Need help with enzyme lab!

  • #1
I'm studying for a lab exam tomorrow and I just want to make sure of some things.

One of the experiments was on the effect of inhibitors on enzyme activity. We were studying the reaction 2 H2O2 (hydrogen peroxide) --> 2 H2O + O2. This reaction uses the enzyme peroxidase. Hydroxylamine (HONH2) is structurally similar to hydrogen peroxide so it competes with it for peroxidase's active site thereby preventing peroxidase from binding with hydrogen peroxide and inhibiting the reaction. But a high enough concentration of substrate with a constant concentration of inhibitor will reduce the inhibition. I used guaiacol as an indicator. It turns colourless to brown when it becomes oxidized and the intensity of the brown is proportional to the amount of oxygen produced. I was supposed to have a used a spectrophotometer to measure the absorbance versus time but I ran out of time in the lab period... We had 10 test tubes:

1. water + guaiacol (indicator)
2. water + hydrogen peroxide (substrate)
3. water + guaiacol + hydroxylamine (inhibitor)
4. water + guaiacol + hydrogen peroxide
5. water + guaiacol + peroxidase (enzyme)
6-10. water + guaiacol + increasing volumes of hydrogen peroxide with each + constant volume of peroxidase + constant volume of hydroxlamine

This is what I think: for #6, there is no inhibitor therefore the absorbance should be high because oxygen will be evidently be produced. For #7-10, the increasing amount of substrate at constant amount of inhibitor should reduce inhibition and therefore absorbance should be lowest for 7 and highest for 10. Is this correct? However, for tubes 1-4, I don't know what the absorbance values would look like. Can anyone tell me this?

Thank you.
 

Answers and Replies

  • #2
178
1
Well, the 1st 4 tubes have no enzyme so there would be no reaction...

I would assume the mixture would remain clear/colorless.
 
  • #3
Ok, is the rest of what I said correct? And what about #5? The enzymes is there but there's nothing to bind to it. This would also remain clear right?
 
  • #4
178
1
Well, you didn't mention tube #5, but yeah there is no substrate so there should also be no reaction in that tube.
 

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