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D. Pharm II Year - Biochemistry and Clinical Pathology

The Oxford College of Pharmacy - I Sessional Examination 2025-26

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PAPER 1 (June 2026)

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SECTION I: LONG ANSWERS

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Q1. Define a cell. Explain prokaryotic and eukaryotic cells in detail with a neat, labeled diagram.

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• Size: 1-10 µm
• No nuclear membrane (no true nucleus)
• Circular, naked DNA (no histones)
• No membrane-bound organelles (no mitochondria, ER, Golgi)
• Ribosomes: 70S (made of 50S + 30S subunits)
• Cell wall present (made of peptidoglycan in bacteria)
• Plasma membrane present
• No mitotic spindle; binary fission for reproduction
• Plasmids may be present (extra-chromosomal DNA)
• Capsule and flagella may be present

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• Size: 10-100 µm
• True nucleus with nuclear membrane (double membrane with pores)
• Linear DNA bound to histone proteins (chromosomes)
• Membrane-bound organelles present
• Ribosomes: 80S (60S + 40S subunits) in cytoplasm; 70S in mitochondria/chloroplasts
• Mitosis and meiosis for reproduction

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PROKARYOTIC CELL                    EUKARYOTIC CELL (Animal)
┌──────────────────────┐           ┌──────────────────────────────┐
│  Capsule             │           │                              │
│  ┌────────────────┐  │           │   Nucleus (Nuclear membrane) │
│  │ Cell wall      │  │           │   ┌──────┐  Nucleolus       │
│  │  ┌──────────┐  │  │           │   │  DNA │                  │
│  │  │ Plasma   │  │  │           │   └──────┘                  │
│  │  │ membrane │  │  │           │   Mitochondria ○            │
│  │  │          │  │  │           │   Rough ER ≋≋≋≋             │
│  │  │ Nucleoid │  │  │           │   Golgi apparatus [][][]    │
│  │  │  (DNA)   │  │  │           │   Ribosomes • •             │
│  │  │ Ribosome │  │  │           │   Lysosomes ○               │
│  │  │  (70S)   │  │  │           │   Cytoplasm                 │
│  │  └──────────┘  │  │           │   Cell membrane             │
│  └────────────────┘  │           └──────────────────────────────┘
│  Flagellum           │
└──────────────────────┘

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Q2. Explain polysaccharides. Write a note on starch and glycogen.

1. Homopolysaccharides - made of same type of monosaccharide
   • Starch, glycogen, cellulose, chitin, inulin
2. Heteropolysaccharides - made of different monosaccharides
   • Hyaluronic acid, heparin, chondroitin sulfate

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• The main storage polysaccharide in plants
• Found in rice, wheat, potatoes, corn
• Made entirely of glucose units

• Linear, unbranched chain
• Glucose units linked by α-1,4-glycosidic bonds
• Helical structure
• Molecular weight: 10,000 - 500,000 Da
• Gives blue-black color with iodine (diagnostic test)

• Branched chain
• Main chain: α-1,4-glycosidic bonds
• Branch points: α-1,6-glycosidic bonds (every 24-30 glucose residues)
• Gives reddish-purple color with iodine

• Salivary and pancreatic amylase hydrolyze α-1,4 bonds → maltose, dextrins
• Eventually broken down to glucose

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• The main storage polysaccharide in animals (humans)
• Stored primarily in liver (6-8% of weight) and muscle (1-2% of weight)
• Synthesized from glucose-1-phosphate via glycogenesis
• Broken down by glycogenolysis

• Highly branched polymer of glucose
• Main chain: α-1,4-glycosidic bonds
• Branch points: α-1,6-glycosidic bonds (every 8-12 residues - more frequent branching than amylopectin)
• Molecular weight: up to 10⁸ Da (much larger than starch)
• Gives reddish-brown color with iodine (less color than starch due to shorter chains)

• Liver glycogen: Maintains blood glucose levels between meals (glucose homeostasis)
• Muscle glycogen: Provides immediate energy for muscle contraction
• Rapidly mobilized during fasting, exercise, or stress

• Glycogen phosphorylase - cleaves α-1,4 bonds (requires Pyridoxal phosphate, vitamin B6)
• Debranching enzyme - cleaves α-1,6 bonds at branch points

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Q3. Define amino acids. Classify them with examples. Discuss the general properties of amino acids.

        H
        |
H₂N — C — COOH
        |
        R (side chain)

• The α-carbon is the central carbon
• R group determines the identity and properties of each amino acid

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• PVT TIM HaLL (mnemonic): Phenylalanine, Valine, Threonine, Tryptophan, Isoleucine, Methionine, Histidine, Arginine (semi-essential), Leucine, Lysine

• Alanine, Asparagine, Aspartate, Glutamate, Glutamine, Glycine, Proline, Serine, Tyrosine, Cysteine

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• Glucogenic: Can be converted to glucose (e.g., Alanine, Glutamate)
• Ketogenic: Converted to ketone bodies (e.g., Leucine, Lysine)
• Both: Phenylalanine, Tyrosine, Isoleucine, Threonine, Tryptophan

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• Amino acids have both acidic (-COOH) and basic (-NH₂) groups
• In solution, they exist as zwitterions (dipolar ions): -NH₃⁺ and -COO⁻ simultaneously
• They can act as both acids and bases - this is called amphoteric behavior

• The pH at which the amino acid carries no net charge (exists as zwitterion)
• At pI: the amino acid does not migrate in an electric field
• pI = (pKa1 + pKa2) / 2
• For most amino acids: pI ≈ 5-6

• All amino acids (except Glycine) have an asymmetric α-carbon and are optically active
• They rotate plane-polarized light
• Most naturally occurring amino acids have L-configuration
• D-amino acids are found in bacterial cell walls

• Amino acids react with ninhydrin reagent to give a purple/violet color (Ruhemann's purple)
• Proline gives a yellow-orange color
• Used to detect and quantify amino acids

• The -COOH group of one amino acid reacts with the -NH₂ group of another
• Water is eliminated (condensation reaction)
• Forms a peptide bond (-CO-NH-)
• This is a covalent bond, planar and rigid, with partial double bond character

• Most amino acids are soluble in water (polar)
• Insoluble in non-polar organic solvents

• Have high melting points due to ionic interactions in zwitterionic form

• Act as buffers in biological systems
• Histidine (pKa ~6.0) is important in buffering blood (hemoglobin)

• Biuret test: proteins (peptide bonds) - purple color
• Xanthoproteic test: aromatic amino acids - yellow color
• Millon's test: tyrosine - red color
• Sakaguchi test: arginine - red color
• Hopkins-Cole test: tryptophan - purple ring
• Lead acetate test: cysteine - black precipitate (H₂S)

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Q4. Explain the composition and functions of enzymes.

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• Made entirely of protein
• e.g., Pepsin, Trypsin, Ribonuclease

Holoenzyme = Apoenzyme + Cofactor

• Apoenzyme: The protein part (inactive alone)
• Cofactor: Non-protein component required for activity
• Holoenzyme: Active complete enzyme

• A specific region on the enzyme where the substrate binds and the reaction occurs
• Composed of:
  • Binding site - holds the substrate
  • Catalytic site - performs the chemical reaction
• Concept explained by:
  • Lock and Key model (Emil Fischer): Rigid complementary fit
  • Induced Fit model (Daniel Koshland): Active site changes shape upon substrate binding (more accepted)

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• Enzymes lower the activation energy (energy required to start a reaction)
• They do NOT change the equilibrium; they only speed up the rate of reaching equilibrium
• Enzymes can increase reaction rates by a factor of 10⁶ to 10¹²

• Absolute specificity: Acts on only one substrate (e.g., urease → only urea)
• Group specificity: Acts on a group of structurally similar substrates (e.g., hexokinase → glucose, fructose, mannose)
• Stereochemical specificity: Acts on only one isomer (e.g., L-amino acid oxidase - only L-amino acids)
• Bond specificity: Acts on a specific type of bond (e.g., esterases cleave ester bonds)

• Key regulatory enzymes (rate-limiting enzymes) control the flux through metabolic pathways
• e.g., Phosphofructokinase (PFK) in glycolysis

• Salivary amylase - digests starch
• Pepsin, Trypsin, Chymotrypsin - digest proteins
• Lipase - digests fats
• Lactase, Sucrase - digest disaccharides

• Protein kinases and phosphatases mediate cellular signaling

• DNA polymerase, DNA ligase, helicase

• Enzymes of glycolysis, TCA cycle, oxidative phosphorylation

• Thrombin, fibrinogen → fibrin

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SECTION II: SHORT ANSWERS (Paper 1)

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Q5. Explain the role of biochemistry in the pharmacy field.

1. Understanding Drug Mechanisms: Most drugs work by interacting with enzymes, receptors, or transporters. Biochemistry explains how drugs inhibit enzymes (e.g., aspirin inhibits COX enzymes), block receptors, or interfere with metabolic pathways.
2. Drug Design (Rational Drug Design): Knowledge of enzyme structure and active sites allows pharmacists to design drugs that fit specifically into enzyme active sites (e.g., protease inhibitors in HIV treatment).
3. Pharmacokinetics: Biochemical processes govern drug absorption (membrane transport), distribution (plasma protein binding), metabolism (cytochrome P450 enzymes), and excretion (ADME).
4. Clinical Biochemistry & Diagnostics: Abnormal levels of enzymes (AST, ALT, ALP), hormones, glucose, and proteins help diagnose diseases and monitor drug therapy.
5. Nutritional Biochemistry: Understanding macro and micronutrient metabolism helps in formulating nutritional supplements and treating deficiency diseases.
6. Biotechnology & Drug Production: Biochemistry underpins production of insulin, vaccines, monoclonal antibodies, and recombinant proteins used as drugs.
7. Toxicology: Understanding biochemical pathways helps predict drug toxicity and adverse effects.

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Q6. Define disaccharides and write the structure of sucrose and maltose.

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• Composed of: α-D-Glucose + β-D-Fructose
• Linkage: α-1,2-glycosidic bond (1→2 bond; C1 of glucose linked to C2 of fructose)
• Non-reducing sugar - both anomeric carbons are involved in the glycosidic bond (no free anomeric -OH)
• Found in: Sugarcane, sugar beets
• Digested by: Sucrase (Invertase) → glucose + fructose ("invert sugar")
• Used as table sugar, sweetener

α-D-Glucose (C1) — O — (C2) β-D-Fructose
     [α-1→2 glycosidic bond]

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• Composed of: α-D-Glucose + α-D-Glucose (two glucose units)
• Linkage: α-1,4-glycosidic bond (C1 of one glucose linked to C4 of another)
• Reducing sugar - one free anomeric hydroxyl group remains
• Found in: Germinating barley (malt), formed during starch digestion
• Digested by: Maltase → 2 glucose molecules
• Used in: Brewing, malted beverages, infant foods

α-D-Glucose (C1) — O — (C4) α-D-Glucose
     [α-1→4 glycosidic bond]
          (free C1-OH = reducing end)

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Q7. Discuss the functions of essential and non-essential amino acids.

• Tryptophan: Precursor of serotonin (neurotransmitter), melatonin (sleep hormone), and niacin (Vitamin B3). Deficiency causes Pellagra-like symptoms.
• Phenylalanine: Precursor of tyrosine → dopamine → epinephrine, norepinephrine. Defect in metabolism → Phenylketonuria (PKU).
• Leucine, Isoleucine, Valine (BCAAs): Primary energy source in muscle. Promote protein synthesis and muscle growth. Important in post-exercise recovery.
• Lysine: Required for collagen synthesis (along with proline). Deficiency → poor wound healing, impaired growth.
• Methionine: Major methyl group donor (S-adenosyl methionine - SAM). Required for cysteine synthesis. Important in lipid metabolism.
• Threonine: Structural component of proteins; important in glycoprotein synthesis.
• Histidine: Precursor of histamine. Important for immune response and gastric acid secretion. Important buffer (in hemoglobin).

• Glycine: Simplest amino acid; component of collagen (most abundant), bile salts, glutathione, porphyrins, creatine; inhibitory neurotransmitter in CNS.
• Alanine: Key gluconeogenic amino acid; glucose-alanine cycle (transport of nitrogen from muscle to liver).
• Glutamate/Glutamine: Major excitatory neurotransmitter (glutamate); important in nitrogen metabolism; glutamine is the most abundant free amino acid in blood.
• Serine: Important in phospholipid synthesis (phosphatidylserine); precursor of glycine and cysteine.
• Tyrosine: Precursor of catecholamines (dopamine, epinephrine), thyroid hormones (T3, T4), melanin. Conditionally essential (from phenylalanine).
• Cysteine: Contains sulfhydryl (-SH) group; important in disulfide bonds (protein structure); precursor of glutathione (antioxidant), taurine.
• Proline: Major structural component of collagen; ring structure gives rigidity to collagen.
• Asparagine/Aspartate: Important in urea cycle, purine synthesis, protein glycosylation.

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Q8. Explain the structure and functions of cholesterol.

• Molecular formula: C₂₇H₄₆O
• Core: Cyclopentanoperhydrophenanthrene nucleus - four fused rings (three 6-membered + one 5-membered)
  • Rings A, B, C are cyclohexane (6-carbon)
  • Ring D is cyclopentane (5-carbon)
• Attached groups:
  • 3-β-hydroxyl group at carbon 3 (head - hydrophilic)
  • Isoprenoid side chain at carbon 17 (8-carbon)
  • Two methyl groups at C10 and C13
  • Double bond between C5 and C6

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1. Cell Membrane Structure:
   • Inserted between phospholipid molecules in the bilayer
   • Regulates membrane fluidity - prevents too rigid or too fluid membranes
   • Important for membrane integrity and selective permeability
2. Precursor of Steroid Hormones:
   • Glucocorticoids (cortisol) - stress response
   • Mineralocorticoids (aldosterone) - sodium/potassium balance
   • Sex hormones: Estrogen, Progesterone, Testosterone
   • Vitamin D (Cholecalciferol/D3) - via 7-dehydrocholesterol in skin
3. Precursor of Bile Acids:
   • Cholesterol is converted to primary bile acids (cholic acid, chenodeoxycholic acid) in liver
   • Bile acids are essential for fat digestion and absorption in the intestine
4. Myelin Sheath:
   • High concentration in myelin, which insulates nerve fibers
5. Lipoproteins:
   • Transported in blood as part of lipoproteins: VLDL, LDL, HDL
   • LDL ("bad cholesterol") carries cholesterol to tissues
   • HDL ("good cholesterol") removes cholesterol from tissues to liver

• High LDL-cholesterol → atherosclerosis → coronary artery disease, stroke
• Statins (HMG-CoA reductase inhibitors) lower cholesterol synthesis

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Q9. Describe the differences between nucleotides and nucleosides with examples.

• Adenosine = Adenine + Ribose
• Guanosine = Guanine + Ribose
• Cytidine = Cytosine + Ribose
• Thymidine = Thymine + Deoxyribose (found in DNA)
• Uridine = Uracil + Ribose (found in RNA)

• AMP (Adenosine monophosphate) - 1 phosphate
• ADP (Adenosine diphosphate) - 2 phosphates
• ATP (Adenosine triphosphate) - 3 phosphates; universal energy currency
• cAMP (Cyclic AMP) - second messenger
• NAD⁺, FAD - coenzymes (contain adenine nucleotide)

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Q10. Discuss factors affecting enzyme activity.

• As temperature increases → enzyme activity increases (more kinetic energy, more collisions)
• Optimum temperature for most human enzymes: 37°C (body temperature)
• Above optimum: denaturation of enzyme protein → loss of activity
• Below optimum: reduced activity (low kinetic energy)
• Rule of thumb: For every 10°C rise (Q10), enzyme activity approximately doubles (up to optimum)

• Each enzyme has an optimum pH at which it works best
• Pepsin (stomach): pH 1-2; Salivary amylase: pH 6.8; Trypsin: pH 8; Alkaline phosphatase: pH 9
• Changes in pH alter ionization of amino acid residues at the active site → altered enzyme-substrate binding
• Extreme pH causes denaturation

• At low [S]: reaction rate increases linearly with [S]
• At higher [S]: rate increases less steeply (enzymes becoming saturated)
• At very high [S]: maximum velocity (Vmax) is reached; all active sites are occupied
• Described by Michaelis-Menten equation: V = Vmax[S] / (Km + [S])
• Km (Michaelis constant) = [S] at which V = Vmax/2; measure of enzyme-substrate affinity (lower Km = higher affinity)

• At constant (saturating) substrate concentration, reaction rate is directly proportional to enzyme concentration
• More enzyme molecules → more active sites → faster reaction

• Competitive inhibition: Inhibitor resembles substrate; competes for active site; increases apparent Km; Vmax unchanged; reversible by excess substrate (e.g., malonate inhibits succinate dehydrogenase)
• Non-competitive inhibition: Inhibitor binds elsewhere (allosteric site); Vmax decreases; Km unchanged; cannot be reversed by excess substrate (e.g., cyanide inhibits cytochrome oxidase)
• Uncompetitive inhibition: Inhibitor binds only to enzyme-substrate complex; both Km and Vmax decrease

• Many enzymes require cofactors (metal ions: Mg²⁺, Zn²⁺, Fe²⁺) or coenzymes (NAD⁺, FAD, CoA) for activity
• Absence of cofactor → reduced or no activity

• Accumulation of products can inhibit enzyme activity by product inhibition (reverse reaction)

• Allosteric enzymes have regulatory sites separate from the active site
• Allosteric activators bind and increase activity
• Allosteric inhibitors bind and decrease activity
• Sigmoidal (S-shaped) V vs [S] curve (not Michaelis-Menten hyperbola)
• Key in metabolic control (e.g., PFK inhibited by ATP, citrate; activated by AMP)

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SECTION III: TWO MARK QUESTIONS (Paper 1)

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• Carbohydrates are polyhydroxy aldehydes or ketones, or substances that yield these on hydrolysis. General formula: Cₙ(H₂O)ₙ
• Classification:
  • Monosaccharides (cannot be hydrolyzed): Glucose, Fructose, Galactose, Ribose
  • Disaccharides (2 monosaccharides): Sucrose, Lactose, Maltose
  • Oligosaccharides (3-10 units): Raffinose, Stachyose
  • Polysaccharides (>10 units): Starch, Glycogen, Cellulose, Chitin

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• Glucose (C₆H₁₂O₆): Aldohexose; exists as α-D-glucose (pyranose ring); Haworth formula shows a 6-membered ring with -OH groups in specific orientations; -OH at C1 is axial (α) or equatorial (β)
• Maltose: Two α-D-glucose units linked by α-1,4-glycosidic bond; reducing sugar; free C1-OH on right glucose
• Galactose (C₆H₁₂O₆): Epimer of glucose at C4 (the -OH at C4 is in opposite direction compared to glucose); component of lactose
• Glycogen: Branched polymer of α-D-glucose; α-1,4 bonds in main chain; α-1,6 bonds at branch points every 8-12 units; tree-like structure; stored in liver and muscle

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• Fatty acids are long-chain monocarboxylic acids (R-COOH) with even number of carbon atoms (usually 16-20 C)
• Saturated fatty acids: No double bonds; solid at room temperature; e.g., Palmitic acid (C16:0), Stearic acid (C18:0) - found in animal fats
• Unsaturated fatty acids:
  • Monounsaturated (MUFA): One double bond (e.g., Oleic acid C18:1 - olive oil)
  • Polyunsaturated (PUFA): Multiple double bonds (e.g., Linoleic acid C18:2, Arachidonic acid C20:4)
• Essential fatty acids (EFA): Linoleic acid (ω-6) and α-Linolenic acid (ω-3) - must be obtained from diet; needed for prostaglandin synthesis, membrane structure, brain function
• Functions: Primary energy storage (β-oxidation yields ATP), membrane structure (phospholipids), signaling (eicosanoids from arachidonic acid)

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• Enzymes that have a regulatory site (allosteric site) separate from the active site
• Binding of a molecule (allosteric effector/modulator) at the allosteric site changes the enzyme's conformation and thus its activity
• Show sigmoidal (S-shaped) kinetics instead of hyperbolic Michaelis-Menten kinetics
• They exhibit cooperativity (binding of one substrate facilitates binding of more)
• Example: ATCase (Aspartate transcarbamoylase), Phosphofructokinase (PFK), Hemoglobin (though not an enzyme)

• A key regulatory mechanism in metabolic pathways
• The end-product of a metabolic pathway inhibits an enzyme (usually the first committed step/rate-limiting enzyme) earlier in the same pathway
• Prevents overproduction of metabolites
• Example: ATP inhibits phosphofructokinase (PFK) in glycolysis; CTP inhibits ATCase; threonine pathway inhibition

A → B → C → D → E (end product)
         ↑         |
         └─────────┘ (end product E inhibits first enzyme)

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PAPER 2 (July 2026)

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SECTION I: LONG ANSWERS

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Q1. Define carbohydrates. Classify them with examples. Explain a few qualitative tests for carbohydrates.

• Trioses (3C): Glyceraldehyde, Dihydroxyacetone
• Pentoses (5C): Ribose, Deoxyribose, Xylulose
• Hexoses (6C): Glucose, Fructose, Galactose, Mannose (most important)

• Sucrose = Glucose + Fructose (non-reducing)
• Lactose = Glucose + Galactose (reducing) - milk sugar
• Maltose = Glucose + Glucose (reducing) - malt sugar

• Raffinose (trisaccharide - in legumes)
• Stachyose (tetrasaccharide)

• Homopolysaccharides: Starch, Glycogen, Cellulose, Chitin, Inulin
• Heteropolysaccharides: Hyaluronic acid, Heparin, Chondroitin sulfate, Blood group substances

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• Reagent: α-naphthol in alcohol + conc. H₂SO₄
• Principle: H₂SO₄ hydrolyzes polysaccharides and dehydrates monosaccharides to furfural (pentoses) or hydroxymethylfurfural (hexoses), which condenses with α-naphthol to give a purple/violet ring
• Result: Violet/purple ring at the interface
• Used for: All carbohydrates (positive test)

• Reagent: Sodium citrate + Sodium carbonate + Copper sulfate
• Principle: Reducing sugars (free aldehyde or ketone groups) reduce Cu²⁺ (blue) → Cu⁺ (red-brick precipitate of Cu₂O)
• Result: Brick-red/yellow/orange precipitate = positive; blue = negative
• Reducing sugars: Glucose, Fructose, Galactose, Maltose, Lactose
• Non-reducing (negative): Sucrose (no free anomeric -OH)

• Similar to Benedict's; Fehling's A (CuSO₄) + Fehling's B (NaOH + potassium sodium tartrate)
• Brick-red precipitate = reducing sugar

• Reagent: Copper acetate in dilute acetic acid (weakly acidic)
• Result: Monosaccharides reduce within 5 minutes (brick-red precipitate); disaccharides react slowly or do not react
• Used to distinguish monosaccharides from reducing disaccharides

• Reagent: Resorcinol in HCl
• Principle: Ketoses dehydrate faster than aldoses with HCl → hydroxymethylfurfural → reacts with resorcinol
• Result: Fructose (ketose) gives deep red/cherry red color within 2 minutes; glucose (aldose) gives faint pink only after prolonged heating

• Reagent: Orcinol + FeCl₃ in HCl
• Principle: Pentoses dehydrate to furfural → reacts with orcinol → green color
• Result: Pentoses (ribose, deoxyribose, xylose) → green/blue-green color; hexoses → brown/red

• Reagent: Iodine solution (I₂/KI)
• Result: Starch → blue-black color; Glycogen → reddish-brown; No change with simple sugars

• Reagent: Phenylhydrazine + sodium acetate + acetic acid
• Result: Glucose, Fructose, Mannose → yellow needle-shaped crystals (glucosazone); Lactose → mushroom-shaped crystals; Maltose → sunflower/hedgehog-shaped crystals
• Used to identify and differentiate sugars

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Q2. What are proteins? List the qualitative tests involved in proteins and add a note on structure of proteins.

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• Reagent: NaOH + dilute CuSO₄
• Principle: Cu²⁺ in alkaline solution forms a complex with peptide bonds (-CO-NH-) → violet/purple color
• Result: Violet/purple = positive for proteins
• Dipeptides are negative; requires at least 2 peptide bonds

• Reagent: Triketohydrindene hydrate (ninhydrin)
• Result: Purple/violet color (Ruhemann's purple) with all α-amino acids and proteins; yellow-orange with proline and hydroxyproline; positive with primary amines

• Reagent: Conc. HNO₃ (warm) + NaOH
• Principle: Nitration of aromatic rings
• Result: Yellow precipitate/color with HNO₃ → turns orange on adding NaOH
• Positive with proteins containing tyrosine, phenylalanine, tryptophan

• Reagent: Mercury in nitric acid (Millon's reagent)
• Result: Red precipitate or red color (due to reaction with hydroxyphenyl group of tyrosine)
• Positive only for proteins/amino acids containing tyrosine

• Reagent: Glyoxylic acid (Hopkins-Cole reagent) + conc. H₂SO₄
• Result: Violet/purple ring at the interface
• Positive only for tryptophan-containing proteins

• Reagent: α-Naphthol + sodium hypobromite (NaOBr)
• Result: Red color
• Positive for arginine (guanidinium group)

• Reagent: NaOH + lead acetate (heat)
• Result: Black precipitate of lead sulfide (PbS)
• Positive for cysteine, methionine (sulfur-containing amino acids)

• Trichloroacetic acid (TCA): Precipitates proteins
• Heavy metals (Hg²⁺, Pb²⁺): Precipitate proteins (protein denaturation)

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• The linear sequence of amino acids in the polypeptide chain
• Maintained by covalent peptide bonds
• Determines all higher orders of structure
• Example: Insulin - A chain (21 aa) + B chain (30 aa) connected by disulfide bridges

• Local folding of the polypeptide chain into regular repeating structures
• Stabilized by hydrogen bonds between -CO and -NH groups of the backbone

• α-Helix: Right-handed helix; 3.6 amino acids per turn; H-bonds between residues 4 apart; side chains project outward; abundant in keratin (hair, nails)
• β-Pleated Sheet: Polypeptide chains arranged side by side; H-bonds between adjacent strands; can be parallel or antiparallel; found in silk fibroin
• β-Turn: Reverses the direction of the polypeptide chain; 4 amino acids; stabilized by H-bond
• Random Coil: Irregular, flexible regions

• Overall 3-dimensional folding of a single polypeptide chain
• Stabilized by: Disulfide bonds (covalent, between Cys residues), Hydrophobic interactions, Ionic bonds (salt bridges), Hydrogen bonds, Van der Waals forces
• Determines the functional shape (active site in enzymes)
• Hydrophobic residues fold inward; hydrophilic residues face outward (toward water)

• Arrangement of multiple polypeptide subunits (protomers) in a protein with more than one chain
• Same forces as tertiary (non-covalent mainly)
• Example: Hemoglobin (2α + 2β subunits); Collagen (3 chains in triple helix); Immunoglobulin
• Exhibits cooperative behavior (e.g., oxygen binding in hemoglobin)

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Q3. Write a note on enzyme inhibitors with examples.

• The inhibitor has a structural similarity to the substrate
• Competes with substrate for the active site
• Inhibitor binds to active site; substrate cannot bind simultaneously
• Inhibition can be overcome by increasing substrate concentration
• Effect on kinetics: Vmax remains unchanged; Km increases (apparent)
• Example: Malonate inhibits succinate dehydrogenase (malonate resembles succinate); Methotrexate inhibits dihydrofolate reductase; Statins competitively inhibit HMG-CoA reductase

• Inhibitor binds to a site different from the active site (allosteric site)
• Can bind to free enzyme and/or enzyme-substrate complex
• Does not prevent substrate binding; but the reaction rate is reduced
• Cannot be overcome by increasing substrate concentration
• Effect on kinetics: Vmax decreases; Km unchanged
• Example: Cyanide inhibits cytochrome oxidase (binds to Fe³⁺); Heavy metals (Pb²⁺, Hg²⁺) inhibit many enzymes by binding to -SH groups

• Inhibitor binds only to the enzyme-substrate complex (ES complex), not free enzyme
• Pulls the reaction toward ES complex formation
• Effect on kinetics: Both Vmax and Km decrease (both reduced proportionally)
• Example: Lithium inhibits inositol monophosphatase

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• Inhibitor forms a covalent bond with the enzyme, permanently inactivating it
• Cannot be reversed by dialysis or dilution
• Examples:
  • Organophosphates (nerve gases - Sarin; insecticides - malathion): Covalently bind to and phosphorylate serine at active site of acetylcholinesterase
  • Aspirin: Covalently acetylates COX (cyclooxygenase); irreversible inhibition of prostaglandin synthesis
  • Penicillin: Covalently inactivates transpeptidase (involved in bacterial cell wall synthesis)
  • Allopurinol: Suicide inhibitor - inhibits xanthine oxidase (used in gout)

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• Inhibitor binds to allosteric site, changing enzyme conformation → decreased activity
• Key in metabolic regulation (see Q10 above)

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Q4. Explain the structure of DNA and RNA.

• Deoxyribose sugar (2'-deoxyribose)
• Nitrogenous bases: Purines - Adenine (A), Guanine (G); Pyrimidines - Cytosine (C), Thymine (T)
• Phosphate groups

• Two antiparallel polynucleotide strands wound around a common axis in a right-handed double helix
• Strands are antiparallel: one runs 5'→3', the other runs 3'→5'
• Strands held together by hydrogen bonds between complementary base pairs:
  • A pairs with T (2 hydrogen bonds)
  • G pairs with C (3 hydrogen bonds)
  • This is Chargaff's rule: A=T and G=C in double-stranded DNA
• The backbone (sugar-phosphate) is on the outside; bases point inward
• Major groove and minor groove alternate along the helix
• Width: ~2 nm; One complete turn: ~3.4 nm (10 base pairs per turn); Rise per base pair: 0.34 nm
• B-DNA is the most common form (physiological conditions); also A-DNA and Z-DNA (left-handed)

• Stores and transmits genetic information
• Template for transcription (RNA synthesis)
• Replication: semiconservative (each strand serves as template)

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• Ribose sugar (has 2'-OH group, unlike DNA)
• Nitrogenous bases: Purines - Adenine (A), Guanine (G); Pyrimidines - Cytosine (C), Uracil (U) (instead of Thymine)
• Phosphate groups

• Cloverleaf structure with 4 stems/loops: Acceptor stem (3'-CCA end - amino acid attachment), D-loop, Anticodon loop (contains anticodon triplet), TψC loop, Variable loop
• L-shaped tertiary structure

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SECTION II: SHORT ANSWERS (Paper 2)

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• A ketohexose (ketone at C2)
• The sweetest naturally occurring sugar
• Found in fruits, honey
• Exists as β-D-fructose in solution (furanose ring - 5-membered)
• Isomer of glucose (epimers differ at C2)
• Haworth formula: 5-membered ring (furanose), with -CH₂OH at C6 and C1

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• Energy currency: ATP is the universal energy currency of all cells; ADP, AMP involved in energy transfer
• Building blocks: Nucleotides are monomers of nucleic acids (DNA and RNA)
• Coenzymes: NAD⁺ (contains adenine nucleotide) - electron carrier in oxidative pathways; FAD; CoA
• Second messengers: cAMP and cGMP - signal transduction (cyclic nucleotides)
• Activated intermediates: UDP-glucose in glycogen synthesis; CDP-diacylglycerol in phospholipid synthesis
• Allosteric regulators: AMP activates, ATP inhibits phosphofructokinase
• Pharmacological agents: Many anticancer drugs are nucleotide analogs (5-fluorouracil, 6-mercaptopurine)

• Stores genetic information for all living organisms
• Self-replication ensures transmission of genetic information to daughter cells (heredity)
• Template for RNA synthesis (transcription)
• Mutations in DNA lead to evolution and genetic diseases

• mRNA: Carries the genetic code from nucleus to ribosome for protein synthesis
• tRNA: Adaptor molecule bringing amino acids during translation
• rRNA: Structural and catalytic component of ribosomes; facilitates peptide bond formation
• Regulatory RNA (miRNA, siRNA): Control of gene expression
• Some RNA molecules have enzymatic activity (ribozymes, e.g., group I introns, RNase P)
• Reverse transcription: In retroviruses (HIV), RNA serves as template for DNA synthesis

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SECTION III: TWO MARK QUESTIONS (Paper 2)

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1. Helps understand disease mechanisms at molecular level
2. Basis for drug design and action (pharmacology)
3. Explains digestion, metabolism, and energy production
4. Basis for clinical diagnostics (blood glucose, LFTs, kidney function tests)
5. Foundation for biotechnology, genetic engineering, and personalized medicine

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• Bial's Test: Pentoses (ribose, xylose) + Orcinol + HCl + FeCl₃ → green/blue-green color. Hexoses give muddy brown color. Used to detect pentoses.
• Seliwanoff's Test: Ketoses (fructose) + Resorcinol in HCl → cherry-red color within 2 minutes. Aldoses (glucose) give faint pink only after prolonged heating. Used to distinguish ketoses from aldoses.

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• Caused by severe protein deficiency with adequate caloric intake
• Typically in children 1-5 years, often after weaning to low-protein diet
• Features: Edema (pitting edema - hypoalbuminemia causes fluid retention), distended abdomen, skin lesions (flaky paint dermatosis), hair changes (flag sign - alternating dark/light bands), fatty liver, apathy, moon-face, growth retardation
• Blood: Low serum albumin, low protein levels

• Caused by severe deficiency of both protein and calories (total caloric malnutrition)
• Features: Severe wasting (loss of muscle and fat), "skin and bones" appearance, no edema, wrinkled skin, "wizened old man" appearance, prominent ribs, loss of subcutaneous fat
• Child is alert but apathetic
• Blood: Near-normal serum albumin, low body weight (< 60% expected weight)

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1. Temperature (optimum 37°C in humans)
2. pH (each enzyme has an optimum pH)
3. Substrate concentration ([S] → Vmax, described by Km)
4. Enzyme concentration
5. Inhibitors (competitive, non-competitive, irreversible)
6. Cofactors and coenzymes (metal ions, vitamins)
7. Product concentration (product inhibition)
8. Allosteric regulators (activators and inhibitors)

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Source references: Harper's Illustrated Biochemistry 32nd Ed; Biochemistry 8th Ed (Lippincott); Basic Medical Biochemistry 6th Ed (Clinical Approach). These answers are aligned with D. Pharm II Year Biochemistry and Clinical Pathology syllabus as per PCI guidelines.