Spectrophotometer Lab

For this lab it was necessary to bring a watch with a second hand, as well as personal protective gear: lab coat, safety goggles, and safety gloves. It was best to work in pairs and one partner needed to be a timekeeper while the other one would record the data. The timekeeper then would announce every 5 second interval, beginning from when the enzyme was added to the tube. On the other hand, the recorder would read and record the absorbance from the spectrometer at the 5 second intervals. This was done for all four experiments so the absorbance could be recorded every 5 seconds for 60 seconds. It was highly important to keep in mind that all the tubes that would enter the spectrophotometer are clean and dry before the substrate and buffer was added.
In the first experiment, optimal temperature for enzymatic activity was tested. Five clean spectrophotometer tubes wereare necessary with the different temperatures labeled on them using a wax pencil (Blank, Room Temp, 35 degrees Celsius, 45 degrees Celsius, and 55 degrees Celsius). Then 1mL of
Again, five clean spectrophotometer tubes were necessary with the different pH numbers labeled on them using a wax pencil (3, 4, 5, 6, and 8), and 1 mL of the buffers with those pH levels was added. Each tube needed 1 mL of substrate mixture which was added. Then the spectrophotometer to 470nm and the first spectrophotometer tube was wiped to be used to blank the spectrophotometer. Then 0.1 mL of enzyme from vial E1 ( 1x10^-7 M peroxidase) was added, then it was shaken with parafilm covering the top. The tube was quickly placed into the spectrophotometer, and it was necessary to be sure not to spill any of the reaction mixture or this experiment would have to be repeated. The absorbance was recorded every 5 seconds for 60 seconds and this process was repeated for each spectrophotometer tube that went in one at a