Soluble, prolonged-acting insulin derivatives. II. Degree of protraction and crystallizability of insulins substituted in positions A17, B8, B13, B27 and B30

It has previously been found that insulins, to which positive charge has been added by substitutions in position B30, thus raising the isolectric point towards pH 7, had a prolonged action when injected as slightly acidic solutions because such derivatives crystallize very readily upon neutralization. Positive charge has now been added by substituting the B13 and A17 glutainic acid residues with glutamines and B27 threonine with lysine or arginine. These substitutions were introduced by site-specific mutagenesis in a gene coding for a single-chain insulin precursor. By tryptic transpeptidation the single-chain precursors were transformed to the double-chain insulin structure, concomitantly with incorporation of residue B30. Thus insulins combining B13 glutamine, A17 glutamine and B27 lysine or arginine with B30 threonine, threonine amide or lysine amide were synthesized. The time course of blood glucose lowering effect and the absorption were studied after subcutaneous injection in rabbits and pigs. The prolonged action of B30-substituted insulins s marked ly enhanced by B27 lysine or arginine substitutions and by B13 glutamine. The B27 residue is located on the surface of the hexamer, so a basic residue in this position presumably promotes the packing of hexainers at neutral pH. The B13 residues cluster in the centre of the hexainer. When the electrostatic repulsive forces from six glutamic acid residues are abolished by substitution with glutamine, a stabilization of the hexamer can he envisaged. The biological potency of insulins was measured in the free fat cell assay and in the mouse blood glucose assay test. A potency factor could be fitted to each substitution, so that the potency of analogs with two or more substitutions can be estimated by multiplication of the corresponding potency factors. A charge-indifferent substitution, B8 glycine to serine, resulted in insulins that crystallize well but have low potencies. A late elution in gradient r.p.-h.p.l.c. indicates that hydrophobic amino acid residues have been exposed as a result of this B8 substitution. This most likely results from distortion of the α-helix cominenc ing at residue B7, permitted only by B8 glycine with dihedral angles (Φ, ψ) of a D-amino acid.