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

Abstract

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.

Alcalase Microarray Base on Metal Ion Modified Hollow Mesoporous Silica Spheres as a Sustainable and Efficient Catalysis Platform for Proteolysis

“…Besides, to further confirmed the immobilization of alcalase, alcalase@HMSS-NH 2 -Fe 3+ was fluorescently labeled by FITC, and an extensive dialysis was performed until no release of free FITC into solution. FITC is an amine reactive fluorescent probe which labels protein by forming a covalent bond between its isothiocyanate group and the primary and secondary amine groups of lysine (Shah et al, 2019). As shown in Figure S2, the distinct green fluorescence in CLSM image of alcalase@HMSS-NH 2 -Fe 3+ provided a persuasive confirmation for the successful immobilization of alcalase.…”

Fatty acids may influence insulin dynamics through modulation of albumin‐Zn2+ interactions

“…[68] Site-specific incorporation of amine-reactive fluorescent molecules into insulin has been mostly achieved through attachment to primary amines A1 (Gly), B1 (Phe) and the ε-amino group of B29 (Lys). The order of reactivity of these amines was found to be B1 > A1 > B29; it has been shown that mono-conjugates of insulin labelled at B1 can be obtained by carefully controlling the labelling conditions, [69] and that B1 conjugates have the same biological activity as native insulin. [70] Thus, examination of Zn 2+ -induced insulin com-plexation, and subsequently, HSA-mediated decomplexation using HSA preparations containing different concentrations of NEFAs, should be possible using fluorescent mono-conjugates of insulin if a method can be developed where the fluorescent properties change upon complexation/decomplexation.…”

Shortcut Approaches to Substance Delivery into the Brain Based on Intranasal Administration Using Nanodelivery Strategies for Insulin

“…In the case of human insulin (Figure 4), the amino groups are 1 Gly in the A-chain and 1 Phe, 22 Arg, and 29 Lys in the B-chain. The reactivity of 1 Phe in the B-chain is the highest among the amino groups of insulin [45]. The hydroxyl groups are 9 Ser, 14 Tyr, and 19 Tyr in the A-chain and 9 Ser and 27 Thr in the B-chain.…”

“…Furthermore, the membrane permeability made a difference depending on the degree of FITC conjugate to insulin. The rank order of the permeability through the Madin-Darby canine kidney cell (MDCK) monolayer was mono-conjugate > unlabeled insulin > tri-conjugate [45]. Tri-conjugate is more hydrophobic than insulin.…”