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    Hi,

    There is a test tube containing milk and NaOH in a water bath at 37.0 degrees C. We add 5cm3 of lipase and 5cm3 of water to the test tube.

    What effect would heating the lipase and water have before adding them?

    We tested multiple test tubes with 10cm3 split 50/50 with lipase and water, lipase and bile, bile and water, boiled bile and lipase and boiled lipase and bile. We timed the time for phenolphthalein to go colourless as the lipase breaks down the fat in milk.

    Why did we add 5cm3 of water with 5cm3 of lipase? Is this because we need to add the same volumes each time otherwise we change multiple variables and will effect the concentration of substrate?

    Why did we need no buffer solution? Is this because the NaOH acts as a buffer already, making the initial pHs the same, providing the optimum pH for lipase?

    THANKS!!
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    Heating would cause the reaction to happen faster as long as the enzyme was not denatured

    yes

    Buffer's are there to make sure the ph is constant therefore making sure the reaction is valid


    Hope that helps
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    (Original post by lianne_s)
    Heating would cause the reaction to happen faster as long as the enzyme was not denatured

    yes

    Buffer's are there to make sure the ph is constant therefore making sure the reaction is valid


    Hope that helps
    So in that case do the enzymes gain more KE due to the temperature rise? Or what happens?
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    well the enzymes work best at their optimum temperature(usually 37-38 degrees) beacause they have a high enough Ek to move fast enough but not a temperature that will cause the hydrogen bonds (these hold them in a shape that allows them to work) to break
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    concerning the buffer, lipase is normally found in the small intestine, hence sligtly alkaline so i think the NaOH (being an alkaline) does act as the buffer...
    err not so sure why you needed to add the 5cm3 of water as well, because if ur keeping the same volume of lipase each time, dont really see the need for the water...dunno maybe to dilute it down so there wasnt excess lipase, so it all reacted and went colourless? not so sure bout that

    ohh for the heatig lipase,
    the lipase gains more kinetic energy, thereofore moves more, increases collisions with the substrate, more enzyme-substarte complexes formed hence heating it in advance (as long as u dont over heat it) would increase the rate of reaction
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    Ok thanks all. I just temperature was more to do with KE of molecules then enzymes but I guess it works both ways! I think the NaOH added makes all the initial pHs the same anyway and provides the optimum pH for the enzyme.
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    Buffer is not the right word to use. What a buffer does is resist pH change, which is not what you are doing in this experiment.

    There is 5cm3 of water added as you need to keep the concentration the same as a different concentration will effect the enzyme kinetics. I don't know how in depth you are going but this reaction would obey Michaelis-Menten kinetics

    \Large V=\frac{V_M.[strike]}{K_M+[strike]}

    If you think, the enzyme IS a molecule - just a very large one.

    The NaOH is added because milk is slightly acidic and as someone else mentioned lipase prefers basic conditions (pH 6.6-9.4, but optimal ~8).
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    (Original post by atheistwithfaith)
    Buffer is not the right word to use. What a buffer does is resist pH change, which is not what you are doing in this experiment.

    There is 5cm3 of water added as you need to keep the concentration the same as a different concentration will effect the enzyme kinetics. I don't know how in depth you are going but this reaction would obey Michaelis-Menten kinetics

    \Large V=\frac{V_M.[strike]}{K_M+[strike]}

    If you think, the enzyme IS a molecule - just a very large one.

    The NaOH is added because milk is slightly acidic and as someone else mentioned lipase prefers basic conditions (pH 6.6-9.4, but optimal ~8).
    Is another reason for NaOH to ensure all initial pHs are the same?
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    (Original post by CHEM1STRY)
    Is another reason for NaOH to ensure all initial pHs are the same?
    All initial pH values would be the same even if you didn't have NaOH...
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    (Original post by atheistwithfaith)
    All initial pH values would be the same even if you didn't have NaOH...
    Hmm ok I thought there may be some fluctuation.
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    (Original post by atheistwithfaith)
    Buffer is not the right word to use. What a buffer does is resist pH change, which is not what you are doing in this experiment.

    There is 5cm3 of water added as you need to keep the concentration the same as a different concentration will effect the enzyme kinetics. I don't know how in depth you are going but this reaction would obey Michaelis-Menten kinetics

    \Large V=\frac{V_M.[strike]}{K_M+[strike]}

    If you think, the enzyme IS a molecule - just a very large one.

    The NaOH is added because milk is slightly acidic and as someone else mentioned lipase prefers basic conditions (pH 6.6-9.4, but optimal ~8).
    Oh I do have one more question, sorry!

    For the heating lipase and water, should I put:

    Heating the substances prior to addition would increase the rate of reaction as the enzymes have more KE so more collisions occur between substrate and enzyme (more ES complexes). However if heated too much the lipase enzyme may denature as the bonds holding the tertiary structure are overcome. The active site will change shape, the substrate will no longer fit and no more ES complexes will form. The rate here would duely fall.
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    (Original post by CHEM1STRY)
    Oh I do have one more question, sorry!

    For the heating lipase and water, should I put:

    Heating the substances prior to addition would increase the rate of reaction as the enzymes have more KE so more collisions occur between substrate and enzyme (more ES complexes). However if heated too much the lipase enzyme may denature as the bonds holding the tertiary structure are overcome. The active site will change shape, the substrate will no longer fit and no more ES complexes will form. The rate here would duely fall.
    It depends on how much the initial volume is (ie. volume of milk + NaOH) because if it is much greater than the volume you are adding (10cm3) then the temperature will quickly equilibriate back to around 37oC. But I wouldn't of thought they would try and catch you out like that.

    If you are adding heated liquids it should heat up the entire medium so both the enzyme and its substrates (the fats in milk) would have more KE.

    You might want to mention what bonds are broken.
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    (Original post by CHEM1STRY)
    Hmm ok I thought there may be some fluctuation.
    Forgot to address this.

    If you added no NaOH at the start then all the tubes would only have milk so would be ~pH 6.7. With NaOH they would all start off at a basic pH (I don't know how much NaOH is in there but lets say...) ~pH 8.

    Bile is slightly alkaline so that would change the pH when you added it however as I mentioned before, NaOH has no buffering capacity so whether NaOH is there or not, the pH will change.
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    (Original post by atheistwithfaith)
    Forgot to address this.

    If you added no NaOH at the start then all the tubes would only have milk so would be ~pH 6.7. With NaOH they would all start off at a basic pH (I don't know how much NaOH is in there but lets say...) ~pH 8.

    Bile is slightly alkaline so that would change the pH when you added it however as I mentioned before, NaOH has no buffering capacity so whether NaOH is there or not, the pH will change.

    so what do you think the purpose of the NaOH was, and did the bile both increase the rate of reaction (by emulsifying the lipids) and create an optimum pH, if it did create an optimum PH, why was the NaOH needed....also i still dont get why u need to add 5cm3 of water and 5cm3 of lipase, if ur just adding 5cm3 of lipase each time, woulnt the conc of lipase always just be the same ?
    thankyou
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    (Original post by _anum)
    so what do you think the purpose of the NaOH was, and did the bile both increase the rate of reaction (by emulsifying the lipids) and create an optimum pH, if it did create an optimum PH, why was the NaOH needed....also i still dont get why u need to add 5cm3 of water and 5cm3 of lipase, if ur just adding 5cm3 of lipase each time, woulnt the conc of lipase always just be the same ?
    thankyou
    Looking at the original post again, I didn't notice that you were using phenolphthalein as an indicator. In that case, I think the NaOH is added because the fatty acids from the breakdown of the fats in the milk will react with it and cause a pH change towards being more acidic. This pH change will cause your indicator to change back colour again (from my memory phenolphthalein goes pink --> colourless, though I haven't done this experiment in quite some time).

    You add the water because you want to eliminate concentration as a variable for lipase, and as concentration is related to volume...
    Conc. = moles x volume
    You will have a different concentration if in Tube 1 you just have 10cm3 of phenolphthalein and NaOH + 5cm3 of lipase (15cm3 total) versus if in Tube 2 you have + 5cm3 of liapse and +5cm3 of bile (20cm3 total).
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    (Original post by atheistwithfaith)
    Looking at the original post again, I didn't notice that you were using phenolphthalein as an indicator. In that case, I think the NaOH is added because the fatty acids from the breakdown of the fats in the milk will react with it and cause a pH change towards being more acidic. This pH change will cause your indicator to change back colour again (from my memory phenolphthalein goes pink --> colourless, though I haven't done this experiment in quite some time).

    You add the water because you want to eliminate concentration as a variable for lipase, and as concentration is related to volume...
    Conc. = moles x volume
    You will have a different concentration if in Tube 1 you just have 10cm3 of phenolphthalein and NaOH + 5cm3 of lipase (15cm3 total) versus if in Tube 2 you have + 5cm3 of liapse and +5cm3 of bile (20cm3 total).

    ahh okayy i see, thannkyou for your help..so the NaOH is there to prevent the colour going back to pink? thankyou again
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    (Original post by _anum)
    ahh okayy i see, thannkyou for your help..so the NaOH is there to prevent the colour going back to pink? thankyou again
    I think the NaOH is there to make the indicator go from pink to colourless, that way you can measure how quickly it takes to change to colourless again and this will be directly proportional to the enzymatic rate.
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    (Original post by atheistwithfaith)
    I think the NaOH is there to make the indicator go from pink to colourless, that way you can measure how quickly it takes to change to colourless again and this will be directly proportional to the enzymatic rate.
    ohhh yeah because milk is already slightly acidic....the NaOH would make the start colour pink, so it will go clear when the fatty acids are produced?...well i hope thats right ! thankyouu
 
 
 
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