Spectrophotometric Activity Measurements

Work of the Gygli group...

Investigation 1: An experimental workflow to provide detailed information of the molecular mechanisms of enzymes is described. This workflow will help in the application of enzymes in technical processes by providing crucial parameters needed to plan, model and implement biocatalytic processes more efficiently. These parameters are homogeneity of the enzyme sample (HES), kinetic and thermodynamic parameters of enzyme kinetics and binding of reactants to enzymes. The techniques used to measure ...

An experimental workflow to provide detailed information of the molecular mechanisms of enzymes is described. This workflow will help in the application of enzymes in technical processes by providing crucial parameters needed to plan, model and implement biocatalytic processes more efficiently. These parameters are homogeneity of the enzyme sample (HES), kinetic and thermodynamic parameters of enzyme kinetics and binding of reactants to enzymes. The techniques used to measure these properties are ...

Specific activity of Gre2p measured by following the change in absorbance of NADPH at 340 nm for the conversion of nitrononane-2,8-dione (NDK).

Kinetic parameters (Km, kcat) of Gre2p measured by following the change in absorbance of NADPH at 340 nm for the conversion of nitrononane-2,8-dione (NDK) or hexane-2,5-dione. Initial rates at different substrate concentrations are measured.

Specific activity of Gre2p measured by following the change in absorbance of NADPH at 340 nm for the conversion of nitrononane-2,8-dione (NDK) using different enzyme concentrations.

Activity (initial rates) of Gre2p were measured followng NADPH absorbance at different substrate [NDK] concentrations. Initial rates are calculated (mM/s) and plotted as a function of [NDK] to allow fitting with a enzyme kinetic model, e.g. Michaelis Menten. These are the raw data obtained directly from the Synergy UV-Vis Spectrophotometer.

Activity (initial rates) of Gre2p were measured followng NADPH absorbance at different substrate [hexane-2,5-dione] concentrations. Initial rates are calculated (mM/s) and plotted as a function of [hexane-2,5-dione] to allow fitting with a enzyme kinetic model, e.g. Michaelis Menten. This is a file that can be opened with the Origin Software for analysis and contains data copied in manually from the xlsx.

Activity (initial rates) of Gre2p were measured followng NADPH absorbance at different substrate [hexane-2,5-dione] concentrations. Initial rates are calculated (mM/s) and plotted as a function of [hexane-2,5-dione] to allow fitting with a enzyme kinetic model, e.g. Michaelis Menten. These are the raw data obtained directly from the Synergy UV-Vis Spectrophotometer.

Specific activity of Gre2p was measured after different treatments (none, on ice, at room temperature, in the ITC, stirred, shaken). These data are used to perform a Selwyn Test to determine if enzyme inactivation or slow-onset inhibition occurs. This is a file that can be opened with the Origin Software for analysis and contains data copied in manually from the xlsx.

Specific activity of Gre2p was measured after different treatments (none, on ice, at room temperature, in the ITC, stirred, shaken). These data are used to perform a Selwyn Test to determine if enzyme inactivation or slow-onset inhibition occurs. These are the raw data obtained directly from the Synergy UV-Vis Spectrophotometer.

Specific activity of Gre2p was measured after incubation with different Tween-20 concentrations (none, 0.01, 0.1, 1.0%). These data are also used to perform a Selwyn Test to determine if enzyme inactivation or slow-onset inhibition occurs. This is a file that can be opened with the Origin Software for analysis and contains data copied in manually from the xlsx.

Specific activity of Gre2p was measured after incubation with different Tween-20 concentrations (none, 0.01, 0.1, 1.0%). These data are also used to perform a Selwyn Test to determine if enzyme inactivation or slow-onset inhibition occurs. These are the raw data obtained directly from the Synergy UV-Vis Spectrophotometer.

Activity (initial rates) of Gre2p were measured followng NADPH absorbance at different substrate [NDK] concentrations. Initial rates are calculated (mM/s) and plotted as a function of [NDK] to allow fitting with a enzyme kinetic model, e.g. Michaelis Menten. This is a file that can be opened with the Origin Software for analysis and contains data copied in manually from the xlsx.

Gre2p activity monitored by NADPH absorbance using different enzyme concentrations. Activity is measured after different storage conditions (treatments) and in presence of different amounts of tween.

A python workflow is used to analyse the data and create a plot where the outcomes of the Selwyn test are plotted. It requires the following directory structure:

./Sewlyn_test_forS19.py ./tween/M1.csv ./tween/M2.csv ./tween/M3.csv ./tween/M4.csv ./treatments/M1.csv ./treatments/M2.csv ./treatments/M3.csv ...

Gre2p activity monitored by NADPH absorbance using different enzyme concentrations. Activity is measured after different storage conditions (treatments) and in presence of different amounts of tween. Also analysis of DLS data of homogeneity of Gre2p samples measured before and after these treatments.

A python workflow is used to analyse the data and create a plot of the data. It requires the following directory structure:

./Script_for_S13andS18.py ./Script_for_S22.py ./Script_for_S15.py

and as ...

SOP describing how to perform specific activity measurements with Gre2p in multiwell plates (96w) using an UV-Vis Plate reader (Synergy H1).

SOP describing how to perform activity measurements (inital rates) with Gre2p in multiwell plates (96w) using an UV-Vis Plate reader (Synergy H1).