We did this both in the absence of an inhibitor and with an inhibitor at two different concentrations, we also determined the mode of inhibition. .
Material and Method.
In experiment 1, for which we determined the optimum incubation time, we used the data from the table 1 below and plotted the two data sets in Excel to create a diagram with the reaction time plotted against the measured product concentration. One where we used the low substrate concentration and one where we used the higher one. After that we checked at which time interval the velocity was constant and after what time the reaction stopped being linear. (Vo) .
Reaction time (min).
Product concentration (µM) (using [S]=0,05 mM).
Product concentration (µM) (using [S]=0,5 mM).
2,5.
8,77.
29,2.
5.
19,7.
58,4.
10.
34,8.
117.
20.
40.
132.
40.
42,5.
143.
Table 1.
In experiment 2, we determined the optimal temperature for AP, alkaline phosphatase. The purpose of this experiment was to see at which temperature was optimal for the enzyme and so we measured the activity of AP at six different temperatures 0, 20, 40, 60, 80, and 100oC. At each of these temperatures we prepared a reaction containing enzyme and also a reaction which was used as a blank and contained no enzyme. We then located heating blocks set at these different temperatures and for the sample at 20oC we used room temperature and for the sample at 0oC we used ice. Before the start of the experiment we kept all samples on ice. This experiment was divided into two parts, the pre-incubation and the incubation part. .
Preincubation: We labeled 12 eppendorf tubes of 1.5ml in a suitable way to indicate the temperature and the content, and also whether it was a tube containing enzyme (Sample) or solely water (Blank). For example Blank: B 0oC, Sample: S 0oC, B 20oC, S 20oC and so on. We added 300μl distilled water + 500μl of Tris buffer pH 9.5 and also 75μl pNPP solution to each tube and we mixed them all well.
