Oxidation and Electron Transport
1. Purpose 1.1 Master the extraction method of animal tissue organelles 1.2 Understand the location of biological oxidation process in the body 3. Understand the different transport pathways through which enzymes remove hydrogen from different substrates
In the process of biological oxidation, the hydrogen removed from the metabolic substrate is handed over to different coenzymes, and then transferred through a series of electron transporters, and finally combined with oxygen to form water. 2. Principle
These oxidoreductases and their coenzymes, which are present on the inner mitochondrial membrane, are arranged sequentially and play the role of transporting electrons or electron protons, which is called the electron transport chain or respiratory chain. On the inner mitochondrial membrane, there are two respiratory chains: 2. Principle
NADH+H+ FMN CoQ Cytochrome O2 FADH2 CoQ Cytochrome O2 2. Principle
In vitro experiments, the protons and electrons carried by the reduced coenzyme can be transferred to an oxidized hydrogen acceptor such as methane blue (blue), which is reduced to a reduced form (colorless). The reaction is as follows: NADH+H+ + oxidized methane (blue) → NAD+ + reduced methane (colorless) FAD + oxidized methane (blue) → FADH2 + reduced methane (colorless) 2. Principle The amount of reduced coenzyme produced in the reaction system can be determined according to the rate of fading of blue
Reagent • Potassium phosphate buffer solution (PBS, 50 mmol/L, pH=7.4). • Methane blue (0.02%) • Glucose solution (90 mmol/L PBS) • Succinic acid solution (90 mmol/L PBS) • Lactic acid solution (90 mmol/L PBS) • NAD+ solution (5 mmol/L PBS)
3. Procedures 3.1 Preparation of liver extract (1) Weigh 2g of fish liver and cut it into minced meat with scissors. (2) Put it in a 100 mL beaker, add 10 mL of ice-cold deionized water, wash, and carefully pour the water. Wash 3 times in the same way. Strain through fine gauze and gently squeeze to remove excess moisture
(3) Transfer the minced meat to an ice-cold mortar, add equal parts fine sand and 2 mL of PBS, grind it in an ice bath until it is paste, add another 15 mL of PBS, and extract (leave for at least 5 min). (4) After double-layer gauze filtration, the filtrate is collected in a test tube and placed in an ice bath for later use. 3. Procedures
3.2 Oxidation of substrates Take 6 test tubes with plugs, numbered, and add each reagent in turn in the following table: 3. Procedures