Complexation of ferrous ions by ferrozine, 2,2′-bipyridine and 1,10-phenanthroline: Implication for the quantification of iron in biological systems

Article abstract of DOI:10.1016/j.jinorgbio.2021.111460

Iron is an essential nutrient for virtually all forms of life. Because of its redox properties and involvement in a wide range of biological processes, a number of qualitative and quantitative chemical tools have been developed to detect reduced (Fe²⁺) and oxidized (Fe³⁺) forms of iron in biomolecules. These types of measurements are not only important in detecting iron species in solution, but also in understanding iron distribution, accumulation, and role in physiological and pathological processes. Here, we use UV–vis spectrophotometry and three common chromogenic reagents, ferrozine, 2,2′-bipyridine, and 1,10-phenanthroline to detect and quantify the concentration of ferrous ions in aqueous solutions, owing to the unique absorption spectra, specific molar absorptivity, and characteristic colors of these Fe²⁺-chelator complexes. Our results show that the kinetics of the formation of the {Fe²⁺-(ferrozine)₃} complex, but not the{Fe²⁺-(bipyridine)₃} or the {Fe(II)-(phenanthroline)₃} complexes depend on the concentration of the iron chelator, requiring up to 20 min to complete when close to stoichiometric ratios are employed. The molar absorptivity values of these complexes under excess chelator concentrations were ~ 10% to 15% higher than reported literature values (i.e. 31,500 ± 1500 M^?1 cm^?1 for ferrozine at 562 nm, 9950 ± 100 M^?1 cm^?1 for 2,2′-bipyridine at 522 nm, and 12,450 ± 370 M^?1 cm^?1 for 1,10-phenanthroline at 510 nm). Our results have important implications when quantifying iron in biological systems and reveal optimal experimental conditions that must be employed for the accurate measurements of ferrous ions, whether free in solution, or after reduction of protein-bound ferric ions.

Full text of DOI:10.1016/j.jinorgbio.2021.111460

Journal of Inorganic Biochemistry 220 (2021) 111460
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Complexation of ferrous ions by ferrozine, 2,2′-bipyridine and 1,10-phe-
nanthroline: Implication for the quantification of iron in biological systems
Gideon L. Smitha, Aliaksandra A. Reutovicha, Ayush K. Srivastavaa, Ruth E. Reicharda,
,
,*
Cass H. Welsha, Artem Melmanb *, Fadi Bou-Abdallaha
a Department of Chemistry, State University of New York at Potsdam, Potsdam, NY 13676, United States of America
b Department of Chemistry & Biomolecular Science, Clarkson University, Potsdam, NY 13699, United States of America
A R T I C L E I N F O
A B S T R A C T
Keywords:
Iron is an essential nutrient for virtually all forms of life. Because of its redox properties and involvement in a
wide range of biological processes, a number of qualitative and quantitative chemical tools have been developed
to detect reduced (Fe2+) and oxidized (Fe3+) forms of iron in biomolecules. These types of measurements are not
only important in detecting iron species in solution, but also in understanding iron distribution, accumulation,
and role in physiological and pathological processes. Here, we use UV–vis spectrophotometry and three common
chromogenic reagents, ferrozine, 2,2′-bipyridine, and 1,10-phenanthroline to detect and quantify the concen-
tration of ferrous ions in aqueous solutions, owing to the unique absorption spectra, specific molar absorptivity,
and characteristic colors of these Fe2+-chelator complexes. Our results show that the kinetics of the formation of
the {Fe2+-(ferrozine)3} complex, but not the{Fe2+-(bipyridine)3} or the {Fe(II)-(phenanthroline)3} complexes
depend on the concentration of the iron chelator, requiring up to 20 min to complete when close to stoichio-
metric ratios are employed. The molar absorptivity values of these complexes under excess chelator concen-
trations were ~ 10% to 15% higher than reported literature values (i.e. 31,500 ± 1500 Mꢀ 1 cmꢀ 1 for ferrozine at
562 nm, 9950 ± 100 Mꢀ 1 cmꢀ 1 for 2,2′-bipyridine at 522 nm, and 12,450 ± 370 Mꢀ 1 cmꢀ 1 for 1,10-phenanthro-
line at 510 nm). Our results have important implications when quantifying iron in biological systems and reveal
optimal experimental conditions that must be employed for the accurate measurements of ferrous ions, whether
free in solution, or after reduction of protein-bound ferric ions.
Ferrozine
2,2′ bipyridine
1,10 phenanthroline
Ferrous-ion complexes
MLCT
Absorption spectra
Iron chelators
1. Introduction
These metal ions-ligand complexes show unique absorption spectra with
characteristic colors in the visible region of the electromagnetic spec-
Iron is one of the most important elements for most forms of life and
plays a significant role in various physiologically important functions,
owing to its distinctive redox properties [1]. However, the high reac-
tivity and interconversion between the multiple oxidation states of iron
can be problematic and can cause cellular damage and apoptosis. One of
the most harmful reactions in biology is the Fenton reaction where Fe2+
ions catalyze the generation of highly toxic reactive chemical species
that can lead to pathological processes including cancer, and neurode-
generative diseases [1–5]. Many chemical tools and assays have been
developed to qualitatively and quantitatively measure the redox status
of iron and its distribution and accumulation in biological systems [6].
One of the most common and frequently used spectroscopic methods to
quantify the concentration of metal ions in aqueous solutions involves
the measurement of absorbance changes using chromogenic reagents.
trum and are due to either metal to ligand charge transfer (MLCT) or
ligand to metal charge transfer bands. Because these metal-ions com-
plexes often have high stability constants and specific molar absorptivity
values, the color changes are directly proportional to the concentration
of metal ions in solution, thus enabling accurate quantification.
Among the most stable and intensely colored ferrous ion complexes
that are suitable for the quantitative determination of iron are 1,10-phe-
nanthroline (ε510 nm = 11,100 Mꢀ 1 cmꢀ 1) [7,8], 4,7-diphenyl-l,l0-phe-
nanthroline (or bathophenanthroline, ε533nm = 22,400 Mꢀ 1 cmꢀ 1) [9],
2, 2′ -bipyridine ε522 nm = 8650 Mꢀ 1 cmꢀ 1) [10], 2,6-bis(2-pyridyl)-pyr-
idine (also known as 2,2′,2′′-terpyridyl, ε552 nm = 11,500 Mꢀ 1 cmꢀ 1
)
[10], 2,4,6-tripyridyl-l,3,5-triazine (or TPTZ, ε593
= 22,600 Mꢀ 1
nm
cmꢀ 1) [11], and 3-(4-phenyl-2-pyridyl)-5,6-diphenyI-l,2,4-triazine (or
PPDT, ε561 nm = 28,700 Mꢀ 1 cmꢀ 1) [12]. All of these reagents contain a
* Corresponding authors.
E-mail addresses: amelman@clarkson.edu (A. Melman), bouabdf@potsdam.edu (F. Bou-Abdallah).
https://doi.org/10.1016/j.jinorgbio.2021.111460
Received 16 January 2021; Received in revised form 5 April 2021; Accepted 5 April 2021
Available online 15 April 2021
0162-0134/© 2021 Published by Elsevier Inc.

G.L. Smith et al.
Journal of Inorganic Biochemistry 220 (2021) 111460
Scheme 1. Chemical structures of ferrozine, ferene, bathophenanthroline disulfonate, {Fe(II)-(Bipyridine)3}, {Fe(II)-(Phenanthroline)3} and {Fe(II)-(Ferrozine)3}.
– –
–
C C N that binds metal
– –
specific and cyclic atomic configuration N
ferrozine for Fe2+ ions [15], many studies have used stoichiometric
amounts of ferrozine, or a slight excess of chelator, to make sure that
Fe2+ ions are fully bound. Surprisingly, while investigating the reductive
mobilization of iron from the major iron storage protein ferritin, we
obtained conflicting results and anomalous absorbance values that were
inconsistent with literature reports and the published molar absorptivity
for the {Fe(II)-(ferrozine)3} complex. This work was therefore under-
taken to examine the complexation reaction of Fe(II) by ferrozine under
conditions close to physiological, and the effect of experimental factors
such as pH, concentration of reagents, buffer, and incubation time on
complex formation. Furthermore, we investigated the complexation ki-
netics of two other frequently used ferrous ion chelators (i.e. 2,2′-
bipyridine and 1,10-phenanthroline) to explore whether the peculiar
behavior observed with ferrozine is also prominent with these other
strong Fe(II) chelators. Given the importance of iron in biology, chem-
istry, and the environment, our results underscore the significance of
proper experimental conditions to accurately detect and quantify the
presence of iron species in aqueous media.
ions, including Fe(II), to form colored complexes.
Perhaps one of the most common and frequently used ferrous iron
chelators is the disodium salt of 3-(2-pyridyl)-5,6-bis(4-phenylsulfonic
acid)-1,2,4-triazine, also referred to as ferrozine (FZ). Since its synthesis
in the 1970 [7], FZ has been extensively used in a variety of applications
to quantify ferrous ions [7,13]. One of the main advantages of FZ over
other reagents is its high-water solubility, high sensitivity, and stability
over a wide range of pH (i.e. pH 4 to 9) [7]. The ferrous-ferrozine
complex exists in a 3:1 ratio of ferrozine to iron and is reported to
have one of the highest molar absorptivity values (i.e. 27,900 Mꢀ 1 cmꢀ 1
at 562 nm) among other commonly used iron complexes. Although
PPDT has a slightly higher molar absorptivity, the problem with that
reagent is its very poor water solubility and very slow kinetics with Fe
(II) in dilute aqueous solutions [12].
The only other highly sensitive and water-soluble ferrous ion
chelator that has similar characteristics to ferrozine is the disodium salt
of 3-(2-pyridy1)-5,6-bis(2-(5-fury1 sulfonic acid))- 1,2,4-triazine (also
known as ferene) (ε593 nm = 34,500 Mꢀ 1 cmꢀ 1) [14]. Structurally, the
only difference between ferrozine and ferene is the presence of an ox-
ygen atom in each of the furyl rings; the two phenyl sulfonate groups in
ferrozine are substituted with sulfonyl furyl groups in ferene (Scheme 1).
Although both molecules behave as bidentate chelating agents via the
pyridyl and 2-triazine nitrogen atoms, the {Fe(II)-(ferene)3}complex
(logK = 14.9) forms a blue color at 593 nm whereas the {Fe(II)-(ferro-
zine)3}complex (log K = 15.7) has a magenta color at 562 nm [7,13–15].
Although ferrozine is not a physiologically relevant ligand, the fer-
rozine assay has been widely used in the detection of ferrous ions in a
variety of samples, including in mineral and biological systems. The
accurate quantification of Fe2+ ions is affected by several factors
including pH, temperature, concentration of reagents, incubation time,
anions such as oxalate, cyanide and nitrite, and also the presence of Fe3+
cations [7,13–20]. Because of the rather high binding affinity of
2. Materials and methods
2.1. Chemicals
All chemicals employed in this study were reagent grade and used
without further purification. MOPS [3-(N-morpholino)propanesulfonic
acid] buffer and 2,2′-bipyridine were purchased from Sigma-Aldrich,
tris-(hydroxymethyl)aminomethanehydrochloride (Tris-HCl) from G-
Biosciences, 1,10 phenanthroline from TCI Chemicals, sodium chloride
and acetic acid from Thermo Fisher Scientific, ferrous sulfate heptahy-
drate and sodium acetate was from J. T. Baker Chemical, and ferrozine
(5,6-diphenyl-3-(2-pyridyl)-1,2,4-triazine-4,4′-disulfonic acid disodium
salt hydrate, > 97% purity) from Alfa Aesar. Fe(II) stock solutions were
freshly prepared immediately before each experiment using pH 2.0
2

G.L. Smith et al.
Journal of Inorganic Biochemistry 220 (2021) 111460
Fig. 1. (A) Kinetics of Fe(II) complexation with ferrozine in 50 mM Mops, 100 mM NaCl, pH 7.4. Conditions: 20 μM Fe(II), 60–600 μM ferrozine. (B) The experiment
was repeated under strict anaerobic conditions using degassed solutions with pure argon gas.
deionized water. Ferrozine stock solutions were prepared in either DI
water or in buffer. Stock solutions of 2,2′-bipyridine and 1,10-phenan-
throline were prepared in a mixture of 50:50 water:ethanol.
Bio (or a Cary 60) spectrophotometer from Agilent Technologies. Unless
otherwise noted, all experiments were conducted at 25.00 ◦C in 50 mM
MOPS and 100 mM NaCl (pH 7.4). For the absorbance measurements,
multiple samples were prepared as indicated in the figure captions and
were left on a rocker, wrapped in aluminum foil, for 30–50 min. For the
UV–vis titrations, multiple microliter injections of a ferrozine stock so-
lution were added to a {Fe(II)-(ferrozine)3} complex prepared in 132
2.2. Absorption spectroscopy and ultraviolet–visible (UV–vis) kinetics
UV–vis absorption spectroscopy was performed on a Varian Cary 50
Fig. 2. (A) UV–vis absorption spectra of different ferrozine-Fe(II) solutions; (B) Plots of the absorbance change at 562 nm vs. ferrozine/Fe(II) and (C) vs. Fe(II)/
ferrozine; (D) Plot of the molar absorptivity (ε) change at 562 nm as a function of ferrozine/Fe(II). Conditions: 90 μM ferrozine in 50 mM Mops, 100 mM NaCl, pH pH
7.4 (or in 132 mM Tris, pH 7.4) with changing Fe(II) concentrations (3–90
μM), at 1 h and 48 h following the preparation of the ferrozine-Fe(II) complexes. The error
bars represent the standard deviation from 5 separate experiments.
3

G.L. Smith et al.
Journal of Inorganic Biochemistry 220 (2021) 111460
Fig. 3. (A) UV–vis absorption spectra of different ferrozine-Fe(II) solutions; (B) Plot of the absorbance change at 562 nm vs. ferrozine/Fe(II) and (C) Plot of the molar
absorptivity (ε) change at 562 nm as a function of ferrozine/Fe(II). Conditions: 20 μM Fe(II) with changing ferrozine concentrations (60–2000 μM). The ferrozine
solution was prepared in 50 mM Mops, 100 mM NaCl, pH 7.4 and the Fe(II) stock solution was prepared in DI water pH 2.0. The error bars represent the standard
deviation from 7 separate experiments.
mM Tris buffer, pH 7.4. The acetate buffer (0.1 M, pH 5.0) was prepared
by dissolving 0.5772 g of sodium acetate in 100 mL of DI water, and then
dissociation of the Fe(II)-ferrozine complex were on the order of 1011
Mꢀ 3 sꢀ 1 and 10ꢀ 5 sꢀ 1 at 25.00 ◦C, respectively, with an equilibrium
association constant of ~5 × 1015 Mꢀ 3. These kinetic experiments were
performed under conditions of excess ferrozine (i.e. 100 times more than
Fe(II)). We thought to examine the kinetics of complexation at pH 7.4
starting at the stoichiometric ratio of 3:1 ferrozine:Fe(II) and up to 10
times higher ferrozine concentration (i.e. 30 ferrozine: 1 Fe(II)). Under
conditions close to stoichiometric ratios (i.e. 3 to 5 ferrozine molecules
per Fe(II) ion), the kinetics of complexation were slow requiring at least
10 min for completion (Fig. 1). At higher ratios (>10 ferrozine:Fe(II)),
the kinetics were instantaneous and exhibited higher absorbance values,
suggesting that excess ferrozine is required for the formation of a stable
{Fe(II)-(ferrozine)3} complex. To make sure that Fe(II) auto-oxidation is
not a factor affecting the absorbance of the slow kinetics, the experiment
was repeated under strictly anerobic conditions, by degassing all solu-
tions for at least 1 h using pure argon gas. The kinetics were then per-
adding a 171 μL of glacial acetic and adjusting the pH to 5.0 using 10 M
HCl. Final reagent concentrations for each experiment are given in the
figure captions.
The kinetics of Fe(II) complexation with ferrozine were followed at
562 nm where the {Fe(II)-(ferrozine)3} species absorbs. To ensure
reproducibility, all experiments were repeated 3 to 10 times, using
freshly prepared solutions. The instrument is zeroed using the ferrozine-
buffered solution as the blank, and the experiment is typically initiated
with the addition of a few microliters of a ferrous sulfate solution, pre-
pared in deionized H2O (pH 2), into the 1 mL ferrozine solution, with a
spin bar rapidly stirring under the conditions stated in the figure cap-
tions. Similar experiments were repeated with 2,2′-bipyridine and 1,10
phenanthroline. Time-dependent absorbance kinetic traces were
analyzed with OriginLab version 8.0 (OriginLab Corp.).
Because a rigorous quantification of Fe(II) ions in solution is critical
to validating molar absorptivity values of all Fe(II)-complexes studied
here, the concentrations of working Fe(II) solutions and the amount of
Fe(II) ions added to solutions of various ligands have been checked by
atomic absorbance spectroscopy (AAS). The AAS data showed that the
concentrations of Fe(II) ions in solution matched the predicted Fe(II)
concentration with >98% accuracy of the expected Fe(II) concentration.
formed in a tightly sealed UV-cuvette to prevent diffusion of
atmospheric oxygen. As can be clearly seen in Fig. 1B, there is virtually
no effect from the presence of O2 in solution on the complexation ki-
netics or the absorbance values of the {Fe(II)-(ferrozine)3} complex,
under our experimental conditions.
3.2. Ferrozine – Fe(II) stoichiometry
3. Results and discussion
In order to determine the binding stoichiometry of Fe(II) to ferrozine,
multiple 1 mL samples were prepared using a constant ferrozine con-
3.1. Fe(II)-ferrozine complexation kinetics
centration (90 μM) and different concentrations of Fe(II) (3 μM to 90
μ
M), such that the ratio of ferrozine to Fe(II) varied between 1 and 30.
The kinetics of Fe(II) complexation with ferrozine has been studied
earlier [15] using rapid stopped-flow mixing and visible spectropho-
tometry at 562 nm in acidic pH. The rate constants for the formation and
The samples were wrapped in aluminum foil and placed on a rocker to
mix gently for a period of 30–50 min before absorbance measurements
were performed. Fig. 2 shows the absorption spectra of 15 different
Fig. 4. (A) UV–vis absorption spectra of different ferrozine-Fe(II) solutions; (B) Plot of the absorbance change at 562 nm vs. ferrozine/Fe(II) and (C) Plot of the molar
absorptivity ( ) change at 562 nm as a function of ferrozine/Fe(II). Conditions: 500 M ferrozine in 50 mM Mops, 100 mM NaCl, pH 7.4 with changing Fe(II)
concentrations (5–166.7 M). The error bars represent the standard deviation from 4 separate experiments.
ε
μ
μ
4

G.L. Smith et al.
Journal of Inorganic Biochemistry 220 (2021) 111460
Fig. 5. (A) Kinetics of Fe(II) complexation with 2,2′-bipyridine at 522 nm and 1,10-phenanthroline at 510 nm. (B) Plot of the molar absorptivity (
function of ligand/Fe(II) ratio. (C, D) Absorbance change of {Fe(II)-(bipyridine)3} and {Fe(II)-(phenanthroline)3} as a function of ligands concentration. Conditions:
60 M Fe(II) and 186, 240, and 600
M 2,2′-bipyridine or 1,10-phenanthroline for. The error bars in (B) represent the standard deviation from 9 separate
experiments.
ε) change as a
μ
μ
samples and their corresponding plots as a function of ferrozine/Fe(II) or
Fe(II)/ferrozine ratios. A clear break at 3:1 ferrozine:Fe(II) is observed
with a molar absorptivity value of 22,500 Mꢀ 1 cmꢀ 1 that slightly in-
creases to ~27,000 Mꢀ 1 cmꢀ 1 at excess ferrozine concentration (i.e. 30
ferrozine:1 Fe(II)).
pattern emerged (Fig. 4) with molar absorptivity (ε) values ranging
between a low ~15,000 Mꢀ 1 cmꢀ 1 at low ferrozine:Fe(II) ratios, to a
saturating value of ~31,000 Mꢀ 1 cmꢀ 1 at ferrozine:Fe(II) ratios >50:1.
These results are consistent with those presented earlier showing that
stable {Fe(II)-(ferrozine)3} complexes are formed in the presence of
excess ferrozine.
To examine the stability of the Fe(II)-ferrozine complexes formed
under the conditions of Fig. 1, the samples were allowed to sit in the dark
for 48 h before the absorbance changes at 562 nm were recorded again.
A similar pattern with a slight increase in the final absorbance over a
period of 2 days was observed, suggesting that under the conditions of
our experiment, the Fe(II)-ferrozine complex is a stable entity that does
not undergo major changes.
Given the structural similarities between ferrozine, ferene, and
bathophenanthroline-disulfonate (BPhen) (i.e. 3 molecules of ligands
bind to 1 Fe(II) ion, and the net charge of – 4 on the {Fe(II)-(BPhen)3} or
the {Fe(II)-(ferene)3} complexes, the repulsion between the negatively
charged BPhen and ferene molecules, due to the six sulfonic groups, will
likely lead to slower kinetics, similarly to what is observed here with
ferrozine.
3.3. Effect of ferrozine and Fe(II) on the molar absorptivity of the Fe(II)-
ferrozine complex
3.4. The kinetics of Fe(II) complexation by 2,2′-bipyridine and 1,10-
phenanthroline
To test how the ferrozine concentration affects the molar absorp-
tivity of the ferrozine–Fe(II) complex, a fixed 20
μ
M Fe(II) concentration
To examine the kinetics of the two other most commonly used Fe(II)
chelators (2,2′-bipyridine and 1,10 phenanthroline), and the effects of
ligand concentration on the Fe(II)-complexes, we designed UV–vis ex-
periments under conditions similar to those employed with ferrozine.
Fig. 5 shows the kinetic traces of Fe(II) complexation with 2,2′-bipyr-
idine and 1,10-phenanthroline at 522 nm and 510 nm, respectively.
Both ligands displayed very rapid kinetics with Fe(II) ions, irrespective
of ligand concentration, although a lower absorbance was observed
when stoichiometric ratios of ~3:1 bipyridine:Fe(II) or phenanthroline:
was added to different ferrozine samples with concentrations ranging
between 60 μM and 2 mM. The data of this experiment are displayed in
Fig. 3 and show saturation at higher ferrozine concentrations. The
absorbance exhibits a plateau at >50:1 ferrozine:Fe(II) ratio with a
molar absorptivity value approaching 31,500 Mꢀ 1 cmꢀ 1(Fig. 3C). The
experiment was then repeated at a fixed ferrozine concentration of 500
μ
M and varying concentration of Fe(II) (from 5 to 166.7 μM) such that
the ratio of ferrozine to Fe(II) ranges between 100 and 3. A similar
5

G.L. Smith et al.
Journal of Inorganic Biochemistry 220 (2021) 111460
Fig. 7. UV–vis absorption spectra of different Fe(II) complexes of ferrozine, bipyridine and phenanthroline (at 38:1 and 20:1 chelator:Fe(II) ratios) in either 132 mM
Tris, pH 7.4, or in 100 mM acetate buffer, pH 5.0. The baselines consist of buffer plus the chelator at either pH.
Fe(II) were used (Fig. 5A). Under these conditions, lower molar ab-
sorptivity values were calculated (Fig. 5B), suggesting that the com-
plexes have not fully formed. When >3:1 ratios were employed,
thermodynamically stable complexes of Fe(II) and bipyridine or phe-
nanthroline are observed, with average molar absorptivity values of
(9950 ± 100 Mꢀ 1 cmꢀ 1 for {Fe(II)-(bipyridine)3} at 522 nm and (12,450
± 370 Mꢀ 1 cmꢀ 1) for {Fe(II)-(phenanthroline)3} at 510 nm (Fig. 5B, C,
D). When larger excess of 2,2′-bipyridine and 1,10-phenanthroline were
employed (up to 100 times molar ratio), no further changes in absor-
bance was observed, suggesting that {Fe(II)-(bipy)3} and {Fe(II)-(phe-
nanthroline)3} have formed stable complexes, consistent with their high
Funding
This work is supported by a National Science Foundation, Division of
Molecular and Cellular Biosciences (MCB) Award 1934666 (F.B.-A.),
and by The Camille & Henry Dreyfus Foundation, Inc., The Henry
Dreyfus Teacher-Scholar Award (F.B.-A., Award TH-16-007).
CRediT authorship contribution statement
Gideon L. Smith: Formal analysis, Investigation, Methodology.
Aliaksandra A. Reutovich: Formal analysis, Investigation, Methodol-
ogy. Ayush K. Srivastava: Formal analysis, Investigation, Methodol-
ogy. Ruth E. Reichard: Formal analysis, Investigation, Methodology.
Cass H. Welsh: Formal analysis, Investigation, Methodology. Artem
Melman: Conceptualization, Formal analysis, Investigation, Methodol-
ogy, Writing - review & editing. Fadi Bou-Abdallah: Formal analysis,
Conceptualization, Funding acquisition, Investigation, Methodology,
Writing - original draft, Writing - review & editing.
formation constants of ~1.2 × 1017 Mꢀ 3 and ~ 2.5 × 1021 Mꢀ 3
,
respectively. Notably, these formation constants are ~22-fold and 5 ×
105-fold higher than that of the {Fe(II)-(ferrozine)3} complex [15].
3.5. Effect of pH
Although the formation of the Fe(II)-ferrozine complex is not
affected by changes in pH [7,15,20], it is unclear whether the formation
of the Fe(II)-bipyridine and Fe(II)-phenanthroline complexes depends
on pH variations. Fig. 7 shows the absorption spectra of 4 different so-
lutions of Fe(II)-ferrozine, Fe(II)-bipyridine, and Fe(II)-phenanthroline
complexes prepared at different chelator:Fe(II) ratios (20:1 and 38:1),
in acetate buffer pH 5.0 and in Tris buffer pH 7.4. The data show that,
similarly to the {Fe(II)-(ferrozine)3} complex, neither {Fe(II)-(bipyr-
idine)3} nor {Fe(II)-(phenanthroline)3} is affected by pH, in the range of
5.0 to 7.4.
Declaration of Competing Interest
None.
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Here, we have shown that the kinetics of Fe(II) complexation by
ferrozine, but not by 2,2′-bipyridine or 1,10-phenanthroline depend on
the concentration of the ligand, or the ligand:Fe(II) ratio. For ferrozine,
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