Re: The Effect Of Temperature On Enzymes

There is no such thing as 'the quantity of temperature'!!!  At the risk of 
sounding superior (I am English!) it is just the effect of temperature!!!
There are many, many web-sites which will give you full details of the 
experiment you want, for it is a standard experiment in the GCSE (= 16+ 
exam) here in the UK.  Use www.Google.co.uk and search under 'GCSE 
coursework science' (or biology), enzymes etc etc.  use the 'pages from 
UK' option.

The egg-white is mainly (besides water) a protein called albumen.  As you 
know, this clots when heated above 75 degrees C (about 165 degrees F) and 
an irreversible change occurs - you cannot 'uncook' an egg.
So, begin by cooking the egg-white anbd then cutting it into small cubes 
about ¼ inch on each side.  Put the same number (10?) into each test-tube.
That is variable 1 (substate concentration).

Each cube has the same size.
That is variable 2 (surface area).

All the egg-white came from the same source (egg or packet!)
That is variable 3 (substrate)

To each test-tube add the same volume of 1% pepsin solution - freshly made 
up (why?).  I suggest 10ml.
That is variable 3 (enzyme concentration)
and variable 4 (volume)
and variable 5 (enzyme)

Since pepsin is found in the stomach and has an optimum pH of 2-3, you 
will need to add the same volume of dil HCl to each tube, and test with 
universal indicator paper (or a pH probe) that the pH is the same in each 
tube.  It won't take much acid to drop the pH - if you overdo it, add a 
drop of dil NaOH solution.
That is variable 6 (pH)

Keep each test-tube in a water-bath at the temperature that you are going 
to investigate.  I suggest 0; 20; 40; 60; 80; and 100 degrees C.  Once you 
have the results, it would probably be worth also doing 25; 30; 35; and 45 
degrees C. (why?) - you'll need to repeat the 20C and 40C runs again (why?)
Since it will take a while for the enzyme and the egg-white to reach these 
temperatures, put them in SEPARATE test-tubes in the water-bath for 10 
mins equilibriate (check temps with a thermometer) then mix them at 'time 
zero'.
That is variable 7 (steady temperature)

Leave the tubes for a while, and shake each the same at regular intervals.
That is variable 8 (agitation)

Now watch what happens.  You need to record the time when ALL the egg-
white has dissolved/broken down. OR you can use an absorption meter to 
record the tubes becoming opaque.  (That's what we use, but they are about 
$200 each, so I do not know if your school/college has them!)
Alternatively, use a black cross on a white background and record the time 
when you can no longer see the cross through the tube.
That is variable 9 (opacity)

Remember to repeat each set-up at least 3 times, so that your results will 
be more RELIABLE (NOT more accurate!)
That is 'variable' 10 - repetitions.

Make sure the same person reads all the tubes to ensure consistency.
That is variable 11 (human error)

That should do for variables! - in the UK we would ask you to explain WHY 
each one of these would affect your results (I'll leave that to you to 
work out!)

Temperature affects all proteins by breaking the hydrogen bonds that hold 
them in a specific shape.  The active site therefore changes shape, the 
substrate no longer fits and the reaction slows down/stops.
The temperature at which this happens varies - the more bonds the higher 
the temperature.  Disulphide bridges also raise the temperature too - they 
are stronger bonds and eggs contain a lot of them (note, it is the ENZYME 
that matters) - hence 'bad egg gas' which is hydrogen sulphide (H2S).
So, lower temperature = more stable enzyme = more active sites = faster 
reaction.

BUT BUT BUT - the higher the temperature the faster the molecules are 
moving (more kinetic energy) so more of them will collide and those 
collisions will be more energetic - so the higher the temperature the 
faster the reaction.  In fact, the rate should double for every 10 degree 
C rise in temperature (Q10 = 2, as the chemists would say!)
So, higher temperatures = faster reaction!

Since these last two points are opposite, there is an OPTIMUM temperature 
where the two effects balance out - normally about 40degrees C
Hope that helps!

(England just won the Rugby World Cup - a game like American football, but 
without the armour!)