Team:GreatBay United/Model
Model
HCV activation test
In our experiment, we included three systems:detection system,cascade system and agglutination system, in which HCV protease is the key protease connecting the cascade system and the aggregation system. We successfully expressed this enzyme. In order to test the activity of this protease, we synthesized a polypeptide fluorescent substrate for this enzyme recognition cleavage check point. This substrate is: (amino acid sequence). At both ends of it, we added the modifying group DABCYL/Glu (EDANS). When the substrate peptide chain is cleaved, the two groups separate and fluoresce. This fluorescence detection condition is ʎex/em 340 and 490nm. In this experiment, fluorescence modified peptide substrate was successfully used to test the enzyme activity of HCV, and Michaelis-Menten equation was calculated by mathematical modeling analysis.
The cascading system includes a very important enzyme: HCV NS3 protease (HCV). HCV serves as a protease that cuts the linker between GFP and AntiGFP, so its activity relates to whether the cascading reaction can happen fastly. Therefore, the test of its activity is necessary. One of the most important indicator of activity of an enzyme is its Km, so in the HCV activation test we figured out the value of Km of HCV, and therefore proves that whether HCV is a good protease for the cascading system.
Fluorescent oligopeptide: A
Fluorescent protein after being activated: B1+B2 (only B1 has fluorescent structure）
Reaction rate: v=-dA/dt=dB1/dt
A———>B1+B2 (catalysed by HCV)
According to Michaelis-Menten equation, the relationship between concentration of substrate and reaction rate is below.
Firstly, we kept the volume of enzyme solution constantly at 5 μL, and changed the concentration of substrate from 0 μM to 150 μM. The results are shown in the graph below.
Secondly, we kept the concentration of the substrate constantly at 5 μM, and changed the volume of the enzyme from 0 μM to 140 μL. The results are drawn in the graph below.
In both graphs the curves intersect, which is caused by the limited accuracy of the microplate reader. As a result, we only take the first 4 points to avoid the error. Also, if we use the Michaelis-Menten equation, and try to estimate Vmax, then the total concentration of the enzyme is not really necessary for drawing the graph. Therefore, we finally kept the volume of the enzyme solution constant, and drew the graph which showed the relationship between the concentration of substrate and fluorescent intensity, then deduced the graph about the relationship between concentration of substrate and reaction rate.
To estimate the value of k2 and Km in the equation, we used microplate reader to generate the following graph (graph 1).
Graph 1. The relationship between the concentration of substrate and fluorescent intensity.
(The temperature was 37 degrees Celsius)
We assume that the rate of change of fluorescent intensity (Rfi) is directly proportional to the rate of the reaction, and the proportional coefficient is m.
Therefore, v=Rfi*k, and v/k equals to the slope of graph 1.
Graph 2. The relationship between the concentration of substrate and reaction rate.
As shown in graph 2, the reaction rate remained constant after the concentration of substrate reaches 25μM. As a result, the enzyme was saturated when the concentration of substrate was 25μM and higher. It could be concluded that the maximum speed (Vmax) is 0.7/k (M*s^(-1)).
According to the conditions above, we wrote an equation to calculate Km.
As a result, the final graph (graph 3) using the Michaelis-Menten equation is below, with comparing to the graph drawned using experimental data.
Graph 3. The relationship between the concentration of substrate and reaction rate (the orange line is drawn using the Michaelis-Menten equation.
In conclusion, the Km is determined to be 8*10(-6), which is not very low, and if the concentration of the substrate is proper, the reaction will process in a relatively high rate.