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enzyme activity laboration

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enzyme activity laboration

Postby Zamzen » Wed Sep 22, 2010 6:15 pm

Ok so i had this laboration that involved enzymes. the first laboration involved the enzyme chymosin and il start with this one. appareantly it can catalyze the protein casein into paracasein, and if there are calcium ions present the parakasein becomes unsolvable which makes the milk coagulate.

basically we have small tubes with milk and we add different types of things inside them. so im thinking that il post what we added in the milk and what i think happened and im thankful if anyone can give me a lead in the right direction if im writing about irrelevant things. all experiments occurs in 37 degrees celcius unless anything else is mentioned.

some information i have about chymosin which i have done in my own research involves that it serves best in acid environments and i will bring this up as well.

1: milk + chyomosin: the milk coagulated at a level of 7/10. Here im thinking that the enzyme catalyzed casein which caused the milk to coagulate and there were ca2+ ions as well so it could coagulate.

2: milk + HCl (2mol/dm^3) + chymosin. so my hypothesis was that this would coagulate the most since its the perfect environemnt for the enzyme, its acidic and good temperature and it did actually coagulate, but there will be a conflict in my theory on test tube nr 6. coagulation lvl 8/10. so here im saying that the pH is good, temp is good and there are ca2+ ions present.

3: milk + chymosin + NaOH 2mol/dm^3: result was no coagulation and im thinking that ca(oh)2 was formed which removed the ca2+ ions and im also thinking that the environment is basic so no coagulation occured since it wasent an ideal environment for the chymosin. 0/10

4: ammonium oxalate, chymosin and milk: result no primary reaction, but when we added CaCl2 it started to coagulate. Here im thinking that the ammonium oxalate binds the ca2+ ions as well as its not acidic enough like the hydrocloric acid thats why the milk doesnt coagulate in the first test. but in the second test ca2+ were added and the milk could coagulate. first coagulation 1/10 second coagulation 6.5/10

5: boiled chymosin + milk: no coagulation: im thinking that we changed the structure of the chymosin so it lost it catalyzing effect. 0/10 coagulation

6: chymosin + milk in fridge: no reaction cause the temperature is to cold, and if there were a reaction it was extremely slow. now here is the part which i have no clue what so ever about. we took this out from the fridge, and placed it in an environment where it was 37 degrees celcius and it coagulated at a level of 10/10.


Does chymosin react with the milks kasein in all these scenarion just in different speeds?

Ok so the second part of the laboration involved the enzyme catalse. we boil 1 potato and place it in a solution of hydrogen peroxide (h2o2), then we put a raw potato in another tube that contains H2O2, and the we put one potato in a solution of CuSO4 (copper sulphate) for a while and the place it in hydrogen peroxide.
Boiled potato: no reaction. my conclusion: we killed the cells and changed the structure of the enzymes when we boiled the potatos and therefor there could be no reaction since the peroxisomes breaks down h2o2 into h2o and water.
normal potato: reaction occured, water and oxygen.
potato in cuso4. Reaction occured, water and oxygen, a bit more than the raw potato.
So what im wondering is why the copper part is relevent. cause a question asks what the effects of high concentration of cu2+ in a cell has. again im clueless but im thinking we changed the cells environment but i tought this would lead to that they would die but appareantly not.

the reserach that i have found states
Any heavy metal ion (such as copper cations in copper(II) sulfate) will act as a noncompetitive inhibitor on catalase. Also, the poison cyanide is a competitive inhibitor of catalase, strongly binding to the heme of catalase and stopping the enzyme's action.

but it didnt look like it inhibited the enzymes action in our experiment.

And i was wondering how i could prove that oxygen was beeing produced since 2H2O2 --> 2H2O + O2. In the experiment i saw bubbles etc. But i guess i can proove that water is beeing produced by measuring the concentration of the hydrogen peroxide but im clueless about the oxygen part.
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Postby canalon » Thu Sep 23, 2010 3:22 pm

You are correct for your first lab.
Tube 6 is just a demonstration of the effect of temperature on enzymatic activity: at 4C the activity is so slow that you cannot measure it, but when you increase to 37C it is even faster than in tube 1. and by doing it in this order you also demonstrate that low temperature (unlike high temp) do not degrade the enzyme, just slow it down, as it is still active when you increase the temp.

I do not know what happened to your Copper treated sample, and why you saw bubbles when it seems that you should not expect some. Maybe the exposition was too short or the concentration was too low... Generally it is accepted that the production of bubbles in H2O2 is a sign of O2 production. I cannot think of simple way to measure the production of O2, but I am sure that there are some (allow the production of a flame in a controlled CO2 or N2 atmosphere?). Measuring the concentartion of water might also be quite tricky.
Patrick

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Postby Zamzen » Thu Sep 23, 2010 3:42 pm

yes but tube 1 was in a 37 degree celcius environment as well. so the expected reaction from tube six would be a similar coagulation level as tube 1 when it was placed in 37 degree celcius after beeing in the fridge.
the concentration of the copper sulphate was 1mol/dm^3.

Thanks alot for youre help.
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Postby canalon » Fri Sep 24, 2010 7:32 pm

I do not know how you measure your coagulation level, but if it is not very strict, the difference might be explained that way.
I cannot comment on the inhibition by copper, as I have no clue what concentration would be sufficient. However when you present your results you can explain that Cu is supposed to be an inhibitor, however you did not see that, and possible explanations are... and such and such complemetary experiment might have been helpful to decide the reason.
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Postby JackBean » Sat Sep 25, 2010 3:06 pm

http://www.brenda-enzymes.org/php/resul ... o=1.11.1.6

apparently, Cu can work both as essential metal for some catalases, yet as inhibitor for others (but I would guess common inhibitory effect of heavy metals), however, what about non-enzymatic decomposition?
http://en.wikipedia.org/wiki/Hydrogen_p ... omposition
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Postby xemilyrdx » Wed Oct 03, 2012 10:44 pm

enzymes II effects of concentration.
Ihave this lab where intro: investigate the influence of enzyme concentration on the rate of starch digestion.
when we did the experiment, the colors never changed in all 5 of the test tubes.
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