Biochemistry and Cell Biology

Biomolecules and Mandatory Food Tests

Living organisms rely on four major groups of organic biomolecules, constructed from chemical elements combined in precise arrangements.

• Carbohydrates: These contain carbon, hydrogen, and oxygen, almost always with hydrogen and oxygen in a 2:1 ratio like water. The basic building block is a single sugar unit, the monosaccharide (glucose being the classic example). When two monosaccharides join together by losing a water molecule, you get a disaccharide like maltose or sucrose; link dozens or hundreds together, and you have a polysaccharide like starch, cellulose, or glycogen. Organisms break them down for fast metabolic energy, while plants weave tough cellulose fibres straight into their protective cell walls.
• Lipids: Lipids also contain carbon, hydrogen, and oxygen, but they carry vastly less oxygen relative to carbon and hydrogen than sugars do. In a standard triglyceride, you will find one glycerol backbone attached to three fatty acid chains. Swap out one of those three fatty acid chains for a phosphate group, and you get a phospholipid instead. Lipids function in long-term energy storage, thermal insulation, organ protection, and membrane architecture.
• Proteins: These always contain carbon, hydrogen, oxygen, and nitrogen, with sulphur appearing in many of them. The structural unit is the amino acid, joined by peptide bonds. Folded proteins take on structural jobs (like keratin in hair) or metabolic jobs (like enzymes and antibodies).
• Vitamins and Minerals: You need to know two representative vitamins: Vitamin C, which dissolves in water, comes from fresh citrus fruits, and prevents scurvy by keeping connective tissues intact; and Vitamin D, which is fat-soluble, comes from oily fish or sunlight on skin, and helps your gut absorb calcium for strong bones and teeth. For minerals, the exam expects you to name iron (the core of haemoglobin for oxygen transport) and calcium (essential for animal bones and teeth, and for the middle lamella cementing plant cell walls together).
• Water: Makes up the bulk of cellular mass, acting as an intracellular solvent, transport medium, reactant in photosynthesis, and contributor to plant turgor.

Mandatory Practical: Food Testing

• Purpose: To detect starch, reducing sugars, protein, and lipids qualitatively, and estimate reducing sugar levels quantitatively.
• Apparatus and materials: Test tubes, boiling water bath, brown paper, dropping pipettes, glucose, starch, vegetable oil, egg white, and specific reagents.
• Method:

1. Starch: Add 2–3 drops of dilute iodine solution to the sample. A positive result turns from yellow/orange-brown to blue-black.
2. Reducing sugars: Add equal volumes of sample and Benedict's reagent. Place the boiling tube into a vigorous water bath and keep it boiling for 5 minutes. A positive result shifts from blue to brick-red.
3. Protein: Add equal volumes of sample and Biuret reagent (sodium hydroxide and copper sulphate). Swirl at room temperature without heating. A positive result turns from blue to purple-violet.
4. Lipids: Rub the sample onto brown paper and allow it to dry completely. A permanent translucent stain confirms lipids.
5. Quantitative reducing sugar estimation: Mix samples with equal volumes of Benedict's solution and heat in a boiling water bath for exactly 5 minutes alongside a water control. Compare the resulting colours (progressing through blue, green, yellow, orange, and brick-red) against a series of standard solutions of known concentration. The test is approximate because concentration is judged by eye against colour standards rather than measured by an analytical instrument.

• Control: Distilled water tested with each reagent; every test remains negative (iodine stays yellow/orange-brown, Benedict's and Biuret stay blue, paper leaves no translucent stain).
• Precautions: Wear safety goggles when handling corrosive Biuret reagent, and handle hot test tubes with a test-tube holder.

Cell Structure, Membrane Transport, and Microscopy

Cells fall into two broad structural categories:

• Prokaryotic cells: These are simple, ancestral cells with neither a nuclear envelope nor internal membrane compartments (bacteria being the prime example). Their genetic material consists of a single loop of DNA free in the cytoplasm, sometimes accompanied by circular plasmids. They possess ribosomes, a cell membrane, and a cell wall not made of cellulose. Bacteria reproduce asexually by binary fission, where the single DNA loop replicates, the cell elongates, and the cytoplasm cleaves into two identical cells.
• Eukaryotic cells: These house their genetic material inside a distinct double-layered nuclear envelope and contain internal specialised compartments such as mitochondria and chloroplasts (plant, animal, and fungal cells all fit here).

Organelles and Cell Comparisons

• Nucleus: Surrounded by a double nuclear membrane with nuclear pores; houses chromosomes made of DNA and protein; controls cellular metabolism and division.
• Ribosomes: Small granular structures free in the cytoplasm or bound to endoplasmic reticulum; the physical site of protein synthesis.
• Mitochondria: Sausage-shaped organelles wrapped in a double membrane, where the inner membrane folds into deep cristae to carry out aerobic respiration and generate ATP.
• Chloroplasts: Present only in green plant cells; contain chlorophyll pigments that absorb light during photosynthesis.
• Cell wall: Exterior to the plant cell membrane; constructed of cellulose fibres; provides structural support and protection while remaining fully permeable.
• Vacuole: Large, permanent fluid-filled compartment containing cell sap; maintains internal cellular turgor.

While plant cells feature a rigid cellulose wall, chloroplasts, a large permanent vacuole, and a fixed shape, animal cells possess an irregular shape, no cell wall, and no chloroplasts.

The Fluid Mosaic Model of the Cell Membrane

The plasma membrane consists of a double layer of phospholipid molecules with protein molecules floating throughout. Each phospholipid features a hydrophilic, polar phosphate head directed towards the aqueous fluid inside and outside the cell, and two hydrophobic, non-polar fatty acid tails pointed inwards towards each other. Cholesterol molecules nestle between the tails in animal cells to stabilise flexibility. The membrane is selectively permeable, regulating molecular movement into and out of the cell.

Transport Across Membranes

• Diffusion: The passive movement of molecules down a concentration gradient from an area of high concentration to an area of low concentration without metabolic energy.
• Osmosis: Water molecules moving from a region of high water concentration to one of lower water concentration through a semi-permeable membrane. The presence of that semi-permeable barrier is the crucial part examiners look for.
• Active transport: The movement of substances against a concentration gradient using energy in the form of ATP and specific carrier proteins (for example, root hair cells taking up mineral ions from dilute soil water).

When a plant cell sits in pure water, water enters the vacuole by osmosis, swelling the cytoplasm and pressing the membrane firmly against the cellulose wall. The resulting internal pressure makes the cell turgid, which supports soft stems and leaves. If placed into a concentrated salt or sugar solution, water leaves the vacuole, causing the cytoplasm to pull away from the cell wall. The cell becomes plasmolysed and the tissue wilts. By contrast, animal cells placed in pure water absorb water until they burst (lysis), and in concentrated solutions they shrivel, because they lack an external wall.

Mandatory Practical: Microscopy and Staining Cells

• Purpose: To prepare, stain, and examine plant and animal cells using a light microscope.
• Apparatus and materials: Light microscope, glass slides, cover slips, mounted needle, cotton buds, onion, forcep, dropper, methylene blue, and iodine solution.
• Method:

1. Animal cells: Scrape the inner cheek lining gently with a clean cotton bud. Smear the cells across a slide, add a drop of water, and apply 1–2 drops of methylene blue. The stain increases optical contrast, colouring nuclei dark blue and cytoplasm pale blue.
2. Plant cells: Peel a thin, transparent epidermal layer from the inner curve of an onion leaf with forceps. Lay it flat on a glass slide and add 1–2 drops of iodine solution to stain nuclei orange and cytoplasm yellow.
3. For both slides, lower the cover slip slowly at an angle using a mounted needle to avoid trapping air bubbles. The cover slip flattens the specimen and protects the objective lens.
4. Place the slide on the stage, turn on the illuminator, and align the lowest power objective lens. Bring the image into approximate focus using the coarse focus knob, then use the fine focus knob to produce a sharp image. Rotate to the high-power objective and focus using only the fine focus knob.

• Magnification calculation: If viewing through a $\times 10$ eyepiece with a $\times 40$ objective lens, total magnification equals $10 \times 40 = \mathbf{\times 400}$.
• Precautions: Place used cotton buds into disinfectant immediately after cheek cell collection. Never focus downwards with the coarse adjustment knob while looking through the eyepiece under high power to avoid cracking the slide.

Enzyme Action and Mandatory Enzyme Investigations

An enzyme is a biological catalyst, composed of folded protein synthesised at ribosomes, that accelerates metabolic reactions without being consumed in the process.

The Induced Fit Theory

The active site on the enzyme has a shape very close to that of the substrate, but not completely rigid. As the substrate enters, the active site moulds itself slightly around it to achieve a tight fit, creating an enzyme–substrate complex. Once the reaction finishes and the products pull away, the active site springs back into its starting shape, ready to take on another incoming substrate molecule.

Enzymes demonstrate sharp specificity. Catabolic enzymes break down complex molecules into simpler units (such as amylase converting starch to maltose, or catalase degrading hydrogen peroxide). Anabolic enzymes synthesise complex molecules from simpler precursors (such as DNA polymerase assembling nucleotides).

Enzyme Graphs and Denaturation

• Temperature: The reaction rate climbs as temperature rises from $0^\circ\text{C}$ to an optimum (around $37^\circ\text{C}$ in human enzymes) because increasing kinetic energy produces more frequent collisions between active sites and substrate molecules. Beyond the optimum, the rate drops steeply to zero. Extreme heat breaks the structural bonds holding the folded protein, permanently altering the active site shape—a process called denaturation.
• pH: Plotting activity against pH produces a bell-shaped curve that peaks at the enzyme's optimum pH. Shifting pH away from this point alters electrical charges on the active site amino acids, reducing substrate binding; extreme pH shifts cause permanent denaturation.
• Substrate concentration: Increasing substrate concentration steadily raises reaction velocity until all enzyme active sites are occupied simultaneously. At this point the curve plateaus, and the enzyme is saturated.

Mandatory Practical: Investigating pH, Temperature, and Denaturation on Catalase

• Purpose: To see how changing pH, altering temperature, or boiling the enzyme alters the rate at which celery catalase breaks down hydrogen peroxide.
• Apparatus and materials: Graduated cylinders, water baths, blended celery (catalase source), hydrogen peroxide, washing-up liquid, and pH buffers.
• Method:

1. In a graduated cylinder, mix $10\text{ cm}^3$ of buffered solution with a drop of washing-up liquid and $5\text{ cm}^3$ of filtered celery extract. Stand the cylinder and a boiling tube of hydrogen peroxide in a water bath at the test temperature to equilibrate.
2. Pour $5\text{ cm}^3$ of hydrogen peroxide into the cylinder. The released oxygen gas is trapped by washing-up liquid to form a column of foam. Record the foam volume produced after 2 minutes.
3. To test pH: Keep temperature constant at $25^\circ\text{C}$ and repeat using buffer solutions across a range including pH 4, 6, 7, 8, 9, and 10. The pH producing the greatest volume of foam represents the optimum pH for the enzyme.
4. To test temperature: Use the optimum pH buffer throughout and run trials across water baths at $0^\circ\text{C}, 10^\circ\text{C}, 20^\circ\text{C}, 30^\circ\text{C}, 40^\circ\text{C}$, and $50^\circ\text{C}$.
5. To test denaturation: Boil one portion of celery extract vigorously for 10 minutes before testing, and compare its activity against unboiled extract under identical conditions.

• Controlled variables: Keep enzyme volume, substrate concentration, and reaction time constant in all trials.
• Result and conclusion: Boiled extract produces negligible foam, showing that excessive heat denatures catalase. Activity displays a distinct peak at the optimum pH and temperature.
• Precautions: Always pull on protective gloves and safety glasses when handling hydrogen peroxide because it burns skin and damages eyesight.

Enzyme Immobilisation and Bioprocessing

An immobilised enzyme is attached to or trapped within an inert, insoluble support material so that it cannot mix freely with the substrate solution.

Mandatory Practical: Immobilising Yeast and Testing its Activity

• Purpose: To immobilise yeast cells in calcium alginate beads and compare their activity with free yeast cells.
• Apparatus and materials: Dried yeast, sodium alginate powder, calcium chloride solution, syringe barrel, retort stand, sucrose solution, and Benedict's reagent.
• Method:

1. Mix $4\text{ g}$ of sodium alginate into $100\text{ cm}^3$ of warm water until dissolved. Stir in a suspension of dried yeast.
2. Draw the yeast-alginate mixture into a syringe. Clamp the syringe over a beaker containing calcium chloride solution.
3. Dispense the mixture drop by drop into the calcium chloride. As each droplet contacts the calcium ions, it hardens into an insoluble alginate bead containing trapped yeast cells.
4. Give the beads around 15 minutes in the calcium chloride bath so the gel matrix cures completely. Once hardened, strain them out and rinse them thoroughly under distilled water to wash away any loose yeast clinging to the exterior surface.
5. Transfer the beads into a syringe barrel fitted with a tap to make a column. Pour $1\%\text{ sucrose}$ solution into the top of the column and collect the product running through the bottom.
6. Test the effluent with Benedict's reagent and heat in a boiling water bath. The appearance of a brick-red precipitate confirms that sucrose has been converted to glucose by the immobilised yeast enzyme (sucrase/invertase).

• Control: Run sucrose through an identical column containing empty alginate beads without yeast; the collected fluid gives a negative Benedict's result.
• Advantages of immobilisation: The enzyme can be recovered and reused repeatedly; the final product contains no contaminating enzyme, cutting downstream purification costs; and immobilised enzymes often exhibit greater thermal stability.
• Bioprocessing: The industrial manufacture of useful products using living cells or enzymes inside large controlled containers known as bioreactors.

Cellular Respiration and Photosynthesis

Metabolism operates through two major energetic pathways: releasing energy by oxidising organic substrates, and capturing solar energy to synthesise carbohydrates.

Cellular Respiration

Aerobic respiration yields ATP by breaking down glucose completely in the presence of oxygen:

Aerobic respiration proceeds through three stages:

1. Glycolysis: This first stage happens out in the cytosol, clear of any organelle. A 6-carbon glucose molecule is cleaved down into two 3-carbon pyruvate molecules without using any oxygen, delivering a small net harvest of 2 ATP.
2. Krebs cycle: Pyruvate diffuses across mitochondrial membranes into the internal matrix, shedding a carbon atom as carbon dioxide to form a 2-carbon acetyl fragment carried by coenzyme A. This acetyl unit drives a cyclic wheel of enzymatic steps where extra carbon dioxide breaks off, an ATP forms, and high-energy hydrogen atoms load onto $\text{NAD}^+$ carriers to yield $\text{NADH}$.
3. Electron transport chain: Located along the inner mitochondrial foldings (cristae). High-energy electrons and hydrogen ions from $\text{NADH}$ pass along a chain of electron carrier proteins. As electrons lose energy, ATP is synthesised. At the end of the chain, oxygen acts as the final electron and hydrogen acceptor, uniting with hydrogen ions to form water.

Without oxygen, cellular respiration cannot enter the mitochondrion, so glycolysis keeps churning on its own in the cytosol. When this anaerobic pathway happens in overworked human muscles, pyruvate absorbs hydrogen from $\text{NADH}$ and turns into lactic acid. In yeast cells, however, the pyruvate sheds a carbon dioxide molecule and converts into ethanol.

Mandatory Practical: Investigating Yeast Fermentation

• Purpose: To produce ethanol by anaerobic respiration in yeast and chemically detect its formation.
• Apparatus and materials: Conical flask, water bath at $30^\circ\text{C}$, glucose solution, dried yeast, liquid paraffin (oil), fermentation airlock, delivery tube, test tube of limewater, potassium iodide, and sodium hypochlorite.
• Method:

1. Dissolve glucose in water and boil the solution vigorously for 5 minutes to expel dissolved oxygen. Cool the flask to room temperature before adding dried yeast, ensuring yeast enzymes are not denatured by heat.
2. Pour a layer of liquid paraffin over the mixture to provide a physical seal against atmospheric oxygen.
3. Fit a rubber bung carrying a water-filled fermentation airlock, and lead a delivery tube from the flask into an external test tube containing clear limewater.
4. Place the flask into a water bath kept at $30^\circ\text{C}$ (optimum temperature for yeast enzymes). Gas bubbles pass through the airlock and turn the limewater milky, confirming carbon dioxide production.
5. Once bubbling stops, filter the yeast suspension. Add potassium iodide solution and sodium hypochlorite solution to the clear filtrate, then warm in a $50^\circ\text{C}$ water bath. The formation of pale yellow crystals (iodoform test) confirms ethanol.

• Control: An identical setup prepared using boiled (killed) yeast; the limewater stays clear and no ethanol crystals form.
• Precautions: Allow boiled glucose to cool fully before adding yeast; ensure the oil seal is continuous.

Photosynthesis

Photosynthesis converts light energy into chemical energy inside chloroplasts:

• Light stage: Occurs across the grana (thylakoid membranes) of the chloroplast. Absorbed light energy excites electrons in chlorophyll. Water molecules undergo photolysis, splitting into protons, electrons, and oxygen gas. Energy is captured to synthesise ATP, and $\text{NADP}^+$ accepts electrons and protons to form $\text{NADPH}$.
• Dark stage: Occurs within the fluid stroma. It is light-independent, relying on enzymes to combine carbon dioxide with hydrogen from $\text{NADPH}$, using ATP energy to construct glucose molecules.

Genetics, Protein Synthesis, and DNA Isolation

Genetic information is carried by nucleic acids that direct the structural and metabolic characteristics of the cell.

DNA and RNA Architecture

A nucleotide consists of a phosphate group, a 5-carbon pentose sugar, and a nitrogenous base. Look closely at a DNA nucleotide and you will find deoxyribose sugar partnered with one of four bases: adenine, thymine, guanine, or cytosine. Two polynucleotide strands wrap into a double helix, linked by hydrogen bonds following the complementary base-pairing rule: A pairs with T, and G pairs with C. In RNA, the sugar is ribose, uracil (U) replaces thymine, and the polymer is single-stranded.

Protein Synthesis

When examiners ask for a gene, they want you to state that it is a distinct stretch of DNA nucleotides carrying the precise code for building a sequence of amino acids into a functional protein. The process has two major stages:

1. Transcription: Inside the nucleus, the DNA double helix unwinds at a gene locus. One strand serves as a template to synthesise a complementary strand of messenger RNA (mRNA). The completed mRNA detaches, exits through a nuclear pore, and moves to the cytoplasm.
2. Translation: The mRNA molecule binds to a ribosome. Transfer RNA (tRNA) molecules, each carrying an amino acid and a specific three-base anticodon, bind sequentially to complementary three-base codons on the mRNA. The ribosome knits the arriving amino acids together using peptide bonds to build an elongated polypeptide, which spontaneously folds into a working protein.

Mandatory Practical: Isolation of DNA from Plant Tissue

• Purpose: To isolate DNA strands from onion (or kiwi) tissue.
• Apparatus and materials: Chopped onion, sodium chloride, washing-up liquid, water bath at $60^\circ\text{C}$, ice bath, blender, coffee filter paper, protease solution, and ice-cold ethanol.
• Method:

1. Mix finely chopped onion with $3\text{ g}$ of sodium chloride and $10\text{ cm}^3$ of washing-up liquid in a beaker. The detergent dissolves the lipid bilayer of cell and nuclear membranes, releasing DNA, while salt makes the released DNA strands clump together.
2. Place the beaker in a water bath at $60^\circ\text{C}$ for exactly 15 minutes. This softens plant cell walls and denatures intracellular DNase enzymes that would otherwise chop DNA into fragments.
3. Cool the mixture immediately in an ice bath for 5 minutes. Rapid cooling prevents high heat from breaking the DNA strands.
4. Blend the mixture for no more than 3 seconds to shear cell walls without breaking the fragile DNA chains, then filter through coffee filter paper into a clean boiling tube to separate cellular debris.
5. Add 2–3 drops of protease enzyme to the filtrate to digest histone proteins bound to the DNA.
6. Tilt the tube and gently run ice-cold ethanol down the inside wall so it forms a distinct layer floating on top of the filtrate. Because DNA is insoluble in ice-cold alcohol, white, stringy DNA precipitates at the boundary between the two layers and can be spooled out using a glass rod.

• Precautions: Keep ethanol chilled on ice until the instant of use, and do not blend for longer than 3 seconds.

Cell Continuity, Division, and Diversity

Cell continuity means all living cells arise from the division of pre-existing cells.

• Chromosomes: Thread-like structures situated in the nucleus consisting of DNA coiled around protein. A diploid ($2n$) cell contains two complete homologous sets of chromosomes (human somatic cells contain 46). A haploid ($n$) cell contains one set of chromosomes (human gametes contain 23).
• Mitosis: Nuclear division that produces two daughter nuclei, each possessing the identical chromosome number and genetic constitution as the parent cell. Mitosis proceeds through four morphological phases: prophase, metaphase, anaphase, and telophase. Mitosis provides for tissue growth, repair, cell replacement, and asexual reproduction.
• Cancer: The loss of control over cell division, causing cells to divide repeatedly by mitosis to form an abnormal mass called a tumour. Carcinogens in our environment, like the chemicals in tobacco smoke, intense ultraviolet sunlight, and certain viruses, can trigger this runaway cell division.
• Meiosis: Nuclear division that yields four genetically distinct daughter nuclei, each containing half the chromosome number ($n$) of the parent diploid cell. Meiosis occurs in reproductive organs to form haploid gametes and introduces genetic variation via independent assortment and crossing over.

Cell Diversity and Organisation

Multicellular organisms depend on cellular differentiation, where cells specialise to carry out specific functions:

• Cell: The fundamental structural and functional unit of life.
• Tissue: A group of similar specialised cells working together to perform a specific job (such as xylem transporting water in plants, or skeletal muscle contracting in animals).
• Organ: A distinct structure composed of two or more tissues working collaboratively (such as a plant leaf or animal stomach).
• Organ system: A collection of coordinated organs operating together to carry out a major bodily function (such as the human digestive system).
• Tissue culture: Growing isolated cells, tissues, or organs in an artificial, sterile nutrient medium outside the living organism. Common applications include micropropagation in agriculture to generate large numbers of identical, disease-free crops, and cultivating sheets of artificial skin for medical graft procedures.

Key terms

Enzyme

A biological catalyst, made of protein, that speeds up the rate of a chemical reaction without being used up in the process.

Active site

The specifically shaped part of an enzyme molecule to which the substrate binds during a chemical reaction.

Denaturation

A change in the shape of a protein or enzyme so that it loses its folded shape and can no longer function, caused by high temperature, extreme pH, or agitation.

Diffusion

The movement of molecules from an area of high concentration to an area of low concentration down a concentration gradient.

Osmosis

The movement of water molecules from a region of high water concentration to a region of low water concentration across a semi-permeable membrane.

Active transport

The movement of substances across a cell membrane against a concentration gradient, requiring energy in the form of ATP.

Cell

The basic structural and functional unit of a living organism.

Tissue

A group of similar cells that carry out the same function.

Metabolism

The sum of all the chemical reactions occurring inside an organism.

Control

An experimental setup identical in every way to the test except that the factor being investigated is absent, used as a baseline for comparison.

Prokaryotic

A cell type that has no true nucleus and lacks membrane-bound organelles, such as a bacterium.

Eukaryotic

A cell type possessing a true nucleus enclosed by a nuclear membrane and distinct membrane-bound organelles.

Immobilised enzyme

An enzyme fixed to or trapped in an inert material so that it cannot mix freely with the substrate.

Turgor

The firm pressure exerted outwards by the fluid-filled vacuole and cytoplasm against the rigid cell wall of a plant cell.