Measurement of Catalase Activity and Intro of Formaldoxime-Week 5

Last week we found the fluorescence from the medium with Pseudomonas putida was way lower than the fluorescence from our control medium. This meant that the P. putida had changed the chemistry of the medium and was breaking down the hydrogen peroxide(H₂O₂) in the medium. In the presence of H₂O₂,  they produced an enzyme called catalase that would break any H₂O₂, and almost every organisms that are expose to oxygen have this enzyme to prevent accumulation of H₂O₂. Knowing that they produces catalase, Matt wanted to test for catalase activities for the first two days of the week. Then Matt introduced me the formaldoxime assay.

On Monday and Tuesday, we measured the activity of catalase from the minimal medium with P. putida. First I added the medium into eight rows of wells on the well plates, and in each of the rows, I only filled eight of the wells. Then I added H₂O₂ at different time points (16, 12, 8, 6, 4, 2, 1, and 0 minutes). One column at each time point. At 0 minutes, I added the mixture amplex-red and HRP (working reagent) at the same time, and then quickly to all of the other columns. The reagent was used stop the reaction of H₂O₂and would make the solution fluoresced. The results did show lower fluorescence when H₂O₂was left to react longer. It could be catalase that was breaking down the H₂O₂ over time, but we weren’t sure. We need further test on that.

Wednesday was a short day because of the midterm presentation. First thing in the morning I prepared any stock solution that was low at the time and then Matt helped me on my presentation. Thankfully the presentation went well. After the presentation, Matt introduced me to the Formaldoxime Assay used to measure total manganese. The working reagent for this assay was a mixture of hydroxylamine hydrochloride and formaldehyde. Hydroxylamine reduced any Mn III and IV to Mn II. A second reagent measured Mn II. It was a mixture of the first working reagent and ammonium hydroxide.

On Thursday, I ran a formaldoxime assay and measured the absorbance every 30 minutes for four hours to see if the absorbance would change over time. Our results showed that the absorbance did not change after four hours. It means we can set up a formaldoxime assay and leave it on the courter up to four hours.

On Friday we were planning to filter out the P. putida that we grew in minimal medium overnight and test the filtrate for hydrogen peroxide. However, the culture did not grow. Then Matt decided to try growing P. putida on minimal medium agar plates with manganese and different concentration of LBB added. After we streaked the plates, they were left in a drawer to grow over the weekend. We would like to see if the P. putida would oxidize manganese or not and at what concentration of LBB would be best for the reaction to occur.

This week was a busiest week so far, but after Matt showed the plans for the next five weeks, it’s going to be busier. This means a lot of work is waiting for me, and it’s going to be fun.