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SPPT1070 BIOCHEMISTRY - Complete Answers

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SECTION - I

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Q-1 (Essay Questions) [10 Marks]

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Q-1(i): Hierarchical Levels of Chromosome Packaging with Diagram - Why Essential for Eukaryotic Cells

Level 1 - Nucleosome (Beads on a String)

• DNA wraps around a core of 8 histone proteins (octamer: 2 each of H2A, H2B, H3, H4)
• ~147 base pairs of DNA wrap around each nucleosome
• Adjacent nucleosomes are connected by linker DNA (~20-80 bp) associated with H1 histone
• This reduces the DNA length ~7-fold
• In this state, chromatin looks like "beads on a string" under electron microscopy

Level 2 - 30 nm Fibre (Solenoid)

• Nucleosomes coil and fold further, assisted by histone H1, to form a 30 nm chromatin fibre
• This provides ~40-fold compaction
• Called heterochromatin (inactive/condensed) or euchromatin (active/open)

Level 3 - Loops and Scaffold

• The 30 nm fibre organizes into loop domains (~50,000 bp each) anchored to a protein scaffold
• Creates a 300 nm fibre (radial loop structure)

Level 4 - Coiled Coils / Condensed Chromosome

• Loops further condense and coil into 700-900 nm structures
• At metaphase, fully condensed chromosome (~1,400 nm) is visible

Diagram

DNA double helix (2 nm)
      ↓
Nucleosomes - "beads on a string" (11 nm)
      ↓
30 nm chromatin fibre (solenoid)
      ↓
300 nm loop domains (radial loops)
      ↓
700 nm condensed fibre
      ↓
Metaphase chromosome (1,400 nm)

Why Essential for Eukaryotic Cells?

1. Physical fit - Without packaging, 2 m of DNA cannot fit in a 6 µm nucleus
2. Gene regulation - Euchromatin (loose) = active transcription; heterochromatin (tight) = gene silencing. Histone modifications (acetylation, methylation) control which genes are active
3. DNA replication and repair - Organized packaging ensures orderly duplication
4. Cell division - Condensed chromosomes are needed for accurate segregation during mitosis/meiosis
5. Protection - Packaging protects DNA from physical damage and random transcription

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Q-1(ii): Differentiate Glycogenesis, Glycogenolysis, and Gluconeogenesis as Components of Carbohydrate Metabolism

Glycogenesis (Glycogen Synthesis)

1. Glucose → Glucose-6-phosphate (hexokinase/glucokinase)
2. Glucose-6-P → Glucose-1-phosphate (phosphoglucomutase)
3. Glucose-1-P + UTP → UDP-glucose (UDP-glucose pyrophosphorylase)
4. UDP-glucose added to glycogen chain by glycogen synthase
5. Branching enzyme creates branch points every 8-12 glucose residues

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Glycogenolysis (Glycogen Breakdown)

1. Glycogen phosphorylase cleaves α-1,4 bonds releasing Glucose-1-phosphate
2. Debranching enzyme removes branch points (releases free glucose)
3. Glucose-1-P → Glucose-6-P (phosphoglucomutase)
4. In liver: Glucose-6-P → Glucose (glucose-6-phosphatase) → released into blood
5. In muscle: No glucose-6-phosphatase → glucose-6-P enters glycolysis directly

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Gluconeogenesis (New Glucose Formation)

1. Pyruvate → Oxaloacetate (pyruvate carboxylase, requires biotin)
2. OAA → PEP (PEPCK, requires GTP)
3. Fructose-1,6-bisphosphate → Fructose-6-phosphate (fructose-1,6-bisphosphatase)
4. Glucose-6-phosphate → Glucose (glucose-6-phosphatase)

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Comparison Table

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Q-2 (Short Notes - Any Two) [10 Marks]

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Q-2(i): Key Differences Between Direct ELISA and Sandwich ELISA with Diagram

Direct ELISA

• Principle: Antigen is coated directly onto the solid phase. A single enzyme-labelled antibody binds directly to the antigen. No secondary antibody is used.
• Steps:
  1. Coat microtiter plate with antigen
  2. Block non-specific binding
  3. Add enzyme-labelled primary antibody
  4. Wash away unbound antibody
  5. Add substrate → color development proportional to antigen amount
• Advantages: Faster, fewer steps, no cross-reactivity from secondary antibody
• Disadvantages: Low sensitivity; primary antibody must be labelled (expensive); no signal amplification

Sandwich ELISA

• Principle: A capture antibody is first coated onto the solid phase. Antigen from sample binds to it. Then a second enzyme-labelled detection antibody binds to a different epitope on the same antigen, forming an antibody-antigen-antibody "sandwich."
• Steps:
  1. Coat plate with capture antibody
  2. Add sample (antigen binds to capture antibody)
  3. Wash
  4. Add enzyme-labelled detection antibody
  5. Wash
  6. Add substrate → colour develops
  7. Measure optical density
• Advantages: High sensitivity (signal amplification possible); high specificity (two antibodies); can detect very low antigen concentrations
• Disadvantages: Requires two antibodies to different epitopes; more steps; more expensive

Diagrams

DIRECT ELISA:
 ___________Plate___________
|  Ag  Ag  Ag  Ag  Ag  Ag  |
|  |   |   |   |   |   |   |
| Ab* Ab* Ab* Ab* Ab* Ab*  |  (* = enzyme-labelled)
 ---------------------------
         ↓ + Substrate
         Color proportional to antigen

SANDWICH ELISA:
 ___________Plate___________
|  Ab  Ab  Ab  Ab  Ab  Ab  |  (capture antibody)
|  |   |   |   |   |   |   |
|  Ag  Ag  Ag  Ag  Ag  Ag  |  (antigen from sample)
|  |   |   |   |   |   |   |
| Ab* Ab* Ab* Ab* Ab* Ab*  |  (detection antibody + enzyme)
 ---------------------------
         ↓ + Substrate
         Color - high sensitivity

Key Differences Table

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Q-2(ii): Protein Metabolism and Significance of Post-Translational Modifications

Protein Metabolism

• Stomach: Pepsin (works at pH 1.5-2) cleaves peptide bonds; HCl denatures proteins
• Pancreatic enzymes: Trypsin, Chymotrypsin, Elastase (endopeptidases); Carboxypeptidases A and B (exopeptidases)
• Intestinal brush border: Aminopeptidases, Dipeptidases
• Final products: Free amino acids, di- and tripeptides absorbed via active transport (Na+-dependent)

• Transamination: Amino group transferred to alpha-ketoglutarate → glutamate (aminotransferases, requires B6/pyridoxal phosphate)
• Oxidative deamination: Glutamate → alpha-ketoglutarate + NH4+ (glutamate dehydrogenase in liver mitochondria)
• Urea cycle: NH4+ is detoxified to urea in liver → excreted by kidneys
• Carbon skeletons enter as pyruvate, acetyl-CoA, TCA intermediates → energy or gluconeogenesis

• Positive (growth, pregnancy, anabolism)
• Negative (starvation, illness, catabolism)
• Neutral (normal adults)

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Post-Translational Modifications (PTMs)

• Abnormal phosphorylation: cancer signalling (e.g., BCR-ABL in CML)
• Defective glycosylation: lysosomal storage diseases, CDG syndromes
• Abnormal ubiquitination: neurodegenerative diseases (Parkinson's, Alzheimer's)
• Defective hydroxylation: scurvy (Vitamin C deficiency)
• Defective carboxylation: bleeding disorders (Vitamin K deficiency)

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Q-2(iii): β-Oxidation vs ω-Oxidation of Fatty Acids - Pathway, Location, and Energy Yield

β-Oxidation (Beta-Oxidation)

1. Fatty acid activated to Fatty acyl-CoA by acyl-CoA synthetase (in cytoplasm, uses 2 ATP equivalents)
2. Transport into mitochondria via carnitine shuttle (carnitine acyltransferase I on outer membrane, rate-limiting step)

1. Oxidation - FAD-dependent: Acyl-CoA → Trans-Enoyl-CoA (produces FADH2)
2. Hydration - Water added: Trans-Enoyl-CoA → L-3-Hydroxyacyl-CoA
3. Oxidation - NAD+-dependent: Hydroxyacyl-CoA → 3-Ketoacyl-CoA (produces NADH)
4. Thiolysis - CoA added: 3-Ketoacyl-CoA → Acetyl-CoA + shortened Acyl-CoA (by 2 carbons)

• 7 cycles of β-oxidation → 7 FADH2 + 7 NADH + 8 Acetyl-CoA
• 7 FADH2 × 1.5 ATP = 10.5 ATP
• 7 NADH × 2.5 ATP = 17.5 ATP
• 8 Acetyl-CoA × 10 ATP (via TCA) = 80 ATP
• Total = 108 ATP - 2 ATP (activation) = 106 net ATP

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ω-Oxidation (Omega-Oxidation)

1. Terminal methyl (-CH3) group → hydroxylated to omega-hydroxy fatty acid (by cytochrome P450, requires NADPH and O2)
2. Omega-hydroxy fatty acid → oxidized to omega-aldehyde (by alcohol dehydrogenase)
3. Omega-aldehyde → oxidized to dicarboxylic acid (by aldehyde dehydrogenase)
4. Dicarboxylic acid can undergo β-oxidation from both ends

• Minor pathway under normal conditions (<5% of fatty acid oxidation)
• Becomes important when β-oxidation is impaired (e.g., Zellweger syndrome - peroxisomal disorder)
• Generates dicarboxylic acids excreted in urine (dicarboxylic aciduria) - diagnostic marker
• Important for metabolism of certain drugs and xenobiotics
• Medium-chain fatty acids are good substrates

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Comparison Table

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Q-3 (Very Short Notes - Any Five) [15 Marks - 3 marks each]

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Q-3(i): Role of Essential Minerals in Maintaining Physiological Homeostasis

• Electrolytes (Na+, K+, Ca2+) maintain resting membrane potentials and cardiac rhythm
• Ca2+ and phosphate regulate bone remodeling (parathyroid-calcitonin axis)
• Trace elements are essential cofactors without which key enzymatic reactions fail

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Q-3(ii): Role of Osmotic Pressure in Nutrient Absorption

1. Driving water absorption: After active transport of nutrients (glucose, amino acids) into enterocytes, the intracellular osmolarity rises. Water follows by osmosis from the intestinal lumen into the cells (and then into portal capillaries), carrying dissolved nutrients along with it (solvent drag).
2. Paracellular transport: Osmotic gradients drive water through tight junctions between enterocytes, taking small dissolved molecules (electrolytes, small nutrients) with it.
3. Glucose and Na+ coupled transport: Na+/glucose cotransporter (SGLT1) moves glucose into cells; intraluminal osmolarity in the fed state drives water absorption which concentrates nutrients and enhances passive absorption.
4. Colloidal osmotic pressure (oncotic pressure): Plasma proteins (albumin) in blood capillaries create a colloid osmotic pressure (~25 mmHg) that draws absorbed nutrients and water from interstitial fluid into capillaries. Hypoalbuminemia → edema and impaired nutrient uptake.
5. Hyperosmolar vs. hypoosmolar solutions: Hyperosmolar solutions (e.g., high-sugar drinks) draw water into the gut lumen (osmotic diarrhea), impairing nutrient absorption. Isotonic solutions (ORS - oral rehydration salts, 245 mOsm/L) optimize absorption.
6. Lymphatic absorption of fats: Dietary fats (chylomicrons) are too large for capillary absorption; they enter lacteals (lymphatics) where lower oncotic pressure allows entry. The hydrostatic pressure gradient drives lymph flow.

• Oral rehydration therapy (ORS) exploits Na+-glucose cotransport and osmotic water absorption
• Osmotic diarrhea (lactulose, sorbitol, Mg2+ salts) results from non-absorbable osmoles drawing water into gut
• Hypoalbuminemia (liver disease, malnutrition) impairs fluid return to capillaries → ascites and edema

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Q-3(iii): Well-Labeled Diagrams of tRNA and mRNA with Major Role in Protein Synthesis

mRNA (Messenger RNA)

5'----[5' Cap (7-methylguanosine)]--[5' UTR]--[AUG]--[CODING SEQUENCE (codons)]--[STOP codon]--[3' UTR]--[Poly-A tail (AAAA...)]----3'

• 5' Cap: 7-methylguanosine added post-transcriptionally; protects mRNA, aids ribosome binding
• 5' UTR: Untranslated region; contains Kozak sequence for ribosome recognition
• Start codon AUG: Codes for methionine; initiates translation
• Coding sequence: Series of triplet codons (each = 3 nucleotides = 1 amino acid)
• Stop codons: UAA, UAG, UGA - terminates translation
• 3' UTR: Regulatory sequences, stability elements
• Poly-A tail: 100-200 adenine nucleotides; protects mRNA from degradation, aids nuclear export and translation

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tRNA (Transfer RNA)

                    [Acceptor Stem]
                   3'-CCA-OH (amino acid attachment site)
                   5'-end
                    |
             [TΨC Arm] --- [D (Dihydrouridine) Arm]
                    |
               [Variable loop]
                    |
             [Anticodon Arm]
                   |||
              [Anticodon] (3 bases complementary to mRNA codon)

• ~73-93 nucleotides; L-shaped 3D structure
• Acceptor stem: 3'-CCA-OH end where specific amino acid is attached by aminoacyl-tRNA synthetase
• Anticodon loop: 3 nucleotide sequence that base-pairs with complementary codon on mRNA (antiparallel, anticodon)
• TΨC arm: Binds to ribosome (50S subunit)
• D arm: Contains dihydrouridine; binds aminoacyl-tRNA synthetase

• tRNA is the adaptor molecule - it decodes mRNA and brings the correct amino acid to the ribosome
• Aminoacyl-tRNA synthetase charges tRNA with the correct amino acid (using ATP → AMP + PPi)
• During translation: charged tRNA enters the A site of the ribosome; anticodon base-pairs with mRNA codon; peptide bond forms; tRNA moves to P site, then E site, then exits

• A site (Aminoacyl): New charged tRNA enters
• P site (Peptidyl): Growing peptide chain attached to tRNA
• E site (Exit): Uncharged tRNA exits the ribosome

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Q-3(iv): Basic Principle of Basal Metabolic Rate (BMR) and Impact on Energy Expenditure

• Energy needed for: heartbeat, breathing, circulation, body temperature maintenance, brain function, kidney filtration, cell repair/maintenance
• Represents ~60-75% of total daily energy expenditure (TDEE)

• Direct calorimetry: Measures heat produced by the body in an insulated chamber
• Indirect calorimetry (standard): Measures O2 consumed and CO2 produced; uses respiratory quotient (RQ = CO2 produced/O2 consumed)
• Harris-Benedict equation: Estimates BMR from height, weight, age, sex
  • Males: BMR = 66 + (13.7 × weight kg) + (5 × height cm) - (6.8 × age)
  • Females: BMR = 655 + (9.6 × weight) + (1.8 × height) - (4.7 × age)

• Total Daily Energy Expenditure (TDEE) = BMR + Physical Activity + Thermic Effect of Food (TEF)
• BMR is the largest component (~60-75%)
• Individuals with high BMR burn more calories at rest → less tendency for obesity
• Reduced BMR (hypothyroidism, starvation) → weight gain and fatigue
• Clinical use: BMR measurement guides nutritional support in ICU patients, guides weight management programs

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Q-3(v): Biochemical Events Involved in Muscle Contraction

Key Proteins

• Thick filaments: Myosin (heavy chain with globular head - ATPase activity)
• Thin filaments: Actin (F-actin), Tropomyosin, Troponin complex (TnT, TnI, TnC)
• Titin: Elastic protein connecting myosin to Z-disk
• Sarcomere: Functional unit of muscle (Z-disk to Z-disk)

Biochemical Steps of Contraction

• Motor nerve action potential → acetylcholine released at neuromuscular junction
• ACh binds nicotinic receptors → depolarization of muscle membrane (sarcolemma)
• Depolarization spreads along T-tubules → triggers L-type Ca2+ channels (DHP receptors)
• DHP receptor activates ryanodine receptor (RyR) on sarcoplasmic reticulum → Ca2+ floods into cytoplasm (from ~0.1 µM to ~10 µM)

• Ca2+ binds Troponin C (TnC) → conformational change
• Troponin-I releases actin inhibition; Tropomyosin shifts, exposing myosin-binding sites on actin

1. Rigor state: Myosin head tightly bound to actin (no ATP)
2. ATP binding: ATP binds myosin head → myosin releases actin
3. ATP hydrolysis: ATP → ADP + Pi; myosin head cocks to high-energy (90°) position
4. Weak binding: Cocked myosin head weakly attaches to next actin binding site
5. Power stroke: Pi released → strong bond formed; myosin head swings to 45° → actin filament slides (thin filament pulled toward center → sarcomere shortens = contraction)
6. ADP release: ADP released; myosin returns to rigor state
7. Cycle repeats as long as Ca2+ and ATP are available

• Motor nerve stimulation stops → Ca2+ pumped back into SR by SERCA pump (ATP-dependent)
• Cytoplasmic Ca2+ falls → Ca2+ dissociates from TnC
• Tropomyosin moves back to block actin binding sites
• Cross-bridges detach → muscle relaxes

Energy Sources for Contraction:

• Immediate: Phosphocreatine (CP) donates phosphate to ADP → ATP (creatine kinase)
• Short-term: Glycolysis (anaerobic) → ATP + lactate
• Long-term: Oxidative phosphorylation (aerobic) - fats and glucose via TCA + ETC

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Q-3(vi): Essential vs. Non-Essential Amino Acids with Their Roles

• Essential amino acids (EAA): Cannot be synthesized by the human body (or in adequate amounts) - must be obtained from diet
• Non-essential amino acids (NEAA): Can be synthesized endogenously from metabolic precursors

Essential Amino Acids (9)

Non-Essential Amino Acids (11)

• Phenylketonuria (PKU): Deficiency of phenylalanine hydroxylase → toxic phenylalanine accumulation → intellectual disability
• Homocystinuria: Methionine metabolism defect
• Maple syrup urine disease: BCAA (Leu, Ile, Val) catabolism defect
• Deficiency of Trp → pellagra (niacin deficiency) in low-protein diets (maize-based)

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Q-3(vii): Classification of Vitamins into Fat-Soluble and Water-Soluble with Physiological Functions

FAT-SOLUBLE VITAMINS (ADEK)

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WATER-SOLUBLE VITAMINS (B complex + C)

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• Fat-soluble: ADEK - stored in liver, need bile, can cause toxicity
• Water-soluble: B1, B2, B3, B5, B6, B7, B9, B12, C - excreted in urine, deficiency faster
• The B vitamins primarily act as enzyme cofactors in metabolic pathways

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Quick Navigation Guide

• For Q1, attempt only one of the two essay options
• For Q2, attempt only two of the three short notes
• For Q3, attempt only five of the seven very short notes
• Draw neat labeled diagrams wherever asked (nucleosome, ELISA, tRNA cloverleaf, sarcomere) - they carry marks
• For the fatty acid oxidation question, clearly state the location (mitochondria vs ER) as that is a key differentiator