Proteins, nucleic acids and other nitrogenous compounds

PROTEINS
Proteins are complex organic compounds of high molecular weight. In common with carbohydrates and fats they contain carbon, hydrogen and oxygen, but in addition they all contain nitrogen and generally sulphur.

Proteins are found in all living cells, where they are intimately connected with all phases of activity that constitute the life of the cell. Each species has its own specific proteins, and a single organism has many different proteins in its cells and tissues. It follows therefore that a large number of proteins occur in nature.

AMINO ACIDS
Amino acids are produced when proteins are hydrolysed by enzymes, acids or alkalis. Although over 200 amino acids have been isolated from biological materials, only 20 of these are commonly found as components of proteins.
Amino acids are characterised by having a basic nitrogenous group, generally an amino group (–NH2), and an acidic carboxyl unit (–COOH). Most amino acids occurring naturally in proteins are of the ␣ type, having the amino group attached to  the carbon atom adjacent to the carboxyl group, and can be represented by the general formula:

                                                                                                               
The exception is proline, which has an imino (–NH) instead of an amino group. The nature of the R group, which is referred to as the side chain, varies in different amino acids. It may simply be a hydrogen atom, as in glycine, or it may be a more
complex radical containing, for example, a phenyl group.

The chemical structures of the 20 amino acids commonly found in natural proteins.

Special amino acids
Some proteins contain special amino acids that are derivatives of common amino acids. For example, collagen, the fibrous protein of connective tissue, contains hydroxyproline and hydroxylysine, which are the hydroxylated derivatives of proline
and lysine, respectively.

                                                                  

Two iodine derivatives of tyrosine, triiodothyronine and tetraiodothyronine (thyroxine), act as important hormones in the body and are also amino acid components of the protein thyroglobulin.



Amino acids commonly found in proteins

Monoamino-monocarboxylic acids

                             

                                              

                              



 

Sulpur-containing amino acids

 

                       

 

Monoamino-dicarboxylic acids and their amine derivatives

                                 

 

                            

 

Basic amino acids

 

                          



 

Aromatic and heterocyclic amino acids

 

                                







 

A derivative of glutamic acid, γ-carboxyglutamic acid, is an amino acid present in the protein thrombin.This amino acid is capable of binding calcium ions and plays an important role in blood clotting.



The amino acid γ-aminobutyric acid functions in the body as a neurotransmitter. It is also found in silage as a fermentation product of glutamic acid.



The sulphur-containing amino acid cysteine also requires special mention. It may occur in protein in two forms, either as itself or as cystine, in which two cysteine molecules are joined together by a disulphide bridge:



 

Properties of amino acids
Because of the presence of an amino group and a carboxyl group, amino acids are amphoteric, i.e. they have both basic and acidic properties. Molecules such as these, with basic and acidic groups, may exist as uncharged molecules, or as dipolar ions
with opposite ionic charges, or as a mixture of these.Amino acids in aqueous solution exist as dipolar ions or zwitter ions (from the German Zwitter, a hermaphrodite):



In a strongly acid solution an amino acid exists largely as a cation, while in alkaline solution it occurs mainly as an anion. There is a pH value for a given amino acid at which it is electrically neutral; this value is known as the isoelectric point.
Because of their amphoteric nature, amino acids act as buffers, resisting changes in pH.
All the α-amino acids except glycine are optically active. As with the carbohydrates , amino acids can take two mirror image forms, D- and L-.
                              

All the amino acids involved in protein structure have an L-configuration of the carbon atom. If supplied in the D-form, some amino acids can be converted to the L-form by deamination of the amino acid to the keto acid and reamination to the
L-form.

Essential amino acids
Plants and many microorganisms are able to synthesise proteins from simple nitrogenous compounds such as nitrates. Animals cannot synthesise the amino group, and in order to build up body proteins they must have a dietary source of amino acids. Certain amino acids can be produced from others by a process known as transamination , but the carbon skeletons of a number of amino acids cannot be synthesised in the animal body; these are referred to as essential or indispensible amino
acids.
Most of the early work in determining which amino acids could be classed as essential was carried out with rats fed on purified diets. The following ten essential amino acids are required for growth in the rat:

Arginine                           Methionine
Histidine                          Phenylalanine
Isoleucine                        Threonine
Leucine                              Tryptophan
Lysine                                 Valine

The chick requires a dietary supply of the ten amino acids listed above but in addition needs a dietary source of glycine. Birds require arginine because their metabolism does not include the urea cycle, which would normally supply this amino acid. The list of essential amino acids required by the pig is similar to that for the rat, with the exception of arginine, which can be synthesised by the pig. It has been reported that rapidly growing animals may respond to arginine because the very active metabolism of the liver results in little of the amino acid being available to the general circulation. Cats require a dietary supply of arginine, owing to their limited ability to synthesise ornithine from glutamate, and a deficiency of arginine results in the accumulation of ammonia from denatured amino acids in the blood. Cats also require the β-sulphonic amino acid taurine in their diet as they are unable to synthesise this from cysteine. Taurine is required for bile acid conjugation. Poultry have a limited capacity to synthesise proline. The actual dietary requirement of certain essential amino acids is dependent upon the presence of other amino acids. For example, the requirement for methionine is partially dependent on the cysteine content of the diet.
In the case of the ruminant, all the essential amino acids can be synthesised by the rumen microorganisms, which theoretically makes this class of animal independent of a dietary source once the rumen microorganisms have become established. However, the supply of amino acids from microbial protein is limiting in quantity and quality for maximum rates of growth in young animals and for maximum milk production.The biological value  of microbial protein is limited by its content of certain essential amino acids, particularly lysine and methionine. For maximum productivity the microbial protein must be supplemented with a supply of dietary amino acids, from foods or synthetic amino acids, in a suitable form that is not degraded by the microorganisms in the rumen.

PEPTIDES
Peptides are built up from amino acids by means of a linkage between the α-carboxyl of one amino acid and the α-amino group of another acid, as shown here:



This type of linkage is known as the peptide linkage; in the example shown, a dipeptide has been produced from two amino acids. Large numbers of amino acids can be joined together by this means, with the elimination of one molecule of water at each linkage, to produce polypeptides.
Besides being important building blocks in the construction of proteins, some peptides possess their own biological activity. Milk, in particular, is a source of many biologically active peptides. The enzymatic hydrolysis of the milk protein casein releases opioid peptides, which have pharmacological activities such as analgesia and sleep-inducing effects. Other peptides derived from casein are involved in calcium flow in tissues and modification of the immune system response. Other milk peptides
stimulate growth of desirable bacteria and suppress harmful bacteria, and some act as growth factors for intestinal cells.

Other peptides, including bombesin, enterostatin, glucagon and leptin, are important in the control of food intake.

Peptides play an important role in the flavour and sensory properties of foods such as yeast extract, cheese and fruit juices.

 

STRUCTURE OF PROTEINS
For convenience the structure of proteins can be considered under four basic headings.
Primary structure

The sequence of amino acids along the polypeptide chain of a protein, as described above, is called the primary structure of the protein.

Secondary structure

The secondary structure of proteins refers to the conformation of the chain of amino acids resulting from the formation of hydrogen bonds between the imino and carbonyl groups of adjacent amino acids, as shown in.
The secondary structure may be regular, in which case the polypeptide chains exist in the form of an α-helix or a β-pleated sheet, or it may be irregular and exist as, for example, a random coil.

Tertiary structure

The tertiary structure describes how the chains of the secondary structure further interact through the R groups of the amino acid residues. This interaction causes folding and bending of the polypeptide chain, the specific manner of the folding giving each protein its characteristic biological activity.

   Configuration of polypeptide chain. Dotted lines represent possible hydrogen bonds.


Quaternary structure

Proteins possess quaternary structure if they contain more than one polypeptide chain. The forces that stabilise these aggregates are hydrogen bonds and electrostatic,or salt bonds formed between residues on the surfaces of the polypeptide chains.

PROPERTIES OF PROTEINS

All proteins have colloidal properties; they differ in their solubility in water, ranging from insoluble keratin to albumins, which are highly soluble. Soluble proteins can be precipitated from solution by the addition of certain salts such as sodium chloride or ammonium sulphate. This is a physical effect and the properties of the proteins are not altered. On dilution the proteins can easily be redissolved.
Although the amino and carboxyl groups in the peptide linkage are non-functional in acid–base reactions, all proteins contain a number of free amino and carboxyl groups, either as terminal units or in the side chain of amino acid residues. Like amino acids, proteins are therefore amphoteric. They exhibit characteristic isoelectric points and have buffering properties.
All proteins can be denatured or changed from their natural state. Denaturation has been defined by Neurath and coworkers as ‘any non-proteolytic modification of the unique structure of a native protein, giving rise to definite changes in chemical, physical or biological properties’. Products of protein hydrolysis are not included under this term. Several agents can bring about denaturation of proteins; these include heat, acids, alkalis, alcohols, urea and salts of heavy metals. The effect of heat on proteins is of special interest in nutrition as this results in new linkages within and between peptide chains. Some of these new linkages resist hydrolysis by proteases produced in the digestive tract and impede their access to adjacent peptide bonds.
Susceptibility of proteins to heat damage is increased in the presence of various carbohydrates, owing to the occurrence of Maillard-type reactions, which initially involve a condensation between the carbonyl group of a reducing sugar with the free amino group of an amino acid or protein. Lysine is particularly susceptible. With increasing severity of heat treatment, further reactions involving protein side chains can occur and result in the browning of foods. The dark coloration of overheated hays and silages is symptomatic of these types of reaction.

CLASSIFICATION OF PROTEINS
Proteins may be classified into two main groups: simple proteins and conjugated proteins.
Simple proteins

These proteins produce only amino acids on hydrolysis. They are subdivided into two groups, fibrous and globular proteins, according to shape, solubility and chemical composition.

Fibrous proteins
These proteins, which in most cases have structural roles in animal cells and tissues, are insoluble and are very resistant to animal digestive enzymes.They are composed of elongated filamentous chains joined together by cross-linkages. The group includes collagens, elastin and keratins.
Collagens are the main proteins of connective tissues and constitute about 30 per cent of the total proteins in the mammalian body. As mentioned earlier, the amino acid hydroxyproline is an important component of collagen. Hydroxylation of proline to hydroxyproline involves vitamin C; if this vitamin is deficient, collagen fibres are weakened and may give rise to gum and skin lesions. The indispensable amino acid tryptophan is not found in these proteins.
Elastin is the protein found in elastic tissues such as tendons and arteries. The polypeptide chain of elastin is rich in alanine and glycine and is very flexible. It contains cross-links involving lysine side chains, which prevent the protein from extending excessively under tension and allow it to return to its normal length when tension is removed.
Keratins are classified into two types. The α-keratins are the main proteins of
wool and hair. The β-keratins occur in feathers, skin, beaks and scales of most birds and reptiles. These proteins are very rich in the sulphur-containing amino acid cysteine; wool protein, for example, contains about 4 per cent of sulphur.

Globular proteins
Globular proteins are so called because their polypeptide chains are folded into compact structures. The group includes all the enzymes, antigens and those hormones that are proteins. Its first subgroup, albumins, are water-soluble and  heat-coagulable and occur in milk, the blood, eggs and many plants. Histones are basic proteins that occur in cell nuclei, where they are associated with DNA . They are soluble in salt solutions, are not heat-coagulable, and on hydrolysis yield large quantities of arginine and lysine. Protamines are basic proteins of relatively low molecular weight, which are associated with nucleic acids and are found in large quantities in the mature male germ cells of vertebrates. Protamines are rich in arginine but contain no tyrosine, tryptophan or sulphur-containing amino acids. Globulins occur in milk, eggs and blood, and are the
main reserve protein in many seeds.

Conjugated proteins
Conjugated proteins contain, in addition to amino acids, a non-protein moietytermed a prosthetic group. Some important examples of conjugated proteins are glycoproteins, lipoproteins, phosphoproteins and chromoproteins.
           Glycoproteins are proteins with one or more heteroglycans as prosthetic  groups. In most glycoproteins the heteroglycans contain a hexosamine, either glucosamine or galactosamine or both; in addition, galactose and mannose may also be present. Glycoproteins are components of mucous secretions, which act as lubricants in many parts of the body. The storage protein in egg white, ovalbumin, is a glycoprotein.
         Lipoproteins, which are proteins conjugated with lipids such as triacylglycerols and cholesterol, are the main components of cell membranes and are also the form in which lipids are transported in the bloodstream to tissues, either for oxidation or for energy storage. They can be classified into five main categories in increasing order of density: chylomicrons, very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL), intermediate-density lipoproteins (IDL) and high-density lipoproteins (HDL) .

NUCLEIC ACIDS
Nucleic acids are high-molecular-weight compounds that play a fundamental role in living organisms as a store of genetic information; they are the means by which this information is utilised in the synthesis of proteins. On hydrolysis, nucleic acids yield a mixture of basic nitrogenous compounds (purines and pyrimidines), a pentose (ri-bose or deoxyribose) and phosphoric acid.
The main pyrimidines found in nucleic acids are cytosine, thymine and uracil. The relationships between these compounds and the parent material, pyrimidine, are shown below:



       

Adenine and guanine are the principal purine bases present in nucleic acids.

                

The compound formed by linking one of the above nitrogenous compounds to a pentose is termed a nucleoside. For example:



D-Ribose                                                                           Adenine                                                                      Adenosine

 

If nucleosides such as adenosine are esterified with phosphoric acid they form nucleotides, e.g. adenosine monophosphate (AMP):



 

Nucleic acids are polynucleotides of very high molecular weight, generally measured in several millions. A nucleotide containing ribose is termed ribonucleic acid (RNA), while one containing deoxyribose is referred to as deoxyribonucleic
acid (DNA).
The nucleotides are arranged in a certain pattern; DNA normally consists of a double-strand spiral or helix. Each strand consists of alternate units of the deoxyribose and phosphate groups. Attached to each sugar group is one of the four bases, cytosine, thymine, adenine or guanine.The bases on the two strands of the spiral are joined in pairs by hydrogen bonds, the thymine on one strand always being paired with the adenine on the other and the cytosine with the guanine. The sequence of bases along these strands carries the genetic information of the living cell. DNA is found in the nuclei of cells as part of the chromosome structure. There are several distinct types of ribonucleic acid, which are defined in terms of
molecular size, base composition and functional properties.They differ from DNA in

 Diagrammatic representation of part of the ladder-like DNA molecule, showing the two strands of alternate phosphate (P) and deoxyribose (D) molecules.The horizontal rods represent the pairs of bases held by hydrogen bonds (represented
by dotted lines).

A = adenine, T = thymine, C = cytosine, G = guanine.



the nature of their sugar moiety and also in the types of nitrogenous base present. RNA contains the pyrimidine uracil in place of thymine.There is evidence to indicate that unlike DNA, most RNA molecules exist in the form of single, folded chains arranged spirally. There are three main forms of RNA, termed messenger RNA, ribosomal RNA and transfer RNA. The functions of these three forms of RNA are dealt with in the protein synthesis section.
Apart from their importance in the structure of nucleic acids, nucleotides exist free as monomers and many play an important role in cellular metabolism.
Although nucleotides are synthesised de novo it appears that this synthesis is not always adequate. In such cases (abrupt early weaning of piglets and times of disease challenge) a dietary supply augments the natural synthesis and enhances immunefunction and the proliferation of cells.
Reference has been made previously to the phosphorylation of adenosine to form adenosine monophosphate (AMP). Successive additions of phosphate residues give adenosine diphosphate (ADP) and then the triphosphate (ATP). The importance of ATP in energy transformations is described.

OTHER NITROGENOUS COMPOUNDS
A considerable variety of nitrogen-containing compounds, other than proteins and nucleic acids, occur in plants and animals. In plants, free amino acids are usually present; those in greatest amount include glutamic acid, aspartic acid, alanine, serine, glycine and proline. Other compounds are nitrogenous lipids, amines, amides, purines, pyrimidines, nitrates and alkaloids. In addition, most members of the vitamin B complex contain nitrogen in their structure.
It is impossible to deal with these compounds in any detail here, and only some of the important ones not previously mentioned will be discussed.
Amines
Amines are basic compounds present in small amounts in most plant and animal tissues. Many occur as decomposition products in decaying organic matter and havetoxic properties.

Amines are basic compounds present in small amounts in most plant and animal tis-
sues. Many occur as decomposition products in decaying organic matter and have
toxic properties.
A number of microorganisms are capable of producing amines by decarboxylation of amino acids.  These may be produced in the rumen under certain conditions and can occur in fermented foods such as cheese, wine, sauerkraut and
sausage. They are termed biogenic amines and may give rise to physiological symptoms; histamine, for example, is an amine formed from the amino acid histidine and in cases of anaphylactic shock is found in the blood in relatively large amounts. Histamine has also been implicated in dietary-induced migraine. Silages in which clostridia have dominated the fermentation usually contain appreciable amounts of amines.
In contrast to the harmful biogenic amines, the polyamines putrescene, spermidine and spermine are necessary for optimal growth and function of cells. They are involved in DNA, RNA and protein synthesis, regulation of gene expression, enzyme activity, cell proliferation and cell signalling.
Several metabolic pathways (e.g. lipid metabolism, creatine and carnitine synthesis) require methyl groups and these can be supplied by choline or methionine. During the process of transmethylation, betaine, a tertiary amine, is formed by the oxidation of choline. Betaine can be added to the diet to act as a more direct supply of methyl groups, thus sparing choline for its other functions of lecithin and acetylcholine formation, and methionine for protein synthesis. Betaine occurs in sugar beet,
 Some important amines and their parent amino acids


and the young leaves may contain about 25 g/kg; it is this amine that is responsible for the fishy aroma frequently associated with the commercial extraction of sugar from beet. In the animal body, betaine may be transformed into trimethylamine, and it is this that gives the fishy taint to milk produced by cows that have been given excessive amounts of sugar beet by-products.
Amides

Asparagine and glutamine are important amide derivatives of the amino acids aspartic acid and glutamic acid. These two amides are also classed as amino acids and occur as components of proteins.They also occur as free amides and play an im-
portant role in transamination reactions.
Urea is an amide that is the main end product of nitrogen metabolism in mammals, but it also occurs in many plants and has been detected in wheat, soya bean,potato and cabbage.


In humans and other primates, uric acid is the end product of purine metabolism and is found in the urine. In subprimate mammals the uric acid is oxidised to allantoin before being excreted.
In birds, uric acid is the principal end product of nitrogen metabolism and thus corresponds, in its function, to urea in mammals.



NITRATES
Nitrates may be present in plant materials and, whereas nitrate itself may not betoxic to animals, it is reduced readily under favourable conditions, as in the rumen,to nitrite, which is toxic. Oat hay poisoning is attributed to the relatively large amounts of nitrate present in green oats.
Quite high levels of nitrate have been reported in herbage given heavy dressings of nitrogenous fertilisers.


ALKALOIDS

These compounds are of particular interest since many of them have poisonous properties. In plants, their presence is restricted to a few orders of the dicotyledons. A number of the more important alkaloids, with their sources, are listed in
Table 4.3. The alkaloid in ragwort, for example, attacks the liver and much of this organ can be destroyed before symptoms appear. Another nutritionally significant source of alkaloids is the fungus ergot, which grows on cereal grains.

 Some important alkaloids occurring in plants