Difference between revisions of "Team:BIT-China/roGFP2-Orp1MichaelisEquationModel"

According to Hypothesis 1, we consider the reaction process of intracellular hydrogen peroxide with roGFP2 as an enzymatic reaction process, which is consistent with the roGFP2-Orp1 Michaelis equation Model.

$$V=\frac{v_{max}[H_{2}O_{2}]}{K_{m}+[H_{2}O_{2}]}$$

Here we define a new parameter OxD_roGFP2 to reflect the degree of oxidation of roGFP2. Meanwhile, facilitate subsequent calculations.

$$OxD_{roGFP2}=\frac{[roGFP2_{ox}]}{[roGFP2_{ox}]+[roGFP2_{red}]}$$

According to Hypothesis 3, we can correlate the redox state of roGFP2 with the fluorescence value measured by the macroscopic observables easily. To establish this relationship, several molecular quantities followed need to be defined. i405_red, i405_ox, i488_red, and i488_ox are the fluorescence intensities contributed by a single roGFP2 molecule at the indicated wavelength and redox state. N_total is the total number of roGFP2 molecules. N_red is the number of reduced roGFP2 molecules. N_ox is the number of oxidized roGFP2 molecules. Based on the assumption that the overall fluorescence intensity is equal to the sum of the fluorescence intensities from the individual roGFP2 molecules, the relationships were given in Table 1.

Table 1. Relationships Between Fluorescence Intensities and Probe Molecules

By substituting the resulting equations (right column of Table 3) into OxD_roGFP2=N_ox/N_total, the relationship between OxD_roGFP2 and the macroscopic observables can be deduced.^[1]

$$oxD_{roGFP2}=\frac{I405\cdot I488_{red}-I405_{red}\cdot I488}{I405\cdot I488_{red}-I405\cdot I488_{ox}+I405_{ox}\cdot I488-I405_{red}\cdot I488}$$

After the above relationship is completed, since the absolute protein concentration determination is difficult, we define the relative reaction rate as

$$V^{'}=\frac{V}{[roGFP2_{ox}]+[roGFP2_{red}]}$$

$$OxD_{roGFP2}=\frac{[roGFP2_{ox}]}{[roGFP2_{ox}]+[roGFP2_{red}]}$$

$$V^{'}=\frac{OxD^{'}-OxD}{t^{'}-t}$$

At the same time, according to Hypothesis 2, roGFP2_total is constant, but the protein concentration could not be measured by us. So we thought that under the same promoter conditions, the protein concentration was basically the same and according to the data provided in the previous literature, it is known that in yeast cells, the Eno2 protein concentration is around 0.5 mM.^[2]

So we could get:

$$[roGFP2_{total}]=0.5\ mM$$

In order to obtain the two constants in the Michealis equation, we measured the reaction rate of the reaction in yeast cells by applying different concentrations of H₂O₂ and using the simulation results of the H₂O₂ Decomposition Model in combination with the function of roGFP2. The results were as follows:

Calculate two constants by double reciprocal mapping.

$$v_{max}=0.015207$$

$$K_{m}=0.451642$$

The relationship between the change in fluorescence intensity of roGFP2 and the concentration of intracellular H₂O₂ is set up successfully.