Food Chemistry

Module 2. Food proteins

Lesson 4
CLASSIFICATION AND PHYSICOCHEMICAL PROPERTIES

4.1 Introduction

Proteins are common constituent of all biological materials, without which life is not possible. They are essential constituent of all living cells. A complex nitrogenous organic compound – a polymer of amino acids - therefore defined as high molecular weight polymers of low molecular weight monomers known as amino acids, which are linked together by peptide bonds. Proteins are polymers of some 20 different amino acids joined together by peptide bonds (primary structure). The amino acid composition establishes the nature of secondary and tertiary structures. These, in turn, significantly influence the functional properties of food proteins and their behaviour during processing.

4.2 Classification of Proteins

Proteins have been classified in many ways. Generally they are classified on the basis of composition, shape of molecules and solubility.

4.2.1 On the basis of composition

On the basis of composition proteins are classified into three groups viz. simple proteins, conjugated proteins and derived proteins.

1. Simple proteins

These are the proteins which consist of only amino acids – They do not contain other class of compounds.

2. Conjugated proteins

These are the proteins which consist of amino acids as well as other class of compounds.

They are further classified into six subgroups.

Table 4.1 Conjugated proteins

Sr. No.	Class	Other compound present	Example
1	Chromoprotein	Coloured pigment	Haemoglobin
2	Glycoprotein	Carbohydrate	Mucin (in saliva)
3	Phosphoprotein	Phosphoric acid	Casein (in milk)
4	Lipoprotein	Lipid	Lipovitelin (in egg yolk)
5	Nucleoprotein	Nucleic acid	Viruses
6	Metalloprotein	Metal	Ciruloplasmin (Cu)

3. Derived proteins

They represent various stages of hydrolytic cleavage of simple or conjugated proteins. e.g. proteoses, peptones, peptides, etc.

4.2.2 On the basis of shape of molecules

On the basis of shape of molecules, proteins are classified into two main groups viz. fibrous proteins and globular proteins.

1. Fibrous proteins-Fibrous proteins are long and thread or ribbon like and tend to lie side by side to form fibers. They are generally insoluble in water as the intermolecular forces in these proteins are rather strong. They serve as the chief structural material of animal tissues. Examples are keratin, myosin, collagen etc.

2. Globular proteins-Globular proteins are spheroidal in shape. They are generally soluble in water or aqueous solution of acids, bases or salts as intermolecular forces in these proteins are relatively weaker. These proteins are generally involved in physiological processes of the animal body. Examples are enzymes, some hormones, haemoglobin, etc.

4.2.3 On the basis of solubility

On the basis of solubility proteins are classified into the following groups.

1. Albumins- These proteins are soluble in distilled water, dilute salt, acid and base solutions. Examples are lactalbumin, egg albumin.

2. Globulins- These proteins are insoluble in distilled water, but soluble in dilute salt, acid and base solutions. Examples are serum globulins and β-lactoglobulin in milk, myosin and actin in meat.

3. Protamine and Histones- These proteins are highly soluble in distilled water. These are small molecules, stable to heat (i.e. not coagulated by heat). Protamine soluble in NH₄OH, whereas histones are insoluble NH₄OH.

4. Glutelins- These proteins are insoluble in distilled water and alcohol but soluble in dilute acid and base solution. Examples are glutenin in wheat, oryzenin in rice.

5. Prolamins- These proteins are insoluble in distilled water, but soluble in dilute acid, dilute base and 70-80% alcohol. Example are zein in corn, gliadin in wheat.

6. Scleroproteins- These proteins are insoluble in most of the solvents like water, dilute acid, dilute base, dilute salt solution etc. They are generally fibrous proteins serving structural and binding purposes. Examples are collagen, elastin, keratin.

4.3 Physicochemical Properties of Proteins

4.3.1 Isoelectric point

The isoelectric point of a protein is that pH at which the net charge on the protein molecule is zero. At isoelectric point protein will not migrate when an electric field is applied. At isoelectric point its ionization is minimum – least soluble. Each protein have its own characteristic isoelectric point – due to difference in amino acids make up. The major milk protein casein has an isoelectric point of 4.6. This character of protein is often made use in the isolation of proteins.

4.3.2 Amphoteric behaviour

Like amino acids, proteins are ampholytes, i.e. they act as both acids and bases. At all but the extremes of pH, possess both positive and negative charged groups. Owing to the presence of carboxylate groups of the acidic amino acids ---- carboxylate group at the end of the chain, most protein solutions are good buffers below pH 5. Similarly owing to the ε-amino groups of lysine, the guanidinium group of arginine and the phenolic hydroxyl group of tyrosine, most proteins are good buffer at pH values above 9. However at neutral pH values, most proteins have limited buffering capacity. This buffering is of great importance in many living tissues.

4.3.3 Ion binding

As ampholytes, proteins can bind both anions and cations. Several ions will form insoluble salts with proteins and this phenomenon is widely used to remove proteins from solutions. e.g. Trichloro acetic acid is used to separate protein nitrogen from non protein nitrogen. It is possible to obtain interactions between proteins and charged macromolecules such as alginates and pectates. These type of complexes have great potential in the food.

4.3.4 Solubility

As would be expected for an ampholyte, protein solubility is markedly dependent on the pH and ionic composition of the solution. Protein solubility is minimal at the isoelectric point since at this pH the net charge on the protein is zero and consequently electrostatic repulsive forces are minimal while interaction between protein molecules is maximal. Relationship between salt concentration and solubility is complex. Globulins which are soluble in 5-10 % salt solutions, are insoluble in water while albumins are readily soluble in both water and dilute salt solutions. However, in concentrated salt solution ; all proteins become less soluble.
The increase in solubility in dilute salt solution observed with globulins is known as “salting in”. It can be explained in terms of the relative affinity of the protein molecules for each other and for the solvent. i.e. the ions of the neutral salt will interact with the protein; thereby decreasing protein-protein interactions and consequently increasing the solubility.
The decreasing solubility of proteins at high salt concentration is known as “salting out”. Dehydration of the protein molecules occur due to the added salt. The large number of salt ions in the solution will ‘hydrate’ and organise water molecules around them, thus reducing the water available for the protein molecules. Since protein solubility depends on whether ‘clustering’ around the hydrophilic groups, the ‘dehydrated’ proteins will precipitate. In an aqueous protein solution not all the water will be ‘free’ as some will be ‘bound’ to the protein via hydration of charged groups and hydrogen bonds.

4.3.5 Swelling

Several native proteins which are not soluble in water may, however, interact with aqueous solution to form swollen, gel like systems, examples being actomyosin and collagen in muscles. There are two mechanisms whereby this swelling occurs.
(i) Osmotic (Donnan swelling) – which is reversible and caused by interactions between ions and charged sites on the protein. To maintain electrical neutrality in the swollen phase, small ions of opposite charge migrate from the solution to the swollen phase. These excess ions in the swollen phase give rise to an osmotic pressure which causes the swelling.
(ii) Lyotropic swelling – which is irreversible and caused by non ionic reagents which act by altering the water structure around the protein, interrupting the hydrogen bonds and / or through direct competition with internal hydrophobic interactions.The swelling of insoluble proteins by these mechanisms will continue until it is restrained by the intermolecular forces between the protein molecules and an equilibrium swollen volume is achieved. Thus, both soluble and insoluble proteins can immobilise water and this ability to bind water is often called their water holding or water binding capacity.

4.3.6 Crystallization

Many of the proteins have been obtained in crystalline condition. Amongst the animal proteins haemoglobin crystallise readily. Many of the enzyme proteins have been crystallized e.g. urease, pepsin, trypsin, catalase etc. The crystallization of protein may be obtained by addition of a salt such as ammonium sulphate or sodium chloride and adjustment towards isoelectric pH. The addition of definite amount alcohol or acetone is occasionally advantageous. The added substances and adjustment to isoelectric pH decrease the solubility of the protein. The protein is also least dissociated at the isoelectric pH and crystallize best in the form of protein salts. The relative ease of crystallization of protein as compared to polysaccharides is due to the high polarity of the protein molecules giving rise to strong field of force which orient the molecules and promote crystal formation.

4.3.7 Optical activity

All the amino acids occurring in nature except glycine, contain one or more asymmetric carbon atom and therefore show optical activity. The rotatory power of amino acid is affected by various factors which influence the degree and the nature of the electrolytic dissociation of the amino acid. These include the concentration of amino acid itself.

• pH of solution.
• The nature of solvent.
• The presence of electrolytes.
• The temperature.

The effect of varying conditions is so large that any statement regarding the specific rotation of an amino acid has little meaning, unless accompanied by the statement of the conditions prevailing in the solution. Optical rotation is an important property of proteins in which they differ widely. This phenomenon results from the presence of asymmetric carbon atom. Specific rotations of proteins obtained at 20^oC and using D-line of sodium are always negative and for globular proteins the values of [α]^D20] are usually within the range of -30^o to -60^o. Denaturation of proteins produces marked increases in optical rotation. Measurement of this property is a sensitive means of following denaturation.

4.3.8 Absorption of ultra violet light

The absorption of ultra violet light with a wavelength of 280 nm is a characteristic of proteins that depends on their content of the aromatic amino acids (tyrosine, tryptophan and phenylalanine).

4.3.9 Refractive index

The refractive index of protein solutions increases linearly with concentration. The difference between the refractive index of a 1 % protein solution and its solvent is called specific refractive increment. Most proteins have a refractive index increment of about 0.0018.