Hill Reaction & Photosynthetic Electron Transport: Photoreduction of 2,6-Dichlorophenolindophenol (DCPIP)
In 1937, Robin Hill demonstrated that isolated chloroplasts illuminated in the presence of an artificial electron acceptor evolve molecular oxygen ($text{O}_2$) in the complete absence of carbon dioxide ($text{CO}_2$). This seminal finding proved that:
1. The light-dependent water-splitting reactions of photosynthesis are physically and chemically distinct from the light-independent enzymatic fixation of $text{CO}_2$ (Calvin-Benson cycle).
2. Water ($text{H}_2text{O}$), not $text{CO}_2$, is the ultimate source of evolved atmospheric oxygen.
In the classical Hill reaction, 2,6-dichlorophenolindophenol (DCPIP) is employed as an artificial, membrane-permeant electron acceptor. In its oxidized state, DCPIP is intensely blue with a prominent absorption maximum at $lambda = 600text{ nm}$ ($epsilon_{600} = 21,000text{ M}^{-1}text{cm}^{-1}$ at $text{pH } 6.5$). Upon photochemical reduction by electrons emerging from Photosystem II and the plastoquinone pool, DCPIP accepts two electrons and two protons to form colorless reducedindophenol ($text{DCPIPH}_2$):
$$text{DCPIP (Blue, Oxidized)} + 2 e^- + 2 text{H}^+ xrightarrow{text{Light, Thylakoids}} text{DCPIPH}_2 text{ (Colorless, Reduced)}$$
$$text{Overall Reaction: } 2 text{ H}_2text{O} + 2 text{ DCPIP} xrightarrow{hnu, text{Chloroplasts}} text{O}_2 uparrow + 2 text{ DCPIPH}_2$$
Mechanism of DCMU Inhibition: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU or Diuron) is a selective herbicide that competes directly with plastoquinone for the $Q_B$ binding site on the D1 reaction center protein of Photosystem II. By blocking electron transfer from $Q_A^-$ to the plastoquinone pool, DCMU completely halts DCPIP photoreduction, confirming that electron flow originates from the photochemical oxygen-evolving complex of PSII.
- Plant Material: Fresh, dark-adapted (1 h) market spinach leaves (*Spinacia oleracea*), destemmed and washed.
- Chloroplast Isolation Buffer (CIB, Ice-Cold): $0.4text{ M Sorbitol}$, $50text{ mM Tricine-KOH}$ ($text{pH } 7.8$), $10text{ mM NaCl}$, $1text{ mM MgCl}_2$, $0.1% (w/v)text{ BSA}$.
- Hypotonic Assay Buffer: $50text{ mM HEPES-KOH}$ ($text{pH } 6.5$), $10text{ mM NaCl}$, $5text{ mM MgCl}_2$ (osmotic shock lyses outer envelope to release naked thylakoids).
- Artificial Acceptor: $0.2text{ mM DCPIP}$ sodium salt in assay buffer (shielded from light).
- Inhibitor: $10text{ mM DCMU}$ stock solution dissolved in $95%$ ethanol (working concentration $10 mutext{M}$).
- Instrumentation: High-speed refrigerated centrifuge ($4^circtext{C}$), mechanical kitchen blender with chilled blades, UV-Vis spectrophotometer ($600text{ nm}$), $100text{ W}$ incandescent or cold-white LED illumination lamp, and water heat filter (to prevent heat transfer).
Homogenize $20text{ g}$ of destemmed spinach leaves in $60text{ mL}$ of ice-cold Isolation Buffer using 3 bursts of 5 seconds in a chilled blender. Filter homogenate through 4 layers of cheesecloth and 1 layer of Miracloth into a chilled beaker. Centrifuge filtrate at $2,000 times g$ for 5 minutes at $4^circtext{C}$. Discard green supernatant, gently resuspend green thylakoid pellet in $10text{ mL}$ of hypotonic assay buffer, and store on crushed ice in darkness.
Extract $50 mutext{L}$ of thylakoid suspension in $4.95text{ mL}$ of $80%$ acetone. Clarify at $3,000 times g$ for 3 minutes and measure $A_{652}$. Calculate total chlorophyll concentration using Arnon’s formula: $text{Total Chl (mg/mL)} = A_{652} times 100 / 34.5$. Adjust thylakoid stock to $0.10text{ mg Chl/mL}$.
Prepare 4 optical glass cuvettes:
• Tube 1 (Light Reaction): $2.7text{ mL}$ buffer + $0.2text{ mL}$ DCPIP + $0.1text{ mL}$ thylakoids (illuminated).
• Tube 2 (Dark Control): Same as Tube 1, wrapped completely in aluminum foil.
• Tube 3 (DCMU Inhibition): Same as Tube 1 + $3 mutext{L}$ of $10text{ mM DCMU}$ ($10 mutext{M}$ final).
• Tube 4 (Heat-Inactivated Control): Same as Tube 1, but using boiled thylakoids ($100^circtext{C}$ for 5 min).
Measure initial absorbance ($t = 0text{ min}$) at $600text{ nm}$ against a water blank. Place illuminated cuvettes $20text{ cm}$ from a $100text{ W}$ light source behind a $5text{ cm}$ water heat bath. Record $A_{600}$ at 2-minute intervals up to 10 minutes. Mix gently by inversion before each spectrophotometric reading.
| Experimental Condition | A₆₀₀ (0 min) | A₆₀₀ (2 min) | A₆₀₀ (6 min) | A₆₀₀ (10 min) | ΔA₆₀₀ / min | Rate (μmol DCPIP · mg Chl−¹ · h−¹) |
|---|---|---|---|---|---|---|
| 1. Light + Active Chloroplasts | 0.785 | 0.642 | 0.365 | 0.118 | 0.0667 | 114.3 μmol · mg−¹ · h−¹ |
| 2. Dark Control | 0.784 | 0.782 | 0.779 | 0.775 | 0.0009 | 1.5 μmol · mg−¹ · h−¹ (Baseline drift) |
| 3. Light + 10 μM DCMU | 0.786 | 0.783 | 0.778 | 0.772 | 0.0014 | 2.4 μmol · mg−¹ · h−¹ (97.9% Inhibition) |
| 4. Boiled Chloroplasts | 0.780 | 0.779 | 0.776 | 0.774 | 0.0006 | 1.0 μmol · mg−¹ · h−¹ (Inactivated) |
Kinetic Hill Reaction Calculation:
Total reaction volume $V = 3.0text{ mL} = 0.003text{ L}$.
Amount of chlorophyll present: $0.1text{ mL} times 0.10text{ mg/mL} = 0.010text{ mg Chl}$.
$$text{Rate} = frac{(Delta A_{600}/text{min}) times 60text{ min/h} times Vtext{ (L)} times 10^6 mutext{mol/mol}}{epsilon_{600} times l times text{mg Chl}}$$
$$text{Rate} = frac{0.0667 times 60 times 0.003 times 10^6}{21,000 times 1.0 times 0.010} = frac{12,006}{105} = mathbf{114.3 mutext{mol DCPIP reduced}cdottext{mg Chl}^{-1}cdottext{h}^{-1}}$$
Photochemical Conclusion: The profound drop in absorbance occurs exclusively when thylakoids receive light energy. The near-complete inhibition ($97.9%$) by $10 mutext{M DCMU}$ confirms that electron flow requires the functional Photosystem II $Q_B$ plastoquinone exchange mechanism.
