Tyrosine autofluorescence as a measure of bovine insulin fibrillation.
The traditional approach to investigating the partial unfolding and fibrillation of insulin, and proteins at large, has involved use of the dyes 1-anilinonaphthalene-8-sulphonic acid (ANS) and Thioflavin T (ThT), respectively. We compare the kinetic profiles of ThT, ANS, light scattering, and intrinsic Tyr fluorescence during insulin fibrillation. The data reveal that the sequence of structural changes (dimers --> monomers --> partially unfolded monomers --> oligomeric aggregates --> fibrils) accompanying insulin fibrillation can be detected directly using intrinsic Tyr fluorescence. The results indicate that at least two distinguishable structural intermediates precede fibril development. There is no evidence of tyrosinate or dityrosine during insulin aggregation. Obtaining such critical information from the protein itself is complementary to existing aggregation probes and affords the advantage of directly examining structural changes that occur at the molecular level, providing concrete details of the early events preceding fibrillation.
Bekard
Innocent B
IB
Department of Chemical and Biomolecular Engineering, The University of Melbourne, Melbourne, Victoria, Australia.
Dunstan
Dave E
DE
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D000818:Animals; D052160:Benzothiazoles; D001703:Biophysics / Q000379:methods*; D002417:Cattle; D007328:Insulin / Q000737:chemistry*; D007700:Kinetics; D008027:Light; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013696:Temperature; D013844:Thiazoles / Q000737:chemistry; D014443:Tyrosine / Q000031:analogs & derivatives / Q000737:chemistry
D000817:Anilino Naphthalenesulfonates; D052160:Benzothiazoles; D007328:Insulin; D013844:Thiazoles; C009462:thioflavin T; D014443:Tyrosine; C027132:1-anilino-8-naphthalenesulfonate; C007543:dityrosine