Insulin partial acid hydrolysis

The studies of Sanger and co-workers (1945-1955) on the amino acid sequence of insulin provide the best example of the use of partial acid hydrolysis for determination of the covalent structure of polypeptides. When oxidized A- or B-chains were submitted to hydrolysis in 11-12 iV HCl... 

Proteins consist of large numbers of amino-acids joined by the p>eptide link —CO —NH — into chains, as shown in the diagram, where R and R" are amino-acid residues. These chains are called peptides and may be broken into smaller chains by partial hydrolysis (see peptides). Proteins may contain more than one peptide chain thus insulin consists of... 

The covalent structure of insulin was established by Frederick Sanger in 1953 after a 10-year effort. This was the first protein sequence determination.237 238 Sanger used partial hydrolysis of peptide chains whose amino groups had been labeled by reaction with 2,4-dinitrofluorobenzene239 to form shorter end-labeled fragments. These were analyzed for their amino acid composition and labeled and hydrolyzed again as necessary. Many peptides had to be analyzed to deduce the sequence of the 21-residue and 30-residue chains that are joined by disulfide linkages in insulin.237 238... 

Clearly the question of whether rearrangements do actually take place in partial hydrolysis experiments can only be solved by experience. If any such reactions occur it would be impossible to interpret results in terms of a unique sequence of amino acids unless the syntheses are completely specific and quantitative, which is most unlikely. In fact, using concentrated acid at low temperatures it has been possible to work out a unique sequence for gramicidin S (Consden et al., 1947b from the peptides identified, and there was no evidence of any peptide that did not fit this sequence. Similarly a unique structure could be determined for the phenylalanyl chains of insulin (p. 54). It thus seems unlikely that any rearrangement occurs under the action of this type of reagent. On the other hand, syntheses have been definitely shown to occur in the presence of proteolytic enzymes. The formation of plastein by the action of pepsin or trypsin on concentrated peptide mixtures has clearly been shown in certain cases to be accompanied by a decrease in amino... 

An early method of sequence determination employs partial hydrolysis of the P. with acid or with nonspecific endopeptidases The resulting hydrolysate contains a large number of relatively small peptides, representing many overlapping regions of the sequence. By analysis of a sufficient number of peptides, and comparison of their amino acid sequences, it is possible (but very laborious) to construct the linear sequence of the original P. The classical determination of the primary structure of the A chain of Insulin (see) was performed in this way. 

To give an example, we utilized this principle in the synthesis of the partially protected C-terminal undecapeptide acid of the human insulin B-chain i-Noc-Gly-Glu(OH)-Arg(N02)-Gly-Phe-Phe-Tyr(Dcbzl)-Thr(Bzl)-Lys(Z)-Thr(Bzl)-0H. From a subsequently activated sample of polymer-supported peptide, 90% of the synthetic material was released by transesterification with N-dimethylaminoethanol in dimethylformamide within 5 hours, whereas in a parallel test from a nonactivated part, it took 70 hours for almost complete detachment of the peptide. After self-catalyzed aqueous hydrolysis of the intermediate 2-dimethylammonium ethyl ester groups and purification of the product, the peptide was obtained in 19% yield calculated over all synthetic operations including the introduction of the handle molecule onto the polymer support [188]. 

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