Presence of Trimers in Glucagon Solution

Glucagon in solution at pH 10.2, at a concentration of 5 mg/ml, at which profiles of optical parameters against concentration show that it is substantially in its associated state, was covalently cross‐linked with dimethyl suberimidate. At a suberimidate concentration of 1 mg/ml the formation of high‐molecular‐weight aggregates, which is observed at higher reagent concentrations, is suppressed, and a largely homogeneous product is eluted from a gel‐filtration column in 6 M guanidine hydrochloride. This is also homogeneous as judged by polyacrylamide‐gel electrophoresis in the presence of sodium dodecylsulphate. Sedimentation equilibrium in 6 M guanidine hydrochloride led to a molecular weight of about 9600, showing that the unique cross‐linked form is the trimer; there is no evidence for dimers or hexamers, which have been inferred from sedimentation‐equilibrium distributions. It is suggested that the trimeric form of high α‐helix content may be equated with the rod‐like trimers making up the asymmetric unit in the crystal.