Michaelis- Menten. Velocity. Initial Velocity. Km and Vmax

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ManiaNiania's picture
Michaelis- Menten. Velocity. Initial Velocity. Km and Vmax

Help urgently needed!
Hey, I've been struggling so much recently to solve following problem, seems like I'm not getting any further. Basically, it's about substrate concentration and enzyme activity. The aim is to plot Michaelis- Menten and find Km and Vmax. And I feel like I'm stuck in one point. During practicals we had 13 tubes and added substrate disodium phenyl phospate  and then enzyme alkaline phospatase. Then the absorbance values were read. So I have concentration of disodium phenyl (expressed in mM), and volume of disodium phenyl added per tube (expressed in ml), volume of alkaline phospatase added per tube (expressed in ml), i have values of absorbance of each tube.
I already calculated substrate concentration (mM) using formula= Conc(mM) x vol added (ml)/ total volume used.
Then I plotted the graph absorbance against subst. concentration. I know if I have this graph I can use gradient and convert abs into conc. But I'm not sure what kind of concentration when I already calculated one. Time of incubation= few minutes. So next step is to divide the result from previous step by time ( i mean divide it by this few minutes; concentration/time). So I'm getting umol phenol/ min. And here's the question- is it already a velocity (or maybe initial velocity) or do I need to divide it by something else (like stock conc. enzyme x volume of enzyme taken per tube) to obtain umol phenol// min/ mg?? I have no idea, please correct me if I've made any mistake above. I guess it's one of the most basic skills I need to have but really I have no clue how to start it. I am guessing I need initial velocity (or velocity) and substrate concentration to plot Michaelis- Menten but no idea how to calculate velocity knowing values I listed at the beginning. I'd be really grateful if anyone could advise me something regarding this problem- like how to calculate velocity, am I correct doing my calculations, do I need more graphs? Whatever that you think that may help. If anyone is willing to help, but don't get my message (i know it's kind of confusing due to my incomprehension of the subject), need more information, any data, graphs or anything that may make it clearer I'm looking forward to hear from you.