Phenolic Compounds - Case 2: potato - Enzyme and sulfite
17 important questions on Phenolic Compounds - Case 2: potato - Enzyme and sulfite
Why is oxygen conversion preferred over photospectometer in measuring PPO activity?
You measure the first reaction of ortho-diphenols into ortho-quinones.
So it is a more direct measurement then measuring the colour compounds formed
What can you tell about PPO (thyrosinase) activity and sulfite form this graph?
- All your substrate is used (not very likely since it did not happen in the test without sulfite)
- The enzyme PPO was inactivated by the sulfate (the real reason, this is proven by having a second adddition of enzyme which lets the reaction go again (prove agains 1).
How can you see if you have a reversible or a irreversible inactivation?
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What does this oxygen consumption test tell you?
(Samples were pre treated with NaHSO3 or with DTT)
In the sample pretreated with NaHSO3 this was not the case. So probably a covelent conversion in the active site took place.
What does an assay with competitive tyrosiante (PPO) inhibiters do?
Why is kojic acid less efficient then tropolene?
Do you have situation A, B, or C, whe you measere inactivation of tyrosinase by a spectophotometer, what is the reason for this?
This is because the enzyme is inactivated by the sulfite and the ortho-dipenols will not react into brown pigments because of the sulfate but into solfu-phenolic molecule.
What will be the line if you add vitamin C and measere it with a spectrophotometer?
What was the hypothesis about why sulfate inactivates PPO?
How can you prove the hypothesis to see if the histine is really changed by sulfite?
How does peptide identification with LC-MS go?
- First the proteins are separated by SDS-gel electrophoresis,
- Cut out the right protein (tyrosinase)
- Digest it with an protease
- Perform an LC-MS.
What is the difference between a mass spectrum of poly-phenols and proteins?
With proteins this can be different. So the m/z value has to be recalculated in order to get the real molar mass.
Why do the peaks are split up in more peaks if you zoom in and what does this tell you?
The 3 different peaks are the same molecule but with a different charge. The peaks also are spit up in 0.5 and 0.3 which indicates that they also contain C13 .
1628/2=814
1628/3=543
How can you use a MS/MS fragmentation of peptides?
The y will count form the carboxyl terminus and the a and b will count form the amino terminus.
The sequence will then be given an you will get it twice. One from the amino terminus and one from the carboxyl terminus side.
Which histine is more likely to be attacked?
HIS263 because it is more exposed.
What are the browning inhibitor options of PPO?
- Sulfite
- Cystein or other thiols
- Vitamin C
- Cu2+ chelators
- Tropolone
- Phenolics
- pH
- Heat
Why can temperature and pH not be used for the potato proteins?
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