Modeling Amyloid Aggregation Kinetics: A Case Study with Sup35NM.

Understanding the aggregation mechanism of amyloid proteins, such as Sup35NM, is essential to understanding amyloid diseases. Significant recent work has focused on using the fluorescence of thioflavin T (ThT), which undergoes a red shift when bound to amyloid aggregates, to monitor amyloid fibril formation. In the present study, the progression of the total mass of aggregates during fibril formation is monitored for initial monomer concentrations in order to infer the relevant aggregation mechanisms. This workflow was implemented using the amyloid-forming fragment Sup35NM under different agitation conditions and for initial monomer concentrations spanning 2 orders of magnitude. The analysis suggests that primary nucleation, monomeric elongation, secondary nucleation, and fragmentation might all be relevant, but their relative importance could not be determined unambiguously, despite the large set of high-quality data. Discriminating between the fibril-generating processes is shown to require additional information, such as a fibril length distribution. Using Sup35NM as a case study, a framework for fitting the parameters of arbitrary amyloid aggregation kinetics is developed based on a population balance model (PBM), which resolves not only the total aggregate mass (monitored experimentally via ThT fluorescence) but the entire fibril length distribution over time. In addition to the rich new set of ThT fluorescence data, we have reanalyzed a previously published aggregate size distribution using this method. With the size distribution, it was determined that in the reanalyzed in vitro experiment, secondary nucleation generated significantly fewer new Sup35NM fibrils than fragmentation. The proposed strategy of applying the same PBM to a combination of kinetic data from fluorescence monitoring and experimental fibril length distributions will allow the inference of aggregation mechanisms with far greater confidence than fluorescence studies alone.

Evaluation of ionic equilibria in mixed-buffer isothermal titration calorimetry and continuously stirred tank reactors.

Determination of an equilibrium pH value in complex aqueous solution and deconvolution of this equilibrium to evaluate phenomena related to mixing, dilution, or progress of reaction is increasingly important in areas ranging from water quality to pharmaceutical formulations and manufacturing. Linearization of pH problems by simple algebraic substitution enables equilibria within complex buffered aqueous solutions to be modeled as an eigenvalue problem. This formulation approach makes rigorous determination of equilibrium pH values and reactor dynamics more accessible than with previous calculation methods, even when activity coefficients and non-ideality are considered. This work demonstrates how such calculations can enable detailed modeling of enthalpic changes in an isothermal titration calorimeter. In support of this work, the acid dissociation constants for three furancarboxylic acids (2-furancarboxylic acid, FA; 5-formyl-2-furancarboxylic acid, FFA; and 2,5-furandicarboxylic acid, FDCA), two of them novel, were determined and compared with multi-wavelength ultraviolet-visible spectrophotometry. The thermodynamic pKa values were determined to be 3.1 for FA, 2.2 for FFA, and 2.1 and 3.4 for the first and second ionization steps of FDCA, respectively.

A high-throughput pH-based colorimetric assay: application focus on alpha/beta hydrolases.

Research involving α/ß hydrolases, including α-amino acid ester hydrolase and cocaine esterase, has been limited by the lack of an online high throughput screening assay. The development of a high throughput screening assay capable of detecting α/ß hydrolase activity toward specific substrates and/or chemical reactions (e.g., hydrolysis in lieu of amidase activity and/or synthesis instead of thioesterase activity) is of interest in a broad set of scientific questions and applications. Here we present a general framework for pH-based colorimetric assays, as well as the mathematical considerations necessary to estimate de novo the experimental response required to assign a 'hit' or a 'miss,' in the absence of experimental standard curves. This combination is valuable for screening the hydrolysis and synthesis activity of α/ß hydrolases on a variety of substrates, and produces data comparable to the current standard technique involving High Performance Liquid Chromatography (HPLC). In contrast to HPLC, this assay enables screening experiments to be performed with greater efficiency.