Insulin primary structure

Fig.l. Insulin. Primary structure of sheep insulin. Human (H) and bovine (B) insulin differ from sheep insulin in the sequence region A8 to 10 in addition, in human insulin, the C-terminal alanine of the B-chain is replaced by threonine. 

Human and horse insulin both have two polypeptide chains, with one chain containing 21 amino acids and the other containing30 amino acids. They differ in primary structure at two places. At position 9 in one chain, human insulin has Ser and horse insulin has Gly at position 30 in the other chain, human insulin has Thr and horse insulin has Ala. How must the DNA for the two insulins differ ... 

The amino group of the N-terminal amino acid residue of a peptide will react with the FDNB reagent to form the characteristic yellow DNP derivative, which may be released from the peptide by either acid or enzymic hydrolysis of the peptide bond and subsequently identified. This is of historic interest because Dr F. Sanger first used this reaction in his work on the determination of the primary structure of the polypeptide hormone insulin and the reagent is often referred to as Sanger s reagent. 

As metabolic pathways became clearer, the detailed study of the enzymes involved was facilitated by the introduction of new procedures for isolation, purification, and characterization of proteins. Developments in chromatography in the early 1940s and the introduction of gel electrophoresis allowed more efficient methods to be used to separate proteins and to analyze their primary structure, so that Sanger was able, by 1953, to report the primary structure of insulin (Chapter 10). 

The promotion by insulin of glucose uptake by muscle and fat cells (adipocytes), of glycogen deposition in liver and muscle, and its stimulation of growth soon emerged as the purified hormone became available for study. Although insulin was crystallized by Abel in 1926, its primary structure established by Sanger in 1953 (see Chapter 10),... 

F ig U re 10.2 The primary structure of bovine insulin. This molecule possesses two polypeptide chains, labeled A and B. These are joined by two disulfide bridges between Cys amino acid residues. There is a third disulfide bridge linking two Cys residues in the A chain. 

Before we take leave of primary structures for proteins, there is one last, important realization. Proteins serving the same function in different species may have different primary structures. For example, the primary structures of bovine, ovine, and human insulins are not quite the same. They are closely related but not identical. Different species have discovered different protein solutions for the same biological problem. 

Now we can ask what is likely to happen to the three-dimensional structure of a protein if we make a conservative replacement of one amino acid for another in the primary structnre. A conservative replacement involves, for example, substitution of one nonpolar amino acid for another, or replacement of one charged amino acid for another. Intnitively, one would expect that conservative replacements would have rather little effect on three-dimensional protein structure. If an isoleucine is replaced by a valine or leucine, the structnral modification is modest. The side chains of all of these amino acids are hydrophobic and will be content to sit in the molecnlar interior. This expectation is borne out in practice. We have noted earlier that there are many different molecnles of cytochrome c in nature, all of which serve the same basic function and all of which have similar three-dimensional structnres. We have also noted the species specificity of insulins among mammalian species. Here too we find a number of conservative changes in the primary structure of the hormone. Although there are exceptions, as a general rule conservative changes in the primary structnre of proteins are consistent with maintenance of the three-dimensional structures of proteins and the associated biological functions. 

The sequence is unique for insulin from a specific species but insulins from different species have slightly different primary structures. 

The primary structure of a protein is its amino acid sequence. During the biosynthesis of insulin in the pancreas, a continuous peptide chain with 84 residues is first synthesized—proinsu/in (see p.160). After folding of the molecule, the three disulfide bonds are first formed, and residues 31 to 63 are then proteolytically cleaved releasing the so-called C peptide. The molecule that is left over (1) now consists of two peptide chains, the A chain (21 residues, shown in yellow) and the B chain (30 residues, orange). One of the disulfide bonds is located inside the A chain, and the two others link the two chains together. 

Various procedures are used to analyze protein primary structure. Several protocols are available to label and identify the amino-terminal amino acid residue (Fig. 3-25a). Sanger developed the reagent l-fluoro-2,4-dinitrobenzene (FDNB) for this purpose other reagents used to label the amino-terminal residue, dansyl chloride and dabsyl chloride, yield derivatives that are more easily detectable than the dinitrophenyl derivatives. After the amino-terminal residue is labeled with one of these reagents, the polypeptide is hydrolyzed to its constituent amino acids and the labeled amino acid is identified. Because the hydrolysis stage destroys the polypeptide, this procedure cannot be used to sequence a polypeptide beyond its amino-terminal residue. However, it can help determine the number of chemically distinct polypeptides in a protein, provided each has a different amino-terminal residue. For example, two residues—Phe and Gly—would be labeled if insulin (Fig. 3-24) were subjected to this procedure. 

Using procedures such as those outlined in this section more than 100 proteins have been sequenced. This is an impressive accomplishment considering the complexity and size of many of these molecules (see, for example, Table 25-3). It has been little more than two decades since the first amino acid sequence of a protein was reported by F. Sanger, who determined the primary structure of insulin (1953). This work remains a landmark in the history of chemistry because it established for the first time that proteins have definite primary structures in the same way that other organic molecules do. Up until that time, the concept of definite primary structures for proteins was openly questioned. Sanger developed the method of analysis for N-terminal amino acids using 2,4-dinitrofluorobenzene and received a Nobel Prize in 1958 for his success in determining the amino-acid sequence of insulin. 

A major problem, until recently, was the determination of the protein primary structure, but with the advent of modern analysis of DNA this has become comparatively easy. One of the first structures to be described was that of insulin which contains 60 amino-acids and has a molecular weight of 12,000. Once the primary structure is known, it is possible to predict the secondary and tertiary structures using additional information obtained through X-ray crystallography of the crystallised protein. 

Unlike polysaccharides, proteins do not have branched chains, but several chains may be linked together via disulphide bridges rather than peptide bonds. The primary structure of ox insulin is shown in Fig. S.A2. The protein consists of two peptide chains which are linked via the formation of the disulphide bridges. Disulphide bridges are formed by the condensation of the thiol groups of two cysteine residues. 

Fig. 5.A2. Protein primary structure. The amino-acid sequence of ox insulin...

I The most important properties of a protein are deter-f mined by the sequence of amino acids in the polypeptide chain. This sequence is called the primary structure of the protein. We know the sequences for thousands of peptides and proteins, largely through the use of methods developed in Fred Sanger s laboratory and first used to determine the sequence of the peptide hormone insulin in 1953. Knowledge of the amino acid sequence is extremely useful in a number of ways (1) it permits comparisons between normal and mutant proteins (see chapter 5) (2) it permits comparisons between comparable proteins in different species and thereby has been instrumental in positioning different organisms on the evolutionary tree (see fig. 1.24) (3) finally and most important, it is a vital piece of information for determining the three-dimensional structure of the protein. 

Preparation of Insulins. Until the early 1980s insulin for therapeutic purposes was produced almost exclusively by extraction from beef and pork pancreases. Between 100 and 400 mg of insulin can be obtained from each kg of pancreatic tissue, and it has been estimated that there would be sufficient supplies of animal insulin to meet the requirements of diabetic patients into the twenty-first century (2). Through modem purification procedures animal insulins can be prepared in essentially pure form, which eliminates the possibility of developing antibodies against impurities in the insulin preparations. However, patients treated with purified insulins still develop antibodies to insulin, suggesting that differences in the primary structures of these insulins might stimulate antibody production. Therefore, enzymatic and biosynthetic methods have been developed for the preparation of therapeutic insulin identical to human insulin. 

The primary structure of a protein is the sequence of its amino acids. For example, the first 10 amino acids in the cytochrome c sequence are Ala-Ser-Phe-Ser-Glu-Ala-Pro-Gly-Asn-Pro, while the first 10 amino acids in the myosin sequence are Phe-Ser-Asp-Pro-Asp-Phe-Gln-Tyr-Leu-Ala. Therefore, the primary structure is just the full sequence of amino acids in the polypeptide chain or chains. Finding the primary structure of a protein is called protein sequencing. The first protein to be sequenced was the hormone insulin. 

In 1944, about the time Sanger determined the primary structure of insulin, two-dimensional paper chromatography became available for analyzing amino acids of protein samples.32 This method allowed Sanger to analyze 20 amino acids in a single run with considerably less sample and time compared to the previous methods. The development of an automated amino acid analyzer in 1958 by Spackman, Stein and Moore had made further progress.33 This first amino acid analyzer performed an analysis with 1 pmol of sample in 20 hours. Due to the continuous improvements made on amino acid analyzers,... 

Insulin (Fig. 6), which belongs to the older-generation polypeptides was discovered by Banting and Best in 1921 and its primary structure was elucidated by Sanger in 1955. In view of the very diverse syntheses and the numerous biological publications on this substance, only a general summary is possible here. 

Over 50% of the aspartic acid of the proteins of hepatic origin is liberated in 16 hours, but the gamma-globulins of extrahepatic origin required 36 hours to reach 50%. The extent to which this reflects certain characteristics of the primary structure is described in similar studies on insulin, ribonuclease, and glucagon (37). The cleavage of the peptide chain at the aspartic acid bonds may release other amino acids when they are between aspartic acid residues, when the aspartic acid is penultimate at either end of the chain, or if the other residues occupy positions of particularly labile sequences not known at this time. The... 

The third approach has been followed predominantly in many laboratories. A comparison of differences in the primary structures of insulins, corticotropins, and cytochromes c from different species (see Table I) may serve as typical examples of these efforts. The data from these studies provide evidence of the extensive structural relationship between functionally identical proteins from different species this relationship decreases with the increased difference in species. 

The most valuable confirmation of this view to date is, without doubt, to be found in the known structures of homologous proteins and peptide hormones, that is compounds of identical biological function isolated from different species. As is well known, the primary structures of the homologous insulins, corticotropins, hypertensins, posterior pituitary hormones, and heme peptide sequences from cytochromes c are closely similar and differ only at certain definite sites in the peptide chains. These can, in particular, serve as a useful point of departure in a search for more general principles governing protein structure, and in the comparison of different proteins. 

An important example ol this behaviour is provided by the reaction of 1-f1uoro-2,H-dinitrobeni eno ai with the terminal amino group of proteins Subsequent acidic hydrolysis yields the yeliow 2.4-dinitrophenyl derivative of the terminal amino acid ol the protein, which can then be identified. With the help of this technique of end-group analysis, Sanger was able to determine the primary structure o insulin. 

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