Amyloid, Aggregation Oligomers, Fibrils

1. Binding of Thioflavin to Amyloid Fibrils
Mr.Halavath Ramesh
Department of Chemistry
Loyola College-Chennai
University of Madras

2.  Many proteins and peptides are able to self-assemble
in solution in vitro and in vivo to form Amyloid like fibrils.
 Misfolding and aggregation of proteins into Amyloid
fibrils.
 Protein fibrils are also called Amyloid aggregates,
composed of insoluble proteins.
 They arise from at least 18 in appropriately folded
version of proteins and polypeptides present naturally in
the body.
 These misfolded structures alter their proper
configuration such that they erroneously interact with one
another or other cell components forming insoluble fibrils.

3.  They have been associated with the pathology of more than 20 serious
human diseases in that abnormal accumulation of Amyloid fibrils in organs
may lead to amyloidosis, and may play a role in various neurodegenerative
dis orders.
 They are self-associating highly repetitive polymers consisting of arrays
of β-sheets that are held together via hydrogen bonding along the peptide
backbone.
 Protein fibrils conformation tests:
1. The Congo Red assay
2. Thioflavin T Spectroscopic assay
3. ANS binding Assay
4. BSB[(trans, trans)-1-Bromo-2,5-bis-(3-hydroxy carbonyl -4-hydroxy)Styryl benzene]

4. 1.Congo Red Assay
Congo red is an organic compound, the sodium salt of 3,3′-([1,1′-biphenyl]-
4,4′-diyl)bis(4-aminonaphthalene-1-sulfonic acid). It is an azo dye. Congo
red is water-soluble, yielding a red colloidal solution; its solubility is greater
in organic solvents. Staining with congo red (CR) is a qualitative method
used for the identification of amyloids in vitro and in tissue sections.

5.  Congo red is used for the visual detection of Amyloid in muscle and nerve fresh
section in patients who have amyloidosis.
 The specificity of this staining results from Congo red's affinity for binding to
fibril proteins enriched in β-sheet conformation.

6. 2.Thioflavin T Spectroscopic assay
ThioflavinT (ThT) has been widely used to investigate Amyloid formation
since 1989.ThT still continues to be a very valuble tool for studying kinetic
aspects of fibrillation and associated inhibition mechanisms. ThT also known
a basic yellow colour is a benzothiozole dye. Thioflavin T consists of a
dimethylated benzothiozole ring connected to a dimethyl amino benzyl ring
through at single C-C bond.

7. Thioflavin T is a benzothiozole dye that exhibits enhanced
fluorescence upon binding to Amyloid fibrils and is commonly
used to diagnose Amyloid fibrils both ex vivo and in vitro.
Formation of Amyloid fibrils underlies a wide range of human
disorders including Alzheimer’s and prion diseases. The
amyloid fibrils can be readily detected thanks to Thioflavin T ,
a small molecule that gives strong fluorescence upon binding to
amyloids.

20.  ThT study is very confusing as I feel. There we need to see the emission
of free and bound.
 They both have different excitation and emission.
 Bound one gives emission around 480nm if you excite at 440nm. The shift
occurs due to binding.
 But here another issue will be the slit width. If it is lower , you will find a
structural emission but in higher slit(5/10) you will find a clear single
emission peak.
 ThT fluorescence to monitor fibrillation.
 Amyloid fibers are likely bound to the surface of the cuvette from a
previous experiments. SDS is often not sufficient to remove them. Try
washing in aqua regia. Many of factors can be the real culprit.
The starting concentration of ThT in your reaction should be 20-25 uM.

21. Intense effort to detect , diagnose, and analyze the kinetic and
structural properties of amyloid fibrils have generated a
powerful toolkit of Amyloid specific molecular probes. Since its
first description in 1959 , the fluorescent dye Thioflavin T
(ThT) has become among the most widely used” gold
standards “ for selectively staining and identifying Amyloid
fibrils both in vivo and in vitro. The large enhancement of its
fluorescence emission upon binding to fibrils makes ThT a
particularly powerful and convenient tool.

22.  ThT study is very confusing assay. There we need to see the
emission of free and bound. They both have different excitation
and emission.
 Bound one give emission around 480nm if you excite at
440nm. The shift occurs due to binding.
 But here another issue will be the slit width. If it is lower, you
will find a structural emission but in higher slit(5/10) you will
find a clear single emission peak.
 ThT fluorescence to monitor fibrillation.