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 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 microM 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, K(d), and a maximal fluorescence increase, DeltaF(max), were calculated at various pHs. The values of K(d) and DeltaF(max) were dependent on pH (K(d) was 9.4 microM 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.
Morimoto
Koichi
K
Department of Biotechnological Science, Kinki University, Kinokawa, Wakayama, Japan. morimoto@waka.kindai.ac.jp
Kawabata
Kazuya
K
Kunii
Saori
S
Hamano
Kaori
K
Saito
Takuya
T
Tonomura
Ben'ichiro
B
D000818:Animals; D052160:Benzothiazoles; D002498:Centrifugation; D002942:Circular Dichroism; D003094:Collagen / Q000378:metabolism* / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D024022:Fibrillar Collagens / Q000378:metabolism* / Q000648:ultrastructure; D005456:Fluorescent Dyes / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D055550:Protein Stability; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013844:Thiazoles / Q000378:metabolism*; D014413:Tuna
D052160:Benzothiazoles; D024022:Fibrillar Collagens; D005456:Fluorescent Dyes; D013844:Thiazoles; C059821:atelocollagen; C009462:thioflavin T; D003094:Collagen