Characteristic, activity and conformational studies of [A6-Ser, A11-Ser]-insulin.

Met-[A6-Ser, A11-Ser]-human proinsulin (Mut-HPI) was prepared in the same way as described previously for Met-human proinsulin (Met-HPI). After trypsin and carboxypeptidase B cleavage and DEAE-Sephadex A25 separation, Met-[A6-Ser, A11-Ser]-human insulin (Mut-HI) and Met-human insulin (Met-HI) were obtained. Their amino-acid compositions are in a good agreement with those expected. Mut-HI keeps full immuno activity, but loses almost all of its receptor binding activity with Met-HI as the control. Some physico-chemical behaviors of Mut-HI have changed to a certain extent. CD studies of mutant insulin demonstrates that the conformation of Met-HI is very similar to that of HI, while that of Mut-HI has altered slightly. The most important observation is that the binding ability of Mut-HI to the receptor has decreased abruptly. These results suggest that though the intra-A chain disulfide bond is deleted, the other two inter-chain disulfide bonds are still correctly paired, and that the intra-A chain disulfide bond is essential for insulin displaying its biological activity.