Insulin primary sequence

By 1953 the complete primary sequence of insulin was known. Sanger could thus conclude that proteins like insulin had unique structures and were, as Staudinger had postulated, defined macromolecules. The primary sequence of other proteins soon followed. 

The three-dimensional structure of insulin remained recalcitrant in spite of the knowledge of its primary sequence. The early crystals had been found by Scott (1936) to contain zinc which could be replaced by other divalent metals. The zinc atom is not heavy enough to be unambiguously distinguishable. Eventually it proved possible to introduce uranyl acetate and uranyl fluoride into the insulin molecule and to obtain the three-dimensional structure, first at 2.8 A resolution and then at 1.9 A (see Blundell, Dodson, Hodgkin, and Mercola, 1972). 

For many years patients with diabetes were treated with insulins that had been isolated from the pancreases of pigs and cows. The primary sequences of these insulins are closely related to the sequence of human insulin. There is only a single difference between the sequences of human and porcine insulins human insulin has a threonine at position B30, and porcine insulin has an alanine. Bovine insulin differs from human insulin at three positions. There is an alanine at the B30 position, an alanine at A8, and a valine at A10. These conservative changes in primary sequence have no apparent effect on biologic activity, although there are slight differences in solubility (7). 

The first primary sequence of a protein (Insulin) established, proving the chemical identity of proteins. Sanger... 

Insulin was the first protein for which the primary sequence was determined. This was done in 1953 by Frederick Sanger, who received the 1958 Nobel Prize in chemistry for his work. Sanger was bom in England in 1918 and received a Ph.D. from Cambridge University, where he has worked for his entire career. He also received a share of the 1980 Nobel Prize in chemistry (Section 27.15) for being the first to sequence a DNA molecule (with 5375 nucleotide pairs). 

Quantum yield of the tyrosine in 029 SSB is obtained by comparing the steady-state fluorescence spectrum to that of aqueous solutions of 5-methoxy-indole (5 MeOI) of known quantum yield ( Oref = 0.28) (Hersberger and Lumry, 1976). Of of 029 SSB = 0.065. This Of value is unusually high for proteins that lack tryptophan in their primary sequence such as insulin for example. Since Of of 029 SSB is approximately 70% of that of A-acetyl-L-tyrosine amide, the authors concluded that 029 SSB tyrosines do not appear to maintain strong interactions with odier residues of the protein... 

The primary sequences of insulin from several species are known, and porcine insulin is the cicsest to that of humans. Their A-chains are identical, and they differ only in their B-chains, with Ala ° (porcine) in place of Thr ° (human). Human and bovine insulin differ in each chain, with Ala and Val ° in the A-Chain (bovine) and... 

Insulin a polypeptide hormone, M, 5,780 (bovine), synthesized in, and secreted by, the B cells of the islets of Langerhans. The first protein primary sequence ever to be elucidated was that of I. (Fig.l) [F. Sanger etal. Biochem. J. 59 (1955) 509-518], I. is the only hormone that decreases the blood glucose concentration. It affects the entire intermediary metabolism, especially of the liver, adipose tissue and muscle. I. increases the permeability of cells to monosaccharides, amino aci and fatty acids, and it accelerates glycolysis, the pentose phosphate cycle, and, in the liver, glycogen synthesis. It promotes the biosynthesis of fatty adds and proteins. These indirect effects on various enzymes and metabolic processes are listed in the tables. 

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. 

Figure 28.6 The primary sequence of the insulin A chain, a short polypeptide of 21 amino acids.

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. 

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. 

Insulin is a large polypeptide of 51 amino acids arranged in a specific sequence and configuration. The primary effect of insulin is to lower blood glucose levels by facilitating the entry of glucose into peripheral tissues. The effects of insulin on energy metabolism, specific aspects of insulin release, and insulin s mechanism of action are discussed here. 

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. 

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