Biochem Lab on Enzymes

[Mandy]

I did a lab on enzymes and while trying to do the Lab write up, i'm not sure how to answer most of the questions, so i'm assuming that I didn't really understand the purpose of the lab.

 

In the first part we were supposed to analyze the dependence of the enzyme activity on the enzyme concentration.

We had 6 test tubes with KOH and another with a sodium acetate buffer, water, and acid phosphotase, heated it, and then added PNPP (which initiates a phosphotase reaction). Then this was added to a test tube with KOH to stop the reaction, and after intrevals of time (0-15 minutes), a certain amount was taken from the previous reaction mixture and added to the next test tube with KOH. We then read the absorbances.

 

If the reaction that takes place is converting PNPP to PNP, is it correct to find the amount of PNP using the beer-Lambert law to find the concentration and then multiply this by the final assay volume to get the moles of PNP?

 

Then when graphing the amount of PNP Vs. time, should this graph be linear since when the first amount of the phosphotase reaction mixture was added to the KOH, the point was to stop the reaction, so when some of this reaction that was supposed to be stopped was taken and added to the next tube with KOH, would that not have an effect because the reaction should have already stopped? Becuase there is a question that asks us to estimate the initial velocity and how long the reaction rate remain constant (but shouldn't it always be constant).

 

Will the velocity for this be moles/time, with the initial velocity being calculated using the first two points?