Biomolecules (Protoplasm) Essentials

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1. Introduction to Biomolecules All living organisms are made up of the same elements and compounds. The relative abundance of carbon and hydrogen is higher in living beings than in the earth's crust. Protoplasm: The living substance within a cell, comprising the cytoplasm and nucleus. It is a complex mixture of biomolecules. 2. Chemical Analysis of Living Tissues (1) Acid Soluble vs. Acid Insoluble Pools To analyze chemical composition, living tissue (e.g., a piece of liver or a plant leaf) is ground in trichloroacetic acid ($Cl_3CCOOH$). The resulting slurry is filtered, yielding two fractions: Filtrate (Acid Soluble Pool): Contains micromolecules (molecular weight 18-800 daltons, e.g., amino acids, simple sugars, nucleotides). Also referred to as the 'cytoplasmic fraction'. Retentate (Acid Insoluble Pool): Contains macromolecules (molecular weight > 10,000 daltons, e.g., proteins, nucleic acids, polysaccharides). Lipids: Although their molecular weight is generally not above 800 Da, lipids are found in the acid insoluble fraction. This is because they form vesicles and are not soluble in the aqueous trichloroacetic acid. Biomolecules: All carbon compounds synthesized by living organisms. (2) Ash Analysis for Inorganic Constituents If a living tissue is burnt, all organic compounds are oxidized to gaseous form ($CO_2$, water vapor) and removed. The remaining residue is called ash , which contains inorganic elements (e.g., $Ca^{++}$, $Mg^{++}$, $Na^+$, $K^+$, $PO_4^{3-}$, $SO_4^{2-}$). Water is the most abundant chemical in living organisms. Average Composition of Cells Component % of total cellular mass Water 70-90 Proteins 10-15 Carbohydrates 3 Lipids 2 Nucleic acids 5-7 Ions 1 Comparison of Elements in Non-living and Living Matter Element Earth's crust (% weight) Human body (% weight) Hydrogen (H) 0.14 0.5 Carbon (C) 0.03 18.5 Oxygen (O) 46.6 65.0 Nitrogen (N) Very little 3.3 Sulphur (S) 0.03 0.3 Sodium (Na) 2.8 0.2 Calcium (Ca) 3.6 1.5 Magnesium (Mg) 2.1 0.1 Silicon (Si) 27.7 Negligible This table highlights that elements like C, H, N, O are significantly more abundant in living matter than in the earth's crust, reflecting their role as building blocks of biomolecules. Inorganic Constituents of Living Tissues (Ionic Form) Component Formula Sodium ion $Na^+$ Potassium ion $K^+$ Calcium ion $Ca^{++}$ Magnesium ion $Mg^{++}$ Chloride ion $Cl^-$ Bicarbonate $HCO_3^-$ Phosphate $PO_4^{3-}$ Sulphate $SO_4^{2-}$ Water $H_2O$ 3. Primary and Secondary Metabolites Metabolites: All biomolecules found in living systems. Primary metabolites: Directly involved in normal growth, development, and reproduction. They have identifiable functions (e.g., amino acids, sugars, nucleotides, fatty acids, vitamins, chlorophylls). Secondary metabolites: Organic compounds not directly involved in primary metabolic processes. Their functions are not always clear for the organism producing them, but many have ecological roles (e.g., defense, attractants) or economic importance for humans (e.g., drugs, pigments, rubber). Found predominantly in plants, fungi, and microbes. Examples of Secondary Metabolites Category Examples Pigments Carotenoids, Anthocyanins Alkaloids Morphine, Codeine, Nicotine, Quinine Terpenoids Monoterpenes, Diterpenes, Gibberellins Essential oils Lemon grass oil, Peppermint oil Toxins Abrin, Ricin Lectins Concanavalin A Drugs Vinblastine, Curcumin, Taxol Polymeric substances Rubber, Gums, Cellulose (structural, but often listed here in context of diverse plant products) 4. Carbohydrates (Saccharides) (1) General Characteristics Compounds of Carbon, Hydrogen and Oxygen. Generally, the ratio of H:O is 2:1 (like water), thus called "hydrates of carbon". General formula: $C_x (H_2O)_y$. Serve as primary energy sources and structural components. Classified based on the number of sugar units upon hydrolysis. (2) Monosaccharides (Simple Sugars) Simplest carbohydrates; cannot be hydrolyzed into smaller sugar units. Contain a free aldehyde (aldose) or ketone (ketose) group. All monosaccharides are reducing sugars because their free aldehyde/ketone group can reduce $Cu^{++}$ to $Cu^{+}$ (basis of Benedict's and Fehling's tests). Can exist as linear chains or, more commonly, as cyclic (ring) structures in aqueous solutions. Six-membered ring: pyranose (e.g., glucose). Five-membered ring: furanose (e.g., fructose, ribose). Anomers: Isomers differing in configuration only at the anomeric carbon ($C_1$ in aldoses, $C_2$ in ketoses) (e.g., $\alpha$-glucose and $\beta$-glucose). Epimers: Isomers differing in configuration at only one chiral carbon atom, other than the anomeric carbon (e.g., glucose and galactose differ at $C_4$). Occur in D- and L- forms (stereoisomers), determined by the configuration of the chiral carbon furthest from the carbonyl group. Most biological sugars are D-forms. Classification of Monosaccharides by Carbon Number Trioses (3C): $C_3H_6O_3$. E.g., Glyceraldehyde (aldotriose), Dihydroxyacetone (ketotriose). Important intermediates in glycolysis. Tetroses (4C): $C_4H_8O_4$. E.g., Erythrose (aldotetrose), Erythrulose (ketotetrose). Pentoses (5C): $C_5H_{10}O_5$. E.g., Ribose (in RNA), Deoxyribose ($C_5H_{10}O_4$, in DNA - exception to general formula), Ribulose, Xylulose. Hexoses (6C): $C_6H_{12}O_6$. E.g., Glucose, Fructose, Galactose, Mannose. Glucose: "Blood sugar", primary energy source. Fructose: "Fruit sugar", sweetest natural sugar. Galactose: Component of lactose (milk sugar). Heptoses (7C): $C_7H_{14}O_7$. E.g., Sedoheptulose. (3) Oligosaccharides Yield 2 to 10 monosaccharide units upon hydrolysis. Monosaccharides are linked by glycosidic bonds (covalent bonds formed by dehydration synthesis). Oligosaccharides are generally water-soluble and sweet (sugars). Disaccharides (2 units): Maltose (Malt sugar): Glucose + Glucose ($\alpha$-1,4-glycosidic linkage). Reducing sugar. Lactose (Milk sugar): Glucose + Galactose ($\beta$-1,4-glycosidic linkage). Reducing sugar. Sucrose (Table sugar): Glucose + Fructose ($\alpha$-1,2-glycosidic linkage). Non-reducing sugar (anomeric carbons are involved in the bond). (4) Polysaccharides (Glycans) Long chains of monosaccharide units (hundreds to thousands). Suffix '---an' is often added (e.g., glucans for glucose polymers). Generally insoluble in water, not sweet, and non-reducing (though one end of a chain can be reducing). Serve as storage compounds or structural components. Form helical structures that can hold iodine ($I_2$) molecules, giving characteristic colors. Types of Polysaccharides (A) Homopolysaccharides (Homoglycans): Composed of only one type of monosaccharide unit. Starch: Primary energy storage in plants. Polymer of $\alpha$-D-glucose. Amylose: Unbranched chain, $\alpha$-1,4-glycosidic linkages. Gives blue color with iodine. Amylopectin: Branched chain, $\alpha$-1,4-glycosidic linkages in linear parts and $\alpha$-1,6-glycosidic linkages at branch points. Gives red-violet color with iodine. Glycogen: Primary energy storage in animals ("animal starch"). Highly branched polymer of $\alpha$-D-glucose. Similar to amylopectin but more branched. Stored mainly in liver and muscles. Gives red-brown color with iodine. Cellulose: Major structural component of plant cell walls. Linear polymer of $\beta$-D-glucose units linked by $\beta$-1,4-glycosidic bonds. Humans cannot digest cellulose due to the lack of cellulase enzyme. Does not give color with iodine (linear structure does not coil). Chitin: Structural polysaccharide found in fungal cell walls and arthropod exoskeletons. Polymer of N-acetylglucosamine units linked by $\beta$-1,4-glycosidic bonds. Second most abundant polysaccharide after cellulose. Inulin: Storage polysaccharide in some plants (e.g., Dahlia, Artichoke). Linear polymer of fructose units linked by $\beta$-1,2-glycosidic bonds. Used to assess kidney function (glomerular filtration rate) as it is freely filtered and not reabsorbed. (B) Heteropolysaccharides (Heteroglycans): Composed of two or more different types of monosaccharide units or their derivatives. E.g., Pectins, Hemicellulose (plant cell walls), Hyaluronic acid, Chondroitin sulfate, Heparin (components of extracellular matrix and connective tissues). 5. Lipids Diverse group of organic compounds that are largely nonpolar and thus insoluble in water, but soluble in organic solvents (e.g., ether, chloroform, benzene). Composed mainly of C, H, O, but with a much lower proportion of oxygen compared to carbohydrates. Do not form true polymers (not built from repeating monomer units like proteins or nucleic acids). Serve as energy storage, structural components of membranes, and signaling molecules. (1) Simple Lipids (Neutral Fats) Esters of fatty acids and glycerol (or other alcohols). Triglycerides (Triacylglycerols): Most common type of fat. Consist of one glycerol molecule esterified with three fatty acid molecules. Formed by dehydration synthesis (esterification). Can be fats (solid at room temp, higher melting point, rich in saturated fatty acids) or oils (liquid at room temp, lower melting point, rich in unsaturated fatty acids). Glycerol is a trihydroxy propane. Fatty acids: Long hydrocarbon chains (4-24 carbons) with a carboxyl group at one end. Saturated fatty acids: Contain only single bonds between carbon atoms (e.g., palmitic acid-16C, stearic acid-18C). Tend to be solid at room temperature. Unsaturated fatty acids: Contain one or more double bonds between carbon atoms. Monounsaturated fatty acids (MUFA): One double bond (e.g., oleic acid - 18C, 1 double bond). Polyunsaturated fatty acids (PUFA): Two or more double bonds (e.g., linoleic acid - 18C, 2 double bonds; linolenic acid - 18C, 3 double bonds; arachidonic acid - 20C, 4 double bonds). Essential fatty acids: Cannot be synthesized by the body and must be obtained from the diet (e.g., linoleic acid, linolenic acid). Waxes: Esters of a long-chain fatty acid and a long-chain alcohol. Form protective coatings (e.g., on leaves, animal fur). (2) Compound (Conjugated) Lipids Contain fatty acids, alcohol, and additional groups. Phospholipids: Major components of cell membranes. Composed of a glycerol backbone, two fatty acids, a phosphate group, and usually a small polar molecule (e.g., choline, ethanolamine) attached to the phosphate. Amphipathic: Possess both hydrophilic (polar head, phosphate group) and hydrophobic (nonpolar tails, fatty acids) regions. This property allows them to form lipid bilayers in aqueous environments. E.g., Lecithin (phosphatidylcholine), Phosphatidylethanolamine. Glycolipids: Contain a sugar group instead of a phosphate group. Found in cell membranes, especially in nerve cells. (3) Derived Lipids Substances derived from simple and compound lipids by hydrolysis. Steroids: Characterized by a distinctive four-ring carbon skeleton (steroid nucleus). E.g., Cholesterol (precursor for other steroids, component of animal cell membranes), steroid hormones (estrogen, testosterone, cortisol), vitamin D, bile acids. Terpenes: Derived from isoprene units. Include fat-soluble vitamins (A, E, K), carotenoids (pigments), essential oils. 6. Proteins (1) General Characteristics From Greek "proteios" meaning "of prime importance" or "holding first place." Most abundant macromolecules in living cells. Essential elements: C, H, O, N, and often S (in cysteine, methionine). Are heteropolymers of amino acids. Perform a vast array of functions: enzymes, structural components, transport, hormones, defense, receptors, movement. (2) Amino Acids (Building Blocks of Proteins) Organic compounds containing both an amino group ($-NH_2$) and a carboxyl group ($-COOH$) attached to the same carbon atom (the $\alpha$-carbon). General structure: A central $\alpha$-carbon bonded to an amino group, a carboxyl group, a hydrogen atom, and a variable side chain (R-group). Amphoteric nature: Can act as both acids (due to -COOH) and bases (due to -$NH_2$). Zwitterionic form: At physiological pH, amino acids exist as zwitterions (dipolar ions) with both groups ionized ($-NH_3^+$ and $-COO^-$), resulting in a net neutral charge at their isoelectric point. There are 20 common standard amino acids that make up proteins. Classification of Amino Acids based on R-group: Nonpolar, aliphatic: Glycine, Alanine, Valine, Leucine, Isoleucine, Methionine, Proline. Aromatic: Phenylalanine, Tyrosine, Tryptophan. Polar, uncharged: Serine, Threonine, Cysteine, Asparagine, Glutamine. Acidic (negatively charged): Aspartic acid, Glutamic acid. Basic (positively charged): Lysine, Arginine, Histidine. Essential amino acids: Cannot be synthesized by the human body and must be obtained from the diet (e.g., Valine, Leucine, Isoleucine, Lysine, Methionine, Phenylalanine, Tryptophan, Threonine). Histidine and Arginine are conditionally essential. Non-essential amino acids: Can be synthesized by the body. All amino acids (except Glycine, which has two H atoms on the $\alpha$-carbon) are chiral and exist as L- and D-stereoisomers. Only L-amino acids are found in proteins. Amino acids are linked by peptide bonds (covalent amide bonds) formed by dehydration synthesis between the carboxyl group of one amino acid and the amino group of another. (3) Levels of Protein Structure Proteins fold into specific three-dimensional structures essential for their function. (A) Primary Structure: The unique linear sequence of amino acids in a polypeptide chain. Determined by genetic information. Held together by peptide bonds. Dictates all higher levels of protein structure. Example: The specific sequence of amino acids in insulin. (B) Secondary Structure: Local spatial arrangement of the polypeptide backbone, stabilized by hydrogen bonds between backbone atoms (not R-groups). $\alpha$-Helix: A common spiral structure, typically right-handed, with about 3.6 amino acid residues per turn. Hydrogen bonds form between the C=O of one peptide bond and the N-H of a peptide bond four residues away. E.g., Keratin (hair, nails), Myosin. $\beta$-Pleated Sheet: Polypeptide chains lie side-by-side in a zig-zag fashion, forming a pleated, sheet-like structure. Hydrogen bonds form between adjacent strands. Can be parallel or antiparallel. E.g., Fibroin (silk). Other structures: $\beta$-turns, random coils. (C) Tertiary Structure: The overall three-dimensional folding of a single polypeptide chain, including the spatial arrangement of all its atoms (including R-groups). Stabilized by various interactions between R-groups: Hydrophobic interactions: Nonpolar groups cluster in the interior. Ionic bonds (salt bridges): Between oppositely charged R-groups. Hydrogen bonds: Between polar R-groups. Disulfide bonds: Strong covalent bonds formed between the sulfhydryl groups of two cysteine residues ($-\text{S-S-}$) - critical for stabilizing many extracellular proteins. Van der Waals forces. Often results in globular proteins (e.g., enzymes, antibodies) that are soluble and compact, or fibrous proteins that are elongated and insoluble. (D) Quaternary Structure: The arrangement of multiple polypeptide subunits (two or more tertiary structures) to form a functional protein complex. Subunits are held together by non-covalent interactions (hydrophobic, ionic, hydrogen bonds) and sometimes disulfide bonds. E.g., Hemoglobin (four subunits: two $\alpha$ and two $\beta$), Immunoglobulins, many enzymes. Protein Denaturation Loss of a protein's native (functional) three-dimensional structure (secondary, tertiary, and quaternary) due to disruption of non-covalent interactions and disulfide bonds. Caused by extreme heat, pH changes, strong acids/bases, organic solvents, heavy metal ions, radiation. Often irreversible, leading to loss of biological activity. Primary structure (peptide bonds) usually remains intact. Examples of Protein Functions Protein Type Function Example Enzymes Catalyze biochemical reactions Trypsin, Amylase, DNA Polymerase Structural Provide support Collagen (connective tissue), Keratin (hair, nails), Actin, Myosin (muscle) Transport Carry substances Hemoglobin (oxygen), Albumin (fatty acids), GLUT-4 (glucose) Hormonal Coordinate bodily activities Insulin, Growth Hormone Defense Protect against disease Antibodies (immunoglobulins) Receptor Respond to chemical stimuli Receptors for hormones, neurotransmitters Motor Movement Actin, Myosin (muscle contraction), Kinesin (intracellular transport) Storage Store amino acids Ovalbumin (egg white), Casein (milk), Ferritin (iron storage) 7. Nucleic Acids (DNA & RNA) Macromolecules found in the acid-insoluble fraction. Polymers of nucleotide monomers. Function as genetic material, storing and transmitting hereditary information, and are involved in protein synthesis. (1) Components of a Nucleotide A nucleotide consists of three parts: A Nitrogenous Base (Heterocyclic compound): Purines (double-ring structure): Adenine (A) and Guanine (G). Pyrimidines (single-ring structure): Cytosine (C), Thymine (T, in DNA only), and Uracil (U, in RNA only). A Pentose Sugar (Monosaccharide): Ribose: In RNA (has -OH group at 2' carbon). 2'-Deoxyribose: In DNA (lacks -OH group at 2' carbon). A Phosphate Group: Derived from phosphoric acid ($H_3PO_4$). Nucleoside: Nitrogenous base + Sugar (without phosphate). E.g., Adenosine (Adenine + Ribose), Guanosine (Guanine + Ribose), Cytidine (Cytosine + Ribose), Uridine (Uracil + Ribose). Deoxyribonucleosides in DNA: Deoxyadenosine, Deoxyguanosine, Deoxycytidine, Deoxythymidine. Nucleotide: Nucleoside + Phosphate group. Also called nucleoside phosphates or "nucleic acids" in their monomeric form. E.g., Adenosine monophosphate (AMP), Guanosine monophosphate (GMP), Cytidine monophosphate (CMP), Uridine monophosphate (UMP). Deoxyribonucleotides in DNA: Deoxyadenosine monophosphate (dAMP), Deoxyguanosine monophosphate (dGMP), Deoxycytidine monophosphate (dCMP), Deoxythymidine monophosphate (dTMP). (2) Polynucleotide Chain Formation Nucleotides are linked together by phosphodiester bonds . A phosphodiester bond forms between the phosphate group attached to the 5' carbon of one sugar and the hydroxyl group on the 3' carbon of the next sugar. This creates a sugar-phosphate backbone with nitrogenous bases projecting outwards. The chain has directionality, with a free 5'-phosphate end and a free 3'-hydroxyl end. (3) Types of Nucleic Acids (A) Deoxyribonucleic Acid (DNA): Contains deoxyribose sugar and bases A, G, C, T. Typically a double helix structure (Watson-Crick model). Two antiparallel polynucleotide strands coiled around a common axis. Strands are held together by hydrogen bonds between complementary base pairs: A pairs with T (2 H-bonds), and G pairs with C (3 H-bonds). Stores genetic information, forms chromosomes. (B) Ribonucleic Acid (RNA): Contains ribose sugar and bases A, G, C, U. Typically a single-stranded molecule, though it can fold into complex 3D structures with internal base pairing. Involved in various aspects of gene expression and protein synthesis. Types of RNA: mRNA (messenger RNA): Carries genetic code from DNA to ribosomes. tRNA (transfer RNA): Carries specific amino acids to the ribosome during protein synthesis. rRNA (ribosomal RNA): Structural and catalytic component of ribosomes. Other regulatory RNAs (e.g., miRNA, siRNA). 8. Enzymes Biological catalysts, almost all are proteins (except some RNA molecules called ribozymes). Speed up biochemical reactions without being consumed in the reaction. Highly specific for their substrates. Have an active site where the substrate binds. Mechanism: Lower the activation energy of a reaction. Sensitive to temperature and pH (optimal activity at specific conditions). Can be regulated by activators and inhibitors. Cofactors: Non-protein components required for enzyme activity. Coenzymes: Organic molecules, often derived from vitamins (e.g., NAD, FAD, Coenzyme A). Metal ions: Inorganic ions (e.g., $Zn^{++}$, $Mg^{++}$, $Fe^{++}$). Apoenzyme: Protein part of an enzyme. Holoenzyme: Apoenzyme + cofactor (the complete, active enzyme). Competitive inhibition: Inhibitor resembles substrate and binds to active site. Non-competitive inhibition: Inhibitor binds to a site other than the active site, altering enzyme conformation.