[High performance liquid chromatography of fluorescent insulin derivatives]

The reversed-phase HPLC of porcine insulin modified by fluorescent labeling with dansyl chloride, fluorescein isothiocyanate, and an N-hydroxysuccinimide ester of 3-carboxyl derivative of Nile Red was studied. Mono-, di-, and tri-Dns-insulins (substituted at residues Gly1 of the A-chain and Phe1 and Lys29 of the B-chain), as well as isomeric 5'- and 6'-fluorescein thiocarbamoyl-Phe1 insulin derivatives were separated on the analytical and semipreparative scale. The results were interpreted in terms of conservation of the globular structure in the modified proteins and their surface-mediated interaction with the reversed-phase sorbent. Observed retention times correlated with the total hydrophobicity of the surface region containing the incorporated label (in the case of monosubstituted derivatives) or with the total hydrophobicity of chromatographic contact regions located between labels (in the case of di- and trisubstituted derivatives).