Simple ITC method for activity and inhibition studies on human salivary α-amylase

Abstract

Isothermal titration calorimetry (ITC) has an increasing significance in enzyme kinetic studies owing to its general applicability and sensitivity. In the present work, we aimed at developing a simple ITC-based screening procedure for the measurement of human salivary a-amylase (HSA) activity. Reaction of two substrates was studied with three independent methods (ITC, HPLC and spectrophotometry). ITC experiments were made using free and chromophore-containing maltooligomers of different length as substrates. Detailed studies revealed that maltoheptaose or longer oligomers could model properly starch and the presence of aromatic chromophore group did not affect the K M values considerably. It is the first time, when ITC was used to investigate of HSA-catalysed hydrolysis of different substrates (2-chloro-4-nitrophenyl-4-O-a-Dgalactopyranosyl-maltoside, maltoheptaose and starch) in the presence of acarbose inhibitor. All measured IC 50 values are in micromolar range (0.9, 18.6 and 29.0 mM, respectively) and increased in parallel with the degree of polymerisation of substrates.

Enzyme Kinetics by Isothermal Titration Calorimetry: Allostery, Inhibition, and Dynamics

“…Alternatively, single-injection assays can be performed with enzyme in the syringe. This variation is preferable for substrates that are poorly soluble, or those that form suspensions rather than solutions, since they can remain at working (diluted) concentration in the sample cell with constant stirring throughout the experiment (Lonhienne et al, 2001;Ali et al, 2013a,b;Commin et al, 2013;Pedroso et al, 2014;Lehoczki et al, 2016;Kaeswurm et al, 2019). Similarly, if very high substrate concentrations (100 s of mM) are needed (e.g., for enzymes with very large K m values), it can be unfeasible to inject sufficient amounts of substrate without generating large injection heat artifacts, related to the large dilutions.…”

Impact of B-Ring Substitution and Acylation with Hydroxy Cinnamic Acids on the Inhibition of Porcine α-Amylase by Anthocyanin-3-Glycosides

“…Therefore, according to our previous study [16], the inhibitory effect for 100 µM was additionally investigated by ITC. Substrate conversion was performed in the cell by a single enzyme addition in the presence or absence (control) of the inhibitor, and the thermal power to maintain a constant temperature (µcal/s), which is proportional to the reaction heat, was recorded over time [18,19,25]. The amplitude of the curve immediately after enzyme injection was reduced; the time span to return to the pre-injection base line, indicating the end of the reaction, was extended in the presence of the anthocyanin-3-glucosides ( Figure 4A) [26]; and the thermogram was converted into a Michaelis Menten diagram ( Figure 4B).…”

“…Foods 2020, 9, x; doi: FOR PEER REVIEW www.mdpi.com/journal/foods which is proportional to the reaction heat, was recorded over time [18,19,25]. The amplitude of the curve immediately after enzyme injection was reduced; the time span to return to the pre-injection base line, indicating the end of the reaction, was extended in the presence of the anthocyanin-3glucosides ( Figure 4A) [26]; and the thermogram was converted into a Michaelis Menten diagram ( Figure 4B).…”

“…As anthocyanins and, in particular, acylated anthocyanins exhibit a strong self-absorption at the corresponding detection wavelength (405 nm), isothermal titration calorimetry (ITC) was used additionally for higher anthocyanin concentrations and the predominantly acylated structures obtained from black carrot. In contrast to the frequently used multiple injection experiment, which provides binding constants between the polyphenols and proteins [17], the simple and quick single injection experimental setup (enzyme addition to the substrate with or without the presence of an inhibitor and followed by reaction tracking) was used [18,19]. This screening approach provides comparable information to UV/Vis detection of the inhibitory effect of polyphenols.…”

Application of ITC-Based Characterization of Thermodynamic and Kinetic Association of Ligands With Proteins in Drug Design

“…It is already accepted that ITC measurement can be used to obtain binding constants ( k m , k cat , and V max ) of enzyme–substrate reactions and inhibitory constant ( K i ) of the enzyme–inhibitor binding, and the employment of this in studies of different enzymes has gained a lot of interest ( Todd and Gomez, 2001 ; Lehoczki et al, 2016 ). However, a putative use of ITC to measure the binding kinetics of ligands to its target remains unaccomplished until Di Trani et al developed a new method, called a kinetic ITC technique, that can measure binding kinetics ( k on and k off ) and the inhibitory constant ( K i ) of inhibitors simultaneously ( Di Trani et al, 2018 ), expanding the application of ITC in characterization of the protein-ligand binding process.…”