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Journal of Biochemistry Advance Access published online on February 9, 2009
Journal of Biochemistry, doi:10.1093/jb/mvp025
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© The authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Characterization of type I collagen fibril formation using thioflavin T fluorescent dye

Koichi Morimoto*, Kazuya Kawabata, Saori Kunii, Kaori Hamano, Takuya Saito and Ben'ichiro Tonomura

Department of Biotechnological Science, Kinki University, Kinokawa, Wakayama 649-6493, Japan

*Address correspondence to: Koichi Morimoto, Department of Biotechnological Science, Kinki University, 930 Nishimitani, Kinokawa, Wakayama 649-6493, Japan, Tel. +81-736-77-3888; Fax. +81-736-77-4754; E-mail: morimoto{at}waka.kindai.ac.jp

Received January 27, 2009; Accepted January 28, 2009


  Abstract

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, {Delta}Fmax, were calculated at various pH. The values of Kd and {Delta}Fmax 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.

Key Words: collagen, fibril formation, thioflavin T, fluorescence


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