Memorándum de entendimiento

Amylinpeptide (25 μM) with and without inhibitors (at varying concentrations) was incubated for 48 hrs at 37 ᵒC prior to the experiment.

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A 7 mg/mL solution of CR was prepared in buffer (5 mM potassium phosphate, 150mM NaCl, pH7.4) and filtered through a 0.2 µm syringe filter immediately prior to use.

The UV-Vis Nanodrop 200c spectrophotometer was first zeroed between 400 and 700 nm at room temperature with a sample of 1 mL phosphate buffer in a disposable cuvette.

To measure the spectrum for CR, 5µL of the CR solution was added to 1 mL phosphate buffer in a disposable cuvette and scanned between 400 and 700 nm.

10 µL of protein sample was then added to disposable cuvette containing 5 µL of CR solution in 1 mL of phosphate buffer and incubated for 30 mins at room temperature. At this stage, a red precipitate becomes visible. The contents of the cuvette were then mixed by pipetting the solution up and down and then the spectrum was recorded, between 400 and 700 nm.

A maximal spectral difference at 540 nm is indicative of amyloid fibrils. This is calculated mathematically by subtracting the CR spectrum from the protein + CR spectrum.

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2.8 Stability Assay using High Performance Liquid Chromatography

High performance liquid chromatography (HPLC) is a more advanced and highly sensitive form of column chromatography where the solvent is forced through under high pressures. This permits the passage of smaller sized particles as well as giving a finer separation of the components of the mixture. Here, reverse-phase HPLC was used to determine the stability of the peptide inhibitors in plasma, and in the presence of various proteolytic enzymes. In this form of HPLC, the silica column is modified to make it non-polar by linking long hydrocarbon chains to its surface. Hydrophobic molecules are adsorbed onto this type of column in the presence of a polar solvent, and are eluted by employing increasing concentrations of a non-polar organic solvent. The HPLC equipment used for these experiments was the Dionex GP50 Gradient pump. The column used for these experiments was the C18 x 2.0mm colunm and the solvent consisted of a gradient produced from 0.01% trifluoro acetic acid in dH2O (Solvent A) versus 0.01% trifluoro acetic acid in acetonitrile (Solvent B). Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) is concerned with the dissociation of molecules by reason of their hydrophobicity.

This dissociation is influenced by the hydrophobic binding of the solute

molecule between the mobile phase and the immobilized hydrophobic ligands

connected to the stationary phase in other words, sorbent. Firstly, the solute

mixture is added to the sorbent in the presence of aqueous buffers, the presence

of organic solvent at the mobile phase elutes the solutes, this can be isocratic,

where there is a constant concentration of organic solvent or it can be by

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a period of time. The solutes are thus eluted in an increasing succession of

molecular hydrophobicity.

RP-HPLC is an especially important approach for peptide and protein analysis

because chromatographs can be altered without difficulty via changes in mobile

phase attributes. Also, RP-HPLC also possesses high resolution for both similar

and disimilar molecules attained through its variety of chromatographic

conditions. In addition, RP-HPLC produces high quality repetitive separations

as well as high recoveries (Aguilar and Hearn, 1996; Mant and Hodges, 1996).

RP-HPLC can however result in irreversible denaturing of protein samples thus

the chances of recovering biologically active materials is greatly diminished.

Human plasma samples were obtained, with ethical approval including informed consent (Oldham Ethics Committee), from Prof. David Mann (University of Manchester). The frozen plasma sample (stored at −80°C) was thawed in a water bath (25°C) for 5 min. To assess the stability of the peptide inhibitors in plasma, 5µl of peptide was added to 95 µl of thawed plasma in a microfuge tube and incubated for 0 hrs, 1hr, 3hrs, 24hrs, 48hrs and 72 hrs at 37°C. To assess the stability of peptides in the presence of proteolytic enzymes (table 2.8), 2 µl of enzyme was added to 98 µL of peptide. After incubation at 0 hrs, 1hr, 3hrs, 24hrs, 48hrs and 72 hrs, 100 μl of sample was injected into the HPLC injector

column and monitored at a flow rate of 1ml/min for a total run time of 40 mins at a linear gradient of 0-60% solution B, with the absorbance measured at a wavelength of 220nm.

67 | P a g e Enzyme Concentration Trypsin 1mg/ml Chymotrypsin 1mg/ml Cathepsin G 1mg/ml Elastase 1mg/ml Thrombin 1mg/ml Kallikrein 1mg/ml Plasmin 1mg/ml Factor X 1mg/ml

Table 2.8: List of proteolytic enzymes used.

2.9 Transmission Electron Microscopy

Solutions of amylin at 25 μM and amylin in the presence of inhibitors at varying concentrations were prepared and incubated in PBS for 48 hrs. After incubation, 5 μl of the mixture was pipetted onto carbon-coated formvar grids held using

forceps and left for 3 mins. The edges of the grids were touched with filter paper to draw off the liquid, then 5 μl of 2% aqueous phosphotungstic acid (adjust pH to 7.3 using 1N NaOH) was applied immediately (before the sample had dried) and left for 1 min. The excess liquid was removed as before, the grid was allowed to dry overnight (in the grid box) before observation. Five fields for each sample were randomly photographed at 5000x magnification, after first examining the grid for uniformity. The negatives were enlarged 3.0x to a final magnification of 15000x. Five photographs were examined for each sample.

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