2.1. Insulin as a Protein

2.1.1 Composition Amino acids

Proteins are made up of molecular units calledamino acids. These monomer units consist of a backbone with an amino group at one end and a carboxylic acid group at the other (NH3-C(R)H-COOH), and a side group, R, on thecarbon. It is the R group that distinguishes one amino acid form the next, gives the acid its name and also defines its properties. These R groups can be of four types: Hydrophobic, Hydrophilic, Basic (+ charged at physiological pH) and acidic (negatively charged at physiological pH). Physiological pH is the pH of the body, which is kept constant at 7.4, except in certain isolated chambers such as the stomach which is between 3 and 4. Because of the four valent bonding structure of the carbon atom, amino acids are optically active, existing in two isomeric forms, the D and L form, but only the L form is found commonly in nature. There are 20 common L-amino acids that make up proteins. Their structures are given on page three of the class handouts. Since the basic and acidic amino acids have ionizable groups, they can be either charged or uncharged depending on the pH of the environment. A basic group can be either neutral or positively charged, and an acidic amino acid can be either neutral or negatively charged. It also follows that a protein, which is composed of a string of linked amino acids, can have different charges at different pH, and that there will be a unique pH at which there is no net charge, that is the sum of the negative charges equals the sum of the positive charges. This point is called the Isoelectric point (IP), and is a important property of a protein. When the pH is above the IP, the protein will have a net negative charge and when it is below the IP it will have a positive charge. At the isoelectric point a protein has minimal solubility because the lack of a net charge means that the protein molecules no longer repel each other, and they have a tendency to clump together and precipitate. The isoelectric point of beef insulin is 5.7 and porcine insulin is 6.0.

2.1.2. Bonding in proteins

The order in which amino acids are linked together in a protein is called the primary structure. Each amino acid can be represented by the first three letters of its name, so that the primary structure can be written out. A complete list of amino acid structures and the three letter code (together with a single letter code that is also used sometimes) is given in the class notes. By convention protein primary sequence is written from the end that contains the NH2- group. For example the primary sequence of the first seven amino acids of the insulin A chain is :

The bond that joins amino acids together is called a peptide bond, and is obtained by elimination of H2O from the acid terminal of one amino acid and the amino terminal of the next.

The resulting peptide bond [] is a rather rigid bond because of the possible resonance structures, and the peptide unit is planar as a result. The peptide bond is also the major target of peptidase enzymes, that is enzymes (natures catalysts) that are specific for breaking the peptide bond. Enzymes themselves are in fact proteins. There are many peptidases in the blood, which normally function to break down waste proteins. This is one of the reasons why insulin and many other protein drugs have a very short half life in the body. Table 2.1.1. lists some half lives of some important peptides in the blood.

Table 2.1.1 Plasma half lives of polypeptide drugs

Polypeptide Molecular weight Half-life
ACTH ~4700 < 5 min.
Angiotensin 1 ~1200 15 sec.
Bradykinin 1060 30 sec.
Calcitonin ~3600 <40 min.
Enkephalins ~600 2 min.



~30,000 0.5-3 hr.
Growth hormone ~22,600 <25 min.
Insulin ~6000 <25 min.
Oxytocin 1007 2 min.



9500 <15 min.
Vasopressin ~1200 4 min.

2.1.3. Three Dimensional Protein Structure and Stability

We have already discussed the Primary Structure of a protein, that is the sequence in which the constituent amino acids are linked together like beads on a necklace. This primary sequence, which is unique to each different protein, is very important in determining what shape the protein adopts. The different amino acids can interact with each other by weak forces such as hydrogen bonding, Van der Waals forces and hydrophobic interactions. This can give the amino acid strand a structure, like twisting the beads of the necklace back on themselves. This structure is called Secondary structure, and it can be in the form of either an-helix, or a so called -sheet. These are illustrated on Figure 2.1. Many fibrous proteins such as collagen, or keratin which is in hair, are composed of intertwined-helices. The -sheet secondary structure is more stable because of the strong hydrogen bonds between chains.

If we again think of our analogy with a necklace, you can imagine that the necklace, with its sequence of beads, and its local defined-helix and -sheet structure, could also be wound up so that beads (amino acids) that are well separated on the necklace can interact. When the amino acid groups are cysteines, this interaction can be covalent, a we saw between the A and B chain on insulin. This longer distance interaction results in what is called the Tertiary Structure. The importance of tertiary structure can be seen when we consider that it is this structure that determines the final shape of the protein. In an enzyme the final shape most likely results in a cleft in the protein which is exactly the correct shape to accommodate the substrate on which that enzymes acts. The example that we have already seen is that of a protease that breaks down a protein. The cleft, known as the active site, is the correct shape to take in the peptide bond, which is then cleaved. For protein hormones, such as insulin, the shape of the insulin results in an area, called the binding site, that will exactly fit into a receptor site on a cell surface. For a class of very large proteins know as immunoglobulins (antibodies), the final shape results in a Y-shaped molecule, in which the top two prong of the Y posses binding sites for a specific antigen. In all these cases the correct shape of the protein is vital for its activity. If the shape is altered or damaged, the protein will not be able to function. It follows that everything must be done to preserve the protein in its active form. Unfortunately proteins are not robust molecules. As was mentioned above, the forces that hold the secondary and tertiary structure together are all weak, such as hydrogen bonding.

Occasionally proteins have other groups attached to them, such as the heme group in the blood protein hemoglobin. These groups are called prosthetic groups. Hemoglobin is also an example of a protein that has yet a fourth level of structure, the Quaternary Structure, in which four folded chains get together to from a multi-chain oligomer. Many of these levels of structure are shown in figure 2.1.1.

Figure 2.1.1. Levels of structure found in proteins

One of the most important take-home messages of this section is that proteins have a very well defined structure, which is vital for their specific activity. The structure is a result of the interactions of amino acids which make up the backbone of the protein molecule. The forces that hold these amino acids together in the secondary through quaternary structures are generally weak force, such as hydrogen bonds. This means that it is easy to break this all important structure down. The fragile nature of proteins has important implications on their handling and delivery.

Another consequence of the fact that the side groups of the amino acids in a protein interact is that protein molecules tend to aggregate when they are in solution. Insulin is particularly bad at forming dimers and trimers at the slightest provocation. The solution can be made to form a precipitate simply upon shaking. This is very unfortunate, since insulin is a prime candidate for delivery from a pump.

2.1.4. Molecular Weight

Proteins usually are made up of more than 50 amino acids. Below 50 they are called polypeptides, and above 50 proteins. Insulin, with 51 amino acids, has a molecular weight of 6000. This is enormous compared with aspirin, which has the same molecular weight as glucose, 180 Daltons. Since most compounds enter the body by passage through membranes or through minute pores, and travel about the tissues by a process of diffusion, this again has important implication in drug delivery.

2.1.5. Immunologic Response

When a foreign protein enters the blood stream, the body mounts what is called an immune response. Antibodies are produced which coat the invader and allow the cells of the immune system to destroy it. In fact there is strong evidence that Diabetes itself is caused by the body producing antibodies to its own cells, the cells in the pancreas which produce insulin. Usually though, antibody production is beneficial to the organism because it helps fight off infection. A vaccine contains proteins of a pathogen, so that the body will produce antibodies, and after a booster shot, the system is primed to mount a very rapid and effective defense against attack by the organism itself. When thinking about proteins as drugs, and how we want to deliver them, we must be aware of the possibility of the body mounting an immune response to the protein and destroying it, or worse giving an allergic reaction after repeated doses. With the development of human insulin, this hazard is greatly reduced for diabetics. Since the gene which expresses the insulin is of human origin, the product of that gene is indistinguishable from that produced by a human. Other researchers have suggested coating foreign proteins with a chemical coat of polyethylene glycol molecules to disguise them from the immune system, or of encapsulating the protein in a tiny capsule called a liposome, that also can evade the immune system.