INVESTIGATION OF THE KINETICS OF INSULIN AMYLOID FIBRILS FORMATION
A.I. Sulatskaya,1,* E.A. Volova,1 Ya.Yu. Komissarchik,1 E.S. Snigirevskaya,1
A.A. Maskevich,2 E.A. Drobchenko,1 I.M. Kuznetsova,1,3 Ê.K. Turoverov 1
1 Institute of Cytology RAS, St. Petersburg, 2 Yanka Kupala Grodno State University, Belarus,
and 3 St. Petersburg State Polytechnical University;
1 e-mail: ansul@mail.ru
Investigation of the structure of ordered protein aggregates - amyloid fibrils, the influence of the native structure of the protein and the environment on the process of
fibrillation is currently the subject of intensive re-search. The present work is devoted to the study of the kinetics of insulin amyloid fibrils formation at low pH values (which
are produced at many stages of the isolation and purification of the protein) using a fluorescent probe thioflavin T (ThT). It has been shown that the increase of fluorescence
intensity of ThT during the formation of amyloid fibrils is described by a sigmoidal curve, in which 3 areas can be distinguished: the lag phase, the growth and the plateau, which
characterize the various stages of fibril formation. Despite the variation in the length of the lag phase at the same experimental conditions (pH and temperature), we have found
its reduction with stirring the solution and seeding. Data obtained using electron microscopy showed that the formed fibrils are long, linear filament having a diameter of ~20 nm.
With increasing incubation time fibril diameter did not change while their length increases to 2-3 |j,m, which was accompanied by a significant increase in the number of aggregates
of fibrils. All the experimental data shows that, regardless of the kinetics of the formation of amyloid fibrils, their properties after the fibrillation process are identical. The results
of this work together with the previously studies of insulin amyloid fibrils might be important for clarification the mechanism of their formation, as well as for the treatment of
amyloidosis associated with the aggregation of insulin.
Key words: amyloid fibrils, insulin, thioflavin T, electron microscopy, fluorescence intensity, absorption spectra
| Back
| Contents
| Main |
|
