Day 3

 Day 3:
Test enzyme activity of mMDH based on the oxidation of
m-Malate + NAD+ <(2)------(mMDH catalyst)-----(1)> beta-NADH +  oxaloacetate + H+
m-Malate is a substrate
Be aware of that oxaloacetate is not stable so the direction (1) should be taken into account.
- Prepare phosphate buffer, and the subtrates following:
* sample 1: 0,2ml-10mM NAD+ 0,2ml-10mM L-Malate + 0,1 opened cells pig heart and 2,5ml buffer phosphate = total 3ml
* sample 2:  0,2ml-20mM NAD+ 0,2ml-20mM L-Malate + 0,1 opened cells pig heart and 2,5ml buffer phosphate = total 3ml
* sample 3:  0,2ml-30mM NAD+ 0,2ml-30mM L-Malate + 0,1 opened cells pig heart and 2,5ml buffer phosphate = total 3ml
* sample 4:  0,2ml-40mM NAD+ 0,2ml-40mM L-Malate + 0,1 opened cells pig heart and 2,5ml buffer phosphate = total 3ml.
Run the spectrometer at 340nm wavelength, running time 1 min.
Note: the cuvet must be up and down after the sample is placed in, the enzyme activity is detected only when the cells  are opened meaning that the protein in this case mMDH is extracted from the cells.
The results: The Absorbance from the spectrometer provides no information about the enzyme acitivity, the reasons the the protein was not extracted from the cell or the pH is so low that ''kill''the protein.
The temperature does not affect the detection of enzymes. After about 30 seconds, the reaction reaches equilibrium, the absorbance is not changed any more.
    The pig heart is stored at -20 degree C, then cut into small pieces that are going to be blended. Because the solid pieces are blended, the cells will be opened effectively.