Interfacial adsorption of insulin conformational changes and reversibility of adsorption.
The adsorption of human insulin to Teflon particles was studied with respect to conformational changes and the reversibility of adsorption was examined by total internal reflection fluorescence (TIRF). Adsorption isotherms for the adsorption of human insulin indicated high affinity adsorption, even at electrostatic repulsive conditions. The plateau value for adsorption was in accordance with a protein layer consisting primarily of insulin monomers. Conformational changes of the insulin upon adsorption, was investigated by circular dicroism (CD) and fluorescence spectroscopy. The results suggested unfolding of adsorbed insulin, as observed by a decrease in alpha-helix and increase in random coil conformation. The changes in protein structure was not only related to the adsorbed species being monomeric, since CD and fluorescence results were different for adsorbed insulin compared to a monomeric analog of human insulin. Furthermore, the thermal stability in the adsorbed state was changed compared to insulin in solution. On the basis of the TIRF studies with FITC-labelled insulin it was not possible to firmly conclude whether exchange between human insulin in the adsorbed state and in solution takes place, due to the limited time range investigated. However, the desorption mechanism appeared to be different with unlabelled insulin in the bulk solution compared to phosphate buffer.
Mollmann
Susanne H
SH
Department of Pharmaceutics and Analytical Chemistry, The Danish University of Pharmaceutical Sciences, Universitetsparken 2, DK-2100-Copenhagen, Denmark. snnh@novonordisk.com
Jorgensen
Lene
L
Bukrinsky
Jens T
JT
Elofsson
Ulla
U
Norde
Willem
W
Frokjaer
Sven
S
D000327:Adsorption; D002942:Circular Dichroism; D004355:Drug Stability; D006801:Humans; D007004:Hypoglycemic Agents / Q000737:chemistry; D007328:Insulin / Q000737:chemistry*; D011138:Polytetrafluoroethylene / Q000737:chemistry; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D012996:Solutions; D013050:Spectrometry, Fluorescence / Q000379:methods; D013696:Temperature
D007004:Hypoglycemic Agents; D007328:Insulin; D011994:Recombinant Proteins; D012996:Solutions; D011138:Polytetrafluoroethylene