Preparation of a crystalline tris-(N-methylpyridinium)-derivative of bovine insulin.

The reaction between 2-chloro-N-methylpyridinium iodide and bovine insulin at pH 8.5 afforded a trisubstituted derivative as main product. Following purification on DEAE-Sephadex A-25, this derivative crystallized from a citrate buffer in the form of large rhombohedra very similar to native insulin. This observation was confirmed by zero-layer X-ray precession photography. Amino acid analysis indicated substitution at the B1-terminus and on two of the four tyrosine residues. In the mouse convulsion assay the derivative showed no biological activity.