Comprehensive Page-by-Page Biochemistry Notes

Page 1

• Slide-only title: Introduction to Biochemistry. No substantive content beyond framing the topic.

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Learning outcomes

• Discuss the definition, aims, scope, and importance of biochemistry
• Explain the role of biochemistry in health sciences
• Enumerate the major elements of the human body
• Describe briefly the major organic biomolecules of the human body
• Identify the important functional groups in biomolecules
• Explain the mechanisms behind the more common chemical reactions in living organisms
• Describe the developments that led to significant progress in the field of biochemistry
• Discuss the process and importance of subcellular fractionation
• Discuss the major components to the experimental approach used in biochemistry

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About Biochemistry

• Biochemistry is the science concerned with the molecular basis of life; chemistry of living organisms.
• It studies the chemical constituents of living cells and the reactions and processes they undergo.
• It encompasses large areas of cell biology, molecular biology, and molecular genetics.
• Its aim is to describe and explain, in molecular terms, all chemical processes of living cells.
• The ultimate goal is a complete understanding at the molecular level of all chemical processes in living cells.
• It also contemplates attempts to understand how life began.

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About Biochemistry – interdisciplinarity and scope

• Biochemistry combines aspects of all fields of chemistry.
• Carbon is the element of life; thus organic chemistry plays a large part in biochemistry.
• Biochemists often study reaction rates (physical chemistry).
• Metals are incorporated in biochemical structures (e.g., iron in hemoglobin) – inorganic chemistry.
• Sophisticated instrumentation is used to determine amounts and structures – analytical chemistry.
• Biochemistry is similar to molecular biology but biochemists focus on the chemical reactions occurring in living systems.

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About Biochemistry – relevance across life sciences

• A knowledge of biochemistry is essential to all life sciences.
• Biochemistry of nucleic acids is central to genetics; genetic approaches have been critical to explaining many areas of biochemistry.
• Physiology overlaps with biochemistry almost completely.
• Immunology uses numerous biochemical techniques.
• Pharmacology and pharmacy require knowledge of biochemistry and physiology.
• Poisons (toxicology) act on biochemical reactions or processes.
• Biochemical approaches are increasingly used to study pathology (inflammation, cell injury, cancer).
• Many microbiology workers employ biochemical approaches almost exclusively.

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About Biochemistry – health and nutrition

• Normal biochemical processes underpin health. The WHO defines health as a state of "complete physical, mental and social well-being and not merely the absence of disease and infirmity."
• From a biochemical viewpoint, health may be a state in which thousands of intra- and extracellular reactions occur at rates compatible with maximal survival in the physiologic state.
• Key prerequisites for health include optimal dietary intake of:
  • Vitamins
  • Nutritionally essential amino acids
  • Nutritionally essential fatty acids
  • Various minerals
  • Water
  • Carbohydrates

Page 7

All diseases have a biochemical basis

• Diseases are manifestations of abnormalities of molecules, chemical reactions, or processes.
• Biochemistry and medicine are reciprocally related.
• Examples (conceptual mapping): nucleic acids, proteins, lipids, carbohydrates relate to genetic diseases, sickle-cell anemia, atherosclerosis, diabetes mellitus.

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Major causes of diseases (part I)

• Physical agents: mechanical trauma, extreme temperature, sudden atmospheric pressure changes, radiation, electric shock.
• Chemical agents: toxic compounds, therapeutic drugs, etc.
• Biologic agents: viruses, bacteria, fungi, higher parasites.
• Lack of oxygen: loss of blood supply, depletion of oxygen-carrying capacity, poisoning of oxidative enzymes.
• Genetic disorders: congenital, molecular.

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Major causes of diseases (part II)

• Immunologic reactions: anaphylaxis, autoimmune diseases.
• Nutritional imbalances: deficiencies or excesses.
• Hormonal imbalances: deficiencies or excesses.
• All listed causes influence various biochemical mechanisms in the cell or body.

Page 10

Uses of biochemical investigations in relation to disease (part I)

• To reveal fundamental causes and mechanisms of diseases (e.g., genetic defects in cystic fibrosis).
• To suggest rational treatment (e.g., diet low in phenylalanine for phenylketonuria, PKU).
• To assist in diagnosis (e.g., CK-MB enzyme in myocardial infarction).

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Uses of biochemical investigations in relation to disease (part II)

• Screening tests for early diagnosis of certain diseases (e.g., measurement of blood T4 or TSH in neonatal congenital hypothyroidism).
• Monitoring progress of diseases (e.g., plasma ALT in infectious hepatitis).
• Assessing responses to therapy (e.g., measurement of CEA in colon cancer patients post-treatment).

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Biomolecules – major elements of the human body

• Carbon, hydrogen, oxygen, and nitrogen are the major constituents of most biomolecules.
• Phosphorus is a component of nucleic acids and other molecules; is widely distributed in its ionized form in the body.
• Calcium plays a key role in innumerable biological processes.
• Potassium, sulfur, sodium, chloride, magnesium, iron, manganese, and iodine are encountered routinely in medical practice (electrolyte imbalances, iron-deficiency anemia, thyroid diseases).

Page 13–16

Table 1-1. Approximate elementary composition of the human body (dry weight basis)

• Carbon: 50%
• Oxygen: 20%
• Hydrogen: 10%
• Nitrogen: 8.5%
• Phosphorus: 2.5%
• Calcium: 4%
• Potassium: 1%
• Sulfur: 0.8%
• Sodium: 0.4%
• Chlorine: 0.4%
• Magnesium: 0.1%
• Iron: 0.01%
• Manganese: 0.001%
• Iodine: 0.00005%

Note: These values refer to the dry weight basis; actual body water content reduces overall percentages in vivo.

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Major complex biomolecules

• The major complex biomolecules in human cells/tissues are:
  • Nucleic acids (DNA and RNA)
  • Proteins
  • Carbohydrates (polysaccharides)
  • Lipids
• These are constructed from simple building blocks:
  • Building blocks of nucleic acids: nucleotides
  • Deoxyribonucleotides for DNA
  • Ribonucleotides for RNA
  • Amino acids for proteins
  • Monosaccharides for polysaccharides
• Example: Glycogen is a principal polysaccharide in human tissues, built from glucose monomers.

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Major complex biomolecules (continued)

• Fatty acids are building blocks for many lipids, but lipids are not polymers of fatty acids.
• Biopolymers: nucleic acids, proteins, and polysaccharides are polymers of repeating building blocks.
• Complex biomolecules are also found in lower organisms; some building blocks differ (e.g., bacteria may lack glycogen or triacylglycerols but have other polysaccharides and lipids).

Page 19

Biomolecules – body composition

• Water is the major component of the body; its amount varies by tissue due to polarity and hydrogen-bonding properties, enabling water as a solvent.
• After water, proteins are the next most abundant component, followed by fats, minerals, and carbohydrates.

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Table 1-2. Normal chemical composition for a 65-kg man

• Mass (kg) | Percent
• Water: 61.6 kg | 40%
• Protein: 11 kg | 17.0%
• Fat: 9 kg | 13.8%
• Minerals: 4 kg | 6.1%
• Carbohydrate: 1 kg | 1.5%
• Note: Water percentage varies across tissues (as low as 22.5% in marrow-free bone) and decreases with body fat.

Page 21–22

Functional groups of organic biomolecules

• Key concept: Chemical properties are determined by functional groups; replacing hydrogen with different functional groups yields different family members.
• Frequently encountered functional groups in biomolecules:
  • Hydroxyl
  • Aldehyde (-CHO)
  • Ketone (-C(=O)-)
  • Carboxyl (-COOH)
  • Ester (-COOR’)
  • Amine (-NH2) / Amido (-CONH2)
  • Sulfhydryl (-SH)
  • Alkenyl (-CH=CH-)

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Functional group: Hydroxyl (-OH)

• Family: Alcohol (R-OH)
• Significance: Polar; participates in hydrogen bonding; found in carbohydrates (e.g., D-glucose).

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Functional group: Aldehyde (-CHO)

• Family: Aldehyde (R-CHO)
• Significance: Polar; participates in hydrogen bonding; found in carbohydrates (aldoses) such as D-glucose.

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Functional group: Ketone (R-CO-R’)

• Family: Ketone (R-CO-R’)
• Significance: Polar; participates in hydrogen bonding; found in carbohydrates (ketoses) such as D-fructose.

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Functional group: Carboxyl (-COOH)

• Example: Glycine has -COOH group; acidic and bears a negative charge when it donates a proton.

Page 26–27

Functional group: Ester (-COOR)

• Family: Ester
• Significance: Polar; participates in hydrogen bonding; found in certain lipids (e.g., triglycerides).
• Diagrams illustrate glycerol with three fatty acids forming triacylglycerol (plus water, illustrating hydrolysis).

Page 28

Functional group: Amine (-NH2) / Amido (-CONH2)

• Example: Glycine shows an amino group; amine is polar and can act as a base.
• Amido group is polar but uncharged under some conditions; participates in hydrogen bonding.

Page 29–30

Functional group: Amido (-CONH2) / Sulfhydryl (-SH)

• Amido: polar; does not bear a charge; participates in H-bonding (as in asparagine).
• Sulfhydryl: thiol; less soluble in water than alcohols; example: cysteine.

Page 31

Functional group: Alkenyl (-CH=CH-)

• Family: Alkene
• Significance: Important structural component in biomolecules such as lipids; can show cis/trans configurations.

Page 32–35

Biochemical Reactions – metabolism overview

• All life processes consist of chemical reactions catalyzed by enzymes; these reactions constitute metabolism.
• Two main types of metabolism:
  • Catabolism: breakdown of molecules into smaller units; oxidation to release energy or feed other reactions.
  • Anabolism: synthesis/building of molecules from smaller units.
• A metabolic pathway is a series of reactions that either synthesizes a complex compound (anabolic) or degrades a compound to end products (catabolic).
• Primary functions of metabolism:
  • Acquisition and utilization of energy
  • Synthesis of molecules needed for cell structure and function (proteins, nucleic acids, lipids, carbohydrates)
  • Removal of waste products
• Despite many reactions, the system is not intractable because: (i) the number of reaction types is relatively small; (ii) mechanisms are relatively simple; (iii) key reactions for energy production and major biomolecule turnover are relatively few.

Page 35–37

Common biochemical reaction types (list)

• Nucleophilic substitution reactions
• Elimination reactions
• Addition reactions
• Isomerization reactions
• Oxidation-reduction (redox) reactions
• Hydrolysis reactions
• Note: In nucleophilic substitution, a nucleophile replaces a leaving group on a substrate.
• Nucleophile = electron-rich species; electrophile = electron-poor species; leaving group = group that departs with electrons.
• Illustration: A + B–X → A–B + X:
  • A is the nucleophile; B–X is the electrophile; X is the leaving group.

Page 38–41

Elimination, Addition, Hydration

• Elimination: formation of a double bond by removing atoms (e.g., dehydration of 2-phosphoglycerate to phosphoenolpyruvate).
• Addition (hydration) reactions: two molecules combine to form a single product; hydration is a common example (e.g., fumarate to malate).
• Isomerization: intramolecular rearrangement (e.g., aldose–ketose interconversion).

Page 43–46

Redox (oxidation–reduction) reactions

• Redox = transfer of electrons from donor (reducing agent) to acceptor (oxidizing agent).
• Rules to determine oxidation state changes:
  • Oxidation when a molecule gains oxygen or loses hydrogen.
  • Reduction when a molecule loses oxygen or gains hydrogen.
• Example sequences include AH2 + O2 → H2O2 + A, and AH2 + NAD+ → NADH + H+ + A, showing reduced and oxidized forms.
• In biological systems, NAD+/NADH is a key redox couple (e.g., pyruvate ↔ lactate; NADH donates electrons to reform NAD+).

Page 47–48

Hydrolysis

• Hydrolysis is the cleavage of a covalent bond by water; often acid- or base-catalyzed.
• Important in digestion (proteins degraded in stomach under acid catalysis).
• ATP phosphate bonds can be broken by hydrolysis to release energy (
  ATP → ADP + Pi).
• Generic hydrolysis: R–C–O–R’ + H2O → R–OH + R’–OH (illustrative).

Page 49–50

Biochemical Methods – cell as the basic unit

• The cell is the fundamental unit of biology (Schleiden, Schwann, Virchow).
• Three developments after WWII spurred biochemistry/cell histology advances:
  • Electron microscope availability
  • Methods to disrupt cells gently while preserving function
  • High-speed refrigerated ultracentrifuge enabling separation of disrupted cell components without overheating
• Electron microscopy revealed many cellular components; disruption and ultracentrifugation allowed in vitro analysis of cellular components.

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Rat hepatocyte as a model

• The rat hepatocyte is heavily studied due to:
  • Availability in large amounts
  • Suitability for fractionation studies
  • Functional diversity
  • Contains major organelles in mammalian cells (nucleus, mitochondria, ER, ribosomes, Golgi, lysosomes, peroxisomes, plasma membrane, cytoskeletal elements)

Page 52–61

Subcellular fractionation – isolation of organelles

• Goal: isolate organelles in relatively pure form, free from significant contamination.
• Core techniques: Extraction, Homogenization, Centrifugation (the standard three-step approach).
• Foundation: Much pioneering work used rat liver.
• Extraction:
  • Harsh conditions can destroy activities; use mild conditions (aqueous solutions, avoid extremes of pH/osmotic pressure, low temperatures ~0–4°C).
  • Many organelles are labile; keep cold to preserve function.
  • STKM buffer: 0.25 M sucrose (isosmotic), 0.05 M TRIS-HCl, K+ and Mg2+ at near-physiologic concentrations.
  • Some solvents are used for lipids/nucleic acids extraction (not as mild as STKM).
• Homogenization:
  • Disrupt cells gently using a pestle in a homogenizing medium (e.g., STKM).
  • Resulting mixture is a homogenate containing intact organelles.
• Centrifugation (differential):
  • Series of centrifugation steps at increasing speeds: e.g.,
  • 600 g for 10 min → Pellet 1: Nuclear fraction; Supernatant 1
  • 15,000 g for 5 min → Pellet 2: Mitochondrial fraction; Supernatant 2
  • 105,000 g for 60 min → Pellet 3: Microsomal fraction; Supernatant 3: Cytosol
  • The three major pellets are not perfectly pure but are enriched in nuclei, mitochondria, and microsomes respectively.
• Validation of fractions:
  • Fractions are evaluated by marker enzymes and chemical markers and supplemented by electron microscopy.
  • Table 1-3 lists organelle markers and major functions (examples below).
• Functional characteristics of major fractions:
  • Nuclear fraction: nuclei, plasma membrane, unruptured cells
  • Mitochondrial fraction: mitochondria, lysosomes, peroxisomes
  • Microsomal fraction: rough and smooth endoplasmic reticulum (RER/SER) and free ribosomes
  • Cytosol: soluble contents
• Modifications and caution:
  • Different homogenization media and centrifugation protocols can yield purer or different organelle preparations.
  • Fractions must be assessed for purity.

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Table 1-3. Major intracellular organelles and their functions (highlights)

• Nucleus: Marker - DNA; Function - site of chromosomes; transcription.
• Mitochondrion: Marker - Glutamate dehydrogenase; Function - CAC and oxidative phosphorylation.
• Ribosome: Marker - High RNA content; Function - protein synthesis.
• Endoplasmic reticulum: Marker - Glucose 6-phosphatase; Function - protein synthesis (RER); lipid synthesis (SER); xenobiotic oxidation (SER).
• Lysosome: Marker - Acid phosphatase; Function - site of hydrolases.
• Plasma membrane: Markers - Na+-K+ ATPase; 5’-nucleotidase; Function - transport; intercellular adhesion/communication.
• Golgi apparatus: Marker - Galactosyltransferase; Function - protein sorting; glycosylation and sulfation.
• Peroxisome: Marker - Catalase; Function - fatty acid and amino acid degradation; hydrogen peroxide metabolism.
• Cytoskeleton: No specific marker; Function - structural support (microfilaments, microtubules, intermediate filaments).
• Cytosol: Marker - Lactate dehydrogenase; Function - glycolysis; fatty acid synthesis.

Page 62–63

Experimental approach – overview

• Three major components:
  1) Isolation of organelles and biomolecules from cells
  2) Determination of structures of biomolecules
  3) Analyses of the function and metabolism of biomolecules
• Isolation of biomolecules often requires purification to homogeneity; Table 1-4 lists major separation/purification methods and typically a sequential combination is used.

Page 64–66

Table 1-4. Major methods used to separate and purify biomolecules

• Salt fractionation (e.g., ammonium sulfate precipitation)
• Chromatography: paper, thin-layer
• Ion-exchange (anion and cation)
• Gas-liquid chromatography (GLC)
• Affinity chromatography
• High-performance liquid chromatography (HPLC)
• Gel filtration (size-exclusion)
• Electrophoresis: paper, agarose, starch gel, polyacrylamide gel
• SDS-PAGE for protein analysis (SDS detergent enables solubilization and separation by molecular weight)
• Ultracentrifugation
• Note: Purification usually requires multiple techniques in sequence; many components can be analyzed with these methods.

Page 65–66

Notes on lipids and structure analysis

• GLC and TLC are used for detailed lipid studies.
• SDS allows solubilization of many proteins previously insoluble, enabling electrophoretic analysis.
• Structural determination methods (Table 1-5) are applied after purification.

Page 66–68

Table 1-5. Principal methods used for determining biomolecule structures

• Elemental analysis
• Ultraviolet, visible, infrared, and NMR spectroscopy
• Chemical degradation (acid/base hydrolysis) to break biomolecule into constituents
• Enzymatic degradation with specific enzymes (e.g., proteases, nucleases, glycosidases)
• Mass spectrometry (MS)
• Sequencing methods (proteins and nucleic acids)
• X-ray crystallography

Page 68–69

Advances in structural determination

• Enzyme specificity and advanced resolution enable structural features to be determined by several approaches.
• High-resolution NMR is increasingly capable for complex carbohydrates and proteins.
• X-ray crystallography remains a leading source of detailed structural information.

Page 69–71

Analysis of biomolecule function and metabolism – experimental preparations

• Early biochemical research was conducted at the whole-animal level (e.g., respiration, fate of nutrients).
• Whole-animal studies are often too complex for definitive answers; simpler in vitro preparations were developed to reduce complexity.
• Table 1-6 summarizes the hierarchy of preparations used in biochemical studies.

Page 70–71

Hierarchy of preparations (continuation)

• Whole-animal studies include organ removal, dietary manipulations, drug/toxin administration, disease models; physiologic but interpretation is complicated by inter-organ interactions.
• Isolated perfused organs (e.g., liver, heart, kidney) allow organ function studies isolated from other organs.
• Tissue slices and isolated cells enable more precise functional studies in a controlled environment.
• Cell homogenates and isolated subcellular organelles allow detailed analysis and portioning of cellular components.
• The level of detail increases with the degree of isolation, but each step requires control and interpretation within its physiological context.

Page 72–73

Table 1-6. Hierarchy of preparations used to study biochemical processes (continuation)

• Methods and comments for each level (from whole-animal to isolated organelles and subfractions)
• Emphasis: Use of increasingly refined in vitro systems to study specific biochemical processes.
• Additional themes: cloning of genes for enzymes and proteins; isolation of the cloned gene to study structure/regulation; potential to reveal amino acid sequences of enzymes/proteins.

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End of presentation

• Final slide: signals the end of the presentation with decorative or filler text.

Summary of key ideas and connections

• Biochemistry sits at the intersection of chemistry and biology, focusing on molecular explanations of life processes, and is foundational to health sciences, genetics, pharmacology, immunology, and toxicology.
• Health is tied to the proper execution and regulation of thousands of biochemical reactions; nutrition and elemental balance are crucial for maintaining health.
• Disease is rooted in biochemical abnormalities, which can be diagnosed, monitored, and treated through biochemical approaches.
• Biomolecules are built from a small set of major elements and functional groups; understanding these groups explains reactivity and properties across sugars, proteins, lipids, and nucleic acids.
• Metabolism integrates catabolic and anabolic pathways; despite complexity, it is organized around a small set of core reaction types (e.g., redox, hydrolysis, additions, eliminations, substitutions, isomerizations).
• Subcellular fractionation and biochemical methods enable the study of cellular components in isolation, using standardized buffers (e.g., STKM) and multi-step centrifugation to enrich organelles.
• A robust toolkit (salt fractionation, chromatography, electrophoresis, MS, NMR, X-ray crystallography) supports purification, structure elucidation, and functional analysis of biomolecules.
• The hierarchical experimental approach—from whole organisms to isolated organelles—allows controlled dissection of complex biological processes while preserving physiologic relevance.

Key formulas and numerical references

• Isolated organelle fractions and centrifugal steps (example):
  • Nuclear fraction pellet at 600\ ext{g} \times 10\ ext{min}
  • Mitochondrial fraction pellet at 15000\ \text{g} \times 5\ ext{min}
  • Microsomal fraction pellet at 105000\ \text{g} \times 60\ ext{min}
• Table 1-1: Major elemental composition (dry weight basis):
  • \text{C} = 50\%; \text{O} = 20\%; \text{H} = 10\%; \text{N} = 8.5\%; \text{P} = 2.5\%; \text{Ca} = 4\%; \text{K} = 1\%; \text{S} = 0.8\%; \text{Na} = 0.4\%; \text{Cl} = 0.4\%; \text{Mg} = 0.1\%; \text{Fe} = 0.01\%; \text{Mn} = 0.001\%; \text{I} = 0.00005\%
• Major functional groups: examples of typical molecules include D-glucose (hydroxyl/aldehyde/ketone groups), glycine (amino/carboxyl), etc., illustrating how functional groups define biomolecule behavior.

Note on ethical/philosophical implications

• The slides focus on foundational biochemical concepts, methods, and applications. Ethical considerations are not explicitly discussed in the presented material; however, the experimental approach sections imply standard laboratory ethics and biosafety principles (e.g., careful handling of biological materials, respect for animal models, and appropriate containment for biochemical agents). Any exam-ready synthesis should acknowledge bioethics and responsible conduct of research as essential context to these methods and applications.