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 Gly(1) of the A-chain and Phe(1) and Lys(29) Of the B-chain), as well as isomeric 5'- and 6'-fluoresceinthiocarbamoyl-Phe(1) 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).