Vol.2, No.2, 85-90 (2010) Natural Science
http://dx.doi.org/10.4236/ns.2010.22013
Metal ion-binding properties of L-glutamic acid and
L-aspartic acid, a comparative investigation
S. A. A. Sajadi
Sharif University of Technology, Institute of Water & Energy, Tehran, Iran;
Received 14 November 2009; revised 9 December 2009; accepted 30 December 2009.
ABSTRACT
A comparative research has been developed for
acidity and stability constants of M(Glu)1,
M(Asp)2 and M(Ttr)3 complexes, which have
been determined by potentiometric pH titration.
Depending on metal ion-binding properties, vital
differences in building complex were observed.
The present study indicates that in M(Ttr) com-
plexes, metal ions are arranged to the carboxyl
groups, but in M(Glu) and M(Asp), some metal
ions are able to build chelate over amine groups.
The results mentioned-above demonstrate that
for some M(Glu) and M(Asp) complexes, the
stability constants are also largely determined
by the affinity of metal ions for amine group.
This leads to a kind of selectivity of metal ions,
and transfers them through building complexes
accompanied with glutamate and aspartate. For
heavy metal ions, this building complex helps
the absorption and filtration of the blood plasma,
and consequently, the excursion of heavy metal
ions takes place. This is an important method in
micro-dialysis. In this study the different as-
pects of stabilization of metal ion complexes
regarding to Irving-Williams sequence have
been investigated.
Keywords: Glutamic Acid; Aspartic Acid; Tartaric
Acid; Divalent Metal Ions; Potentiometric Titration;
Acidity and Stability Constants.
1. INTRODUCTION
It is known that metal ions are important for numerous
biochemical reactions. For example, enzymes work only
in the presence of such metal ions. The metal ion com-
plexes of many amino acids have been investigated [1-6].
Functionalized membranes represent a field with multi-
ple applications. Examination of specific metal-macro-
molecule interactions on these surfaces indicates an ex-
cellent method for characterization of these materials.
Ion exchange, chelation, and electrostatic interactions
form the basis of metal sorption. The behavior of various
materials functionalized with polypeptides and other
molecules is a topic of interest because of its applica-
tions in affinity separations, biosensors, and other appli-
cations including site-specific interactions [7]. An exam-
ple of the latter involves the removal of heavy metals
from aqueous solutions [8-11]. These sorbents are made
of a variety of materials containing different functional
groups. The advantage of affinity separations is that they
may be tailored for the desired selectivity and capacity.
The functionalization of materials is of vital importance
for the production of new materials with specific proper-
ties. The characterization of these new materials is also
critical. Previous investigations showed that for the
“harder” ligands, the ionic term dominates and their
binding energies are affected by changes in covalency
over the series. It is the competition between these be-
haviors that produces the Irving-Williams series in sta-
bility constants [12]. L-glutamate and L-aspartate are
key molecules in cellular metabolism (Figure 1). In hu-
mans, dietary proteins are broken down by digestion into
amino acids, which serve as metabolic fuel for other
functional roles in the body. Based on above-mentioned,
the essential role of Glu is interesting to study the interaction
between other metal ions with Glu and related compounds.
2. EXPERIMENTAL
2.1. Materials
The L-glutamic acid and L-aspartic acid (extra pure) was
purchased from Merck, Darmstadt, Germany. The nitrate
salt of Na+, Ca2+, Mg2+, Mn2+, Co2+, Cu2+, and Zn2+ (all
pro analysis) were from Merck. All the starting materials
were of reagent grade and used without further purifica-
tion. Potassium hydrogen phthalate and standard solu-
tions of sodium hydroxide (titrasol), nitric acid, EDTA
and of the buffer solutions of pH 4.0, 7.0 and 9.0 were
all from Merck. All solutions were prepared with deion-
ized water. Water was purified by Milil-Q water purifi-
cation system, de-ionized and distillated.
1L-Glutamic acid; 2L-As
p
artic acid; 3L-Tartaric acid

S. A. A. Sajadi / Natural Science 2 (2010) 85-90
86
(b)
(a)
(d)
(c)
Figure 1. Chemical structures of (a, b) L-glutamic acid; (c)
L-aspartic acid and; (d) tartaric acid.
2.2. Ph Titrations
Reagents
Carbonate-free sodium hydroxide 0.03 M was pre-
pared and standardized against sodium hydrogen phtha-
late and a standard solution of nitric acid 0.5 mM. M(II)
nitrate solution (0.03 M) was prepared by dissolving the
above substance in water and was standardized with
standard solution of EDTA 0.1 M (triplex).
2.3. Apparatus
All pH titrations were performed using a Metrohm 794
basic automatic titrator (Titrino), coupled with a thermo-
stating bath Hero at 25C (±0.1C) and a Metrohm com-
bined glass electrode (Ag/AgCl). The pH meter was
calibrated with Merck standard buffer solutions (4.0, 7.0
and 9.0).
2.4. Procedure
For the determination of acid dissociation constants of
the ligand L, an aqueous solution (0.03 mM) of the pro-
tonated ligand was titrated with 0.03 M NaOH at 25C
under nitrogen atmosphere and ionic strength of 0.1 M,
NaNO3. For the determination of binary (a ligand and
Cu2+) system, the ratios used were 1:1, Cu(II) : Ligand
and 1:1, Cu(II) : L, 0.3 mM. This solution was titrated
with 0.03 M NaOH under the same conditions men-
tioned above. Each titration was repeated seven times in
order to check the reproducibility of the data.
2.5. Calculation
The acid dissociation constants, 2()
H
H
L
Kand ()
H
H
L
K for
H2(L) were calculated by an algebraic method. The
equilibria involved in the formation of 1:1 complex of L
and a divalent metal ion may be expressed as Eqs.(7-
10).
3. RESULTS AND DISCUSSION
The potentiometric pH-titrations (25C, 0.1 M, NaNO3)
were carried out to obtain the acidity and stability con-
stants which are summarized in Tables 1 and 2.
Acidity constants
L-glutamate O2CCH2CH2CH(NH2)CO2 and L-
aspartate ions (L2-), O2CCH2CH(NH2)CO2, are
two-basic species, and thus they can accept two protons,
given H2(L), for which the following de-protonation
equilibriums are hold:
H2(L) H++H(L) (1)
2()
H
H
L
K= [H(L)-][H+]/[H 2(L)] (2)
H(L)H++L2 (3)
()
H
H
L
K= [L2][H+]/[H(L)] (4)
The two proton in H2(L) are certainly bound at the
terminal acetate and amino groups, i.e., it is released
from HO2CCH2CH2CH(NH3
+)CO2 – H
2Glu according to
Equilibriums (1) and (2). It is known as zwitter-ion, and
is also closed to the de-protonation of acetate groups
which occurs at the terminal acetate groups of tartaric
acid [6,13]. L2 can release one more proton from the
terminal acetate group. Hence, here due addition to
Equilibrium (3) should be considered, which takes place
above a pH ≈ 2 (see Figure 2).
H3(L)+H++H2(L) (5)
3()
H
H
L
K=[H2(L)][H+]/[H3(L)+] (6)
Here, the aforementioned reaction is not considered
further.
3.1. Stability of Binary and Ternary
Complexes
If we abbreviate for simplicity associating with Ca2+,
Mg2+, Mn2+, Co2+, Cu2+, and Zn2+ with M2+, then one
may write the following two Equilibriums (4) and (5):
M2++H(L)M(H;L)+ (7)
(;)
M
M
HL
K= [M(H; L)+]/[M2+][ H(L) ] (8)
M2++(L)2M(L) (9)
()
M
M
L
K= [M(L)]/[M2+][L2] (10)
3.2. Potentiometric Analyses
These results are summarized in Tables 1 and 2. The data

S. A. A. Sajadi / Natural Science 2 (2010) 85-90
87
(a) Cu-Glu
(b) Cu-Ttr
(c) Cu-Asp
Figure 2. Schematic structures of the species with interactions
according to Equilibrium (5) for a) Cu(Glu), b) Cu(Asp) and c)
Cu(Ttr). The structure in the right part of the figure was drawn
with the programCS Chem 3D, version 3.5, from Cambridge
Software Corporation.
Table 1. Negative logarithm of the acidiity constants of
H3(Asp)+, H2(Ttr)+, and H3(Glu)+ at 25C, 0.1 M, NaNO3
*,
Equilibriums (1), (2) & (3).
Asp Ttr Glu Site
1.99** 3.09±0.07 2.05±0.13 -CO2H
pKa 3.72±0.03 4.19±0.05 4.37±0.03 -CO2H
9.90±0.03  9.98±0.02 -NH3
*The given errors are three times the standard error of the mean value
or the sum of the propabable systematic errors. **[6]
Table 2. Comparison of the stability constants of binary com-
plexes of Asp, Ttr and Glu with M2+ at 25C, I = 0.1 M,
NaNO3
*.
No.Species log ()
M
M
Asp
K log()
M
M
Ttr
K log()
M
M
Glu
K
1 Mg2+ 2.50±0.06 1.90±0.05 1.82±0.06
2 Ca2+ 1.26±0.06 1.80±0.051 1.41±0.02
3 Mn2+ 3.91±0.03 4.08±0.08 3.19±0.08
4 Co2+ 6.69±0.06 3.27±0.08 4.15±0.09
5 Cu2+ 8.78±0.02 3.65±0.07 7.70±0.09
6 Zn2+ 5.35±0.06 2.69±0.07 5.84±0.03
*The given errors are three times the standard error of the meanvalue
or the sum of the propabable systematic errors. 1[6,14]
Figure 3. Irving-Willams sequence-type plot for the 1:1 com-
plexes of Ca2+, Mg2+, Mn2+, Co2+, Cu2+, and Zn2+ formed with.
a) Tartarate (Ttr2); b) oxalate (Ox2); c) glutamate (Glu2); d)
glycine (Gly); e) aspartate (Asp2); The data are taken from
Table 2 and [15], they represent also the stability constants of
M2+ complexes of L (25C, 0.1 M, NaNO3).
in Table 1 present the acidity constants pKa (()
H
H
L
K,
2()
H
H
L
K, and 3()
H
H
L
K) of L-aspartic acid, L-tartaric acid, and
L-glutamic acid (Eqs.(1-6)). L-aspartic acid can release
totaly three protons, two protons from carboxyl groups
and one from amin group. L-tartaric acid contains no
amino group, so two reported acidity constants regard to
carboxyl groups. In case of L-glutamic acid two protons
are released from carboxyl groups and one proton from
amin group, which were showed as regarding sites. As we
can see from these results, the stability constants of the bi-
nary complexes, such as M(L) (Figure 3) were refined
separately using the titration data of this system in a 1:1,
ligand: M2+ ratio in the same conditions of temperature and
ionic strength (according Eqs.(9-10)), as they were in good
agreement with reported value [6,14]. We didn’t receive
reasonable results for (;)
M
M
HL
K. The stability constants of
Table 2 show the following trends. The obtained order for
Ttr is Ca2+  Mg2+ Mn2+> Co2+ Cu2+ Zn2+. The corre-
sponding order for Glu and Asp is Ca2+  Mg2+ Mn2+<
Co2+ Cu2+ Zn2+. The last observed stability order for
Glu and Asp follows the Irving-Williams sequence [15].

S. A. A. Sajadi / Natural Science 2 (2010) 85-90
88
Figure 4. Relationship between log
M
M
L
K
and p
H
H
L
K
for Ca2+, Mg2+, Mn2+, Co2+, Cu2+, and Zn2+ 1:1 com-
plexes of glutamate (Glu2), tartarate (Ttr2) and aspar-
tate (Asp2). All plotted equilibrium constant values
refer to aqueous solutions at 25C, 0.1 M, NaNO3 (Ta-
ble 1, second row & Table 2).
Figure 5. Relationship between log
M
M
L
K
and p
H
H
L
K
for
Ca2+, Mg2+, Mn2+, Co2+, and Cu2+, 1:1 complexes of
glutamate (Glu2) and aspartate (Asp2). All plotted
equilibrium constant values refer to aqueous solutions at
25C, 0.1 M, NaNO3 (Table 1, second row & Table 2).
As we can use from Figure 3, glutamate and aspartate
chelate metal ions weakly via the amino nitrogen and
carbonyl oxygen. A stronger chelation occurs upon am-
ide nitrogen bound hydrogen by some metal ions such as
Cu2+. This reaction occurs in neutral pH conditions (pH≈
7) with Cu2+. A crystal structure of M2+ chelate with a
structure analogous has been studied [16].
In Figure 4, we can consider the relationships be-
tween log
M
M
L
K
and p
H
H
L
K
for Ca2+, Mg2+, Mn2+, Co2+,
Cu2+, and Zn2+ 1:1 complexes of glutamate (Glu2), as-
partate (Asp2) and tartarate (Ttr2). The data are taken
from Table 2. They also represent the stability constant
values.
All the plotted equilibrium constant values refer to
aqueous solution at 25°C, I=0 M (NaNO3). As one can
see, this relationship is not linear. The interesting point is
that in case of hard metal ions such as Ca2+ and Mn2+,
the results for Ttr show a maximum ▲ and in case of
softer metal ions such as Co2+, Cu2+, and Zn2+ , they
show a minimum▼. This is an indication, that for softer
metal ions an additional interaction such as with amino
group exists, which we can not find by Ttr.
In Figure 5, we can consider the relationships be-
tween log
M
M
L
K
and p
H
H
L
K
for Ca2+, Mg2+, Mn2+, Co2+,
and Cu2+ 1:1 complexes of glutamate (Glu2) and aspar-
tate (Asp2). However, the most important question is
that: Is there a correlation between complex stability and
donor groups’ basicity? In other words, is there a linear
relationship between log
M
M
L
K
and p
H
H
L
K
? Based on
Figure 5, it seems that this is the case, where the data
pairs for the systems of several metal ions are plotted. It
is interesting to point out, that in contrast to increasing
of basicity from Asp to Glu, show the stability of re-
garding complexes a decreasing trend.
Now, we are able to compare the stability constants of
two species M(Glu) and M(Asp). It could easily distin-
guish that those constants of M(Asp) are generally larger
than those of the corresponding M(Glu) species. This
increased stability of the difference between the stability
constants as defined in Eq.(11):
∆logK = logK(Asp)–logK(Glu) (11)
Positive amount of ∆logK indicates that the M-Glu
complexes in case of Mg, Mn and Cu are less stable than
M-Asp complexes. These decreases of the stability, from
Asp to Glu are based on larger ring size of Glu in M-Glu
complexes. The calculated results of ∆logK for Mg, Mn
and Cu are 0.68, 0.72 and 1.08, respectively.
The standard Gibbs free energy change for the
reaction is related to the following equilibrium constant
ΔG = 2.303 RT logK (12)
As results, we received the according calculated val-
ues for Gibbs free energy change 3.88, 4.11 and 6.16
kJmol-1 for Mg, Mn and Cu, respectively. If we compare

S. A. A. Sajadi / Natural Science 2 (2010) 85-90
89
these values with the plotted results in Figure 5, we dis-
tinguish the decrease of stability constants from Asp – to
Glu complexes, which refers to weaker interaction be-
tween metal ions and regarding ligands. This is a se-
quence of well-known ring size of the chelate. In case of
M-Asp complexes is seven-member chelate ring and in
M-Glu complexes is eight-member chelate ring. In case
of Ca2+ the difference is within error range.
According to Irving and Williams, the order was as a
consequence of the fact that the two parameters, which
serve as a guide to the magnitude of the ionic (electro-
static) and covalent interactions (the reciprocal of the
metal ionic radius and the sum of the first two ionization
energies, respectively), both increase monotonically
throughout the series from Mn to Cu and then decrease
from Cu to Zn. Thus, if water is replaced from
[M(OH2)n]2+ by a ligand of better electron-donating
power, then the gain in stability will increase with the
ionization potential of the metal. If H2O is replaced by a
ligand with a formal negative charge, the stability gain
through electrostatic forces will increase as the radius of
the metal cation decreases.
A series of metal-varied [ML(SC6F5)] model com-
plexes related to blue copper proteins have been studied
by a combination of absorption [12]. Thus, the “softer”
thiolate ligand can have comparable covalent and ionic
contributions to bonding, and these compensate to pro-
duce little change in the binding energy over the series
of metal ions (open squares in Figure 6). For the
“harder” ligands (F-, OH-, H2O, etc.), the ionic term
dominates and their binding energies are affected by
changes in covalency over the series (open circles in
Figure 6). It is the competition between these behaviors
that produces the Irving-Williams series (solid circles in
Figure 6) in stability constants.
Biological systems have the ability to selectively bind
to metals taking advantage of the array of protein bind-
ing functionalities [17]. Short chain synthetic biopoly-
mers also have unique, strong and selective binding pro-
perties offered by their constituent amino acids.
Interactions between aspartic acid (Asp) and cytidine-
5-monophosphate (CMP) in metal-free systems as well
as the coordination of Cu(II) ions with the above ligands
were studied. The composition and overall stability con-
stants of the species formed in those systems were de-
termined [18]. Amino acid chelated minerals, also re-
ferred to as chelated minerals or mineral chelates, are
minerals that have been chemically engineered to be-
come more bio-available to our body. Amino acids act as
carriers to ship the much-needed minerals to the destina-
tion (the small intestine) where consumption takes place.
Elixir Industry has tested many self-claimed “mineral
chelates” available on the market and found that most of
them are merely mixtures of amino acids and inorganic
minerals [19]. Why are amino acid chelated minerals
superior to common inorganic minerals? Chelated min-
erals help protect vitamin stability. Inorganic metal ions
may serve as a catalyst to further the oxidation and deg-
radation of vitamins. Chelated minerals, on the other
hand, are well shielded by bonded organic ligands,
which we can consider in Figure 3. They will not come
in contact with vitamin molecules; thus, the vitamins
will be protected from oxidation and degradation. And
since magma precipitation is prevented, chelated miner-
als will not absorb vitamins and cause them to become
non-absorbable, problems that common inorganic min-
eral are known to cause. As we can see, in Figure 3, Ttr
is not able to build three dentate chelate like Asp, so that
metal ions are not enough shielded. As consequence
metal ions can take part in substitution reactions.
There are different types of amino acids. L-aspartic
acid plays a vital role in energy production and is a ma-
jor excitatory neurotransmitter. It is involved in building
DNA (genetic structures in cells), in carbohydrate me-
tabolism & protein metabolism. It helps detoxify ammo-
nia in the body, helps reduce fatigue and depression, and
it also supports liver protection. Mineral Chelates (for
example AbSolu) are chelates of L-aspartic acid and one
of the minerals such as calcium, magnesium and zinc.
Figure 6. (a) Relative formation energies (kcal mol-1) of the
metal-thiolatecomplexes, [ML(SC6F5)], and the metal-fluoride
complexes, [ML(F)], andtheir difference, ¢Ef.; (b) Ionic com-
ponent of the ML+-SC6F5 - bonding energy, Eionic, and the
difference, Eint - Eionic. Edward I. Solomon et al., Inorg.
Chem. 44 (2005) 4947-4960.

S. A. A. Sajadi / Natural Science 2 (2010) 85-90
90
There are different types of amino acids. Nutrition
scientists selected L-aspartic acid based on many addi-
tional benefits that come with it. Most of the competi-
tors’ products contain two or more crystalline water in
their molecules. Theoretically those products should not
be referred to as “chelates” and the bonding (if indeed
exists) of amino acid molecules to mineral ions is vul-
nerable. These products are anhydrous chelates of two L-
aspartic acid molecules and a single metal ion. Based on
the results of this work, we can draw the conclusion, that
hard metal ions just with identical stability constants-
could have similar interaction with glutamate. Even
based on these, results of acidity constants reported (Ta-
ble 2) glutamamic acid occurs in high organism in form
of glutamate. Earlier works have reported the structure
of glutamate complexes with some metal ions [20] such
as Co2+, Cu2+ and Zn2+. These metal ions are able to have
additional interactions with glutamate. This leads to
building of macro-chelate (Figure 3).
4. CONCLUSIONS
It is shown, that Glu has identical complex properties as
Asp i.e. the both amino acids have similar structure. The
only difference is also the size of amino acids. The pre-
vious studies have shown, that Glu has special applica-
tions [21].
The glutamate industry have found that manufactured
free glutamic acid, in the form of monosodium gluta-
mate (MSG), hydrolyzed vegetable proteins, etc., etc.,
when added to the processed foods, masks off flavors
and makes the blandest and cheapest foods taste won-
derful. An other interesting point is the pharma- cologi-
cal application of new generation of glutamate and as-
partate complexes and it seems essential to understand
their reaction mechanisms.
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