Enzyme Activity Assay (Attempt)

On Wednesday (March 6th, 2013), Eun Ji and I caught up where we left last week and worked on enzyme activity assay.  Enzyme activity measures how much enzymes is present in a reaction and how active an enzyme is under certain conditions. Eun Ji showed me her initial result of the assay. She expected that the graph should look like that of the red line, with the reaction rate eventually level off when all the substrates are converted to products. Yet, instead, hers look like the blue line. 

Why did this happen? We still don't know. Yet, we have come up with some hypothesis:

• 3GT binds with the substrate instead of cyanidin Cl --> can't react
• either enzyme or the intermediate molecules denatured under the condition
• reverse reaction (The model below illustrates the enzyme action. E=enzyme, S = substrate, P=products. The main idea is that intermediate molecules can react reversely back to substrate while a small portion goes on to the second reaction to produce the final product.)

Enzyme assay examines the following control factors:

• salt concentration
• enzyme-substrate ratio
• pH
• inhibition (inhibitors decrease enzyme activity)
• activators (increase enzyme activity)
• temperature (most denatured in high temp.)

The two enzymes we examined are 3GT and ANS, and this time we examined the effect on enzyme activity under pH 6 and 7. We loaded our samples and some supplements in a 96-well plate. We spent quite a while recalculating the concentration because we had previously messed our calculation for the concentration of the standard. Also, the standard solution for some reason wouldn't dissolve until we diluted it to 10mM. Luckily, we recognized something wrong before we add everything and were able to solve them:) 

Division of the plate

Once all the substances were added, Eun Ji added HCl to the 0 min to stop the reaction (control), and removed them to 4 microtubes. The rest samples were put in the incubators. When it reached 15 min, Eun Ji would repeat the same thing she did with 0 min, and so on. Because we were short of time I wasn't able to see the complete process. Later Eun Ji would put this test tubes in a spectrophotometer to analyze the enzyme activity and construct a graph.

Since I didn't quite understand the whole process, the information above included some outside research. Nevertheless, I found some interesting facts about kuromanin Cl (the product). Kuromanin Cl belongs to anthocyans family. In a paper the Koffas group previously published, Anthocyanins are "red, purple, or blue plant pigments that belong to the family of polyphenolic compounds collectively called flavonoids". Their antioxidant properties give them the economic value in food dye.

Where does this fit one our map?

I will keep doing some research about the process, and I hope I can discuss this with my mentor next time!