Binding of phenol to R₆ insulin hexamers

Small amounts of phenolic compounds are being used as preservatives in pharmaceutical insulin preparations. It has been shown previously that these compounds bind to specific sites on the insulin hexamer and act as allosteric effectors, inducing a transformation of the T₆ hexamer to the R₆ hexamer, via a T₃R₃ intermediate. In this article, the crystal structures of eight different insulin derivatives, all in the phenol-containing R₆ form, are analyzed with respect to their phenol-binding sites. While six phenol molecules are normally bound per insulin hexamer, one of the engineered insulins appears to contain only three phenols but yet exists in an R₆ conformation. This observation provides additional evidence for an inherent nonequivalence of the two trimers in the insulin hexamer. The unusual observation of a seventh phenol molecule bound to the hexamer of crystalline A21Gly-B31,B32Arg₂ insulin (HOE 901), a long-acting derivative currently undergoing phase III clinical trials, provides a partial explanation for its protracted activity.