How To Analyse Chemical Composition?

To understand the chemical composition of living tissues, specifically the organic compounds, scientists perform chemical analysis. A common method involves grinding living tissue (e.g., vegetable, liver) in trichloroacetic acid. This creates a thick slurry, which is then strained through a filter (like cheesecloth or cotton) to separate it into two fractions:

• **Acid-soluble pool (Filtrate):** Contains thousands of small organic compounds with molecular weights ranging from approximately 18 to 800 daltons (Da). These are generally referred to as **micromolecules** or **biomolecules**.
• **Acid-insoluble fraction (Retentate):** Contains four main types of large organic compounds: proteins, nucleic acids, polysaccharides, and lipids. These are generally referred to as **macromolecules** or **biomacromolecules**, with molecular weights of ten thousand Da or more, except for lipids.

Specialized analytical techniques are used to isolate, purify, determine the molecular formula, and deduce the structure of specific organic compounds from living tissues. These carbon compounds obtained from living tissues are collectively called **biomolecules**.

Living organisms also contain inorganic elements and compounds. To analyse inorganic composition, a small amount of living tissue is weighed (wet weight), dried completely to remove water (giving dry weight), and then completely burnt to oxidise carbon compounds into gases (CO$_2$, water vapour), leaving behind **ash**. This ash contains inorganic elements (e.g., calcium, magnesium) and inorganic compounds (e.g., sulphate, phosphate), which can also be found in the acid-soluble pool.

From a biological perspective, the organic constituents are classified into categories like amino acids, nucleotide bases, fatty acids, sugars, etc.

• **Amino acids:** Organic compounds with an amino group (-NH$_2$) and an acidic carboxyl group (-COOH) attached to the same alpha ($\alpha$) carbon atom. They are substituted methanes with four substituent groups on the $\alpha$-carbon: hydrogen, carboxyl group, amino group, and a variable **R group**. The nature of the R group determines the type of amino acid. There are 20 types of amino acids that make up proteins. Examples of R groups include hydrogen (Glycine), methyl group (Alanine), hydroxymethyl (Serine). Amino acids can be acidic (e.g., glutamic acid), basic (e.g., lysine), or neutral (e.g., valine) based on the number of amino and carboxyl groups. Aromatic amino acids have aromatic rings in their R group (e.g., tyrosine, phenylalanine, tryptophan). Amino acids are ionizable due to the -NH$_2$ and -COOH groups and exist as zwitterions at neutral pH.

• **Lipids:** Generally water-insoluble. They can be simple fatty acids (carboxyl group attached to an R group hydrocarbon chain, e.g., palmitic acid with 16 carbons, arachidonic acid with 20 carbons). Fatty acids can be saturated (no double bonds) or unsaturated (one or more C=C double bonds). Glycerol is a simple lipid (trihydroxy propane). Many lipids are formed by esterification of fatty acids to glycerol, forming monoglycerides, diglycerides, and triglycerides (fats and oils, distinguished by melting point). Phospholipids contain phosphorous and a phosphorylated organic compound, found in cell membranes (e.g., lecithin).

• **Nitrogen bases:** Heterocyclic rings containing nitrogen. Examples: Adenine (A), Guanine (G) - Purines (double ring); Cytosine (C), Uracil (U), Thymine (T) - Pyrimidines (single ring).
• **Sugars (Carbohydrates):** Monosaccharides (simple sugars, e.g., glucose, ribose) and disaccharides.

• **Nucleosides:** Formed when a nitrogen base is attached to a sugar (ribose or deoxyribose). Examples: Adenosine (Adenine + ribose), Guanosine, Thymidine, Uridine, Cytidine.
• **Nucleotides:** Formed when a phosphate group is esterified to a nucleoside (nitrogen base + sugar + phosphate). Examples: Adenylic acid, Guanylic acid, Cytidylic acid, Uridylic acid, Thymidylic acid. Nucleic acids (DNA and RNA) are polymers of nucleotides.

Inorganic constituents in living tissues (Table 9.2 in text) include ions (Na$^+$, K$^+$, Ca$^{++}$, Mg$^{++}$), water (H$_2$O), and compounds (NaCl, CaCO$_3$, phosphates, sulphates).

Primary And Secondary Metabolites

Biomolecules in living organisms can be broadly classified into primary and secondary metabolites.

• **Primary Metabolites:** Compounds that have identifiable functions and play known, essential roles in the normal physiological processes of an organism (e.g., amino acids, sugars, nucleotides, lipids found in animal tissues). They are directly involved in growth, development, and reproduction.
• **Secondary Metabolites:** Thousands of compounds (e.g., alkaloids, flavonoids, rubber, essential oils, antibiotics, pigments, scents, gums, spices) found especially in plant, fungal, and microbial cells, which are not directly involved in the primary metabolic processes necessary for the host organism's immediate survival. Their specific roles in the host are not always fully understood, though some have ecological importance (e.g., defense). Many secondary metabolites are of significant use to human welfare (e.g., as drugs, spices, pigments, rubber).

Some Secondary Metabolites (Table 9.3 in text):

Pigments	Carotenoids, Anthocyanins, etc.
Alkaloids	Morphine, Codeine, etc.
Terpenoides	Monoterpenes, Diterpenes etc.
Essential oils	Lemon grass oil, etc.
Toxins	Abrin, Ricin
Lectins	Concanavalin A
Drugs	Vinblastin, curcumin, etc.
Polymeric substances	Rubber, gums, cellulose

Biomacromolecules

**Biomacromolecules** are large organic compounds found in living organisms, with molecular weights of ten thousand Da or above. They are found in the acid-insoluble fraction after chemical analysis.

The four main types of organic compounds in the acid-insoluble fraction are: **proteins, nucleic acids, polysaccharides, and lipids**.

With the exception of lipids, these macromolecules are **polymeric substances**, formed by the joining of many smaller monomer units (building blocks).

Lipids are an exception; although their individual molecular weights are small (less than 800 Da), they are found in the acid-insoluble fraction because they are often arranged into structures like cell membranes and other cellular membranes. When tissue is ground, these membranes break into pieces and form vesicles that are not water-soluble and thus separate with the acid-insoluble pool. Lipids themselves are not polymers, but they are considered part of the macromolecular fraction due to their association with insoluble cellular structures.

The acid-soluble pool primarily represents the cytoplasmic composition of micromolecules. The acid-insoluble fraction represents the macromolecules from the cytoplasm and organelles. Together, the acid-soluble and acid-insoluble fractions represent the entire chemical composition of living tissues.

In terms of abundance, **water** is the most abundant chemical in living organisms, making up 70-90% of the total cellular mass (Table 9.4 in text). Proteins, carbohydrates, lipids, nucleic acids, and ions are present in smaller percentages.

Average Composition of Cells (Table 9.4 in text):

Component	% of the total cellular mass
Water	70-90
Proteins	10-15
Carbohydrates	3
Lipids	2
Nucleic acids	5-7
Ions	1

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Proteins

**Proteins** are complex biomacromolecules that perform a wide variety of functions in living organisms. They are **polypeptides**, which are linear chains of **amino acids** linked together by **peptide bonds**.

A peptide bond is formed between the carboxyl group of one amino acid and the amino group of the next amino acid, with the removal of a water molecule.

Each protein is a polymer made up of amino acid monomers. Since there are 20 different types of amino acids (e.g., glycine, alanine, lysine, tryptophan), proteins are **heteropolymers** (made of different repeating units) rather than homopolymers (made of only one type of repeating unit).

Some amino acids are considered **essential** for human health and must be obtained from the diet. Others are **non-essential**, as the body can synthesize them. Dietary proteins are sources of essential amino acids.

Proteins perform diverse functions (Table 9.5 in text):

• **Structural:** Provide support and shape (e.g., collagen, keratin). Collagen is the most abundant protein in the animal world.
• **Enzymatic:** Catalyse biochemical reactions (e.g., trypsin, RuBisCO). RuBisCO (Ribulose bisphosphate Carboxylase-Oxygenase) is the most abundant protein in the entire biosphere.
• **Transport:** Move molecules across membranes or within the body (e.g., GLUT-4 for glucose transport, haemoglobin for oxygen transport).
• **Hormonal:** Some hormones are proteins (e.g., insulin).
• **Immunological:** Antibodies fight infectious agents.
• **Receptors:** Receive signals (smell, taste, hormones, etc.).

Some Proteins and their Functions (Table 9.5 in text):

Protein	Functions
Collagen	Intercellular ground substance (structural)
Trypsin	Enzyme (digestion)
Insulin	Hormone (regulates blood sugar)
Antibody	Fights infectious agents (immune response)
Receptor	Sensory reception (smell, taste, hormone, etc.)
GLUT-4	Enables glucose transport into cells

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Polysaccharides

**Polysaccharides** (carbohydrates) are another class of biomacromolecules found in the acid-insoluble fraction. They are long chains of **monosaccharides** (simple sugars) linked together by glycosidic bonds.

Examples of polysaccharides:

• **Cellulose:** A structural polysaccharide in plant cell walls. It is a **homopolymer** consisting only of glucose monomers linked by $\beta$-glycosidic bonds. Cellulose does not form complex helices and does not bind iodine (no blue colour). Paper and cotton are made of cellulose.
• **Starch:** A storage polysaccharide in plant tissues (e.g., in potatoes, grains). It is a polymer of glucose, existing in two forms: amylose (linear, helical) and amylopectin (branched). Starch forms helical secondary structures that can hold iodine molecules, resulting in a blue colour ($I_2$ test).
• **Glycogen:** A storage polysaccharide in animal tissues (e.g., liver, muscles). It is a highly branched polymer of glucose, similar in structure to amylopectin but more branched. It is often depicted with a reducing end and a non-reducing end with branches.

• **Inulin:** A polymer of fructose.
• **Chitin:** A complex structural polysaccharide forming the exoskeletons of arthropods and the cell walls of fungi. It is a homopolymer of N-acetyl glucosamine (a modified sugar).

More complex polysaccharides can be formed from amino-sugars and chemically modified sugars.

Nucleic Acids

**Nucleic acids** are biomacromolecules found in the acid-insoluble fraction. They are **polynucleotides**, polymers made up of repeating monomer units called **nucleotides**. Nucleic acids function as the **genetic material** of living organisms.

A nucleotide consists of three chemically distinct components:

1. A **heterocyclic compound** (a nitrogen base: Adenine, Guanine, Uracil, Cytosine, or Thymine).
2. A **monosaccharide pentose sugar** (Ribose in RNA, 2'-deoxyribose in DNA).
3. A **phosphoric acid** or phosphate group.

Nucleotides are linked together by phosphodiester bonds to form polynucleotide chains (nucleic acids). There are two main types of nucleic acids:

• **Deoxyribonucleic acid (DNA):** Contains deoxyribose sugar and the bases A, G, C, and T. DNA is typically double-stranded and stores genetic information.
• **Ribonucleic acid (RNA):** Contains ribose sugar and the bases A, G, C, and U. RNA is typically single-stranded and involved in protein synthesis and gene expression.

Structure Of Proteins

Proteins are heteropolymers of amino acids. Unlike simple molecular formulae or 2D structures, protein structure is described at four levels of organization, reflecting its complex 3D shape necessary for function.

1. **Primary Structure:** The linear sequence of amino acids in the polypeptide chain. It specifies the order in which the 20 different amino acids are linked by peptide bonds. The left end is the N-terminal amino acid (with free amino group), and the right end is the C-terminal amino acid (with free carboxyl group).
2. **Secondary Structure:** Local folding patterns of the polypeptide chain, stabilized by hydrogen bonds between amino acid backbones. Common secondary structures include the **alpha ($\alpha$)-helix** (coiling similar to a spiral staircase, usually right-handed in proteins) and **beta ($\beta$)-pleated sheets** (polypeptide chains lying parallel or anti-parallel, folded into pleated structures).

3. **Tertiary Structure:** The overall three-dimensional folding of a single polypeptide chain. It results from interactions (hydrogen bonds, ionic bonds, disulphide bonds, hydrophobic interactions) between the R groups of amino acids in different parts of the chain. This folding forms a specific compact 3D shape, often resembling a hollow woolen ball with crevices or pockets. The tertiary structure is essential for the protein's biological activity (e.g., forming the active site of an enzyme).

4. **Quaternary Structure:** The assembly of multiple polypeptide chains (subunits) to form a functional protein. Not all proteins have a quaternary structure, only those composed of two or more polypeptide subunits. It describes how these individual folded subunits are arranged relative to each other. Example: Adult human haemoglobin (Hb) has four subunits: two identical alpha ($\alpha$) subunits and two identical beta ($\beta$) subunits, arranged in a specific manner.

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Enzymes

**Enzymes** are biological catalysts that are essential for carrying out biochemical reactions in living cells at high rates. Most enzymes are **proteins**, but some nucleic acids (called **ribozymes**) also exhibit catalytic activity.

Like other proteins, enzymes have primary, secondary, and tertiary structures. When you look at the tertiary structure of an enzyme, you find specific crevices or pockets formed by the folding of the polypeptide chain. One such pocket is the **active site**.

The **active site** of an enzyme is a three-dimensional region (a crevice or pocket) where the **substrate** (the molecule undergoing the reaction) binds. The active site has a unique shape that is complementary to the shape of its specific substrate, ensuring enzyme specificity.

Enzyme catalysts differ from inorganic catalysts (which work efficiently at high temperatures and pressures) because enzymes are typically sensitive to temperature and pH. Most enzymes are denatured (lose their structure and activity) at high temperatures (above 40°C for many, though enzymes from thermophilic organisms can be stable at higher temperatures) and outside their optimal pH range.

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Chemical Reactions

A chemical reaction involves the breaking of old chemical bonds and the formation of new ones, leading to the transformation of substances. The **rate** or **velocity** of a chemical process is the amount of product formed per unit time.

Enzymes dramatically increase the rate of biochemical reactions. They do this by lowering the **activation energy** required for the reaction to occur.

Consider the reaction S $\to$ P, where a substrate S is converted to a product P. For this conversion, S must pass through a transition state, which is an unstable, high-energy intermediate structure. The difference in energy between the substrate and the transition state is the **activation energy**.

Enzymes facilitate the reaction by binding to the substrate at the active site and stabilizing the transition state or providing an alternative reaction pathway with a lower activation energy. This makes the conversion of substrate to product much easier and faster.

A multi-step chemical reaction catalyzed by a series of enzymes is called a **metabolic pathway** (e.g., glycolysis, where glucose is converted to pyruvic acid through ten enzyme-catalyzed steps).

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How Do Enzymes Bring About Such High Rates Of Chemical Conversions?

Enzymes achieve high reaction rates by lowering the activation energy of the reaction. They do this through a process involving the binding of the substrate to the enzyme's active site.

The steps in enzyme action (catalytic cycle):

1. **Substrate Binding:** The substrate (S) binds to the active site of the enzyme (E), forming an **enzyme-substrate complex (ES)**. The active site's shape allows specific binding of the substrate.
2. **Catalysis:** The binding of the substrate causes the enzyme to undergo a slight conformational change, fitting more tightly around the substrate (induced fit). Within the active site, the enzyme facilitates the breaking of bonds in the substrate or the formation of new bonds, converting the substrate into product(s) (P). This occurs via a transition state structure.
3. **Product Release:** The enzyme releases the product(s) from the active site.
4. **Enzyme Regeneration:** The free enzyme is then ready to bind another molecule of substrate and repeat the catalytic cycle.

The formation of the ES complex is crucial for lowering the activation energy and speeding up the reaction. The enzyme remains unchanged after the reaction.

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Nature Of Enzyme Action

The catalytic process follows the sequence: E + S $\to$ ES $\to$ EP $\to$ E + P.

The enzyme-substrate (ES) complex is a transient, highly reactive state essential for catalysis. The enzyme-product (EP) complex is an intermediate formed before the product is released.

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Factors Affecting Enzyme Activity

Several factors can influence the rate of an enzyme-catalyzed reaction by affecting the enzyme's structure (especially tertiary structure) and the binding of the substrate to the active site.

• **Temperature:** Enzyme activity is highest at an **optimum temperature**. Activity decreases both below and above the optimum. Low temperatures typically preserve the enzyme in an inactive state. High temperatures denature (unfold) the protein structure of the enzyme, permanently destroying its catalytic activity.

• **pH:** Enzyme activity is also highest at an **optimum pH**. Activity decreases outside this narrow range. Extreme pH values can cause denaturation. Different enzymes have different optimum pH values (e.g., pepsin works best in acidic stomach conditions, trypsin in alkaline intestinal conditions).

• **Concentration of Substrate:** At a fixed enzyme concentration, increasing the substrate concentration initially increases the reaction velocity (rate). As substrate concentration increases, more active sites are occupied, and the reaction rate increases. However, the rate eventually reaches a maximum velocity (V$_{max}$) and plateaus. This happens because all the active sites of the enzyme molecules are saturated with substrate, and the enzyme is working at its maximum capacity. Further increase in substrate concentration does not increase the rate unless more enzyme is added.

• **Inhibitors:** Specific chemicals called inhibitors can bind to the enzyme and decrease or shut off its activity.
  • **Competitive inhibitor:** Resembles the substrate in molecular structure and competes with the substrate for binding to the active site. This reduces the number of enzyme-substrate complexes formed, decreasing the reaction rate. Example: Malonate inhibiting succinic dehydrogenase (malonate resembles succinate). Competitive inhibitors are used to control bacterial pathogens.
  • Non-competitive inhibitor: Binds to a site on the enzyme different from the active site, altering the enzyme's conformation and reducing its activity.

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Classification And Nomenclature Of Enzymes

Thousands of enzymes have been classified into six major classes based on the type of reaction they catalyze. Each class is further divided into subclasses. The systematic naming involves a four-digit number (Enzyme Commission number) and a name indicating the substrate and reaction type.

The six major classes of enzymes:

1. **Oxidoreductases/Dehydrogenases:** Catalyze oxidation and reduction reactions (transfer of electrons or hydrogen atoms) between two substrates.
2. **Transferases:** Catalyze the transfer of a functional group (other than hydrogen) from one substrate to another.
3. **Hydrolases:** Catalyze hydrolysis reactions – breaking bonds (ester, ether, peptide, glycosidic, C-C, C-halide, P-N bonds) by adding water.
4. **Lyases:** Catalyze the removal of groups from substrates by mechanisms other than hydrolysis, often resulting in the formation of double bonds.
5. **Isomerases:** Catalyze the inter-conversion of isomeric forms (optical, geometric, positional isomers) of a molecule.
6. **Ligases:** Catalyze the joining together (ligation) of two molecules, forming bonds like C-O, C-S, C-N, P-O, often coupled with ATP hydrolysis.

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Co-Factors

Some enzymes require non-protein components called **co-factors** to be catalytically active. The protein portion of such an enzyme is called the **apoenzyme**. The apoenzyme plus the co-factor constitute the complete, catalytically active enzyme, called the **holoenzyme** (Holoenzyme = Apoenzyme + Co-factor).

Co-factors can be of three types:

1. **Prosthetic groups:** Organic compounds that are tightly bound (often covalently) to the apoenzyme. Example: Haem (an iron-containing porphyrin ring) is a prosthetic group in enzymes like peroxidase and catalase, where it is part of the active site.
2. **Co-enzymes:** Organic compounds that are loosely or transiently associated with the apoenzyme, typically binding only during the course of catalysis. Many coenzymes are derived from vitamins (e.g., NAD and NADP contain niacin). Coenzymes often function as carriers of groups (like hydrogen or functional groups) between substrates.
3. **Metal ions:** Inorganic ions required by some enzymes for activity. Metal ions can form coordination bonds with amino acid side chains at the active site and also with the substrate, helping to stabilize the enzyme-substrate complex or facilitate the reaction. Example: Zinc is a cofactor for carboxypeptidase.

The catalytic activity of an enzyme is lost if the co-factor is removed, highlighting their crucial role.

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