Characterization of Type I Collagen Fibril Formation Using Thioflavin T Fluorescent Dye

Collagen is composed of fibrils that are formed by self-assembly of smaller units, monomers which are triple-helical polypeptide. However, the mechanism of fibril formation at the level of individual molecules has remained to be clarified. We found that the fluorescence of thioflavin T, which has be...

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Bibliographic Details
Published in:Journal of biochemistry (Tokyo) 2009-05, Vol.145 (5), p.677-684
Main Authors: Morimoto, Koichi, Kawabata, Kazuya, Kunii, Saori, Hamano, Kaori, Saito, Takuya, Tonomura, Ben'ichiro
Format: Article
Language:English
Subjects:
Animals
Centrifugation
Circular Dichroism
collagen
Collagen - metabolism
Collagen - ultrastructure
Electrophoresis, Polyacrylamide Gel
fibril formation
Fibrillar Collagens - metabolism
Fibrillar Collagens - ultrastructure
fluorescence
Fluorescent Dyes - metabolism
Hydrogen-Ion Concentration
Protein Stability
Spectrometry, Fluorescence
Temperature
Thiazoles - metabolism
thioflavin T
Tuna
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Summary:Collagen is composed of fibrils that are formed by self-assembly of smaller units, monomers which are triple-helical polypeptide. However, the mechanism of fibril formation at the level of individual molecules has remained to be clarified. We found that the fluorescence of thioflavin T, which has been widely used as a specific dye for amyloid fibrils, also increased by binding with fibrils of atelocollagen prepared from yellowfin tuna skin. There was a linear correlation between the fluorescence increase and the amount of atelocollagen within a collagen concentration range of 0-0.15 mg/ml at pH 6.5 with 50 μM thioflavin T. In contrast, neither actinidain-processed collagen that keeps monomeric nature nor heat-denatured collagen could cause the fluorescence increase of thioflavin T at all. The relationship between the fluorescence increase and thioflavin T concentration was fit to a theoretical binary binding curve. An apparent dissociation constant, Kd, and a maximal fluorescence increase, ΔFmax, were calculated at various pHs. The values of Kd and ΔFmax were dependent on pH (Kd was 9.4 μM at pH 6.5). The present finding demonstrates that thioflavin T specifically binds to collagen fibrils and may be used as a sensitive tool for the study of collagen structure.
ISSN:0021-924X
1756-2651
DOI:10.1093/jb/mvp025