BIOCHEMISTRY

LECTURE
INTRODUCTION TO THE BIOCHEMISTRY
I. Introduction
II. Biomolecules HEALTH
III. Biochemical reactions  WHO defines health as a state of “complete
IV.Biochemical methods physical, mental and social well-being and not
I. INTRODUCTION merely the absence of disease and infirmity”
BIOCHEMISTRY  Normal biochemical processes are the basis of
 Science concerned with the molecular basis of health
life  Health may be a situation in which all of the
 Chemistry of living organisms thousands of intra- and extracellular reactions
 Science concerned with the chemical constituents that occur in the body are proceeding at rates in
of living cells and with the reactions and proportion with its maximal survival in the
processes that they undergo physiologic state.
 It encompasses large areas of cell biology, MAJOR PREREQUISITE FOR THE MAINTENANCE
molecular biology, and molecular genetics. OF HEALTH IS THE OPTIMAL DIETARY INTAKE OF
A NUMBER OF CHEMICALS
Its aim is to describe and explain, in molecular
1. Vitamins
terms, all chemical processes of living cells,
2. Nutritionally essential amino acids
Complete understanding at the molecular level of
3. Nutritionally essential fatty acids
all chemical processes associated with living
4. Various minerals
cells, and attempt to understand how life began
BIOCHEMISTRY IN THE FIELD OF SCIENCE
5. Water
CHEMISTRY 6. Carbohydrates
BIOCHEMICAL BASIS
 Biochemistry combines aspects of all the fields of
chemistry  All disease have biochemical basis
ORGANIC CHEMISTRY  All diseases are manifestations of abnormalities
of molecules, chemical reactions, or processes
 Because carbon is the element of life, organic
 Hence, a reciprocal relationship between
chemistry plays a large part in biochemistry.
PHYSICAL CHEMISTRY biochemistry and medicine exists
 Many times biochemists study how fast reactions
occur.
INORGANIC CHEMISTRY
 Often metals are incorporated into biochemical
structures (such as iron in hemoglobin).
ANALYTICAL CHEMISTRY
 Biochemists use sophisticated instrumentation to MAJOR CAUSES OF DISEASE
determine amounts and structures. PHYSICAL AGENTS
MOLECULAR BIOLOGY
 Mechanical trauma, extreme temperature,
 Biochemistry is similar to molecular biology.
sudden changes in atmospheric pressure,
 Both fields study living systems at the molecular radiation, electric shock
level. CHEMICAL AGENTS
 However, biochemists concentrate on the
 Certain toxic compounds, therapeutic drugs, etc.
chemical reactions that are occurring. BIOLOGIC AGENTS
LIFE SCIENCES
 Biochemistry of nucleic acids is at the heart of  Viruses, bacteria, fungi, higher forms of parasites
genetics; in turn, the use of genetic approaches LACK OF OXYGEN
has been critical in explaining many areas of  Loss of blood supply, depletion of the oxygen-
biochemistry. carrying capacity of blood, poisoning of oxidative
 Physiology overlaps with biochemistry almost enzymes
completely. GENETIC DISORDERS
 Immunology uses numerous biochemical  Congenital, molecular
techniques. IMMUNOLOGIC REACTIONS
 Pharmacology and pharmacy requires a sound  Anaphylaxis, autoimmune diseases
knowledge of biochemistry and physiology. NUTRITIONAL IMBALANCES
 Poisons, which fall under toxicology, act on  Deficiencies, excesses
biochemical reactions or processes HORMONAL IMBALANCES
 Biochemical approaches are being used  Hormonal deficiencies, excesses
increasingly to study the basic aspects of USES OF BIOCHEMICAL INVESTIGATIONS AND
pathology, such as inflammation, cell injury, and LABORATORY TESTS IN RELATION TO DISEASES
cancer. TO REVEAL THE FUNDAMENTAL CAUSES AND
 Many workers in microbiology employ MECHANISMS OF DISEASES
biochemical approaches almost exclusively.  Demonstration of the nature of the genetic
defects in cystic fibrosis
TO SUGGEST RATIONAL TREATMENT OF DISEASES
 Use of a diet low in phenylalanine for the
treatment of phenylketonuria (PKU)

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TO ASSIST IN THE DIAGNOSIS OF SPECIFIC - In the case of glycogen, which is the principal
DISEASES polysaccharide found in human tissues, that
 Use of the enzyme creatine kinase MB (CK-MB) monosaccharide is glucose.
in the diagnosis of myocardial infarction (heart  Fatty acids may be considered to be the building
attack). blocks of many lipids; however, lipids are not
TO ACT AS SCREENING TESTS FOR THE EARLY polymers of fatty acids
DIAGNOSIS OF CERTAIN DISEASES  In contrast, nucleic acids, proteins, and
 Use of measurement of blood T4 or TSH in the polysaccharides are biopolymers because they
neonatal diagnosis of congenital hypothyroidism. are composed of repeating units of their building
TO ASSIST IN MONITORING THE PROGRESS OF blocks.
CERTAIN DISEASES  These complex molecules are also generally
 Use of the plasma enzyme alanine found in lower organisms, although the building
aminotransferase (ALT) in monitoring the blocks in certain cases may differ.
progress of infectious hepatitis.  For example, bacteria do not contain glycogen or
TO ASSIST IN ASSESSING THE RESPONSES OF triacylglycerols, but contain other polysaccharides
DISEASES TO THERAPY and lipids.
 Use of measurement of blood carcinoembryonic CHIEF COMPONENTS OF THE HUMAN BODY
antigen (CEA) in certain patients who have been  Water constitutes the major component, although
treated for colon cancer. its amount varies widely among different tissues
II. BIOMOLECULES  Its polar nature and ability to form hydrogen
MAJOR ELEMENTS OF THE HUMAN BODY bonds allow water to function as a solvent of the
 Carbon, hydrogen, oxygen, and nitrogen are the human body
major constituents of most biomolecules  Protein follows water as the next abundant
 Phosphorous is a component of nucleic acids and component, followed by fat, minerals, and finally,
other molecules, and is widely distributed in its carbohydrates.
ionized form in the human body
 Calcium plays a key role innumerable biologic Table 1-2. Normal chemical composition for a
processes 65-kg man
 Potassium, sulfur, sodium, chlorine, magnesium,
iron, manganese, and iodine are encountered on Mass (kg) Percent
an almost daily basis in medical practice in
dealing with patients with electrolyte imbalances Water* 40 61.6
(K+, Na+, Cl-, and Mg2+), iron-deficiency anemia
(Fe2+), and thyroid diseases (I-) Protein 11 17.0

Table 1-1. Approximate elementary composition Fat 9 13.8

of the human body (dry weight basis)
Minerals 4 6.1
Element Percent Element Percent
Carbohydrate 1 1.5
Carbon 50 Potassium 1
*The value of water can vary widely among
Oxygen 20 Sulfur 0.8 different tissues, being as low as 22.5 percent in
Hydrogen 10 Sodium 0.4 marrow-free bone. The percentage of water also
diminishes as body fat increases.
Nitrogen 8.5 Chlorine 0.4 FUNCTIONAL GROUPS OF ORGANIC
BIOMOLECULES
Calcium 4 Magnesium 0.1
 Chemical properties of organic molecules are
Phosphorous 2.5 Iron 0.01 determined by specific arrangement of atoms
called functional groups
Manganese 0.001  Different families of organic compounds result
Iodine 0.00005 when hydrogen atoms on organic molecules are
replaced by different functional groups
MAJOR COMPLEX BIOMOLECULES IMPORTANT FUNCTIONAL GROUPS IN ORGANIC
 Nucleic acids (DNA and RNA), proteins, BIOMOLECULES
carbohydrates (polysaccharides), and lipids Hydroxyl Amino
are the major complex biomolecules found in the Aldehyde functional group Amido
cells and tissues of higher animals, including Ketone functional group Sulfhydryl
humans Carboxyl Alkenyl
- These molecules are constructed from simple Ester functional group
biomolecules, which are referred to as building HYDROXYL
blocks
 The building blocks of nucleic acids are
nucleotides Functional group: Hydroxyl (-OH)
 Deoxyribonucleotides for DNA Family name: Alcohol (R-OH)
 Ribonucleotides for RNA Significance: Polar and participates in H-bond
 Amino acids are the building blocks of proteins formation found in carbohydrates
 Polysaccharides are made up of simple
carbohydrates (monosaccharides)

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ALDEHYDE FUNCTIONAL GROUP ALKENYL

Functional group: Alkenyl (-CH=CH-)

Functional group: Aldehyde functional group (-CHO) Family name: Alkene (R-CH=CH-R’)
Family name: Aldehyde (R-CHO) Significance: Important structural component of
Significance: Polar and participates in H-bond many biomolecules, such as in lipids.
formation found in carbohydrates (aldoses) III. BIOCHEMICAL REACTIONS
KETONE FUNCTIONAL GROUP ENZYME
 All life processes consist of chemical reactions
that are catalyzed by enzymes.
METABOLISM
 Chemical reactions in a living system are
collectively known as metabolism
Functional group: Ketone functional group (C-CO-C) TWO TYPES OF METABOLIC PROCESSES:
Family name: Ketone (R-CO-R’) CATABOLISM
Significance: Polar and participates in H-bond  Is a set of metabolic pathways that breaks down
formation found in carbohydrates (ketoses) molecules into smaller units, which are either
CARBOXYL oxidized to release energy, or used in other
reactions
ANABOLISM
 Is a set of metabolic pathways that construct or
build molecules from smaller units
Functional group: Carboxyl (-COOH) In other words, the combined catabolic and
Family name: Carboxylic acid (R-COOH) anabolic processes constitute metabolism
Significance: Polar and participates in H-bond METABOLIC PATHWAY
formation weakly acidic and bears a negative charge  A metabolic pathway is a series of reactions
when it donates a proton responsible for the synthesis of a more complex
ESTER FUNCTIONAL GROUP compound from one or more simple compounds
(anabolic), or for the degradation of a compound
to its end product (catabolic).
PRIMARY FUNCTIONS OF METABOLISM:
 Acquisition and utilization of energy
Functional group: Ester functional group (-COOC-)  Synthesis of molecules needed for cell structure
Family name: Ester (R-COOR’) and functioning (i.e., proteins, nucleic acids, lipids,
Significance: Polar and participates in H-bond, and carbohydrates)
formation found in certain lipids  Removal of waste products
AMINO MOST FREQUENT REACTIONS ENCOUNTERED IN
BIOCHEMICAL PROCESSES ARE THE FOLLOWING:
Nucleophilic substitution reactions
Elimination reactions
Addition reactions
Isomerization reactions
Functional group: Amino (-NH2) Oxidation-reduction (redox) reactions
Family name: Amine (R-NH2) Hydrolysis reactions
Significance: Polar and participates in H-bond NUCLEOPHILIC SUBSTITUTION REACTION
formation, weakly basic and bears a positive charge
 As the name suggests, one atom or group is
when it accepts a proton
AMIDO substituted for another

 In the reaction, the attacking species (A) is called

a nucleophile (“nucleus-lover”)
Functional group: Amido (-CONH2)  Nucleophiles are most commonly negatively-
Family name: Amide (R-CONH2) charged atoms or groups
Significance: Polar, but does not bear a charge  However, neutral species with unshared electrons
participates in H-bond formation can also act as nucleophiles
SULFHYDRYL  Electrophiles (“electron-lover”) are atoms or
groups that are transferred from one nucleophile
to another
Functional group: Sulfhydryl (-SH)  In the example, nucleophile A is attracted to
Family name: Thiol (R-SH) electrophile B
Significance: Does not participate in H-bond  As the new bond forms between A and B, the old
formation, less soluble in water than alcohols one between B and X breaks
 The outgoing nucleophile, X, is called a leaving
group

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ELIMINATION REACTION
 In an elimination reaction, a double bond is formed
when atoms in a molecule are removed.

 Although it is not always easy to determine

whether biomolecules have gained or lost
 The removal of H2O from biomolecules with a
electrons, there are two simple rules which may
hydroxyl functional group is a commonly
be used to determine whether a molecule has
encountered elimination reaction. An example is
been oxidized or reduced:
the dehydration of 2-phosphoglycerate to form
phosphoenolpyruvate.  Oxidation has occurred if a molecule gains
ADDITION REACTION oxygen or loses hydrogen
 Reduction has occurred if a molecule loses
 Hydration is one of the most common addition
oxygen or gains hydrogen
reactions. The hydration of the metabolic HYDROLYSIS
intermediate fumarate to form malate is a typical
example.  Hydrolysis is the cleavage of a covalent bond by
water

 Hydrolytic reactions, which usually involve

nucleophilic substitution either at a saturated
carbon or a carbonyl carbon, may be catalyzed
by an acid or a base
ISOMERIZATION REACTION  Digestion of many food molecules involves
hydrolysis
 Isomerization reactions involve the intramolecular
shift of atoms or groups  For example, proteins are degraded in the
stomach in acid-catalyzed reaction
 One of the most common biochemical
isomerization reactions is the interconversion  Another important example is breaking the
between aldose and ketose sugars phosphate bonds of ATP
 Energy obtained during this reaction is used to
drive many cellular processes
IV. BIOCHEMICAL METHODS
 The cell is the basic unit of biology
HISTORY OF CELL
 In the 19th century, the cell was established as
the fundamental unit of biologic activity by
Schleiden and Schwann, and other pioneers such
as Virchow
OXIDATION-REDUCTION REACTION  However, in the years immediately after WWII,
 Also called redox reaction three developments helped usher in a period of
 Redox reactions occur when there is transfer of unparalleled activity in biochemistry and cell
electrons from a donor (called the reducing histology
agent) to an electron acceptor (called the  Increasing availability of the electron microscope
oxidizing agent)  Introduction of methods that permit disruption of
 When reducing agents donate their electrons, cells under relatively mild condition that
they become oxidized preserved function
 As oxidizing agents accept electrons, they  Increasing availability of the high-speed,
become reduced refrigerated ultracentrifuge, which is capable of
 The two processes always occur simultaneously generating centrifugal forces sufficient to
separate the components of disrupted cells from
one another without overheating them
 Use of the electron microscope revealed many
previously unknown or poorly observable cellular
components
 Disruption and ultracentrifugation permitted the
isolation and analysis of cellular components in
vitro
RAT HEPATOCYTE
 The rat hepatocyte is probably the most studied
of all cells from a biochemical standpoint due to
the following reasons:
- Availability in relatively large amounts
- Suitability for fractionation studies
- Diversity of functions

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 - Contains the major organelles found in  The supernatant from each step is subjected to
eukaryotic cells (nucleus, mitochondria, centrifugation in the next step
endoplasmic reticulum, free ribosomes,  Overall, the procedure provides three pellets,
Golgi apparatus, lysosomes, peroxisomes, named the nuclear, mitochondrial, and
plasma membrane, and certain cytoskeletal microsomal fractions
elements)
SUBCELLULAR FRACTIONATION
 In order to study the function of any organelle in
detail, it is necessary to isolate it in relatively pure
form, free of significant contamination by other
organelles.
 Subcellular fractionation is the usual process to
achieve this, and it requires three physical
techniques:
- Extraction
- Homogenization
- Centrifugation  None of these fractions, though, are composed of
 Much of the pioneering work in this area was absolutely pure organelles
done using rat liver.  However, it has been well established by electron
EXTRACTION microscopy and measurements of marker
 To isolate and study a specific organelle (or enzymes and chemical components that the
molecule), it is necessary to extract it from the major constituents of each of these three
cells in which it is located fractions are nuclei, mitochondria, and
 Most organelles and biomolecules are labile and microsomes, respectively
subject to loss of biologic activities  A marker enzyme or chemical (refer to table 1-3)
 Hence, they must be extracted using mild is one that is almost exclusively confined to one
conditions, i.e., employment of aqueous particular organelle, such as acid phosphatase to
solutions, and avoidance of extremes of pH and lysosomes and DNA to the nucleus
osmotic pressure, and of high temperatures  Thus, the marker serves to indicate the presence
 Most procedures for isolating organelles are or absence in any particular fraction of the
performed at about organelle in which it is contained
0 to 4°C  The nuclear fraction contains mostly nuclei, and
 Significant losses of activity can occur at room plasma membrane and unruptured cells
temperature, partly due to the action of various  The mitochondrial fraction contains mostly
enzymes that are released when cells are mitochondria, and lysosomes and peroxisomes
disrupted as well
 A common solution for extraction of organelles is  The microsomal fraction (microsomes) contains
STKM, which consists of 0.25 M sucrose mostly a mixture of SER, RER, and free
(isosmotic), adjusted to pH 7.4 by 0.05 M TRIS ribosomes
(tris-[hydroxymethyl]aminomethane) HCl buffer,  The contents of the final supernatant correspond
and K+ and Mg2+ at near physiologic to those of the cytosol (cell sap)
concentrations  Modifications of this basic approach, such as
 Not all solvents used for extraction are as mild as using different homogenization media or different
STKM protocols or methods of centrifugation, have
 For example, organic solvents are used for the permitted the isolation in more or less pure form
extraction of lipids and nucleic acids of all organelles listed in table 1-3 (next slide)
HOMOGENIZATION  The described procedure is applicable in general
 To extract an organelle from cells, it is first to most organs and cells
necessary to disrupt the cells under mild  However, cell fractions of this type must be
conditions assessed by measuring marker enzymes and
 Organs and their cells may be conveniently chemicals, and by the electron microscope
disrupted by the process of homogenization
 During homogenization, a manually operated or
motor-driven pestle is rotated within a glass tube
of suitable dimensions containing minced
fragments of the organs under study, and a
suitable homogenizing medium, such as STKM
 The controlled rotation of the pestle exerts
mechanical shearing forces on cells and disrupts
them, releasing their constituents into the STKM
solution
 The resulting solution, which contains many intact
organelles, is called a homogenate
CENTRIFUGATION
 Fractionation of the contents of the homogenate
is achieved by differential centrifugation
 The classic differential centrifugation method
uses a series of three different centrifugation
steps at successively greater speeds
 Each step yields a pellet and a supernatant

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 Table 1-3. Major intracellular organelles and their Table 1-4. Major methods used to separate and purify
functions biomolecules

Organelle or
Marker Major Functions Salt fractionation (e.g., precipitation with ammonium sulfate)
Fraction

Site of
Chromatography Paper Thin-layer
chromosomes;
Nucleus DNA
site of
transcription Ion exchange
(anion and
Gas-liquid
Glutamate Citric acid cycle, cation
Mitochondrio
dehydrogena oxidative exchange)
n
se phosphorylation
High-performance
Affinity
High content liquid
Ribosome Protein synthesis
of RNA

Protein synthesis Gel filtration

(RER); synthesis of
Endoplasmic Glucose 6- various lipids Electrophoresis Paper High-voltage
reticulum phosphatase (SER), oxidation of
many xenobiotics
(SER) Agarose gel Cellulose acetate
Acid Site of many
Lysosome
phosphatase hydrolases Starch gel Polyacrylamide gel
Transport of
+ +
molecules in and SDS-
Na -K
Plasma out of cells; polyacrylamide
ATPase;
membrane intercellular gel
5’-
nucleotidase adhesion and
communication Ultracentrifugation

Intracellular
Most of these methods are suitable for analyzing the
sorting of
components present in cell extracts and other biochemical
Golgi Galactosyltra proteins;
materials. The sequential use of several techniques generally
apparatus nsferase glycosylation
permits purification of most biomolecules.
reactions;
sulfation reactions  Isolation of biomolecules
 Gas-liquid chromatography (GLC) and thin-layer
Degradation of chromatography (TLC) allow the extensive study
certain fatty acids
of lipids
Catalase; and amino acids;
 Analysis of membrane and many other proteins
Peroxisome Uric acid production and
oxidase degradation of
was extremely difficult until the introduction of
hydrogen
sodium dodecyl sulfate-polyacrylamide gel
peroxide electrophoresis (SDS-PAGE)
 The introduction of the detergent SDS permitted
None;
Microfilament,
the “solubilization” for electrophoresis the many
recognized proteins that were previously insoluble
microtubules,
Cytoskeleton by EM or
intermediate  Determination of the structure of biomolecules
electrophore  After a biomolecule has been purified, it is then
filaments
sis necessary to determine its structure
Lactate  This should allow detailed correlations to be
Glycolysis, fatty made between structure and function
Cytosol dehydrogena
acid synthesis  Table 1-5 lists the major methods used to analyze
se
EXPERIMENTAL APPROACH
the structures of biomolecules
 Isolation of biomolecules
 As in the case of organelles, determination of the
function of any biomolecule initially requires its
isolation in pure form
 Table 1-4 lists the major methods that are used to
separate and purify biomolecules
 A combination of successive use of several of
them is almost always necessary to purify a
biomolecule to homogeneity (i.e., freedom from
contamination by any other biomolecule)

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 Table 1-6. Hierarchy of preparations used to study
Table 1-5. Principal methods used for determining the biochemical processes (continuation)
structures of biomolecules
Method Comments
Tissue slice Liver slices have been
Elemental analysis especially used
Removes the sliced tissue
from other influences, but the
preparations tend to
Ultraviolet, visible, infrared, and NMR spectroscopy deteriorate within a few hours,
partly because of inadequate
supply of nutrients
Use of acid or alkaline hydrolysis to degrade the Use of whole Particularly applicable to blood
biomolecule under study into its basic constituents cells cells, which can be purified
relatively easily
Use of cells in tissue culture is
Use of a battery of enzymes of known specificity to indispensable in many areas of
degrade the biomolecule under study (e.g., proteases, biology
nucleases, glycosidases)
Homogenate Ensures a cell-free preparation
Specific compounds can be
added or removed (e.g., by
Mass spectrometry (MS)
dialysis), and their effects
studied
Can be fractionated by
Specific sequencing methods (e.g., for proteins and centrifugation to yield
nucleic acids) individual cell organelles
Isolated cell Extensively used to study the
organelles function of mitochondrion, the
X-ray crystallography endoplasmic reticulum,
ribosomes, etc.

 Analysis of the function and metabolism of Subfractionation Extensively used; for example,
biomolecules of organelles in studies of mitochondrial
 Initial biochemical research on humans and function
animals was performed at the level of the whole
animal
 Examples were studies of respiration and of the Table 1-6. Hierarchy of preparations used to study
fate of ingested compounds biochemical processes (continuation)
 It became apparent that the whole animal was too
complex to permit definitive answers to many Method Comments
questions
Isolation and A vital part in the analysis of
 Accordingly, simpler in vitro preparations were
characterization any chemical reaction or
developed that removed many of the of metabolites pathway
complications experienced at the level of the and enzymes
whole animal
 Table 1-6 summarizes the various preparations Cloning of genes Isolation of the cloned gene is
that are now available to study biochemical for enzymes and vital for studying the details of
processes proteins its structure and regulation
It can also reveal the amino
acid sequence of the enzyme
or protein for which it codes

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