FITC labeling of human insulin and transport of FITC-insulin conjugates through MDCK cell monolayer

Fluorescein isothiocyanate-labeled insulin (FITC-insulin) has been widely used for bioanalytical applications. Due to the high cost of commercial FITC-insulin and tedious labeling procedures described in the literature, there is still a need to develop a cost effective, reliable and quick labeling method for insulin. The purpose of the present work was to develop a quick and affordable method for FITC labeling of human insulin and to determine the effect of different conjugations of FITC to human insulin on its permeability through the MDCK cell monolayer. FITC labeling of insulin gives mono-, di- or tri-conjugates depending on the reaction time and the molar ratio of FITC:insulin. Mono-conjugate with unlabeled insulin, mixture of di- and tri-conjugate, and tri-conjugate with very little amount of di-conjugate were synthesized in less than 4 h. Degree of conjugation had an effect on the permeability of insulin through the MDCK cell monolayer. Mono-conjugate had higher permeability than the unlabeled insulin due to increase in partition coefficient. However, tri-conjugate showed lower permeability than the unlabeled insulin due to the increase in molecular weight.