3rd year practicals - Measurment of Lactase Dehydrogenase Activity

Hello Student,

today we will work with the Lactase Dehydrogenase (LDH). In our experiment we had been assaying activity of the Lactase Dehydrogenase in 7 different samples: homogenate, Supernatant and Pellet 1, Supernatant and Pellet 2 and Supernatant and Pellet 3.
To measure the LDH activity we were looking at its substrate NADH (see the picture below). Maximum adsorbance for NADH is 340nm. Beacause NADH is used in this reaction by the LDH we expect that adsorbance will decrease with time of experiment.

To prepare your lab report you need to determine the LDH activity in each sample provided and discuss on the results obtained.

To determine the LDH activity you will need your "fancy" adsorbance plots that you have obtained during the practical (see the picture below).

From the adsorbance plot you have to read out the change of the adsorbance that happened over one minute. To perform that we ... (see the picture below).

In the first step we tick out six vertical boxes (each of them is "worth" 10s). As you see adsorbance change over time. To get the change of the adsorbance draw a line down from the top of the curve (1min time point) to the bottom of it (the zero time point). The sector on the x-axis is your change in adsorbance over one minute. Remember that each of the small boxes is equal to 0.1.

When you read out change in adsorbance/min for each sample we construct table like this, where you put your result (see below).

To calculate the Enzyme Activity we are going to use a Lambert Beer Law. We know that enzyme activity is described as the turn over of the amount of substrate over time.Additionally, from the Lambert Beer Law we know that adsorbance of compont X is proportional to its concentration (see below).

After you get the Enzyme Activity of each sample you put it in the Table and discuss your results:) Remember to give the lab reports till tomorrow evening (if someone need more time for that please leave them till Sunday evening or bring it to me monday).

That would be it:) Not so bad ha?

Till Next Time NUIG:)

Cya Soon

Maciek GGSTEAM