Circular dichroic and immunologic studies of structure relations of insulin and derivatives

Abstract

Desoctapeptide-and desalanine-desasparagine insulins have similarly altered circular dichroic spectra compared to insulin. In contrast, the results of immunological studies clearly demonstrate significant conformational differences between these two types of structurally modified insulin molecules. Removal of alanine B-30 alone from insulin yields a derivative indistinguishable from insulin. It is concluded, therefore, that changes in structure of desalaninedesasparagine-insulin are due to the removal of Asn A-21 and not Ala B-30. The presence of a one-way cross-reaction between insulin and antibodies against desalanine-desas-T A he structural and functional integrity of the insulin molecule is dependent on the interaction of the COOH-terminal B chain with the core of the molecule (Carpenter, 1966; Arquilla and Bromer, 1967; Arquilla et al., 1972;Hodgkin and Mercola, 1972). This interaction results in a hydrophobic center rich in aromatic amino acids (Arquilla et al., 1969;Hodgkin and Mercola, 1972). Specific molecular alterations which affect the interaction of the COOH-terminal B chain with the core of the molecule are, therefore, of interest. The two derivatives most widely studied in this regard have been desalanine (B-30)-desasparagine-(A-21)-insulin and desoctapeptide (B-23 to B-30)-insulin, both of which have attenuated biologic activity (Carpenter, 1966;Arquilla et al., 1969), as well as similarly altered circular dichroic (CD) spectra (Morris et al., 1970), immunologic properties (Arquilla et al., 1969), and ability to aggregrate (Goldman, 1967).We previously proposed that the structural role of Asn A-21 may involve stabilization of the COOH-terminal B chain via electrostatic interaction with the guanidinium residue of Arg B-22, suggesting that deletion of Asn A-21, or the octapeptide of the COOH-terminal B chain may induce similar conformational changes.In order to investigate possible structural similarities between insulin, desoctapeptide-insulin, and des-Ala-des-Aspinsulin, antisera prepared against each of them were tested for cross-reactivity. These experiments have shown that desoctapeptide-and des-Ala-des-Asp-insulins differ immunologically and hence, structurally in spite of their similar circular dichroic spectra. In addition, we demonstrate an antigenic determinant which is expressed in des-Ala-des-Asp-insulin but not in native insulin.

[21 ‐ Arginine ‐ A] Insulin

“…This arrangement results in the formation of a salt bridge between these two residues and favorably disposes the -NHof the A'' asparagine and the -COof the BZ3 glycine for hydrogen bond formation (Blundell et al, 1972). Data obtained from degradative and synthetic studies (Carpenter, 1966;Brugman & Arquilla, 1973;Arquilla & Stanford, 1972;Cosmatos el al., 1975Cosmatos el al., , 1976Blundell et al, 1972) strongly suggest that the A' ' asparagine, through the abovementioned interactions, is critically involved in the maintenance of a conformation of insulin compatible with high biological activity. In the [Arg"-A] insulin, however, the A21 arginine still retains the A' ' -B" salt bridge-and A' ' -BZ3 hydrogen bond-forming capabilities.…”

Differences in Humoral Insulin-Antibody Response Among Inbred Lou/M Rats and Epitope Presentation Differences in ELISA and Radioimmune Titration

“…Apparently only 1 or possibly 2 of the 28 hyperimmunized Lou/M rats were able to produce a humoral immune response to such cryptic epitopes. We have presented evidence for the presence of such cryptic epitopes (29).…”

Cell-Mediated Immunity to Insulin and its Polypeptide Chains in Insulin-Treated Diabetics

“…Recent studies by A rquilla et al [1969], Brugman and Arquilla [1973], and others have indicated that the immunological specificity of the humoral response to insulin is directed to conformational sites on the intact molecule. Our results with the blastogénie test have shown that some patients' lymphocytes also respond to B chains, and, in rather fewer cases, to A chains.…”