Partition column chromatography of insulin: production and separation of transformation products.

As a result of a general study on the partition column chromatography of insulin, it was noted that a number of commercial samples of crystalline insulin gave elution curves which indicated the presence of at least two components in the eluted fractions (3, 4). The solvent systems used in these studies were similar to those that had been reported by Harfenist and Craig in their countercurrent distribution studies on insulin (5-8). The latter workers had reported the fractionation of bovine insulin into two components (A and B) which appeared to differ from one another in the number of amide groups (8). The present report describes improvements in the technique of partition column chromatography of insulin that resulted in the separation of up to four components from some samples of commercial crystalline insulin. Several of these components were found to be formed from the main component when it was treated with acid and are designated transformation products. The preparation and separation of these components are described. In recent years several other chromatographic fractionations of insulin have been reported. These reports include the work of Dickinson (9), who detected an acid transformation of insulin by the use of chromatography on calcium phosphate gel, of Boardman (10, ll), who used resin-coated diatomaceous earth, of Cole (1‘2, 13), who used a cation exchange resin and eluted with buffers containing urea, of O’Donnell and Thompson (14, 15), who used diethylaminoethyl-cellulose along with urea-containing buffers, and of Volini and Mitz (16), who also used diethylaminoethyl-cellulose. The chemical differences between the fractions revealed by these chromatographic procedures have not been reported. Probably some of the procedures are detecting the same type of heterogeneities revealed in partition column chromatography.