Human islet amyloid polypeptide (hIAPP) is the principal constituent of islet amyloid, a characteristic pathologic feature of unknown etiology in non-insulin-dependent diabetes mellitus (NIDDM). To evaluate factors affecting hIAPP amyloidogenesis, we developed an assay that depends on altered fluorescence characteristics of thioflavine T bound to amyloid. Synthetic hIAPP,]7, verified foramino acid composition and purity, was dissolved in dimethylsulfoxide (1-3 mM). Amyloid formation was induced by dilution (1:20) into 10 mM Tris.HCl (pH 7.4), 100 mM NaCl in the presence or absence of 0.3% bovine serum albumin (BSA) and/or 0.1 % triton X100(TX). Fluorescence (X= 450 nm and X, = 482 nm) was measured at 30 minute intervals for 2 hours and hourly thereafter. Regression analysis was used to assess the rate of amyloid formation versus time and the data were analyzed by multivariate ANOVA using a Bonferroni t test to assess individual comparisons.. No time-dependent increase in the fluorescence signal was observed in the absence of TX. Inclusion of TX at concentrations above the critical micelle concentration resulted in a time-dependent increase in fluorescence of 9.8 arbitrary units x hr"1 (p < 0.05). BSA alone was without effect; however, it acted synergistically with TX, resulting in a rate of fluorescence increase of 28.6 arbitrary units x hr1 (p < 0.05). The specificity of the assay was verified by electron microscopy, revealing characteristic amyloid fibrils (9-12 nm width) that were strictly correlated with fluorescence and by absence of fibrils or fluorescence with non-amyloidogenic rat IAPP. TX/BSA may prevent non-specific hIAPP aggregation or binding to plasticware, allowing ordered fibril assembly. Amyloid formation was directly dependent on the concentration of IAPP (50-150 (iM), temperature (4-37 °C), maximal at pH > 7.4 and increased by both Na+ and K* ions (50-200 mM). In conclusion, we have: (i) developed a sensitive assay for quantifying the rate of hIAPP amyloid formation, (ii ) defined the conditions for achieving specific in vitro fibril formation, and (iii) applied this assay to define the impact of peptide and salt concentration, pH and temperature on amyloid formation. This assay will be valuable in studying the various factors that influence islet amyloidogenesis and will contribute to our knowledge of the pathogènes is of NIDDM.
|Original language||English (US)|
|Journal||Journal of Investigative Medicine|
|State||Published - Jan 1 1996|
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)