Formation of amyloid fibrils from kidney bean 7S globulin (Phaseolin) at pH 2.0.
The amyloid fibrils formed by heating 1.0% (w/v) kidney bean phaseolin (7S globulin) solution at pH 2.0 with an ionic strength of 20 mM at 85 degrees C were characterized using transmission electron microscopy (TEM), atomic force microscopy (AFM), binding of thioflavin T (Th T) and Congo Red dyes, and circular dichroism spectroscopy. The morphology of the formed fibrils was closely dependent upon heating time from 15 to 720 min. The diameters of the fibrils formed at various times were similar, but the mean contour length progressively increased with heating time. The Th T maximum fluorescence also progressively increased with heating time. The heating process caused remarkable changes in secondary, tertiary, and quaternary conformations of the phaseolin, but the extents of the changes were closely related to the heating time. With a short heating time (e.g., 15 min), the beta-strand content decreased from 38.7 to 22.9%, but further heating resulted in recovery of beta-strand structure. The tertiary and quaternary conformations gradually became flexible and unfolded upon heating. Gel electrophoresis analysis indicated that heating disrupted the polypeptides of phaseolin, leading to the formation of fragments with lower molecular mass (e.g., <10 kDa after 360 min). The results suggest that the amyloid fibril formation of phaseolin (7S globulin) involved the disruption of its polypeptides, as well as conformational changes at secondary, tertiary, and quaternary structural levels. This appears to be the first direct observation of amyloid fibrils from legume 7S storage globulin.
Tang
Chuan-He
CH
Department of Food Science and Technology, South China University of Technology, Guangzhou 510640, People's Republic of China. chtang@scut.edu.cn
Zhang
Ye-Hui
YH
Wen
Qi-Biao
QB
Huang
Qingrong
Q
D000682:Amyloid / Q000737:chemistry*; D005916:Globulins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D009994:Osmolar Concentration; D027805:Phaseolus / Q000737:chemistry*; D010940:Plant Proteins / Q000737:chemistry*; D011487:Protein Conformation; D055550:Protein Stability
D000682:Amyloid; D005916:Globulins; D010940:Plant Proteins