BIO201 Lab Six: The Hill Reaction

Focus: One of the vital reactions of photosynthesis, known as the Hill reaction.
Importance: Characterized as an energetically interesting reaction critical to photosynthetic processes.
Laboratory Procedure: Follow along with the lab manual for detailed steps.
Note Regarding Spectrophotometer: No spec readings need to be recorded from this video; however, recording is necessary during actual lab sessions.

Definition of Photosystems: Protein-pigment complexes within chloroplasts that harvest energy from sunlight.
- Specifically highlighted: Photosystem II (P680).
Function: Energy harvested is used to excite electrons, released into the chloroplast's electron transport system.
Result: Production of ATP through the excitation of electrons.

Oxidation Process: Loss of electrons from P680 results in it becoming positively charged and oxidized.
Re-reduction Requirement: P680 requires electrons to be replenished to continue photosynthesis.
Source of Electrons: The Hill reaction involves the oxidation of water (photolysis).
- Sub-process: Also referred to as splitting water.
- End Result: This reaction is essential for the generation of molecular oxygen in the atmosphere, although it is largely unfavorable and not fully understood how plants perform it.

DCPIP: A chromophore used to track the Hill reaction.
- Function: Accepts the electrons released during the oxidation of water.
- Properties: The oxidized form absorbs light; upon reduction, the color changes, indicating reaction progress.
Monitoring Reaction Progress: The effectiveness of the reaction is followed by measuring the decrease in absorbance.

PPE Requirements: Always wear gloves, lab coat or apron, and safety goggles.
Chloroplast Source: Purified chloroplasts from spinach, obtained via differential centrifugation.
Temperature Control: Ensure all materials remain on ice and in the dark to prevent any premature reactions.

Purpose: Concentrated chloroplasts must be diluted for accurate spectrophotometric measurements.
Dilution Process:
- Measure specified volumes of concentration using graduated beakers.
- Return remaining chloroplasts to ice and dark storage.
- Prepare three clean test tubes and serological pipettes.

Initial Steps: Turn on the spectrophotometer and wait for it to initialize.
Setting parameters:
- Choose "Spec 200 e modern interface" from the home menu.
- Ensure it displays "live display" with measurement mode set to absorbance (ABS).
- Set wavelength to 600 nm using the knob. Fine-tune it using arrow keys.

Blanking the Instrument:
- Fill a test tube with water/buffer as the blank.
- Place it in the sample compartment and close.
- Press the zero button to auto zero the machine, which should read zero absorbance after calibration.

Control Sample Preparation:
- One sample is wrapped with aluminum foil to serve as the dark control.
- Pipette 3.5 mL of diluted chloroplasts into each test tube.
- Mix by adding DCPIP to start the reaction (500 µL added).
Using pipette techniques:
- Steady the pipette with the opposite finger while pipetting.
Post-addition: Cover tubes with parafilm and invert them to mix.

Zero Time Point Measurement:
- Insert the dark control in its foil wrapper; take note of the absorbance stabilization before recording it in a lab notebook.
- Document readings of both tubes (dark control and experimental).
- Store samples under the light source and set a timer for two minutes.
Repeat Measurements:
- Continue measuring every two minutes until the reaction reaches a plateau, indicating no further change in DCPIP absorbance.

Transition to Part Two: Clean up and prepare new experimental set-ups for herbicide DCMU tests.
Labeling Test Tubes: Three new test tubes labeled as control (C), low concentration DCMU (Low), and high concentration DCMU (High).
Sample Preparation: Pipette 3.5 mL of diluted chloroplasts into each of two tubes while storing the chloroplasts on ice.

Stock Solution Concentration: 0.1 mM DCMU (High concentration).
Creating Low Concentration: Aim for 10 µM DCMU; determine dilution from stock solution:
- Calculation: Convert 0.1 mM to µM = 100 µM. To achieve 10 µM, it requires a 10-fold dilution.
- Use a microcentrifuge tube to perform the dilution. 100 µL water combined with 11 µL stock yields low concentration.

Selection of Micropipettors: Use the appropriate size for the required volume:
- 100 µL: Use p200, display should read 100.
- 11 µL: Use p20, display should read 110 (for proper volume).
Remember to close the pipette tip box after obtaining tips and correctly dispose of them after use.

Introduction of Treatment:
- Control tube receives 100 µL of water for equal variable standards.
- Add 100 µL of low (10 µM) and high (0.1 mM) DCMU into respective tubes while ensuring sample mixing by inverting.
Post-addition: Cover samples and ice storage to mitigate photosynthesis impact. Wait approximately 10 minutes for DCMU diffusion.

Blanking the Spectrophotometer: Re-blank the spec before adding DCPIP by repeating the original calibration steps.
Measurement Process: Add DCPIP (500 µL) to each sample at temperature zero, cover and mix.
Synchronize samples under light source, timing and continue absorbance measurements every two minutes until saturation is observed.
Clean Up Procedures: Turn off spectrophotometer, dispose of all lab materials correctly and wash tubes as specified.
Waste Disposal: Ensure liquid waste goes to the appropriate beaker, and routine cleaning of tools and pipette tips occur throughout.
Follow through till all lab materials are returned properly, minimizing contamination risk throughout procedures.

To end the session, ensure gloves are removed, safely dispose of, and thoroughly wash hands post-lab.