Peptides and proteins

Chapter 5

Peptides and proteins

From the biological point of view the most important reaction which the amino acids undergo is condensation and the formation of peptide bonds (page 39). This reaction is responsible for a vast spectrum of compounds containing anything from two to many hundreds of amino acid residues. The smaller members of the group containing less than 10–20 residues are classed as oligopeptides while above this they are known as polypeptides. It is customary, however, to refer to polypeptides which have a molecular weight of more than 10000 as proteins and those with lower values simply as peptides. This distinction is arbitrary but convenient. The average molecular weight of the amino acid residues present in polypeptides is rather less than 110 (Table 4.1), so that a molecular weight of 10000 is roughly equivalent to 100 residues.

Table 5.1

Some biologically active peptides of animal origin

Substance No. of amino acid residues Source Activity
Glutathione 3 Wide distribution Maintenance of -SH groups
Oxytocin 9 Posterior pituitary Contraction of uterus. Let-down of milk
Vasopressin 9 Posterior pituitary Vasoconstrictor, Antidiuretic
Calcitonin 10 Thyroid Lowering of blood Ca2+. Inhibition of bone resorption
Gastrin 17 Pyloric region of stomach Secretion of HCl by stomach
Glucagon 29 α-Cells of pancreatic islets Raising of blood sugar. Increase of glycogen breakdown in liver
ACTH 39 Anterior pituitary Release of adrenal cortical hormones
Parathormone 84 Parathyroid Mobilization of Ca2+.
      Increase of blood Ca2+


Although the mammalian peptides normally contain only those amino acids that are found in proteins linked by normal peptide bonds, some of the peptides produced by micro-organisms show unusual features such as the presence of ornithine and of D-amino acids.


Peptides are widely distributed in nature and show a great range of biological activities. Most tissues contain them and more than 30 have been found in the mammalian nervous system. They are also present in plants, fungi and bacteria and include some of the most biologically active compounds known. Some of the antibiotics, e.g. penicillin, gramicidin and chloramphenicol, are essentially peptide in character as are some extremely toxic substances, e.g. the fungal poisons amanitin and phalloidin. The botulinus, tetanus and black widow spider toxins are all polypeptide neurotoxins. A list of some of the best characterized peptides is given in Table 5.1. They include hormones, released from the hypothalamus, posterior pituitary, gut and pancreas and neuropeptides such as the opioids and tissue growth factors.

The mammalian peptides often occur in families which may be products of a single gene or of closely related multiple genes. Peptides are currently arousing considerable interest because neuropeptides are now thought to play a part not only in pain perception, in feeding and temperature control but also in memory and learning ability! More is said about the neuropeptides and tissue growth factors in Chapter 24.


Proteins comprise more than half the solid matter of the body. They are responsible for virtually all the reactions which occur within the tissues as well as being of structural importance. Proteins are macromolecules and have colloidal properties but their molecules are not necessarily very large. Their molecular weights range from an arbitrary lower limit of 10000 to several million and from its DNA content it has been estimated that the human body contains some 100000 different types of protein. Proteins are, moreover, usually unique to the species from which they are derived so that human serum albumin is distinct from that of a duck, dog, hen or horse and, although similar in structure and function, they have slight differences in composition and are immunologically distinct. From this it can be seen that proteins must exist in almost infinite variety and some idea of the range of their functions may be obtained from Table 5.2. Nevertheless all proteins are derived from the same selection of 20 amino acids which are joined by peptide linkages into long unbranched polypeptide chains. Their main distinguishing characteristics lie in the precise selection of amino acids and the sequence or order in which they follow one another in the polypeptide chain which is genetically determined.

Table 5.2

Functional classification of proteins

1. Enzyme and enzyme precursors

2. Structural proteins:

3. Transport proteins:

4. Receptor proteins

5. Hormones:

6. Contractile proteins: actomyosin of muscle

7. Lubricants: glycoproteins present in saliva and dental plaque and generally in secretions of the gastrointestinal, respiratory and urinary tracts

8. Storage proteins:

9. Chromosomal protein:

10. Immunity-conferring proteins: immunoglobulins of various types

11. Bactericidal proteins: lysozyme present in tears, nasal mucus and other secretions and also in egg-white

12. ? Memory proteins: long term memory in higher animals may be encoded in some brain proteins

13. Virus proteins: these form a protective coat round the viral nucleic acid

14. Controlling proteins: e.g. nerve growth factor

15. Miscellaneous: e.g. proton conductance protein of brown adipose tissue


Although the polypeptide chains are linear and unbranched, their component amino acids interact with one another in a number of different ways causing the molecules to assume highly specific shapes and/or combinations of chains. Interactions may occur

Proteins can be broadly divided into two categories – fibrous and globular.

Fibrous proteins

These include nearly all the structural proteins which have molecules with a high axial (length/width) ratio. Most of them are very insoluble and metabolically unreactive. They include the various keratins found in the skin and its appendages, the fibrin of blood clots, and collagen and elastin the protein constituents of connective tissues. Myosin the fibrous protein of muscle contains both fibrous and globular regions and is both metabolically reactive and relatively soluble.

In the fibrous proteins a large number of molecules of the same type associate to form sheets or bundles. Association of the molecules occurs in the first instance as the result of the formation of weak non-covalent bonds but, subsequently, strong covalent bonds develop which convert the structure into a rigid insoluble aggregate usually of considerable strength. The structure of keratin is dealt with on page 401, fibrin on page 386 and collagen and elastin in Chapter 27.

Globular proteins

There are an enormous number of soluble proteins each of which fulfils a specific function or functions. They are usually more or less globular in shape and have highly characteristic conformations which are designed to bring particular reactive chemical groups together or to force them apart. To do this the molecules must be folded in a complex manner in order to produce a specific shape and charge pattern. The larger proteins often consist of two or more separate chains which may or may not be identical. Such proteins are said to be oligomeric and their constituent chains are known as subunits or monomers. Oligomeric proteins usually have more complex functions than single chain proteins. They are often able to react appropriately to physiological changes and hence play an important part in the control of metabolism (Chapter 23). The folded shapes of the globular proteins are inherently flexible and are able to undergo subtle changes which relate to their biological function. The normal biologically active form of such proteins is known as the native form and when major changes in its three-dimensional characteristics are induced by some external agent and cause a marked reduction in its biological activity, the protein is said to be denatured (page 63).

The ionization of proteins

Proteins in aqueous solution behave as giant polyions, with positively and negatively charged groups projecting from the molecule at various points. These are associated with various small counterions which help to maintain local and general electrical neutrality. The charged groups on a protein markedly affect its behaviour and the configuration which it adopts, since like charges repel and unlike ones attract each other.

Nearly all the ionizable groups of proteins are contributed by the side chain groups of amino acid residues because all but the terminal α-amino and α-carboxyl groups are involved in peptide linkages which do not ionize. The properties of the main ionizable groups in proteins are summarized in Table 5.3.

Table 5.3

The ionizable groups of proteins

Ionizable group Acid form pKa value at 25°C Conjugate base
α-COOH (C-terminal) -COOH 3·0–3·2 -COO
β-COOH (Asp) -COOH 3·0–4·7 -COO
γ-COOH (Glu) -COOH 4·4 -COO
Imidazole (His) image 6·2–7·6 image
α-amino (N-terminal) image 7·6–8·4 -NH2
ɛ-amino (Lys) image 9·4–10·6 -NH2
Sulphydryl (Cys) -SH 9·1–10·8 -S
Phenolic hydroxyl (Tyr) -OH 9·8–10·4 -O
Guanidinium (Arg) image 11·6–12·6 image


Below pH 1·5 the protein is fully protonated and has maximum positive charge, since at this pH it possesses no negatively charged groups and a maximum number of cationic groups on the basic amino acid residues. As the pH is increased, hydrogen ions are lost so that its charge, originally highly positive, is progressively reduced to zero, after which the protein molecule gradually acquires an increasing net negative charge. Each type of ionizable group has its own characteristic pK value and a buffering effect centred at the pK value and extending about 1·5 units on either side of it.

Proteins thus act as buffers at many regions of pH and make a significant contribution to total buffering in cells and tissues. Most importantly, the imidazole side chains of the histidine residues lose their positive charge between pH 6·5 and 8·0 and, since this change in ionization occurs within the physiological range, it is highly significant both as a buffering factor and in causing changes in charge distribution and molecular configuration in living cells and tissue.

Proteins, like amino acids, have an isoelectric point at which they are ‘self-neutralized’ and have zero net charge. The isoelectric point is largely determined by the ratio of the free acidic (Asp and Glu) to basic (Lys and Arg) amino acid residues. The majority of proteins contain a preponderance of the acidic amino acids, that is Glu + Asp, so that their isoelectric point (pI) is less than 7·0, and they are negatively charged at neutral pH. On the other hand, basic proteins such as the histones, which have a marked preponderance of lysine and arginine, are positively charged at neutral pH. Proteins with different isoelectric points will interact, and a basic protein will tend to form a precipitate with an acidic one. Insulin protaminate formed by combination of insulin (pI 5) with protamine (pI 12) is relatively insoluble and is more slowly absorbed in the body; consequently it has a more prolonged action than ordinary insulin. In the cell nucleus, basic histones are bound to the nucleic acids and may function as regulators of gene activity (Chapter 21).

Protein purification and identification

Before the structure and properties of a protein can be studied it must be obtained in a pure condition. Protein purification is laborious and difficult for three reasons.

The first stage in the isolation of a protein which is not already in solution, e.g. in plasma or milk, is to release it from cell structures. Cells may be disrupted by homogenization, exposure to hypo-osmotic solutions or to ultrasonic vibrations, or by drying them to a powder with acetone at low temperatures. This latter process also serves to remove lipids and facilitates subsequent extraction of the protein.

Once the cells have been disintegrated, the proteins may be extracted with a dilute buffer solution of appropriate pH and ionic strength. From this crude extract means must be found of isolating the required protein from others present. Methods for separating proteins include differential precipitation, ion-exchange chromatography, electrophoresis, gel filtration and ultracentrifugation.

One of the difficulties encountered in protein purification is how to test for the desired protein among other protein contaminants. This is relatively simple if the protein is an enzyme or a hormone, since the specific activity of the preparation may be followed. Specific activity, defined as the activity per unit weight of total protein, increases as the protein becomes progressively purer until no further purification occurs. This may result either because purification has been completed or because the method is not effecting any further purification. The problem of determining whether or not a protein is pure is extremely difficult since ordinary criteria (such as melting point determination) cannot be applied, and, although crystalline preparations of proteins can sometimes be obtained, these may nevertheless still be mixtures. A further complication arises because polypeptide chains may interact specifically with other polypeptide chains. Criteria of purity for proteins are therefore necessarily negative, a protein being assumed to be pure or at least homogeneous if it cannot be shown to be impure. With the development of the highly sensitive technique SDS-PAGE (sodium dodecyl sulphate–polyacrylamide gel electrophoresis) if the protein shows as a single band, it is almost certainly homogeneous. This method which is the one which is now most commonly used depends mainly on molecular size.

Since the only criterion of protein purity is consistent failure to detect inhomogeneity, a protein should be tested by at least two methods that depend on different molecular characteristics as the basis of separation. Thus techniques such as ion-exchange chromatography or electrophoresis, which depend on charge differences, should be used in conjunction with others such as ultracentrifugation and gel filtration, which are based on differences in molecular size and shape.

Affinity chromatography

This valuable technique which may be used to purify certain proteins is very simple in principle. It depends on the highly specific binding affinity of pairs of compounds such as enzymes and inhibitors, hormones and their receptors or antigens and antibodies. It is first necessary to attach covalently one of the pair of high-affinity compounds (ligands) to an insoluble matrix without interfering with its specific binding properties. This may not be easy but, once it has been achieved, the high-affinity material to which, for example, a trypsin inhibitor has been attached, can be packed into a column; then, when an appropriately buffered solution containing trypsin is passed through the column the trypsin is strongly bound to the resin via the inhibitor while the impurities pass through it freely. The trypsin may subsequently be eluted from the solid phase, e.g. by changing the buffer in the column to one having a pH at which the inhibitor has no affinity for trypsin. This liberates the trypsin which is eluted in a pure state. The method can be used not only for the separation of proteins but also for peptides, nucleic acids and polysaccharides or any other substance which takes part in highly specific interactions with a substance which can be covalently linked to an appropriate stationary phase.

The size and shape of protein molecules

Methods which give absolute values for the molecular weight of a protein depend upon determination of their osmotic pressure, their rate of diffusion or their rate of sedimentation in the ultracentrifuge. Such methods, though simple in theory, require expensive equipment and meticulous technique for accurate results. However, under reducing conditions which break -S-S- bridges and with the use of marker proteins of known molecular weight the SDS-PAGE method mentioned earlier provides a means by which the approximate molecular weight of a protein may be simply and rapidly determined.

The molecular weight of a single molecular species is, of course, independent of the method used for its determination. However, owing to the tendency of protein molecules to associate and form aggregates, e.g. gels and fibres, or complexes with other cell constituents such as lipids and nucleic acids, one of the problems of the protein chemist is to decide whether the entities under any given conditions represent a single molecular species or a molecular aggregate. For example, it has been established that the molecule of haemoglobin is made up of four separate polypeptide chains that may only be separated under conditions which are outside the physiological range. The individual chains are consequently regarded as subunits of the haemoglobin molecule. It is now recognized that many large proteins are built up of smaller subunits (Table 5.4). The subunits are held together by non-covalent bonds which are weak enough to be broken by reagents, such as urea, that are unable to break covalent bonds. The construction of large units from a number of smaller ones is a sound building principle which increases the flexibility of the resulting structure and reduces the likelihood of errors. Thus if any single unit is defective either it has little effect on the structure as a whole or it may be rejected in favour of a normal one.

Table 5.4

Molecular weights and subunit constitution of various proteins

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Protein Molecular weight Number of subunits
Ribonuclease 12600 1
Lysozyme (egg white) 13900 1
Myoglobin 16900 1
Malate dehydrogenase 66300 2
Glycerol-1-phosphate dehydrogenase 78000 2
Creatine kinase 80000 2
Enolase 82000 2
α-Amylase 97600 2
Haemoglobin 64500 4
Hexokinase 102000 4
Lactate dehydrogenase 150000 4
Fumarase 194000 4
Catalase 232000 4
Pyruvate kinase 237000 4
Glucose-6-phosphate dehydrogenase 240000 6
Mitochondrial ATPase 284000 10
Phosphorylase a 370000 4
Glutamine synthetase

Dec 10, 2015 | Posted by in General Dentistry | Comments Off on Peptides and proteins
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