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Topic: Kinetics: Finding Tyrosinase level and creating double-reciprocal plot  (Read 2207 times)

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Offline batillia

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Forgive me, I've always been terrible at kinetics.

So in our last lab we combined a phosphate buffer, L-Dopa, and tyrosinase (5 trials with different amounts of tyrosinase). For each trial, we took the absorbance values (470nm) every 30 seconds.

We then did the same thing with a constant amount of tyrosinase and a cinnamic acid inhibitor, but varying levels of dopa.

Now I'm trying to figure out the tyrosinase levels in the first experiment.

I graphed the 5 trials where it is absorbance vs time (mins).

I've tried the equation c=A/El and got an answer in M/min for each of the 5 trials which I converted to μmoles/min. My book tells me to also calculate μmoles of product formed per minute, but I'm not sure what that means...

I am also told to graph rate (μmoles/min) vs enzyme concentration in each assay (mg) but I'm also not sure what the latter is.

For the second experiment, I can analyze a double reciprocal plot... I just may have trouble getting that plot. It asks for L-Dopa concentration per assay, which might be the only thing I'm stuck on. Is that the substrate concentration?

Any help is appreciated.

Offline Babcock_Hall

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Re: Kinetics: Finding Tyrosinase level and creating double-reciprocal plot
« Reply #1 on: November 17, 2012, 05:27:29 PM »
L-Dopa is the substrate.  It is not clear from your message what the chromophore is, the one that produces absorbance at 470 nm.  Does absorbance increase with time?  If so, then the chromophore is either the product or something related to the product.  It sounds as if you calculated a rate in µmol/min, which is exactly what you were asked to do, unless I am missing something.

It should be easy to calculate the amount of tyrosinase in each reaction as long as you know the stock concentration of the enzyme and the volume that was added to each reaction.  What do you expect to see when you vary the concentration of enzyme?

Offline batillia

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Re: Kinetics: Finding Tyrosinase level and creating double-reciprocal plot
« Reply #2 on: November 17, 2012, 05:53:12 PM »
Evidently, I'm an idiot who didn't even think about the stock concentrations ^^;

The enzyme activity was assayed by monitoring the oxidation of L-Dopa to dopachrome.

But now I'm confused about something else. Concentration of tyrosinase is given as 100 units/ml where 1 unit represents the amount of enzyme that causes an A280 change of 0.001 using tyrosine as a substrate... How am I supposed to convert that into usable units.

Offline Babcock_Hall

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Re: Kinetics: Finding Tyrosinase level and creating double-reciprocal plot
« Reply #3 on: November 17, 2012, 06:15:58 PM »
I am stumped with respect to your last question.

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