Biochem & Chemical Tests - Quick Review Notes

Amino Acids & Buffers

  • Amino acids: NH₃⁺ on α-amino group and COO⁻ on α-carboxyl at certain pH; side chains vary in pKa.

  • Forms of amino acids by pH:

    • Form I (fully protonated): net +1.

    • Form II (isoelectric point, pI): net zero.

    • Form III (fully deprotonated): net −1.

  • Triprotic amino acids (D, E, H, K, R) have 3 buffering regions due to three ionizable groups.

  • Glycine is achiral (no chiral center); all others are chiral.

  • pI concept: pH at which net charge = 0; used for extraction and separation.

  • 2D electrophoresis separates by mass and pI.

  • Lysine example: pKa values ~2.18 (carboxyl), ~8.95 (α-amino), ~10.79 (side chain); pI ≈ (8.95 + 10.79)/2 ≈ 9.9.

  • Aromatic amino acids absorb UV at 280 nm: Trp > Tyr > Phe; used for quantification.

Protein Structure & Composition

  • Aliphatic/nonpolar residues drive hydrophobic cores; β-branched residues (V, I, L) strengthen hydrophobic packing.

  • Proline disrupts hydrogen bonding in helices (no amide H-bond donor).

  • Aromatic residues (F, Y, W) absorb UV; W absorbs the most.

Secondary & Higher-Order Structure (Key features)

  • Alpha helices: C=O(i)–N–H(i+4) hydrogen bonds; transmembrane helices common.

  • Beta sheets: parallel or antiparallel strands connected by H-bonds.

  • Quaternary structure: multiple polypeptide chains; cooperativity possible in multimeric proteins.

  • Proline and glycine influence turns and flexibility; glycine is highly flexible.

  • Hydrogen bonding and electrostatics govern folding and stability.

Post-Translational Modifications & Phosphorylation

  • Serine, threonine, and tyrosine can be phosphorylated via nucleophilic attack on a phosphate.

  • Phosphorylation forms phosphoester bonds.

Hemoglobin, Myoglobin & Cooperativity

  • Hemoglobin (Hb) is a tetramer (2 α, 2 β); oxygen binding is cooperative.

  • Myoglobin is a monomer; acts as O₂ storage in muscle.

  • Cooperativity described by Hill coefficient: Hb ≈ 2.5–3; Myoglobin ≈ 1.

  • Allosteric states: T (deoxyhemoglobin) and R (oxyhemoglobin).

  • CO₂ binding forms carbaminohemoglobin; CO₂ and O₂ can influence each other (competitive binding at heme vicinity).

  • Higher affinity for O₂ shifts saturation curve left (allosteric effect).

Enzymes, Kinetics & Inhibition

  • Enzyme catalysis types: Oxidoreductases, Transferases, Hydrolases, Lyases, Isomerases, Ligases.

  • Mechanisms: acid-base, covalent, and metal ion catalysis.

  • Michaelis–Menten kinetics:

    • v=V<em>max[S]K</em>m+[S]v = \frac{V<em>{max} [S]}{K</em>m + [S]}

    • Km = substrate concentration at half-maximal velocity; low Km = high affinity; high Km = low affinity.

    • Vmax = maximum rate at saturating substrate.

  • Catalytic efficiency: K<em>catK</em>m\frac{K<em>{cat}}{K</em>m} with K<em>cat=V</em>max[E]totK<em>{cat} = \frac{V</em>{max}}{[E]_{tot}}

  • Lineweaver–Burk (linear) form: 1v=K<em>mV</em>max⋅1[S]+1Vmax\frac{1}{v} = \frac{K<em>m}{V</em>{max}} \cdot \frac{1}{[S]} + \frac{1}{V_{max}}

  • Enzyme inhibition types (affect Vmax and/or Km):

    • Competitive: binds free enzyme; increases Km; Vmax unchanged.

    • Uncompetitive: binds ES; decreases Km and Vmax; lines parallel.

    • Noncompetitive: binds E or ES; decreases Vmax; Km unchanged (pure noncompetitive).

    • Mixed: binds E and ES with varying affinities; Vmax decreases; Km may increase or decrease.

  • Kinetics visuals: Eadie–Hofstee/Lineweaver–Burk depict changes in Vmax and Km.

Enzyme Regulation & Activity

  • Kinetics and allosteric regulation explain sigmoidal vs hyperbolic binding curves (Hill coefficient >1 implies cooperativity).

Nucleotides, Nucleic Acids & Transcription

  • Nucleotides components: base (Purine A, G; Pyrimidines C, T, U), ribose/deoxyribose, phosphate.

  • Purines: A, G; Pyrimidines: C, T (DNA) or U (RNA).

  • Sugar types: deoxyribose (DNA) lacks 2'-OH; ribose (RNA) has 2'-OH; RNA is more labile to base-catalyzed hydrolysis.

  • DNA structure: phosphodiester backbone; antiparallel double helix; base pairing A–T (2 H-bonds) and G–C (3 H-bonds).

  • Histone packaging: DNA wrapped around histone octamers forming nucleosomes (10 nm); higher-order folding to chromatin.

  • RNA types: mRNA, rRNA, tRNA; also siRNA, miRNA, piRNA; RNA processing includes splicing (hnRNA).

  • Transcription: RNA polymerase reads template strand 3'→5' to synthesize 5'→3' mRNA; sense (coding) vs antisense (template).

  • 2'-OH in RNA makes it more prone to hydrolysis than DNA; RNA often mono-cistronic in eukaryotes, polycistronic in prokaryotes.

DNA Damage & Repair (Mutagenesis)

  • Exogenous mutagens:

    • Pyrimidine dimers from UV light; repair via photoreactivation (photolyases) uses visible light.

    • Alkylation: methyl/ethyl groups alter base bulk and expression (cigarette smoke example).

    • Bulky adducts from carcinogens distort DNA shape.

  • Endogenous: spontaneous deamination (C → U; 5-methylcytosine → T), depurination (apurine release) creating AP sites.

  • Mutations can be transitions (purine↔purine, pyrimidine↔pyrimidine) or transversions (purine↔pyrimidine).

Carbohydrates, Sugars & Polysaccharides

  • Monosaccharides: aldoses vs ketoses; ring forms: furanose (5-member) and pyranose (6-member).

  • D-forms predominate in nature.

  • Anomeric carbon can be α or β; α has OH on C1 down; β has OH up (in Haworth projection).

  • Disaccharides:

    • Lactose: galactose + glucose; β-1,4 glycosidic bond.

    • Maltose: two glucose units; α-1,4 glycosidic bond.

    • Sucrose: glucose + fructose; α-1,2 glycosidic bond.

  • Starch: amylose (linear, α-1,4) and amylopectin (branched, α-1,6 branches); iodine binds to amylose helical regions (Lugol’s test).

  • Cellulose: β-1,4 linkages; humans cannot digest.

  • Glycogen: highly branched α-1,4 with α-1,6 branches; stored in liver & muscle.

  • Chitin: monomer N-acetyl-D-glucosamine; β-1,4 linkages; in fungi exoskeletons.

  • Glycosaminoglycans (GAGs): repeating disaccharides (acidic sugar + amine sugar) with negative charge; highly hydrated, form ECM matrices; six classes include chondroitin sulfate, keratan sulfate, dermatan sulfate, hyaluronic acid, heparin, heparan sulfate.

  • Glycolipids: carbohydrate + lipid (glycosphingolipids); ABO blood group antigens often on glycolipids; important in infection and genetic disorders.

Biochemical Tests (Qualitative/Quantitative)

  • Biuret test: qualitative for proteins; blue → purple with peptide bonds in presence of Cu²⁺.

  • Bradford assay: protein quantity via Coomassie blue binding (spectrophotometric readout).

  • Ninhydrin test: detects amino acids; colorimetric changes (purple for most AAs, yellow for proline).

  • Benedict’s test: detects reducing sugars (monosaccharides and some disaccharides); color changes depend on reducing capacity; sucrose is non-reducing.

  • Lugol’s solution: starch presence; blue/black color with amylose due to helical inclusion of iodine.

  • Tollens & Fehling tests: aldehydes and reducing sugars; qualitative precipitation/color changes.

Oxyhemoglobin, Carbon Dioxide Transport & Allostery

  • Hb displays cooperativity; sigmoidal oxygen binding curve; Hill coefficient describes cooperativity (Hb ~2.5–3; Mb = 1).

  • Deoxyhemoglobin (T state) vs oxyhemoglobin (R state); CO2 binds to N-termini, forming carbamates; allosteric effects influence affinity.

  • CO stabilizes the R state, shifting O₂ saturation left; CO and O₂ can compete at the heme/nearby site.

Transcription & Translation (RNA World)

  • DNA is transcribed to RNA (mRNA, hnRNA, rRNA, tRNA, siRNA, miRNA, piRNA).

  • 2'-OH in RNA makes it more chemically reactive and less stable than DNA.

  • RNA processing includes splicing of hnRNA to produce mature mRNA.

  • Transcription uses a template strand; sense vs antisense nomenclature matters for coding sequences.