**2.2 The chelate effect**

314 Soybean Physiology and Biochemistry

the Cosmetic Ingredient Review Expert Panel found that these ingredients are safe as used

Ethylenediaminetetraacetic acid (EDTA) is a very effective chelating agent but has the disadvantage that is quite persistent in the environment owing to its low biodegradability. For that reason different chelating agents were investigated, such as [S,S,] ethylenediaminedisuccinic acid, iminodisuccinic acid, methylglycine diacetic acid, etc. but the problem is the dependence of the pH on the extraction efficiency. (Tandy et al., 2004) Major industrial processes involve the sequestration of metal ions in an aqueous solution. In the textile industry, this prevents metal ion impurities from modifying colors of dyed products. In the pulp and paper industry, EDTA inhibits the ability of metal ions, especially Mn2+, to catalyze disproportionate amounts of hydrogen peroxide, which is used in "chlorine-free bleaching." Similarly, EDTA is added to some foods as a preservative or stabilizer to prevent a catalytic oxidative discoloration which is catalyzed by metal ions. Oral exposures have been noted to cause reproductive and developmental effects (Elliot & Brown, 1989). The same study by Lanigan also found that both dermal exposure to EDTA in most cosmetic formulations and inhalation exposure to EDTA in aerosolized cosmetic formulations would produce systemic effects below those seen to be toxic in oral dosing

A crucial factor to be considered in comparing studies on chelating agent is the pH of the extraction solution. While extraction was investigated at various pH values in some studies (Elliot & Brown, 1989 ,; Pichtel, 1998; Pichtel, 1997; Kim, 2003; Ghestem, 1998), some only stated the pH of the solution (Reed, 1996; Cline, 1995; Van Benschoten, 1997), while others did not consider pH at all (Pichtel, 2001; ). In general, the lower the pH of the chelating

The history and chemistry of the industrial use of natural products and their derivatives have a rich technological tradition. Many modern products, such as plastics, fuels, chemical intermediates and fibers, find their origins in natural products derived from plants and animals. Given the recent social emphasis on the environment and resource renewability, utilizing natural materials as potential resources for industrial products receives a ready

Together, the oil and protein contents of dry soybeans account for about 60% of the weight; protein being 40% and oil 20%. The remainder consists of 35% carbohydrate and about 5% ash. Most soy protein is a relatively heat-stable storage protein. This heat stability enables the manufacture of soy food products requiring high temperature cooking, such as tofu, soy

This article focuses on the application of natural "green" textured soya extract as a

The word chelation is derived from Greek, meaning "claw." The ligands lie around the

The IUPAC definition of chelation is the formation or presence of two or more separate bindings between a polydentate (multiple bonded) ligand and a single central atom. Usually these ligands are organic compounds and are called chelants, chelators, chelating agents, or

agent solution, the greater is the extraction efficiency of the toxic metals.

substitute for EDTA in its role as a metals-sequestering agent in foods.

welcome. Among the most versatile of raw materials is the soybean. (Liu, 1997)

in cosmetic formulations.

studies (Lanigan & Yamarik, 2002).

milk and textured vegetable protein (soy flour).

**2. Antecedents** 

**2.1 What is a chelating agent?** 

sequestering agents. (IUPAC)

central atom like the claws of a lobster.

The increased stability of complexes containing chelating ligands over those containing comparable monodentate ligands can be envisaged as having the following physical basis. Suppose we have a metal ion in solution, and we attach to it a monodentate ligand, followed by a second monodentate ligand, figure 1. These two processes are completely independent of each other. But suppose we have a metal ion and we attach to it one end of a chelating ligand (it is reasonable to assume that when we put a chelate ligand onto a metal, it happens in a stepwise fashion, i.e. one end attaches first and then the other end). The point is that the attachment of the second end of the chelate is now no longer an independent process: once one end is attached, the other end, rather than floating around freely in solution, is anchored by the linking group in reasonably close proximity to the metal ion, and is therefore more likely to join onto it than a comparable monodentate ligand would be.

Fig. 1. Complexes formation.

The figure 2 shows the EDTA ligand binding to a central copper ion.

Fig. 2. Copper ion complexes with EDTA.

Amino acids are classified into different ways base don polarity, structure, nutricional requirement, metabolic fate, etc.

Generally used classification is based on polarity. Based on polarity amino acids are classified into four groups.


Chelates of glycine with cations such as iron, zinc and copper have been fully studied. The chelates usually contain two moles of ligand (glycine) and one mol of metal as demonstrated in the figure 3.

Fig. 3. Chelate of glycine with some metal M.

Consider the two equilibriums, in an aqueous solution, between the copper (II) ion, Cu2+ and ethylenediamine (en) on the one hand and methylamine, MeNH2 on the other.

$$\mathrm{Cu^{2+} + en} \rightleftharpoons [\mathrm{Cu(en)}]^{2+} \tag{1}$$

$$\text{Cu}^{2+} + 2\text{ MeNH}\_2 \rightleftharpoons [\text{Cu(MeNH}\_2)\_2]^{2+} \tag{2}$$

In (1) the bidenate ligand ethylene diamine forms a chelate complex with the copper ion. Chelation results in the formation of a five–member ring. In (2) the bidentate ligand is replaced by two monodentate methylamine ligands of approximately the same donor power, meaning that the enthalpy of formation of Cu—N bonds is approximately the same in the two reactions. Under conditions of equal copper concentrations and when the concentration of methylamine is twice the concentration of ethylenediamine, the concentration of the complex (1) will be greater than the concentration of the complex (2). The effect increases with the number of chelate rings so the concentration of the EDTA complex, which has six chelate rings, is much higher than a corresponding complex with two monodentate nitrogen donor ligands and four monodentate carboxylate ligands. Thus, the phenomena of the chelate effect are a firmly established empirical fact.

The thermodynamic approach to explaining the chelate effect considers the equilibrium constant for the reaction: the larger the equilibrium constant, the higher the concentration of the complex.

The formation of a chelant compound is an equilibrium reaction as shown in the reaction (3)

aM n+ + b L < ==> c ML (3)

Metal Ligand Metal-chelate

The reaction rates of the forward and reverse reactions are generally not zero but, being equal; there are no net changes in any of the reactant or product concentrations. Since forward and backward rates are equal:

$$\mathbf{k}\_1 \begin{bmatrix} \mathbf{M} \mathbf{r}^\flat \end{bmatrix} \mathbf{\hat{u}} \begin{bmatrix} \mathbf{L} \end{bmatrix} \mathbf{\hat{v}} = \mathbf{k}\_2 \begin{bmatrix} \mathbf{M} \mathbf{L} \end{bmatrix} \mathbf{\hat{c}} \tag{4}$$

and the ratio of the rate constants is also a constant, now known as an equilibrium constant.

$$K = \frac{[ML]^c}{[M^{n+}]^a [L]^b} \tag{5}$$

The concentration of ligand does not change during the reaction. For that reason the equilibrium constant can be expressed only in function of metal ion and metal-complex, as showing in the equation 6.

$$K = \frac{[ML]^c}{[M^{n+}]^a} \tag{6}$$

#### **2.3 Common chelating agents**

316 Soybean Physiology and Biochemistry

Amino acids are classified into different ways base don polarity, structure, nutricional

Generally used classification is based on polarity. Based on polarity amino acids are

• *Non-polar amino acids.- They have equal number of amino and carboxyl groups and are neutral. These amino acids are hydrophobic and have no charge on the 'R' group. The amino acids in this group are alanine, valine, leucine, isoleucine, phenyl alanine, glycine, tryptophan, methionine* 

• *Polar amino acids with no charge.- These amino acids do not have any charge on the 'R' group. These amino acids participate in hydrogen bonding of protein structure. The amino acids in this* 

• *Polar amino acids with positive charge.- Polar amino acids with positive charge have more amino groups as compared to carboxyl groups making it basic. The amino acids, which have positive charge on the 'R' group, are placed in this category. They are lysine, arginine and histidine.*  • *Polar amino acids with negative charge.- Polar amino acids with negative charge have more carboxyl groups than amino groups making them acidic. The amino acids, which have negative charge on the 'R' group are placed in this category. They are called as dicarboxylic mono-amino* 

Chelates of glycine with cations such as iron, zinc and copper have been fully studied. The chelates usually contain two moles of ligand (glycine) and one mol of metal as demonstrated

M

O - - - - - N

Consider the two equilibriums, in an aqueous solution, between the copper (II) ion, Cu2+

Cu2+ + en [Cu(en)]2+ (1)

 Cu2+ + 2 MeNH2 [Cu(MeNH2)2]2+ (2) In (1) the bidenate ligand ethylene diamine forms a chelate complex with the copper ion. Chelation results in the formation of a five–member ring. In (2) the bidentate ligand is replaced by two monodentate methylamine ligands of approximately the same donor power, meaning that the enthalpy of formation of Cu—N bonds is approximately the same in the two reactions. Under conditions of equal copper concentrations and when the concentration of methylamine is twice the concentration of ethylenediamine, the concentration of the complex (1) will be greater than the concentration of the complex (2). The effect increases with the number of chelate rings so the concentration of the EDTA complex, which has six chelate rings, is much higher than a corresponding complex with two monodentate nitrogen donor ligands and four monodentate carboxylate ligands. Thus,

and ethylenediamine (en) on the one hand and methylamine, MeNH2 on the other.

the phenomena of the chelate effect are a firmly established empirical fact.

N - - - - - O

O

C

CH2

*group are - serine, threonine, tyrosine, cysteine, glutamine and aspargine.* 

*acids. They are aspartic acid and glutamic acid.* 

O

Fig. 3. Chelate of glycine with some metal M.

C

H C2

requirement, metabolic fate, etc.

classified into four groups.

*and proline.* 

in the figure 3.

There are many chelating agents used in the industry as Na, Ca-ethylenediaminetetraacetic (EDTA), diethylenetriaminepentaacetic acid (DTPA), nitriloacetic acid, ethylene glycolbis8aminoethyl)tetraacetic acid (EGTA), D,L-mercaptosuccinic acid (MSA), meso-2-3 dimercaptopropanesuccinic acid (DMSA), D,L-2,3-dimercaptopropane-1-sulfonic acid (DMPS), penicillamine (PA), N-acetylpenicillamine (NAPA), vitamins as: thiamine (B1), pyridoxine (B6), cobalim (B12) and ascorbic acid, and many more. The most common is EDTA.

#### **2.4 Naturals chelating agents**

Virtually all biochemicals exhibit the ability to dissolve certain metals cations. Thus, proteins, polysaccharides, and polynucleic acids are excellent polydentate ligands for many metal ions. In addition to these adventitious chelators, several biomolecules are produced to specifically bind certain metals. Histidine, malate and phytochelatin are typical chelators used by plants. (U Kramer, 1996; Jurandir, 2006 & Suk-Bomg Há, 1999)

Virtually all metalloenzymes feature metals that are chelated, usually to peptides or cofactors and prosthetic groups (Lippard & Berg, 1994). Such chelating agents include the porphyrin in hemoglobin and chlorophyll. Many microbial species produce water-soluble pigments that serve as chelating agents, termed sideropho. For example, species of *Pseudomonas* are known to secrete pycocyanin and pyoverdin that bind iron. Enterobactin, produced by E. coli, is the strongest chelating agent known.

In earth science, chemical weathering is attributed to organic chelating agents, *e.g.* peptides and sugars that extract metal ions from minerals and rocks. (Michael) Most metal complexes in the environment and in nature are bound in some form of chelate ring, *e.g.* with a humic acid or a protein. Thus, metal chelates are relevant to the mobilization of metals in the soil, the uptake and the accumulation of metals into plants and micro-organisms. Selective chelating of heavy metals is relevant to bioremediation *e.g.* removal of 137Cs from radioactive waste. (Prasad, 2001)
