3rd year biochemistry practicals - Alkaline phosphatase inhibition kinetics

Hi Students,

This week you have been working with the Alkaline Phosphatase again but this time we included an inhibitor in our reaction. This practical is about measuring the inhibitor constant (Ki) of the beta-glycerophosphate. As I already said this practical was very similar to the previous one. You had to incubate the enzyme with different concentrations of the substrate and different concentration of the beta-glycerophosphate (our inhibitor). Because some of the work necessary to prepare the lab report was already done last week, I will be refering to the 3rd year biochemistry practical - Alkaline phosphatase post, which you can find on this site as well.

Ok, lets begin mad scientists:)

OH REMEMBER AGAIN, I DO MAKE UP ALL THOSE NUMBERS. IT IS JUST TO GIVE YOU IDEA HOW TO DO YOUR CALCULATIONS:)

First we construct Table 1 which look like this:

We use the adsorbances to calculate the Vo value using the same formula as previously:

Vo = 1000 x 3 x (Adsorbance -blank)/(18600 x 5)

Remember to use the Extinction Coefficient value of 18600 microM/cm

1. We convert our Adsorbance - blank values to Vo and we put them in another Table 2:

2. In the next step we plot the Vo (microM/min/ml) values against the substrate concentration [mM] to get the Michaelis-Menten plots, like here:

3. In the next step using the values from the Table 2 we calculate the 1/Substrate Concentration and 1/Vo (as in the previous lab report), getting the Table 3:

4. We plot the 1/Vo versus the 1/[S] (where [S] - substrate concentration) to obtain the Lineweaver-Burk plots like these here:

To get the trendlines we go right-click on the data points and choose the option Add Trendline, where in Bookmark called Type you choose Linear. Then you change the Bookmark to Options and tick the Display Equation on Chart. In the option forecast you can change the length of the trendline.

We will use the equations provided to go further with our lab report but first we have to go back to the Velocity Equation for the Competetive Inhibition:

1/V = (1/ Vmax) + (Km/Vmax) x (1 + [I]/Ki) x 1/[S]

where:

V - reaction velocity

Vmax - maximum reaction velocity

Km - Michealis Menten constant

[I] - inhibitor concentration

Ki - inhibition constant

[S] - substrate concentration

Because we have ploted our 1/V against the 1/[S] it means that we can express this equation like that:

y = c + mx

where:

y = 1/V

c = 1/Vmax

m = (Km/Vmax) x (1 + [I]/Ki)

x =  1/[S]

5. From the properties of the linear plot we know that m is our slope. Using the equations provided (by Excel:) we construct the Table 4:

Using the Table 4 we construct another plot Slope (for each [I]) against the [I], and we get this:):

Again we go and add the Trendline (with the equation) which will help us to calculate the Ki. From the theory we know that x-intercept of the Dixon plot gives us the -Ki value. So using the equation:

y = 89.082x + 1094.3

 From the linear function we know that to find the x-intercept the y must equal 0, so we substitute the 0 for y:

0 = 89.082x + 1094.3

89.082x = -1094.3

x = -1094.3/89.082

x = -120.491

and we know that x-intercept = -Ki so:

-Ki = -120.491

Ki = 120.491 [mM]

6. In the next step we will try to get the Ki value in the different way. Using the equations from the Lineweaver-Burk Plots we calculate the Km (exaclty the same as previously but thhis time it is actually Kmapp because includes the inhibition effect) and we put it in the Table 5:

Then we plot the Kmapp vs the [I] and using the Excell we get the equation of the trendline again. The x-intercept is our Ki.

You should end up with two different Ki values. You compare and discuss them:)

This was easy huh?

CyA Next Time:)

Maciek GGS