扬州大学：《生物化学 Biochemistry》课程教学课件（讲稿）Experiments for Bichemistry 2_electron transport

Oxidation and Electron Transport

Oxidation and Electron Transport

1.Purpose1.1 Master the extraction method of animal tissueorganelles1.2 Understand the location of biological oxidationprocess in the body3. Understand the different transport pathways throughwhichenzymes remove hydrogen from differentsubstrates

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

2.PrincipleIn the process of biological oxidation, the hydrogenremoved from the metabolic substrate is handedover to different coenzymes, and then transferredthrough a series of electron transporters, and finallycombined with oxygen to form water

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

2.PrincipleThese oxidoreductases and their coenzymes, whichare present on the inner mitochondrial membrane, arearranged sequentially and play the role of transportingelectrons or electron protons, which is called theelectron transport chain or respiratory chain.On the inner mitochondrial membrane, there are tworespiratorychains:

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

2.PrincipleNADH+H+.-FMNCoQCytochrome0.02FADH, CoQCytochrome

NADH+H+ FMN CoQ Cytochrome O2 FADH2 CoQ Cytochrome O2 2. Principle

The amount of reduced coenzyme producedinthereaction systemcanbe determinedaccording to the rate of fading of blue2.PrincipleIn vitro experiments, the protons and electrons carriedby the reduced coenzyme can be transferred to anoxidized 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) )FADH, + reduced methane (colorless)

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)

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.Procedures3.1Preparationofliverextract(1) Weigh 2g of fish liver and cut it into minced meat withscissors(2) Put it in a 100 mL beaker, add 10 mL of ice-colddeionized water, wash, and carefully pour the water. Wash 3times in the same way. Strain through fine gauze and gentlysqueeze to remove excess moisture

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.Procedures(3) Transfer the minced meat to an ice-cold mortar,add equal parts fine sand and 2 mL of PBS, grind it inan 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 iscollected in a test tube and placed in an ice bath forlater use

(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.Procedures3.2 Oxidation of substratesTake 6 test tubes with plugs, numbered, and add eachreagent in turn in the following table:

3.2 Oxidation of substrates Take 6 test tubes with plugs, numbered, and add each reagent in turn in the following table: 3. Procedures