MW ~ 80 kD) and are synthesized
primarily in the liver (1, 19). Structurally, transferrin
consists of a single polypeptide chain with N-terminal and C-terminal lobes. Each lobe contains
two domains that are joined by a short peptide to create a hydrophobic metal binding site (1, 11,
20). Transferrin reversibly binds two Fe(III) ions with high affinity (Ka = ~ 1020 M-1); the amino
acid residues coordinating iron in each lobe include two tyrosine, an aspartate, and a histidine
residue, along with a bidentate carbonate anion (CO32-) (Figure 3) (21). In this coordinating
environment, the positive charge of the Fe(III) is balanced by the negative charges from the
tyrosine and aspartate residues; the negative charge of the carbonate anion is balanced by
neighboring positive charges on nearby amino acids (22). Transferrin can also bind other metal
ions, including bismuth(III), gallium(III), indium(III), aluminum(III), copper(II), manganese(II),
zinc(II), nickel(II), and ruthenium(III); however, transferrin binding affinity is highest for iron
(20).
Fe3+
N
NP
P
O
OO
O
P
O
PO
O
Figure 3. The amino acid residues that bind iron in the C- and N-terminal lobes of transferrin; the protein backbone is indicated by P.
Fe3+
N
NP
P
O
OO
O
P
O
PO
O
Figure 3. The amino acid residues that bind iron in the C- and N-terminal lobes of transferrin; the protein backbone is indicated by P.
18
Conformational changes occur in transferrin that are associated with iron binding or
release. When iron is bound, each domain moves to enclose iron in the metal binding site.
Similarly, upon release of iron, the metal-binding domains move apart; these “closed” and
“open” conformations have been observed through crystallography (Figure 4) (11, 23). Once
iron is bound to transferrin it is internalized by the cell through a process known as
receptor-mediated endocytosis; iron is then released and either used or stored in ferritin (24).
Researchers have studied iron removal from transferrin to gain a better understanding of
its mechanism of iron binding, and also how iron is stored and transported throughout the body.
Additionally, iron availability has been implicated as a limiting nutrient for bacterial infections
since the very high affinity of transferrin for iron prevents elevated levels of non-protein bound
iron, thereby limiting bacterial growth (25, 26). Investigating the mechanism of bacterial iron
Figure 4. The crystal structure of ovotransferrin: apo-ovotransferrin (left) and diferric ovotransferrin (right);
carbonate anion not shown. Structures were obtained from the Protein Data Bank with identifiers 1AOV and 1DOT,
respectively.
19
sequestration during infection may have significant implications in the treatment and prevention
of serious bacterial infections, including tuberculosis and peptic ulcers (27, 28).
Non-protein-bound iron also can react with naturally-occurring hydrogen peroxide to
generate reactive oxygen species (ROS), including superoxide radical (O2•-) and hydroxyl radical
(•OH), in the Fenton reaction (18):
FeII + H2O2 FeIII + •OH + OH-.
ROS have been implicated in increased oxidative stress and development of cancer,
inflammatory, cardiovascular, and neurodegenerative diseases (29-32). Iron chelating agents
may provide a way to prevent oxidative damage by keeping the non-protein-bound iron
concentration low thereby decreasing oxidative stress. For example, because long-term
transfusion therapy is required to treat β−thalassemia, a genetic blood disorder that causes
decreased hemoglobin production, a continual increase in iron concentration occurs in patients
since humans have no mechanism for iron excretion (33, 34). Similarly, hemochromatosis, a
genetic disorder associated with elevated iron levels due to increased intestinal iron absorption,
results in fatigue, diabetes, heart disease, and death (7). Chelation drugs must be used to remove
this excess iron, and continuing efforts are focused on developing new chelating agents that
would have improved properties over currently used drugs (35).
Several chelating agents are used for treatment of iron overload. For example,
desferrioxamine B (Desferral; Figure 5) is available to treat iron overload diseases, such as
hemochromatosis and β-thalassemia (10, 36). Desferral is a very effective iron chelator with
relatively low toxicity; however, it is expensive and can only be administered by continuous
intravenous infusion, resulting in poor patient compliance (8, 10). Another drug used to treat
iron overload is 1,2-dimethyl-3-hydroxy-4(1H)-pyridone, also known as Deferiprone (Figure 5)
20
(35). Deferiprone can be administered orally rather than intravenously; unfortunately, the high
toxicity of this compound has led the FDA to ban the use of Deferiprone in the United States
(10).
Testing chelators for their ability to kinetically remove iron from transferrin is an
effective method for evaluating their potential usefulness as chelation drugs. This laboratory
experiment will consist of monitoring iron loading into transferrin and then studying the kinetics
of iron removal by the drug Deferiprone using UV-vis spectroscopy.
Experimental procedure
Preparation of Fe(NTA)2 solution. Due to the low solubility of Fe(III) at pH 7.4, the
iron is loaded into transferrin using iron nitrilotriacetic acid, Fe(NTA)2, stock solution. You will
prepare Fe(NTA)2 by dissolving 45 μmol of FeCl3·6H2O and 90 μmol of nitrilotriacetic acid in 2
mL of 1 M HCl, and adjusting the pH to 4.0 with 1M NaOH solution, and diluting to 10 mL.1,2
Iron loading of transferrin. Dissolve human apotransferrin (20 mg, ~ 0.25 μmol; from
Sigma) in 3 mL of Tris buffer (Tris 50 mM, NaCl 150 mM, NaHCO3 20 mM; adjust pH to 7.4),
and take a UV/vis spectrum from 200 nm to 800 nm; be sure that you also collect a UV-vis
spectrum of Tris buffer as a blank at the same wavelengths. Calculate the concentration of
transferrin by measuring the protein absorbance at 280 nm and using an extinction coefficient of
87,200 M-1cm-1 (8).3 (Your concentration of apotransferrin should be between 50-100 μM.)
Figure 5. Chemical structure of Deferiprone (left) and Desferrioxamine B (right).
N O
OH(CH2)5
N
(CH2)2
NH(CH2)5H3N
OOH
O
NOOH
(CH2)2
NH(CH2)5
O
NCH3
OOH
Figure 5. Chemical structure of Deferiprone (left) and Desferrioxamine B (right).
N O
OH(CH2)5
N
(CH2)2
NH(CH2)5H3N
OOH
O
NOOH
(CH2)2
NH(CH2)5
O
NCH3
OOH
N O
OH(CH2)5
N
(CH2)2
NH(CH2)5H3N
OOH
O
NOOH
(CH2)2
NH(CH2)5
O
NCH3
OOH
21
Add Fe(NTA)2 solution (0.4 equivalents per transferrin protein) to 2700 μL of
apotransferrin, and, after waiting five minutes, take a UV/vis spectrum from 200 nm to 800 nm.4
You should repeat this process of Fe(NTA)2 addition and acquisition every five minutes until no
further changes in the UV/vis spectra are observed. Since the exact molarity of transferrin
cannot be accurately measured by weighing, it may take up to 3.2 equivalents to completely iron
load transferrin. The percentage of iron loading in the transferrin can be estimated by measuring
the absorbance at 470 nm using an extinction coefficient of 4,860 M-1cm-1 (8). Remove unbound
iron by passing the transferrin solution through a Sephadex G25-column (PD-10 column,
Pharmacia Biotech) equilibrated and eluted with the Tris buffer (pH = 7.4). You should collect
only the orange band to ensure purity. The protein concentration and iron loading should be re-
determined after the protein is collected. Your solutions can then be stored at 4 °C until further
use.5
Iron removal. Carry out iron removal studies from diferric transferrin at 37 °C and pH
7.4 in Tris buffer (50 mM Tris, 150 mM NaCl, and 20 mM NaHCO3). Prepare a stock solution
of the Deferiprone (commercially available from VWR) by dissolving the desired amount of
Deferiprone in 1 mL of Tris buffer, heating this solution and 700 μL of your diferric transferrin
solution to 37 °C, and allowing 10 minutes for temperature equilibration.6,7 At this point, you
should add 300 μL of the chelator solution to the transferrin solution (final volume = 1 mL; 120
equivalents of Deferiprone to transferrin protein)8 and monitor the kinetics of iron removal from
transferrin by UV/Vis at 460 nm with scans every second for 50 minutes (3000 scans).
Using the Sephadex G25-column (PD-10)
When using the Sephadex G25-column (PD-10), cut off the bottom tip of the column,
remove the top cap, and pour off the liquid. Add several milliliters of Tris buffer to the column,
22
wait for it to completely load onto the column, and discard the flow-through solution. Once
equilibrated with Tris buffer, your iron-loaded transferrin solution should be immediately added
to the column. Once your transferrin solution is completely loaded onto the column, collect only
the orange band by adding several more milliliters of Tris buffer to elute the transferrin solution.9
Notes
1. pH should be above 2. If you exceed pH 4, Fe(OH)3 precipitates.
2. When adjusting pH of Fe(NTA)2, a precipitate may occur; however, as pH is increased
(with shaking), the solid will re-dissolve.
3. Dilution by a factor of 20 is usually required to get the absorbance below 1.
4. Use the calculated apotransferrin concentration when determining equivalents of
Fe(NTA)2.
5. Solutions of transferrin, stored properly at 4 ºC, can be used for approximately 3 months.
6. When preparing the Deferiprone solution, be sure that all of the Deferiprone is dissolved
prior to use; this may require several minutes of mixing.
7. These experiments can also be conducted at room temperature (25˚C). If experiments are
performed at 25˚C, heating the Deferiprone and diferric transferrin solutions and
temperature equilibration are not required.
8. The ideal final concentration of transferrin should be between 20-100 μM.
9. Be sure to collect only the orange band from the Sephadex column so that dilution of the
transferrin solution does not occur.
23
Calculations
The concentration of transferrin is calculated using the formula A = εlc. Where A is
absorbance at 280 nm, ε is the extinction coefficient (87,200 M-1cm-1), l is the path length of the
cuvette used (1 cm), and c is the concentration of transferrin (8). When determining
concentration, be sure to account for the absorbance of Tris buffer and include dilution factors.
The percentage of iron loading in transferrin is also calculated using the above equation;
however, absorbance is determined at 470 nm and the extinction coefficient used is 4,860
M-1cm-1 (8).
The kinetic data can be fit to the equation: At = A∞(1-e-kt) + Aoe-kt. Where At is
absorbance at time t, A∞ is the final absorbance, Ao is the absorbance due to diferric transferrin
alone, and k is the observed rate of reaction (kobs). Ao should be determined by performing a
blank run with transferrin in buffer without the Deferiprone chelator. Fitting the kinetic data to
the above equation should be completed using a spreadsheet program; you can find instructions
for performing these calculations in the Data analysis section.
Data analysis
You will need Microsoft Excel or an equivalent spreadsheet program in order to graph
iron loading and removal. Your UV-vis spectra should be saved as text or other compatible files
and opened in the spreadsheet program. When opening a text file in Excel, select fixed width in
Step 1 of the Text Import Wizard. Create line breaks in Step 2 so that there are 2 columns
containing wavelength (Column A) and absorbance (Column B); once line breaks are generated,
select finish. For iron loading, the absorbance for the Tris buffer blank should be entered into
Column C and subtracted from the absorbance of the apotransferrin and iron-loaded transferrin
24
solutions; enter data into Column D using the equation: =(B#-C#). Plot the corrected
absorbance of apotransferrin and iron loaded transferrin.
To determine the pseudo-first-order rate constant for iron removal from transferrin (kobs),
you must generate an additional spreadsheet containing time (Column A) and absorbance
(Column B). Follow the same procedure when opening text files in Excel. Similarly, the
absorbance for Tris buffer should be entered into Column C and subtracted from the absorbance;
enter data into Column D using the equation: =(B#-C#).
When determining the rate of iron removal from transferrin, enter Ao and A∞ in Columns
E and F. Determine ln[(At-A∞)/(Ao-A∞)] from the equation given in the procedure using the
spreadsheet program equation: =LN((D#-F#)/(E#-F#)). Once the rates have been calculated,
find -kobs by plotting ln[(At-A∞)/(Ao-A∞)] versus time: =LN((D#-F#)/(E#-F#))/(A#). The true
second-order rate constant (k2) can be calculated using the formula kobs = k2[L]/(1 + k2/kmax[L]),
where [L] is the final concentration of Deferiprone chelator used in the iron removal experiment,
and kmax is 1.35 × 10-3 s-1 (8).
Safety and hazards
When handling chemicals during this laboratory experiment gloves, adequate ventilation,
and protective eyewear and clothing are required. You should avoid contact with eyes and skin,
and do not ingest laboratory chemicals. Special care should be taken when handling human
apotransferrin due to the possibility of infectious agents being present. This material should be
handled at the bio-safety level 2 as recommended for any potentially infectious human serum or
blood specimen in the CDC/NIH manual "Biosafety in Microbiological and Biomedical
Laboratories", 1999, or can be found at the following web address:
25
http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4s3.htm. Additionally, after each three-hour
laboratory session, your transferrin solutions should be stored at 4˚C.
Laboratory questions
1. Describe the structure of human serum transferrin and how iron is coordinated in this
protein. How do the various families of transferrin proteins function in humans, and why
are they important?
2. Describe in detail the function of transferrin. How is ferric transferrin is taken up by a
cell, and how is iron released from the protein?
3. Why is the conformational change that occurs during iron binding to transferrin
important?
4. Why is it important to remove excess unbound iron from the iron-loaded transferrin using
column chromatography before determining the kinetics of iron removal?
5. From your experiments, what is the average observed rate constant for iron removal (kobs)
by Deferiprone?
6. Based on the calculated second-order rate constants for iron removal (k2) from transferrin
by Deferiprone, do you believe that Deferiprone is an effective iron chelating drug?
Compare your calculated second-order rate constant for iron removal (k2) by Deferiprone
to that of other iron chelating ligands (Reference: Turcot, I.; Stintzi, A.; Xu, J.; Raymond,
K. N. J. Biol. Inorg. Chem. 2000, 5, 634-641.)
Instructor notes
Pre-laboratory assignment
26
It is recommended that a pre-laboratory assignment is given to students prior to the first
three-hour laboratory session. The pre-laboratory assignment is optional; however, it is strongly
suggested for reducing laboratory time, ensuring completion of the laboratory experiment, and
providing students with a greater understanding of the mathematical calculations required for this
experiment. For this assignment, students may be provided with molecular weights for each
chemical listed in the laboratory procedure; however, it is not required.
It is also suggested that students explore the Protein Databank (PDB) as an additional
pre-laboratory assignment. The PDB is a worldwide collection of 3-dimensional structural data
of biological macromolecules, including proteins and nucleic acids. Exploring this database will
allow students to investigate more thoroughly the apo- and diferric ovotransferrin protein
structures given in the laboratory background, while familiarizing themselves with this very
useful resource. Furthermore, students should locate an additional transferrin protein or other
iron-containing protein found in humans, and discuss its function and structure. Students can
access the PDB at http://www.rcsb.org/pdb/home/home.do. If students appear to have difficulty
navigating the PDB, additional instruction in the form of a laboratory experiment developed by
Ship et al. may be useful (37). Alterations to the directions for the pre-laboratory assignments
can be made at the discretion of the instructor.
Alternative experiments
The experimental procedure dictates that kobs for iron removal should be determined at
both 37˚C (human body temperature) and room temperature (25˚C). However, if the required
isotemperature thermal modulator is unavailable or too costly, this can be determined only at
27
25˚C. Additionally, this modified procedure allows instructors to reduce experimental length
because temperature equilibration is not necessary for experiments conducted at 25˚C.
In addition to iron, transferrin can also bind a variety of other metal ions, including
bismuth(III), gallium(III), indium(III), aluminum(III), copper(II), manganese(II), zinc(II),
nickel(II), and ruthenium(III) (20). This provides an alternative experiment that would greatly
complement the present study by enabling students to compare the various rates of metal
removal by Deferiprone; students would determine if Deferiprone is more efficient at removing
iron or other metals from transferrin, and gain some insight into the efficacy of Deferiprone as a
metal-chelating agent. Similarly, performing several UV-vis kinetic experiments with transferrin
loaded with different metals ions, will allow students to observe comparative metal binding
affinity of transferrin. Experimental procedures for loading transferrin with metals other than
iron can be found in Turcot, et al. and Li, et al. (8, 38).
Polyacrylamide gel electrophoresis (PAGE) is another technique that students can use to
analyze iron loading and removal from transferrin, and this procedural modification would
provide an alternative experiment for a biochemistry laboratory. PAGE is technique that
separates proteins based on their electrophoretic mobility, including molecular weight and
protein folding (39). Using this technique, students can estimate the molecular weight of
transferrin, and compare their observed value to the known value (~ 80 kDa) (21). Students can
also determine the distribution of iron in the N-terminal and C-terminal lobes, and determine iron
removal from each lobe by Deferiprone. Laboratory experiments using PAGE to analyze
transferrin proteins have not been published for educational purposes; however, procedures
detailing the use of PAGE for protein analysis can found in papers by Hamilton, Williams, and
Makey and their coworkers (11, 40-42).
28
Laboratory preparation
Students can work in small groups or individually. Each group or individual should be
provided with equipment and chemicals found in the Chemicals and equipment per student
section.
Solution preparation
Solution preparation by the instructor is only necessary for 1 M NaOH and 1 M HCl; all
other solutions should be prepared by the student. Please read Safety and hazards prior to
solution preparation.
Instrumental directions
A UV-vis spectrophotometer is required for this laboratory experiment. An
isotemperature thermal modulator is also required for temperature regulation of the kinetics
experiments; however, this instrumentation is optional (see the Alternative experimental
procedures section for experimental modifications). Also, kinetics software that enables one
wavelength detection capabilities over time greatly simplifies kinetic data acquisition.
Prior to starting each three-hour laboratory session, instructors should turn on the
spectrophotometer and allow the instrument to warm up for approximately 20-30 minutes; if
using an isotemperature thermal modulator also allow approximately 20-30 minutes for
temperature equilibration. The desired spectra should be taken at the wavelengths specified in
the experimental procedure; the appropriate blank spectra should also be obtained in addition to
the experimental spectra.
29
Tips for success
Notes are provided to students to ensure that the laboratory experiment is properly
executed; however, some additional areas where difficulties may occur can be seen in the
Troubleshooting section of the lab summary.
Answers to student questions
1. Transferrin is made up of approximately 700 amino acids (80kDa), and contains both α-
helices and β-sheets. The structure consists of two lobes known as the C- and N-terminal lobes
and are held together by a short peptide, which creates a hydrophobic metal binding site. For
iron coordination, the amino acids that bind iron(III) include two tyrosine, an aspartate, and a
histidine residue, along with a bidentate carbonate anion (CO32-) that is not part of the protein,
known as the synergistic anion. The Fe(III) is bound in an octahedral geometry.
There are several families of transferrin proteins found in humans, including serum transferrin,
lactotransferrin, and melanotransferrin. Serum transferrin carries iron in the bloodstream, while
lactotransferrin is found in milk, tears, and saliva and serves as an antimicrobial agent.
Melanotransferrins are expressed in low levels in normal tissues but higher levels in melanomas
and are required for iron metabolism and cell proliferation. Transferrin proteins are very
important for binding and distribution of iron in the human body.
2. Humans acquire iron from the food that we ingest. Once iron is absorbed, serum transferrin
binds iron in the blood, thus solubilizing and sequestering the extremely insoluble Fe(III) ion,
and allowing transport throughout the body. To be transported into cells, iron-loaded transferrin
binds to the transferrin receptor protein on the outer membrane of a cell, and is internalized in a
30
process known as receptor-mediated endocytosis. During this process, a portion of the
membrane containing the transferrin bound to the receptor protein folds in and pinches off to
form a vesicle inside the cell. Inside the cell, the vesicle forms an endosome, and the pH inside
the vesicle is lowered (by ATP-driven proton pumps). Once the pH is lowered in the vesicle, the
synergistic carbonate is protonated and can no longer bind to the iron. This dramatically lowers
the stability of transferrin-bond iron, allowing the iron to be released from the protein.
3. When iron is not bound to transferrin (in the apo form of the protein), both lobes exist in the
‘open’ conformation. When iron is bound in each lobe, the protein conformation changes to a
‘closed’ lobe conformation. This change in protein conformation allows for easier access of
iron(III) to the iron binding site in the apo form of transferrin, and increases the stability of iron
binding in the ‘closed’ conformation of the lobes. The conformational change is also important
for the cooperativity seen for iron binding and removal in transferrin.
4. Removal of excess unbound iron prior to determining the rate of iron removal is important
because Deferiprone will coordinate to unbound iron, thus giving an inaccurate rate of iron
removal from transferrin.
5. The average kobs for iron removal is determined by students from their collected data.
6. Students should compare their measured k2 of iron removal from transferrin by Deferiprone
to the rate reported by Turcot et al. (7.8 × 10-2 M-1min-1); conversion of their rate from s- 1 to
min-1 will be required for comparison. Students should also comment on why differences or
similarities in the rates of iron removal are seen.
Based on their measured average rate of iron removal, students should give their opinion
about the effectiveness of Deferiprone as an iron chelating drug and explain the reasons for their
31
opinions. Answers should discuss their measured values for Deferiprone iron removal in
comparison to rates reported for other iron chelators in Turcot, et al. Rates of iron removal from
transferrin using other chelators determined by are given in Table 1.
Table 1. Rate constants (k2) for iron removal from transferrin using various iron chelators (8).
Iron Chelators Calculated k2 (M-1min-1)
Enterobactin 8.1
Desferrioxamine B ~ 0
TRENCAM 10
BU-O-3,4-HOPO 4.5
5LIO-3,2-HOPO 7.2
TRENCAM-3,2-HOPO 23
TRPN-3,2-HOPO 5.9
TREN-Me-3,2-HOPO 16
TREN-1,2,3-HOPO 6.3
Deferiprone 7.8
Chemicals and equipment per student
UV/vis spectrophotometer with kinetics software
UV-vis temperature-regulating water bath (optional)
Cuvettes (3 mL and 1mL)
32
Automatic pipettor (100 to 1000 microliters)
Timer
~ 7-10 Plastic conical tubes (15 mL)
Conical tube rack
1 Box pipette tips (100 to 1000 microliter volume)
Marking pen
pH paper
Beaker (250 mL)
Thermometer
Nitrilotriacetic acid (25 mg)
FeCl3·6H2O (12 mg)
Human Apotransferrin (20 mg)
1,2 dimethyl-3 hydroxy-4 (1H)-pryidone (Deferiprone; ~50mg)
Tris buffer: tris base (73 mg), NaCl (105 mg), NaHCO3 (20 mg) (pH = 7.4)
Sephadex G25-column (PD-10)
1 M HCl
1 M NaOH
ddH2O
CAS registry
1. NaOH: 1310-73-2
2. HCl: 7647-01-0
3. FeCl3⋅6H2O: 10025-77-1
33
4. Nitrilotriacetic acid (NTA): 18662-53-8
5. Human apotransferrin: 11096-37-0
6. NaCl: 7647-14-5
7. NaHCO3: 144-55-8
8. Tris (base): 77-86-1
9. Deferiprone: 30652-11-0
Safety and hazards
When handling chemicals during this laboratory experiment gloves, adequate ventilation,
and protective eyewear and clothing are required. Avoid contact with eyes and skin, and do not
ingest laboratory chemicals. When making 1 M HCl from concentrated HCl, prepare solutions
in a fume hood with the hood shield down as far as possible. Transferrin solutions can be stored
at 4˚C and can be used for up to 3 months. Due to the possibility of infectious agents being
present, human apotransferrin should be handled at the bio-safety level 2 as recommended in the
CDC/NIH manual "Biosafety in Microbiological and Biomedical Laboratories", 1999, or can be
found at the following web address: http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4s3.htm.
To avoid these biosafety issues, bovine serum transferrin can be substituted for human serum
transferrin for these experiments. If this substitution is made, the extinction coefficients for the
apotransferrin and ferric transferrin are 85,000 M-1cm-1 at 278 nm and 4500 M-1cm-1 at 463 nm,
respectively (16). Slight differences in the rate of iron removal from bovine serum transferrin
may occur; however, these should not be significant due to structural similarities with human
transferrin.
34
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