Lab 7 - Bio. oxidation Flashcards
What are the examinations?
- Demonstatrion of bio. oxidation by measuring oxygen consumption. We use 2,6-dichlorophenolindophenol.
- Detection of cytochrome a+a3 in viro, by using p-phenylenediamine as H-donor.
- Detection of cytochrome a+a3 in situ, by using Nadi´s reagent.
- Detection of xanthine oxidase.
What are the methods to measure oxygen consumption of cells and tissues?
- Manometric method
- Electrodes showing the changes in the partial pressure of oxygen
- Redox dyes and redox indicators, that has diff. colour in reduced and oxidized form.
How do we demonstrate the bio. oxidation by using 2,6-dichlorophenolindophenol?
It act´s as a redox dye, by accepting H from reduced flaboproteins (FMNH2, FADH2). In oxidized form it is blue, when reduced it is colourless.
What materials are used in “demonstatrion of bio. oxidation by measuring oxygen consumption”?
- Heart muscle
- Phosphate buffer
- 2,6-dichlorophenolindophenol
- Glucose solution
- Potassium succinate solution
- Lactate solution
- NAD+ in potassium phosphate buffer
What does the time of fading depen on, in “Demonstatrion of bio. oxidation by measuring oxygen consumption”?
How fast and how many reduced NAD+ and FAD are prod. during the oxidation, and how fast they can pass the H2 to the redox dye.
How is energy released in the resp.chain? Who are the H-donors?
By burning hydrogen into water. Reduces coenzymes and prosthetic groups (NADH+H+, FADH2) of dehydrogenases are H-donors.
How do we detect cytochrome a+a3 in vitro?
P-phenylenediamine acts as a H.donor and will be oxidized into violet coloured p-phenylenediimine.
What can inhibit the last enzyme of the resp. chain (cytochrome a+a3) ?
Cyanide
What are the materials in “detection of cytochrome a+a3 in vitro”?
- Kidney
- P-phenylenediamine
- Phosphate bugger
- KCN sol.
Where are the mitochondria found in the n.c. in the brain?
In the cortex
How do we detect cytochrome a+a3 in situ?
Nadi´s reagent diffusing in brain slices reacts with cytochrome oxidase and changes into dark violet colour.
What are the materials in “detection of cytochrome a+a3 in situ”?
- Pig brain
- Nadi´s reagent (p-phenylenediamine and alpha-naphtol)
- KCN
- Phosphate buffer
What is xanthine oxidase?
Plays a role in degradation of purine bases. An enzyme, aerobe degrydrogenase, that can foward the H carried by it´s prostetic group (FADH2) directly to the molecular oxygen.
Which two reactions are catalysed by xanthine oxidase?
- Hypoxanthine->xanthine->uric acid
2. Formaldehyde->aldehyde-hidrate-(methylene blue)->formic acid
How is xanthine oxidase indicated? What can inhibit the reaction?
Discolourization. Not KCN, because the enzyme can oxidise H without cytochrome too, but allopurinol.
