Peptides

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

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.

1. between different parts of the same chain

2. between two or more chains which may be of the same or different types

3. between a polypeptide chain and a non-protein molecule or molecules. This latter type of interaction produces the so-called conjugated proteins, e.g. haemoglobin, the lipoproteins of cell membrances and the nucleoproteins of the chromosomes. The non-protein part of the molecule is known as the prosthetic group.

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)		6·2–7·6
α-amino (N-terminal)		7·6–8·4	-NH2
ɛ-amino (Lys)		9·4–10·6	-NH2
Sulphydryl (Cys)	-SH	9·1–10·8	-S−
Phenolic hydroxyl (Tyr)	-OH	9·8–10·4	-O−
Guanidinium (Arg)		11·6–12·6

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.

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.