BIOCHEMISTRY-LABORATORY

INTRODUCTION

Laboratory Paraphernalia
⎯ Beaker = used to hold, mix, and heat liquids; comes in different sizes
⎯ Erlenmeyer flask = used to hold and mix chemicals; the small neck is to facilitate mixing without
spilling
⎯ Florence flask = used for swirling liquids; used as a container to hold liquids
⎯ Graduated cylinder = used to measure a precise volume of a liquid, with graduation or measurements
⎯ Pipette = used to transport a measured volume of liquid, often as a media dispenser
⎯ Aspirator = suction device used along with the pipette
⎯ Burette = a graduated glass tube with a tap at one end, for delivering known volumes of a liquid,
especially in titrations
⎯ Burette holder = u sed to hold and secure a burette on a stand
⎯ Ball and stick = used for modelling molecular structure
⎯ Volumetric flask = used to prepare solutions to an accurate volume; have sizes up to 1000 mL
⎯ Spot plate = also called as a reaction plate; is a laboratory tool made either from ceramics which
consists of small holes
⎯ Reagent bottles = used to hold liquid substances; comes in amber (used to for liquids that are light
sensitive) or colorless
⎯ Spatula = used for scraping, transferring, or applying powders and paste like chemicals or treatments
⎯ Evaporating dish = used for the evaporation of solutions and supernatant liquids, and sometimes to
their melting point
⎯ Crucible and cover = made of high temperature-resistant materials, usually porcelain; used for
conducting high-temperature chemical reactions
⎯ Watch Glass = a circular concave piece of glass used in chemistry as a surface to evaporate a liquid,
to hold solids while being weighed, for heating a small amount of substances
⎯ Test tube = used to hold and mix liquids
⎯ Test tube rack = used to hold several test tubes at one time
⎯ Test tube brush = used to wash the test tube
⎯ Test tube holder = used to hold a test tube, particularly when hot
⎯ Stirring rod = used to mix and transfer chemicals
⎯ Funnel = used to transfer liquids or fine-grained materials into containers with small openings. Also
used for filtration
⎯ Ignition tube or combustion tube = bigger test tube; a heavy-walled test tube of hard glass for
examining the behavior of heated substances
⎯ pH paper = used to determine if a solution is acidic, basic, or neutral
⎯ Litmus paper = used to determine if a solution is acidic, basic, or neutral; specifically, in form of
blue or red colored paper indicator
⎯ Filter paper = used to separate fine solid particles from liquids or gases
⎯ Digital multimeter = a test tool used to measure two or more electrical values
⎯ Stopwatch = used to measure the amount of time
⎯ Tirrill burner = allows adjustment of both the air supply and the gas supply; has collar and
regulator
⎯ Bunsen burner = a small adjustable gas burner used in laboratories; neither have collar or regulator
⎯ Iron stand = supports the iron ring when heating substances or mixtures in a flask or beaker
⎯ Iron clamp = used to hold glassware on the iron ring
⎯ Iron ring = supporting apparatus above the work surface
⎯ Wire gauze = used to support a container, such as a beaker, on a ring stand while it is being heated;
may have a fiberglass or ceramic center
⎯ Tripod = a three-legged platform used to support flasks and beaker
⎯ Clay triangle = used to support a crucible being heated by a Bunsen burner or other heat source
⎯ Extension clamp = holds test tube when heating in iron stand; allow placement of apparatus at various
distances from the frame or rod
⎯ Platform balance = used for measuring mass
⎯ Beaker tongs = used to handle hot beakers
⎯ Crucible tongs = used to handle hot crucible
⎯ Forceps = used to pick up or hold small objects
 ⎯ Mortar and Pestle = used to crush and grind materials
⎯ Thermometer = used to measure temperature
⎯ Wash bottle = used to rinse pieces of glassware and to add small quantities of water
⎯ Alcohol burner = used to heat chemicals in beaker or test tube
⎯ Dropper = used to transfer small amount of liquids

General Laboratory Safety Procedures

= Laboratory safety is the key to reducing injury and illness.
= There are many exposures in the laboratory that pose a hazard to your health and you may have never
considered them as a hazard before.
= It is important that you are aware of the potential dangers that may threaten your health or life.

Introduction to Laboratory
⎯ Working in a laboratory can be an exciting experience.
⎯ It can also pose many threats and hazards that a traditional classroom does not.
⎯ That is why it is important to know your surroundings. Know where the exits to your room are.
⎯ Your supervisor will go over the emergency action plan including the escape route procedures for your
room.
⎯ Know your surroundings.
= It is also recommended to be aware of the fire extinguishers in location to your laboratory.
= Campus personnel are highly encouraged to not fight fires. In the event of a fire, the first
response is to evacuate the area and notify the fire department!
= Do not wait any longer than necessary to call, time is of essence!
= Know where the fire alarm is in proximity to your laboratory. Is it right down the hall or is it
in the stairwell?
⎯ Many laboratories contain hazardous substances.
= A hazardous substance is defined as a material/substance that poses a physical or health hazard.
This includes both chemicals and biological agents.
= A biohazard is defined as any organism that is capable of replication and can cause disease in
human, animal, or plant.
⎯ There are differences between a physical hazard and a health hazard.
= Health hazards has the following characteristics: Carcinogen, Toxic or highly toxic, Reproductive
Toxins, Irritants, Corrosives, etc.
= Physical hazards have the following characteristics: Explosive Flammable, Oxidizer, Organic
peroxide, Compressed gas, Combustible liquid, Unstable (Reactive), Water-reactive
⎯ When physical hazards and health hazards exist, it is very important to know where the eye
wash/safety shower is located. Unexpected accidents do occur and knowing where to go at the time of
an emergency can reduce injury/illness.
⎯ First aid kits have a variety of quick relief items.
⎯ If more than first aid is needed, it is recommended to go to Student Health Services for further
treatment.
⎯ In an event that would require more than first aid to be treated, report it to the EHS office within
the next 24 hours.
⎯ When there are chemical, biological, or radioactive agents being used, an emergency spill kit should
be available.
⎯ Each laboratory has a telephone in a designated area for use. The emergency contact numbers are
posted near the phone in every laboratory on ISU campus.

Hazards in Lab (Know what Hazards are present)

⎯ Each lab is faced with different hazards. There could be exposure to biological, chemical, or
radioactive material, which may pose a variety of physical and/or health hazards.
⎯ A biological hazard includes an organism or material of biological origin that could potentially
cause harm to humans, animals, or plants.
⎯ Chemicals can pose a significant hazard.
= They should be limited to the use under a properly working fume hood.
= Chemicals can release hazardous fumes which not only harm the environment, but they can be a major
health threat.
= They must be handled carefully and disposed properly.
⎯ The following guidelines have been established to minimize the hazards in a laboratory setting.
⎯ It is important to take responsibility for your actions and to keep in mind that irresponsible acts
could have lasting future effects.
⎯ As the hazards increase, the risks increase, and the responsibility must increase.
Lab Attire
= No open-toed shoes
= No shorts unless a lab coat is used
= Restrain hair when working with hazardous materials
= Remove protective clothing in public
= Use the proper Personal Protective Equipment for the job

Personal Habits
Personal habits play a large role in minimizing hazards. The following measures must be taken:
⎯ Do not eat, drink, smoke, chew gum or apply cosmetics, or remove/insert contact lenses while in the
laboratory.
⎯ Do not store food or beverages in the lab or in chemical refrigerator.
⎯ Do not mouth pipette.
⎯ Wash hands before leaving laboratory or after handling contaminated material.

Safe Practices
These safe practices should be followed to ensure safe working conditions:
⎯ Do not use chipped or cracked glassware.
⎯ When working with hazardous materials, have a second person nearby.
⎯ Know emergency procedures.
⎯ Keep the laboratory neat and clean.
⎯ Use hazardous chemicals under a fume hood and biohazardous materials under a biosafety cabinet (BSC)
⎯ Decontaminate as needed.
⎯ All procedures should be performed to minimize aerosol.

Guidelines
⎯ Never perform unauthorized experiments
⎯ No food or drink in the laboratory
⎯ Never remove chemicals or equipment from the laboratory
⎯ Never use chemicals from unlabeled container
⎯ In heating test tubes, never point the mouth of the test tube or Erlenmeyer flask at yourself or
another person; and always direct the mouth away from people.
⎯ Never heat a closed container, the increasing pressure in the container can cause the contents to
spray out the top, or the container shatters, spreading shards of glass
⎯ Keep flammable substances away from heat sources.
⎯ Clean up all spills immediately.
= Never return spilled or unused material to reagent bottle
= Solids should be swept up and placed in the waste disposal container for that lab
= Small liquid spills should be diluted with lots of water and mopped up with a cloth towel
= Large spills must be handled by the instructor
⎯ Begin First Aid immediately
= Alert the instructor.
= If chemicals have come in contact with the eyes, begin eye-washing immediately.
= Remove clothes that have chemical contamination.
= Move everyone away from the area to avoid further contamination.
⎯ Report all injuries to your instructor immediately.
⎯ Know details or locations of your vital laboratory information.

Recording Laboratory Information

⎯ Title
⎯ Aim
⎯ Methods
⎯ Results
⎯ Observations
⎯ Interpretation
⎯ Conclusions
Rubric
⎯ Aims and methods
⎯ Results
⎯ Conclusions
* Always write the date on top right side
* Right side of notebook is for texts, left side is for the non-textual data (figures)
* Back of notebook, for scribbles and scratch (quick calculations)
* Never wrote on piece of paper

Organization
Practical report (Published report/Research paper)
⎯ Why you carried out experiment?
⎯ What you aim to find out?
⎯ How did you find it out?
⎯ What are the results that you have obtained and interpretation?
⎯ Conclusion you have drawn

Standard report
⎯ Title = Measurement of creativity
⎯ Introduction = Why we did the lab experiment?
⎯ Methods = How did we did the experiment?
⎯ Results = What happened?
⎯ Discussions and Conclusions = What we found out?
 BIOCHEMISTRY-LABORATORY
BASIC PRINCIPLES

Biochemistry of Solution in Water

⎯ Water is the most abundant substance in living systems, making up 70% or more of the weight of most
organisms.
⎯ The first living organisms on Earth doubtless arose in an aqueous environment, and the course of
evolution has been shaped by the properties of the aqueous medium in which life began.
⎯ The attractive forces between water molecules and the slight tendency of water to ionize are of
crucial importance to the structure and function of biomolecules
⎯ The water molecule and its ionization products, H and OH, profoundly influence the structure, self-
assembly, and properties of all cellular components, including proteins, nucleic acids, and lipids.
⎯ The non-covalent interactions responsible for the strength and specificity of “recognition” among
biomolecules are decisively influenced by the solvent properties of water, including its ability to
form hydrogen bonds with itself and with solutes.

Acids and Bases pH Measurement

⎯ The pH Scale designates the H+ and OH- concentrations
⎯ The ion product of water, Kw, is the basis for the pH scale.
⎯ It is a convenient means of designating the concentration of H (and thus of OH-) in any aqueous
solution in the range between 1.0 M H and 1.0 M OH-.
⎯ The term pH is defined by the expression; the symbol p denotes “negative logarithm of.” Note that
the concentration of H must be expressed in molar (M) terms.
⎯ The value of 7 for the pH of a precisely neutral solution is not an arbitrarily chosen figure; it is
derived from the absolute value of the ion product of water at 25 degree Celsius, which by convenient
coincidence is a round number.
⎯ Solutions having a pH greater than 7 are alkaline or basic; the concentration of OH- is greater than
that of H. Conversely, solutions having a pH less than 7 are acidic. Keep in mind that the pH scale
is logarithmic, not arithmetic.
⎯ To say that two solutions differ in pH by 1 pH unit means that one solution has ten times the H
concentration of the other, but it does not tell us the absolute magnitude of the difference.
⎯ There are pH values of some common aqueous fluids. For example, a cola drink (pH 3.0) or red wine (pH
3.7) has an H concentration approximately 10,000 times that of blood (pH 7.4).
⎯ Measurement of pH is one of the most important and frequently used procedures in biochemistry.
⎯ The pH affects the structure and activity of biological macro-molecules; for example, the catalytic
activity of enzymes is strongly dependent on pH.
⎯ Measurements of the pH of blood and urine are commonly used in medical diagnoses.
⎯ The pH of the blood plasma of people with severe, uncontrolled diabetes, for example, is often below
the normal value of 7.4; this condition is called acidosis.
⎯ In certain other diseases the pH of the blood is higher than normal, a condition known as alkalosis.
⎯ Extreme acidosis or alkalosis can be life-threatening.

Virtual Laboratory Experiment

⎯ Solutions consists of a solute and a solvent. Solutions are clear and homogenous, have a variable
composition, do not settle, maybe separated by physical means and pass-through filter papers.
= Solutions may be labelled as dilute or concentrated, as saturated, or unsaturated, which are
relative terms and do not indicate any definite amount of solute and solvent.
= Definite concentrations of solutions can be expressed as percent by weight, percent by volume,
parts per million (ppm), molarity, molality, or normality.
= Most health professions use percent by weight or percent by volume while chemists use molarity in
expressing concentrations.
= Percent by weight is the grams of solute per 100 grams of solvent, while percent by volume is the
millilitre (mL) of solute per 100 mL of solvent. A molar solution contains 1 mole of solute per
liter (L) of solution.
= Compounds such as NaCl contain many ions. Ions with opposite charges are attracted to each other,
leading to the formation of ionic bonds within the crystals of the compound.
= When NaCl dissolves in water, the individual ions dissociate and move about independently in the
solution.
= Pure distilled water does not conduct electricity.
 = After ions dissolve in water, the solution becomes a good conductor of electric current. This is
due to the presence of the mobile charged particles, called ions that have been released in the
solution.
= Solutes that release ions in solution are called electrolytes and they conduct electric current.
= Molecules such as sugar contain polar covalent bonds and are polar molecules.
= When crystals of sugar dissolve in water, the molecules of sugar move about independently of other
sugar molecules.
= The covalent bonds that link the atoms together in one sugar molecule do not dissociate.
= No ions are released, and the sugar molecules are not charged. A sugar solution does not conduct
current.
= Solutes of this type are non-electrolytes and they do not conduct electricity.
- Virtual Lab: https://phet.colorado.edu/en/simulation/concentration

⎯ The pH scale is used to rank solutions in terms of acidity or basicity (alkalinity).

= Since the scale is based on pH values, it is logarithmic, meaning that a change of 1 pH unit
corresponds to a ten-fold change in H+ ion concentration.
= The pH scale is often said to range from 0 to 14, and most solutions do fall within this range,
although it is possible to get a pH below 0 or above 14.
= Anything below 7.0 is acidic, and anything above 7.0 is alkaline, or basic.
= pH scale, ranging from 0 (very acidic) to 14 (very basic/alkaline) and listing the pH values of
common substances.
= The pH inside human cells (6.8) and the pH of blood (7.4) are both very close to neutral.
= Extreme pH values, either above or below 7.0, are usually considered unfavorable for life.
= However, the environment inside your stomach is highly acidic, with a pH of 1 to 2. How does the
stomach get around this problem?
= The answer: Disposable cells! Stomach cells, particularly those that come in direct contact with
stomach acid and food, are constantly dying and being replaced by new ones. In fact, the lining of
the human stomach is completely replaced about every seven to ten days.
- Virtual Lab: https://phet.colorado.edu/en/simulation/ph-scale

⎯ The electrolytes are divided into three groups: acids, bases, and salts.
= Acids are compounds that can release hydrogen ions in an aqueous solution. Such compounds usually
contain hydrogen linked to an element or group.
= Some common acids are hydrochloric acid (HCl), sulfuric acid (H2SO4), nitric acid (HNO3) and
phosphoric acid (H3PO4).
= Bases also called alkalis release the hydroxide ion (OH-) in aqueous solution.
= Some common bases are sodium hydroxide (NaOH), potassium hydroxide (KOH), calcium hydroxide
(Ca(OH)2), and ammonium hydroxide (NH4OH).
= Salts are electrolytes that release ions other than H+ or OH- in solution.
= Some common salts are sodium chloride (NaCl), calcium sulfate (CaSO4) and ammonium chloride
(NH4Cl).
- Virtual Lab: https://phet.colorado.edu/en/simulation/acid-base-solutions
 BIOCHEMISTRY-LABORATORY
BIOCHEMICAL PROCESSES

Transport along Cell Membrane

⎯ Plasma membranes must allow certain substances to enter and leave a cell, while preventing harmful
material from entering and essential material from leaving.
⎯ In other words, plasma membranes are selectively permeable; they allow some substances through but
not others.
⎯ If they were to lose this selectivity, the cell would no longer be able to sustain itself, and it
would be destroyed.
⎯ Some cells require larger amounts of specific substances than do other cells; they must have a way of
obtaining these materials from the extracellular fluids.
⎯ This may happen passively, as certain materials move back and forth, or the cell may have special
mechanisms that ensure transport.
⎯ Most cells expend most of their energy, in the form of adenosine triphosphate (ATP), to create and
maintain an uneven distribution of ions on the opposite sides of their membranes.
⎯ The structure of the plasma membrane contributes to these functions, but it also presents some
problems.
⎯ A physical space in which there is a different concentration of a single substance is said to have a
concentration gradient.
⎯ Substances move through the plasma membrane in basically two ways; passively or actively.
⎯ In passive processes, substances are transported across the membrane without any energy input from
the cell.
⎯ In active processes, the cell provides the metabolic energy (ATP) that drives the transport process.

Passive Processes
⎯ Diffusion is an important means of passive membrane transport for every cell of the body. The other
passive transport process, filtration, generally occurs only across capillary walls.
= Diffusion is the process by which molecules (and ions) move away from areas where they are more
concentrated (more numerous) to areas where they are less concentrated (with fewer of them).
= All molecules possess kinetic energy, or energy of motion, and as the molecules move about randomly
at high speeds, they collide and change direction with each collision.
= The greater the difference in concentration between the two areas, the faster diffusion occurs.
= Because the driving force (source of energy) is the kinetic energy of the molecules themselves, the
speed of diffusion is affected by the size of the molecules (the smaller the faster) and temperature
(the warmer the faster).
= The hydrophobic core of the plasma membrane is a physical barrier to diffusion.
= However, molecules will diffuse through the plasma membrane if any of the following are true:
- The molecules are small enough to pass through the membrane’s pores (channels formed by
membrane proteins).
- The molecules are lipid-soluble.
- The molecules are assisted by a membrane carrier.
= Several factors affect the rate of diffusion:
- Extent of the concentration gradient: The greater the difference in concentration, the more
rapid the diffusion. The closer the distribution of the material gets to equilibrium, the
slower the rate of diffusion becomes.
- Mass of the molecules diffusing: More massive molecules move more slowly, because it is more
difficult for them to move between the molecules of the substance they are moving through;
therefore, they diffuse more slowly.
- Temperature: Higher temperatures increase the energy and therefore the movement of the
molecules, increasing the rate of diffusion.
- Solvent density: As the density of the solvent increases, the rate of diffusion decreases.
The molecules slow down because they have a more difficult time getting through the denser
medium.
= 2 types of Diffusion:
= Simple Diffusion
- The unassisted diffusion of solutes through the plasma membrane (or any selectively
permeable membrane) is called simple diffusion. Solutes transported this way are lipid-soluble
(such as fats, fat-soluble vitamins, oxygen, carbon dioxide).
 = Facilitated Diffusion
- Facilitated diffusion provides passage for certain needed substances (notably glucose) that
are both lipid-insoluble and too large to pass through the membrane pores, or charged, as in
the case of chloride ions passing through a membrane protein channel.
- Although facilitated diffusion follows the laws of diffusion—that is, the substances move
down their own concentration gradients—a protein membrane protein channel is used, or a
membrane protein that acts as a carrier is needed to move glucose and certain other solutes
passively across the membrane into the cell.
⎯ Osmosis
= Diffusion of water through a selectively permeable membrane such as the plasma membrane is
specifically called osmosis.
= Because water is highly polar, it is repelled by the (nonpolar) lipid core of the plasma membrane,
but it can and does pass easily through special pores called aquaporins (“water pores”) created by
proteins in the membrane
= Osmosis into and out of cells is occurring all the time as water moves down its concentration
gradient. The movement of water across the membrane occurs quickly.
= Anyone administering an IV (intravenous, into the vein) solution must use the correct solution to
protect the patient’s cells from life-threatening dehydration or rupture.

Active Transport
⎯ Active transport mechanisms require the use of the cell’s energy, usually in the form of adenosine
triphosphate (ATP).
⎯ Active transport uses ATP to energize its protein carriers, which are called solute pumps.
⎯ If a substance must move into the cell against its concentration gradient, that is, if the
concentration of the substance inside the cell must be greater than its concentration in the
extracellular fluid, the cell must use energy to move the substance.
⎯ Some active transport mechanisms move small-molecular weight material, such as ions, through the
membrane.
⎯ Because cells contain proteins, most of which are negatively charged, and because ions move into and
out of cells, there is an electrical gradient, a difference of charge, across the plasma membrane.
⎯ The interior of living cells is electrically negative with respect to the extracellular fluid in
which they are bathed; at the same time, cells have higher concentrations of potassium (K+) and lower
concentrations of sodium (Na+) than does the extracellular fluid.
⎯ Thus, in a living cell, the concentration gradient and electrical gradient of Na+ promotes diffusion
of the ion into the cell, and the electrical gradient of Na+ (a positive ion) tends to drive it
inward to the negatively charged interior.

Sodium-Potassium Pump
Bulk (Vesicular) Transport
= Some substances cannot get through the plasma membrane by active or passive transport.
= Vesicular transport, which involves help from ATP to fuse or separate membrane vesicles and the cell
membrane, moves substances into or out of cells “in bulk” without their crossing the plasma membrane
directly.
⎯ Exocytosis
= “Out of the cell”
= Is the mechanism that cells use to actively secrete hormones, mucus, and other cell products or to
eject certain cellular wastes.
= The product to be released is first “packaged” (typically by the Golgi apparatus) into a secretory
vesicle.
= The vesicle migrates to the plasma membrane, fuses with it, and then ruptures, spilling its
contents out of the cell.
= Exocytosis involves a “docking” process in which docking proteins on the vesicles recognize plasma
membrane docking proteins and bind with them.
= This binding causes the membranes to “corkscrew” together and fuse
⎯ Endocytosis
= “Into the cell”
= Includes those ATP-requiring processes that take up, or engulf, extracellular substances by
enclosing them in a vesicle
= Once the vesicle is formed, it detaches from the plasma membrane and moves into the cytoplasm,
where it typically fuses with a lysosome and its contents are digested (by lysosomal enzymes).
= However, in some cases, the vesicle travels to the opposite side of the cell and releases its
contents by exocytosis there
⎯ Phagocytosis
= “Cell-eating”
= The endocytosis process that engulfed substances which are relatively large particles, such as
bacteria or dead body cells, and the cell separates them from the external environment by pseudopods
= Certain white blood cells, such as the macrophage, and other “professional” phagocytes of the body
act as scavenger cells that police and protect the body by ingesting bacteria and other foreign
debris.
= Hence, phagocytosis is a protective mechanism—a way to “clean house”—not a means of getting
nutrients.
⎯ Pinocytosis
= “Cell-drinking”
= The cell “gulps” droplets of extracellular fluid.
= The plasma membrane indents to form a tiny pit, or “cup,” and then its edges fuse around the
droplet of extracellular fluid containing dissolved proteins or fats
= Unlike phagocytosis, pinocytosis is a routine activity of most cells. It is especially important in
cells that function in absorption (for example, cells forming the lining of the small intestine).
⎯ Receptor-Mediated Endocytosis
= The main cellular mechanism for taking up specific target molecules.
= In this process, receptor proteins on the plasma membrane bind exclusively with certain substances.
= Both the receptors and high concentrations of the attached target molecules are internalized in a
vesicle, and then the contents of the vesicle are dealt with in one of the ways.
= Although phagocytosis and pinocytosis are important, they are not very selective compared to
receptor-mediated endocytosis.
= Specific substances taken in by receptor-mediated endocytosis include enzymes, some hormones,
cholesterol, and iron. Unfortunately, flu viruses exploit this route to enter and attack our cells.
 BIOCHEMISTRY-LABORATORY
CARBOHYDRATES
Molecular Structures
= The stoichiometric formula (CH2O)n, where n is the number of carbons in the molecule represents
carbohydrates. In other words, the ratio of carbon to hydrogen to oxygen is 1:2:1 in carbohydrate
molecules.
= This formula also explains the origin of the term “carbohydrate”: the components are carbon (“carbo”) and
the components of water (hence, “hydrate”).
= Scientists classify carbohydrates into three subtypes: monosaccharides, disaccharides, and
polysaccharides.
⎯ Monosaccharides
= Monosaccharides (mono = “one”; sacchar = “sweet”) are simple sugars, the most common of which is
glucose.
= In monosaccharides, the number of carbons usually ranges from three to seven.
= Most monosaccharide names end with the suffix -ose.
= If the sugar has an aldehyde group (the functional group with the structure R-CHO), it is an
aldose, and if it has a ketone group (the functional group with the structure RC(=O)R'), it is a
ketose.
= Depending on the number of carbons in the sugar, they can be trioses (three carbons), pentoses
(five carbons), and/or hexoses (six carbons).

= The chemical formula for glucose is C6H12O6. In humans, glucose is an important source of energy.
= During cellular respiration, energy releases from glucose, and that energy helps make adenosine
triphosphate (ATP).
= Plants synthesize glucose using carbon dioxide and water, and glucose in turn provides energy
requirements for the plant.
= Humans and other animals that feed on plants often store excess glucose as catabolized (cell
breakdown of larger molecules) starch.
= Galactose (part of lactose, or milk sugar) and fructose (found in sucrose, in fruit) are other
common monosaccharides.
= Although glucose, galactose, and fructose all have the same chemical formula (C6H12O6), they differ
structurally and chemically (and are isomers) because of the different arrangement of functional
groups around the asymmetric carbon. All these monosaccharides have more than one asymmetric carbon.
 = Glucose, galactose, and fructose are isomeric monosaccharides (hexoses), meaning they have the same
chemical formula but have slightly different structures. Glucose and galactose are aldoses, and
fructose is a ketose.
= Monosaccharides can exist as a linear chain or as ring-shaped molecules. In aqueous solutions they
are usually in ring forms (Figure 3.6).
= Glucose in a ring form can have two different hydroxyl group arrangements (OH) around the anomeric
carbon (carbon 1 that becomes asymmetric in the ring formation process).
= If the hydroxyl group is below carbon number 1 in the sugar, it is in the alpha (α) position, and
if it is above the plane, it is in the beta (β) position.

⎯ Disaccharides
= Disaccharides (di = “two”) form when two monosaccharides undergo a dehydration reaction (or a
condensation reaction or dehydration synthesis).
= During this process, one monosaccharide's hydroxyl group combines with another monosaccharide's
hydrogen, releasing a water molecule and forming a covalent bond.
= A covalent bond forms between a carbohydrate molecule and another molecule (in this case, between
two monosaccharides). Scientists call this a glycosidic bond (Figure 3.7).
= Glycosidic bonds (or glycosidic linkages) can be an alpha or beta type. An alpha bond is formed
when the OH group on the carbon-1 of the first glucose is below the ring plane, and a beta bond is
formed when the OH group on the carbon-1 is above the ring plane.
 = Common disaccharides include lactose, maltose, and sucrose (Figure 3.8).
= Lactose is a disaccharide consisting of the monomer’s glucose and galactose. It is naturally in
milk.
= Maltose, or malt sugar, is a disaccharide formed by a dehydration reaction between two glucose
molecules.
= The most common disaccharide is sucrose, or table sugar, which is comprised of glucose and fructose
monomers.

⎯ Polysaccharides
= A long chain of monosaccharides linked by glycosidic bonds is a polysaccharide (poly = “many”).
= The chain may be branched or unbranched, and it may contain different types of monosaccharides. The
molecular weight may be 100,000 daltons or more depending on the number of joined monomers.
= Starch, glycogen, cellulose, and chitin are primary examples of polysaccharides.
= Plants store starch in the form of sugars. In plants, an amylose and amylopectic mixture (both
glucose polymers) comprise these sugars.
= Plants can synthesize glucose, and they store the excess glucose, beyond their immediate energy
needs, as starch in different plant parts, including roots and seeds.
= The starch in the seeds provides food for the embryo as it germinates and can also act as a food
source for humans and animals.
= Enzymes break down the starch that humans consume.
= For example, an amylase present in saliva catalyzes, or breaks down this starch into smaller
molecules, such as maltose and glucose. The cells can then absorb the glucose.
= Glucose starch comprises monomers that are joined by α 1-4 or α 1-6 glycosidic bonds. The numbers
1-4 and 1-6 refer to the carbon number of the two residues that have joined to form the bond.
= As Figure 3.9 illustrates, unbranched glucose monomer chains (only α 1-4 linkages) form the starch,
whereas amylopectin is a branched polysaccharide (α 1-6 linkages at the branch points).

= Glycogen is the storage form of glucose in humans and other vertebrates and is comprised of
monomers of glucose. Glycogen is the animal equivalent of starch and is a highly branched molecule
usually stored in liver and muscle cells.
= Whenever blood glucose levels decrease, glycogen breaks down to release glucose in a process call
glycogenolysis.
= Cellulose is the most abundant natural biopolymer. Cellulose mostly comprises a plant's cell wall.
This provides the cell structural support. Wood and paper are mostly cellulosic in nature.
= Glucose monomers comprise cellulose that β 1-4 glycosidic bonds link (Figure 3.10).

= As Figure 3.10 shows, every other glucose monomer in cellulose is flipped over, and the monomers
are packed tightly as extended long chains.
= This gives cellulose its rigidity and high tensile strength which is so important to plant cells.
While human digestive enzymes cannot break down the β 1-4 linkage, herbivores such as cows, koalas,
and buffalos are able, with the help of the specialized flora in their stomach, to digest plant
material that is rich in cellulose and use it as a food source.
= In some of these animals, certain species of bacteria and protists reside in the rumen (part of the
herbivore's digestive system) and secrete the enzyme cellulase.
= The appendix of grazing animals also contains bacteria that digest cellulose, giving it an
important role in ruminants' digestive systems.
 = Cellulases can break down cellulose into glucose monomers that animals use as an energy source.
= Termites are also able to break down cellulose because of the presence of other organisms in their
bodies that secrete cellulases.
= Carbohydrates serve various functions in different animals.
= Arthropods (insects, crustaceans, and others) have an outer skeleton, the exoskeleton, which
protects their internal body parts (as we see in the bee in Figure 3.11).
= This exoskeleton is made of the biological macromolecule chitin, which is a polysaccharide-
containing nitrogen.
= It is made of repeating N-acetyl-β-d-glucosamine units, which are a modified sugar.
= Chitin is also a major component of fungal cell walls.
= Fungi are neither animals nor plants and form a kingdom of their own in the domain Eukarya.

Benefits of Carbohydrates
⎯ Most people are familiar with carbohydrates, one type of macromolecule, especially when it comes to
what we eat.
⎯ To lose weight, some individuals adhere to “low-carb” diets. Athletes, in contrast, often “carb-load”
before important competitions to ensure that they have enough energy to compete at a high level.
⎯ Carbohydrates are, in fact, an essential part of our diet. Grains, fruits, and vegetables are all-
natural carbohydrate sources that provide energy to the body, particularly through glucose, a simple
sugar that is a component of starch and an ingredient in many staple foods.
⎯ Carbohydrates also have other important functions in humans, animals, and plants.
⎯ Some diets completely forbid carbohydrate consumption, claiming that a low-carbohydrate diet helps
people to lose weight faster.
⎯ However, carbohydrates have been an important part of the human diet for thousands of years.
⎯ Artifacts from ancient civilizations show the presence of wheat, rice, and corn in our ancestors’
storage areas.
⎯ As part of a well-balanced diet, we should supplement carbohydrates with proteins, vitamins, and
fats.
⎯ Calorie-wise, a gram of carbohydrate provides 4.3 Kcal. For comparison, fats provide 9 Kcal/g, a less
desirable ratio.
⎯ Carbohydrates contain soluble and insoluble elements. The insoluble part, fiber, is mostly cellulose.
Fiber has many uses.
⎯ It promotes regular bowel movement by adding bulk, and it regulates the blood glucose consumption
rate.
⎯ Fiber also helps to remove excess cholesterol from the body. Fiber binds to the cholesterol in the
small intestine, then attaches to the cholesterol and prevents the cholesterol particles from
entering the bloodstream.
⎯ Cholesterol then exits the body via the feces.
⎯ Fiber-rich diets also have a protective role in reducing the occurrence of colon cancer.
⎯ In addition, a meal containing whole grains and vegetables gives a feeling of fullness. As an
immediate source of energy, glucose breaks down during the cellular respiration process, which
produces ATP, the cell's energy currency.
⎯ Without consuming carbohydrates, we reduce the availability of “instant energy”.
⎯ Eliminating carbohydrates from the diet may be necessary for some people, but such a step may not be
healthy for everyone.

Virtual Laboratory Experiments

There will be three virtual laboratory experiments that you will engage in for our topic on the
Biochemistry of Carbohydrates: Qualitative Analysis of Carbohydrates, Yeast Fermentation and Hydrolysis of
Sucrose and Starch.
⎯ Qualitative Analysis of Carbohydrates
= In the first virtual simulation, you will be conducting several tests for the presence or absence
of carbohydrates in several compounds qualitatively. Specifically, you will be contrasting
monosaccharides, disaccharides and polysaccharides based on their properties and reactions to general
and specific tests performed for carbohydrates and identifying and classifying an unknown sample of
carbohydrate by comparing the results of the unknown to the various tests with that of the known test
substances.
Virtual laboratory:
https://www.youtube.com/watch?v=ojhdTFmkY1c
https://www.filscihub.com/community/blogpost-biolabs
biomolecules?fbclid=IwAR2HeEqaVQqPjXBiiF4rrtI9TTIKlPRoh1aNFLwmFs8wziIsga6VNHRVKAs
Tests and Reactions for Carbohydrates
= Molisch test is a general test for carbohydrates, and monosaccharides will give a positive test rapidly.
Disaccharides and polysaccharides will slowly hydrolyse to produce monosaccharides in the acidic medium of
the test and will also give a positive test. The test is on the basis that pentoses and hexoses are dehydrated
by concentrated Sulphuric acid to form furfural or hydroxymethylfurfural, respectively. These products
condense with α-naphthol to form purple condensation product.
= Fehling's test forms the reduction test of carbohydrates. Fehling’s solution contains blue alkaline cupric
hydroxide solution, heated with reducing sugars gets reduced to yellow or red cuprous oxide and is
precipitated. Hence, formation of the yellow or brownish-red colored precipitate helps in the detection of
reducing sugars in the test solution.
= Benedict’s test is used to detect the presence of reducing sugars. Any monosaccharide or disaccharide that
can form a free aldehyde group in solution will react with Benedict’s reagent to form a brick-red, brown,
green, or occasionally yellow precipitate. A colored solution with no precipitate is a negative result. As in
Fehling’s test, free aldehyde or keto group in the reducing sugars reduce cupric hydroxide in alkaline medium
to red colored cuprous oxide. Depending on the concentration of sugars, yellow to green color is developed.
All monosaccharides are reducing sugars as they all have a free reactive carbonyl group. Some disaccharides,
like maltose, have exposed carbonyl groups and are also reducing sugars, but less reactive than
monosaccharides.
= Seliwanoff test will distinguish between ketohexoses and aldohexoses. The difference in the times required
for the red color to appear is used to distinguish ketohexoses from aldohexoses. It is a color reaction
specific for ketoses. When concentrated HCl is added, ketoses undergo dehydration to yield furfural derivatives
more rapidly than aldoses. These derivatives form complexes with resorcinol to yield deep red color. The
test reagent causes the dehydration of ketohexoses to form 5-hydroxymethylfurfural. 5-hydroxymethylfurfural
reacts with resorcinol present in the test reagent to produce a red product within two minutes (reaction not
shown). Aldohexoses reacts so more slowly to form the same product.
= Barfoed’s test is used to distinguish between reducing monosaccharides and reducing disaccharides. Its color
reaction is like Benedict’s reagent. Barfoed's test is used to detect the presence of monosaccharide (reducing)
sugars in solution. Barfoed's reagent, a mixture of ethanoic (acetic) acid and copper (II) acetate, is combined
with the test solution and boiled. A red copper (II) oxide precipitate is formed will indicates the presence
of reducing sugar. The reaction will be negative in the presence of disaccharide sugars because they are
weaker reducing agents. This test is specific for monosaccharides. Due to the weakly acidic nature of Barfoed's
reagent, it is reduced only by monosaccharides.
= Iodine test is used to detect the presence of polysaccharides. Starch will form a dark-blue complex with
iodine. This test is used for the detection of starch in the solution. The blue-black color is due to the
formation of starch-iodine complex. Starch contains polymer of α-amylose and amylopectin which forms a complex
with iodine to give the blue-black color.
= Bial test differentiates pentoses from hexoses. A blue color is positive test for a pentose. They react
with Bial’s reagent and are converted to furfural. Orcinol and furfural condense in the presence of ferric
ion to form a colored product. Appearance of green colour or precipitate indicates the presence of pentoses,
and formation of muddy brown precipitate shows the presence of hexoses. All other colors are considered
negative for pentoses.
= Osazone test results when the ketoses and aldoses react with phenylhydrazine to produce a phenylhydrazone
which further reacts with another two molecules of phenylhydrazine to yield osazone. Needle-shaped yellow
osazone crystals are produced by glucose, fructose, and mannose, whereas lactosazone produces mushroom shaped
crystals. Crystals of different shapes will be shown by different osazones. Flower-shaped crystals are produced
by maltose.

⎯ Yeast Fermentation Experiment

= In the second virtual lab exercise, you will perform simulation on how fermentation process is
possible in yeast, using carbohydrate compounds as essential component.

The History of Beer and Wine Production

Over the course of human history, and using a system of trial, error, and careful observation, different
cultures began producing fermented beverages. Mead, or honey wine, was produced in Asia during the Vedic
period (around 1700–1100 BC), and the Greeks, Celts, Saxons, and Vikings also produced this beverage. In
Egypt, Babylon, Rome, and China, people produced wine from grapes and beer from malted barley. In South
America, people produced chicha from grains or fruits, mainly maize; while in North America, people made octli
(now known as "pulque") from agave, a type of cactus (Godoy et al. 2003).
At the time, people knew that leaving fruits and grains in covered containers for a long-time produced
wine and beer, but no one fully understood why the recipe worked. The process was named fermentation, from
the Latin word fervere, which means "to boil." The name came from the observation that mixtures of crushed
grapes kept in large vessels produced bubbles, as though they were boiling. Producing fermented beverages was
tricky. If the mixture did not stand long enough, the product contained no alcohol; but if left for too long,
the mixture rotted and was undrinkable. Through empirical observation, people learned that temperature and
air exposure are key to the fermentation process.
 Wine producers traditionally used their feet to soften and grind the grapes before leaving the mixture
to stand in buckets. In so doing, they transferred microorganisms from their feet into the mixture. At the
time, no one knew that the alcohol produced during fermentation was produced because of one of these
microorganisms — a tiny, one-celled eukaryotic fungus that is invisible to the naked eye: yeast. It took
several hundred years before quality lenses and microscopes revolutionized science and allowed researchers to
observe these microorganisms.
Reference: https://www.nature.com/scitable/topicpage/yeast-fermentation-and-the-making-of-beer-14372813/

Yeast and Fermentation

In the seventeenth century, a Dutch tradesman named Antoni van Leeuwenhoek developed high-quality
lenses and was able to observe yeast for the first time. In his spare time Leeuwenhoek used his lenses to
observe and record detailed drawings of everything he could, including very tiny objects, like protozoa,
bacteria, and yeast. Leeuwenhoek discovered that yeast consist of globules floating in a fluid, but he thought
they were merely the starchy particles of the grain from which the wort (liquid obtained from the brewing of
whiskey and beer) was made (Huxley 1894). Later, in 1755, yeast was defined in the Dictionary of the English
Language by Samuel Johnson as "the ferment put into drink to make it work; and into bread to lighten and swell
it." At the time, nobody believed that yeast was alive; they were just organic chemical agents required for
fermentation.
In the eighteenth and nineteenth centuries, chemists worked hard to decipher the nature of alcoholic
fermentation through analytical chemistry and chemical nomenclature. In 1789, the French chemist Antoine
Lavoisier was working on basic theoretical questions about the transformations of substances. In his quest,
he decided to use sugars for his experiments, and he gained new knowledge about their structures and chemical
reactions. Using quantitative studies, he learned that sugars are composed of a mixture of hydrogen, charcoal
(carbon), and oxygen.
Lavoisier was also interested in analyzing the mechanism by which sugarcane is transformed into alcohol
and carbon dioxide during fermentation. He estimated the proportions of sugars and water at the beginning of
the chemical reaction and compared them with the alcohol and carbon dioxide proportions obtained at the end.
For the alcoholic reaction to proceed, he also added yeast paste (or "ferment," as it was called). He concluded
that sugars were broken down through two chemical pathways: Two-thirds of the sugars were reduced to form
alcohol, and the other third were oxidized to form carbon dioxide (the source of the bubbles observed during
fermentation). Lavoisier predicted (according to his famous conservation-of-mass principle) that if it were
possible to combine alcohol and carbon dioxide in the right proportions, the resulting product would be sugar.
The experiment provided a clear insight into the basic chemical reactions needed to produce alcohol. However,
there was one problem: Where did the yeast fit into the reaction? The chemists hypothesized that the yeast
initiated alcoholic fermentation but did not take part in the reaction. They assumed that the yeast remained
unchanged throughout the chemical reactions.
Reference: https://www.nature.com/scitable/topicpage/yeast-fermentation-and-the-making-of-beer-14372813/
Virtual laboratory:
http://www.bch.cuhk.edu.hk/vlab2/animation/fermentation/index.html
http://www.bch.cuhk.edu.hk/vlab2/#2/slide4

⎯ Starch Hydrolysis
= The third virtual lab experiment has something to do with the process of hydrolyzing sucrose and
starch, representative carbohydrate forms and you will be tasked to identify chemical changes that
will happen to these as they undergo reactions with water.

Starch Hydrolysis Test

Complete hydrolysis of polysaccharides and disaccharides produces monosaccharide. While the reaction
involves water as the actual reactant, catalysts are required for the reaction to proceed at an appreciable
rate. In the experiment, some of these catalysts will be studied namely, salivary amylase and dilute acid,
HCl. Tests done in the preceding experiment will be used to determine the products of hydrolysis.
Hydrolysis of starch progresses in several stages with a-D-glucose as the final product. Starch gives
an intense, brilliant blue-black color with iodine, whereas intermediate products of hydrolysis called dextrins
give red to red-brown color. As hydrolysis proceeds, the products give no color with iodine.
References:
https://www.youtube.com/watch?v=zFhMbXSgve8
https://www.youtube.com/watch?v=6i7KSyAwplc
 BIOCHEMISTRY-LABORATORY
PROTEINS
Protein
⎯ The term "protein" is derived from the Greek word "proteios", meaning of first importance. It is the
most abundant of cellular components making up 70% of the organic matter of a cell.
⎯ It is also the first most extensively studied biomolecule simply because all expressed traits or
phenotype is due to a particular protein component.
⎯ Hence any change in the organisms' characters such as in mutation represents a change in the protein
responsible for these traits.
⎯ The simplest unit is the amino acid. Proteins are, therefore, polymers of this repeating unit linked
together by peptide bonds.
⎯ The properties and biological functions of proteins is dependent on the amino acids making up the
protein.
⎯ Thus, to understand protein structure and function, one has first to understand the properties and
features of the repeating unit, amino acids.
⎯ Proteins are one of the most abundant organic molecules in living systems and have the most diverse
range of functions of all macromolecules.
⎯ Proteins may be structural, regulatory, contractile, or protective. They may serve in transport,
storage, or membranes; or they may be toxins or enzymes. Each cell in a living system may contain
thousands of proteins, each with a unique function.
⎯ Their structures, like their functions, vary greatly. They are all, however, amino acid polymers
arranged in a linear sequence.

Types and Functions of Proteins

⎯ Enzyme
= Enzymes, which living cells produce, are catalysts in biochemical reactions (like digestion) and
are usually complex or conjugated proteins.
= Each enzyme is specific for the substrate (a reactant that binds to an enzyme) upon which it acts.
The enzyme may help in breakdown, rearrangement, or synthesis reactions.
= We call enzymes that break down their substrate’s catabolic enzymes. Those that build more complex
molecules from their substrates are anabolic enzymes, and enzymes that affect the rate of reaction
are catalytic enzymes.
= Note that all enzymes increase the reaction rate and, therefore, are organic catalysts. An example
of an enzyme is salivary amylase, which hydrolyzes its substrate amylose, a component of starch.
⎯ Hormones
= Hormones are chemical-signaling molecules, usually small proteins, or steroids, secreted by
endocrine cells that act to control or regulate specific physiological processes, including growth,
development, metabolism, and reproduction.
= For example, insulin is a protein hormone that helps regulate the blood glucose level. Table 3.1
lists the primary types and functions of proteins.

= Proteins have different shapes and molecular weights. Some proteins are globular in shape, whereas,
others are fibrous in nature. For example, hemoglobin is a globular protein, but collagen, located in
our skin, is a fibrous protein.
= Protein shape is critical to its function, and many different types of chemical bonds maintain this
shape. Changes in temperature, pH, and exposure to chemicals may lead to permanent changes in the
protein's shape, leading to loss of function, or denaturation.
= Different arrangements of the same 20 types of amino acids comprise all proteins. Two rare new
amino acids were discovered recently (selenocystein and pirrolysine), and additional new discoveries
may be added to the list.
Amino Acids
⎯ Amino acids are the monomers that comprise proteins. Each amino acid has the same fundamental
structure, which consists of a central carbon atom, or the alpha (α) carbon, bonded to an amino group
(NH2), a carboxyl group (COOH), and to a hydrogen atom. Every amino acid also has another atom or
group of atoms bonded to the central atom known as the R group (Figure 3.22).

Figure 3.22 Amino acids have a central asymmetric carbon to which an amino group, a carboxyl group, a
hydrogen atom, and a side chain (R group) are attached.
⎯ Scientists use the name "amino acid" because these acids contain both amino group and carboxyl-acid-
group in their basic structure.
⎯ As we mentioned, there are 20 common amino acids present in proteins. Nine of these are essential
amino acids in humans because the human body cannot produce them, and we obtain them from our diet.
For each amino acid, the R group (or side chain) is different.

⎯ The chemical nature of the side chain determines the amino acid's nature (that is, whether it is
acidic, basic, polar, or nonpolar).
⎯ For example, the amino acid glycine has a hydrogen atom as the R group. Amino acids such as valine,
methionine, and alanine are nonpolar or hydrophobic in nature, while amino acids such as serine,
threonine, and cysteine are polar and have hydrophilic side chains.
⎯ The side chains of lysine and arginine are positively charged, and therefore these amino acids are
also basic amino acids. Proline has an R group that is linked to the amino group, forming a ring-like
structure. Proline is an exception to the amino acids standard structure since its amino group is not
separate from the side chain (Figure 3.23).
⎯ A single upper-case letter or a three-letter abbreviation represents amino acids. For example, the
letter V or the three-letter symbol val represent valine. Just as some fatty acids are essential to a
diet, some amino acids also are necessary. These essential amino acids in humans include isoleucine,
leucine, and cysteine. Essential amino acids refer to those necessary to build proteins in the body,
but not those that the body produces. Which amino acids are essential varies from organism to
organism.
⎯ The sequence and the number of amino acids ultimately determine the protein's shape, size, and
function.
 ⎯ A covalent bond, or peptide bond, attaches to each amino acid, which a dehydration reaction forms.
One amino acid's carboxyl group and the incoming amino acid's amino group combine, releasing a water
molecule. The resulting bond is the peptide bond (Figure 3.24).

Figure 3.24 Peptide bond formation is a dehydration synthesis reaction. The carboxyl group of one amino
acid is linked to the incoming amino acids amino group. In the process, it releases a water molecule.
⎯ The products that such linkages form are peptides. As more amino acids join to this growing chain,
the resulting chain is a polypeptide.
⎯ Each polypeptide has a free amino group at one end. This ends the N terminal, or the amino terminal,
and the other end has a free carboxyl group, also the C or carboxyl terminal. While the terms
polypeptide and protein are sometimes used interchangeably, a polypeptide is technically a polymer of
amino acids, whereas the term protein is used for a polypeptide or polypeptides that have combined,
often have bound non-peptide prosthetic groups, have a distinct shape, and have a unique function.
⎯ After protein synthesis (translation), most proteins are modified. These are known as post-
translational modifications. They may undergo cleavage, phosphorylation, or may require adding other
chemical groups. Only after these modifications is the protein completely functional.

Protein Structure
= As we discussed earlier, a protein's shape is critical to its function. For example, an enzyme can bind
to a specific substrate at an active site.
= If this active site is altered because of local changes or changes in overall protein structure, the
enzyme may be unable to bind to the substrate. To understand how the protein gets its final shape or
conformation, we need to understand the four levels of protein structure: primary, secondary, tertiary, and
quaternary.
⎯ Primary Structure
= Amino acids' unique sequence in a polypeptide chain are its primary structure. For example, the
pancreatic hormone insulin has two polypeptide chains, A and B, and they are linked together by
disulfide bonds. The N terminal amino acid of the A chain is glycine, whereas the C terminal amino
acid is asparagine (Figure 3.25). The amino acid sequences in the A and B chains are unique to
insulin.

Figure 3.25 Bovine serum insulin is a protein hormone comprised of two peptide chains, A (21 amino acids
long) and B (30 amino acids long). In each chain, three-letter abbreviations that represent the amino
acids' names in the order they are present indicate primary structure. The amino acid cysteine (cys) has a
sulfhydryl (SH) group as a side chain. Two sulfhydryl groups can react in the presence of oxygen to form a
disulfide (S-S) bond. Two disulfide bonds connect the A and B chains together, and a third helps the A
chain fold into the correct shape. Note that all disulfide bonds are the same length, but we have drawn
them different sizes for clarity.
= The gene encoding the protein ultimately determines the unique sequence for every protein. A change
in nucleotide sequence of the gene’s coding region may lead to adding a different amino acid to the
growing polypeptide chain, causing a change in protein structure and function.
 = In sickle cell anemia, the hemoglobin β chain (a small portion of which we show in Figure 3.26) has
a single amino acid substitution, causing a change in protein structure and function. Specifically,
valine in the β chain substitutes the amino acid glutamic.
= What is most remarkable to consider is that a hemoglobin molecule is comprised of two alpha and two
beta chains that each consist of about 150 amino acids. The molecule, therefore, has about 600 amino
acids. The structural difference between a normal hemoglobin molecule and a sickle cell molecule—
which dramatically decreases life expectancy—is a single amino acid of the 600. What is even more
remarkable is that three nucleotides each encode those 600 amino acids, and a single base change
(point mutation), 1 in 1800 bases causes the mutation.

Figure 3.26 The beta chain of hemoglobin is 147 residues in length, yet a single amino acid substitution
leads to sickle cell anemia. In normal hemoglobin, the amino acid at position seven is glutamate. In sickle
cell hemoglobin, a valine replaces glutamate.
= Because of this change of one amino acid in the chain, hemoglobin molecules form long fibers that
distort the biconcave, or disc-shaped, red blood cells and causes them to assume a crescent or
“sickle” shape, which clogs blood vessels (Figure 3.27).
= This can lead to myriad serious health problems such as breathlessness, dizziness, headaches, and
abdominal pain for those affected by this disease.

Figure 3.27 In this blood smear, visualized at 535x magnification using bright field microscopy, sickle
cells are crescent shaped, while normal cells are disc shaped. (credit: modification of work by Ed Uthman;
scale-bar data from Matt Russell)
⎯ Secondary Structure
= The local folding of the polypeptide in some regions gives rise to the secondary structure of the
protein. The most common are the α-helix and β-pleated sheet structures (Figure 3.28).
= Both structures are held in shape by hydrogen bonds. The hydrogen bonds form between the oxygen
atom in the carbonyl group in one amino acid and another amino acid that is four amino acids farther
along the chain.
 Figure 3.28 The α-helix and β-pleated sheet are secondary structures of proteins that form because of
hydrogen bonding between carbonyl and amino groups in the peptide backbone. Certain amino acids have a
propensity to form an α-helix, while others have a propensity to form a β-pleated sheet.
= Every helical turn in an alpha helix has 3.6 amino acid residues. The polypeptide's R groups (the
variant groups) protrude out from the α-helix chain. In the β-pleated sheet, hydrogen bonding between
atoms on the polypeptide chain's backbone form the "pleats".
= The R groups are attached to the carbons and extend above and below the pleat's folds. The pleated
segments align parallel or antiparallel to each other, and hydrogen bonds form between the partially
positive nitrogen atom in the amino group and the partially negative oxygen atom in the peptide
backbone's carbonyl group.
= The α-helix and β-pleated sheet structures are in most globular and fibrous proteins and they play
an important structural role.
⎯ Tertiary Structure
= The polypeptide's unique three-dimensional structure is its tertiary structure (Figure 3.29). This
structure is in part due to chemical interactions at work on the polypeptide chain.
= Primarily, the interactions among R groups create the protein's complex three-dimensional tertiary
structure. The nature of the R groups in the amino acids involved can counteract forming the hydrogen
bonds we described for standard secondary structures.
= For example, R groups with like charges repel each other and those with unlike charges are
attracted to each other (ionic bonds). When protein folding takes place, the nonpolar amino acids'
hydrophobic R groups lie in the protein's interior, whereas the hydrophilic R groups lie on the
outside.
= Scientists also call the former interaction types of hydrophobic interactions. Interaction between
cysteine side chains forms disulfide linkages in the presence of oxygen, the only covalent bond that
forms during protein folding.

Figure 3.29 A variety of chemical interactions determine the proteins' tertiary structure. These include
hydrophobic interactions, ionic bonding, hydrogen bonding, and disulfide linkages.
= All of these interactions, weak and strong, determine the protein's final three-dimensional shape.
When a protein loses its three-dimensional shape, it may no longer be functional.
⎯ Quaternary Structure
= In nature, some proteins form from several polypeptides, or subunits, and the interaction of these
subunits forms the quaternary structure.
= Weak interactions between the subunits help to stabilize the overall structure.
= For example, insulin (a globular protein) has a combination of hydrogen and disulfide bonds that
cause it to mostly clump into a ball shape. Insulin starts out as a single polypeptide and loses some
internal sequences in the presence of post-translational modification after forming the disulfide
linkages that hold the remaining chains together.
= Silk (a fibrous protein), however, has a β-pleated sheet structure that is the result of hydrogen
bonding between different chains.
= Figure 3.30 illustrates the four levels of protein structure (primary, secondary, tertiary, and
quaternary).
 Figure 3.30 Observe the four levels of protein structure in these illustrations. (credit: modification of
work by National Human Genome Research Institute)
Denaturation and Protein Folding
= Each protein has its own unique sequence and shape that chemical interactions hold together. If the
protein is subject to changes in temperature, pH, or exposure to chemicals, the protein structure may
change, losing its shape without losing its primary sequence in what scientists call denaturation.
= Denaturation is often reversible because the polypeptide's primary structure is conserved in the process
if the denaturing agent is removed, allowing the protein to resume its function.
= Sometimes denaturation is irreversible, leading to loss of function. One example of irreversible protein
denaturation is frying an egg. The albumin protein in the liquid egg white denatures when placed in a hot
pan. Not all proteins denature at high temperatures.
= For instance, bacteria that survive in hot springs have proteins that function at temperatures close to
boiling.
= The stomach is also very acidic, has a low pH, and denatures proteins as part of the digestion process;
however, the stomach's digestive enzymes retain their activity under these conditions.
= Protein folding is critical to its function. Scientists originally thought that the proteins themselves
were responsible for the folding process.
= Only recently researchers discovered that often they receive assistance in the folding process from
protein helpers, or chaperones (or chaperonins) that associate with the target protein during the folding
process.
= They act by preventing polypeptide aggregation that comprise the complete protein structure, and they
disassociate from the protein once the target protein is folded.

Virtual Lab Experiments

⎯ Bradford Assay Simulation
= The Bradford protein assay was developed by Marion M. Bradford in 1976. It is a quick and accurate
spectroscopic analytical procedure used to measure the concentration of protein in a solution. The
reaction is dependent on the amino acid composition of the measured proteins. The principle of this
assay is that the binding of protein molecules to Coomassie dye under acidic conditions results in a
color change from brown to blue.
Reference: https://www.ruf.rice.edu/~bioslabs/methods/protein/bradford.html
Virtual Laboratory: https://www.youtube.com/watch?v=o1_A11D6oPU
⎯ Qualitative Analysis of Amino Acids
The following are the different qualitative tests or analysis of amino acids and proteins being done
in the laboratory.
= Xanthoproteic test, the aromatic rings on the side chains of proteins or free amino acids become
substituted with nitro groups when treated with concentrated nitric acid. The nitrated products are
yellow both in solution and as precipitate. When the solution is made with basic sodium hydroxide,
the color becomes more intense and the color ranges from yellow to orange to brown.
= Biuret test is a general test for proteins due to the presence of the peptide bond. The positive
result gives a violet-pink complex due to the coordination of cupric ion with the lone pair of the
amide nitrogen and oxygen form water in basic solution.
= Ninhydrin test involves several complex reactions between the amino group of the alpha carbon and
the ninhydrin solution. Two ninhydrin molecules cause the removal of the alpha amino group from an
amino acid or protein to form a blue-violet complex.
= Hopkin’s Cole test involves the condensation of tryptophan to form a violet colored complex upon
reaction with glyoxylic acid and concentrated sulfuric acid due to the presence of the indole ring.
= Millon’s test if positive for proteins containing a phenol group in the side chain such as that of
tyrosine. The positive test results in the formation of a flesh to red colored precipitate which is
due to the mercury salt produced in the reaction.
Virtual Laboratory: https://www.youtube.com/watch?v=wmhmAESv72E
⎯ Separation of Amino Acids by Thin Layer Chromatography
= Thin layer chromatography is planar chromatography belonging to the family of chromatographic
methods used for separation and determination of substances. It allows to carry out qualitative and
sometimes also quantitative analysis of chemical components in complex mixtures. Substances to be
determined are transferred onto the layer of sorbent as a spot or band using a capillary,
microsyringe or special applicator. Sorbent may comprise of a layer (0,05...0,2 mm thick) of fine-
grain material (silicagel, cellulose, aluminium oxide, ion exchange resin) on a solid support (glass,
plastic, metal). Instead of sorbent, porous materials (paper, plastic etc) can be used as well.
Spots of samples are applied on the plate in a straight line called starting line. The mobile
phase starts moving upwards due to capillary forces when the plate edge is inserted into
the eluent in chromatographic chamber. While the eluent is moving the components of sample separate
from each other because of the interactions between the molecules of eluent, sorbent and substances
to be separated. The elution process is stopped when the eluent has traveled up the plate until 5-10
 mm from the upper edge of the chromatographic plate. This eluent front is marked and this line is
called stop line. The elution distance, h0, is the distance between the starting line and the stop
line.
= After the chromatographic separation, all the components of the analyzed mixture are located
somewhere between the starting line and stop line. In order to visualize the separated substances (if
they are not colored) different techniques are used: chemical reactions, adding fluorescence
indicators to the sorbent layer during the process of preparation of the plates or spraying the
plates with fluorescent solutions and then observing under ultraviolet lamp. As the distances
traveled by the different substances differ, their mobilities can be used for qualitative analysis.
Therefore, the distance between the starting line and the center of the spot of substance hx (mm)
characterizes each substance. Retention factor, RF, provides better way to identify
substances. Provided that exactly the same amount of sample is applied, the intensity of the spot
can be used for quantitative determination: the bigger the amount of substance in the mixture, the
more intensive is the spot. Also, the size of the spot can give quantitative information – the bigger
the spot, the bigger the content of this compound in the mixture. Intensity of the spots is evaluated
by comparing with the intensities of analyte spots with known amounts visually or using densitometer
(device for measuring optical density).
Virtual Laboratory: https://www.youtube.com/watch?v=tDaKxskUwA0
⎯ Protein Denaturation
= Denaturation of proteins involves the disruption and possible destruction of both the secondary and
tertiary structures. Since denaturation reactions are not strong enough to break the peptide bonds,
the primary structure (sequence of amino acids) remains the same after a denaturation process.
Denaturation disrupts the normal alpha-helix and beta sheets in a protein and uncoils it into a
random shape.
= Denaturation occurs because the bonding interactions responsible for the secondary structure
(hydrogen bonds to amides) and tertiary structure are disrupted. In tertiary structure there are four
types of bonding interactions between "side chains" including: hydrogen bonding, salt bridges,
disulfide bonds, and non-polar hydrophobic interactions. which may be disrupted. Therefore, a variety
of reagents and conditions can cause denaturation. The most common observation in the denaturation
process is the precipitation or coagulation of the protein.
Reference: http://chemistry.elmhurst.edu/vchembook/568denaturation.html
Virtual Laboratory: https://www.labster.com/simulations/protein-denaturation/
Laboratory Activity at home: https://askabiologist.asu.edu/activities/breaking-proteins
⎯ Protein Sequencing
= Protein sequencing refers to methods for determining the amino acid sequence of proteins (or
peptides) and analysis of the sequence, for example to infer protein conformation. Techniques include
mass spectrometry and the Edman degradation reaction as well as prediction of the protein sequence
from the encoding DNA or mRNA sequence.
= The characteristic of each protein depends on its unique amino acid sequence (Links to an external
site.) (Garrett and Grisham, 2013). The first protein sequencing was achieved by Frederic Sanger in
1953 bovine insulin (Links to an external site.) for which he was awarded the Nobel Prize in 1958.
Protein sequencing is used to identify the amino acid sequence and its conformation. The
identification of the structure and function of proteins is important to understand cellular
processes.
= There are several applications of protein sequencing
- Identification of the protein family to which a particular protein belongs and finding the
evolutionary history of that protein.
- Prediction of the cellular localization of the protein based on its target sequence.
- Prediction of the sequence of the gene encoding the particular protein.
- Discovering the structure and function of a protein through various computational methods
and experimental methods.
Reference: https://www.sciencedirect.com/book/9780128046593, 2018
Virtual Laboratory: www.bch.cuhk.edu.hk/vlab2
⎯ Enzyme Kinetics
= Enzymes are protein catalysts that accelerate the rates at which reactions approach equilibrium.
Enzyme kinetics is the branch of biochemistry that deals with a quantitative description of this
process, mainly, how experimental variables affect reaction rates. The variables that are studied
include the concentrations of the enzymes, substrates (reactants), products, inhibitors, activators,
the pH, temperature, and ionic strength.
= A complete kinetic analysis (together with complementary studies of equilibrium ligand binding,
isotope exchange, covalent modification of amino acid side chains, etc.) can disclose most of the
functional characteristics of a particular enzyme. These include
- The specificity and affinities of the ligand subsites,
- The order in which substrates bind and products leave,
 - The enzyme species that are intermediates in the overall reaction,
- The magnitudes of component rate constants,
- The possible identities of active-site residues,
- The mode of action of an inhibitory drug, and
- How the enzyme might be regulated in vivo.
= Enzyme kinetics combined with related approaches can show how the functional properties of a mutant
or engineered enzyme compare to those of its wild-type parent. Many of the equations of enzyme
kinetics are also applicable to other saturable biological processes, for example, membrane transport
and receptor–ligand interactions.
Virtual laboratory:
https://www.youtube.com/watch?v=EF9PUR76y6w
https://www.youtube.com/watch?v=7uB2o1UglMk
- Ever wonder why some folks handle their alcohol better than others? This introductory video to
Michaelis-Menten dynamics explains the enzyme behavior behind why you're left feeling pukey after a
few drinks.
Reference: https://www.youtube.com/watch?v=rCVRC-AQ54M
Practice Quiz on Enzyme:
https://quizizz.com/join/quiz/5f414e4964f17f001b5441e8/start?referrer=5a98f7012b086c001a91b644

BIOCHEMISTRY-LABORATORY
LIPIDS
Lipids
⎯ Include a diverse group of compounds that are largely nonpolar in nature.
⎯ This is because they are hydrocarbons that include mostly nonpolar carbon–carbon or carbon–hydrogen
bonds. Non-polar molecules are hydrophobic (“water fearing”), or insoluble in water.
⎯ Lipids perform many different functions in a cell. Cells store energy for long-term use in the form
of fats. Lipids also provide insulation from the environment for plants and animals (Figure 3.12).
⎯ For example, they help keep aquatic birds and mammals dry when forming a protective layer over fur or
feathers because of their water-repellant hydrophobic nature.
⎯ Lipids are also the building blocks of many hormones and are an important constituent of all cellular
membranes.
⎯ Lipids include fats, oils, waxes, phospholipids, and steroids.

Figure 3.12 Hydrophobic lipids in aquatic mammals' fur, such as this river otter, protect them from the
elements. (credit: Ken Bosma)

Fats and Oils

⎯ A fat molecule consists of two main components—glycerol and fatty acids. Glycerol is an organic
compound (alcohol) with three carbons, five hydrogens, and three hydroxyl (OH) groups.
⎯ Fatty acids have a long chain of hydrocarbons to which a carboxyl group is attached, hence the name
“fatty acid.”
⎯ The number of carbons in the fatty acid may range from 4 to 36. The most common are those containing
12–18 carbons.
⎯ In a fat molecule, the fatty acids attach to each of the glycerol molecule's three carbons with an
ester bond through an oxygen atom (Figure 3.13).
 Figure 3.13 Joining three fatty acids to a glycerol backbone in a dehydration reaction forms
triacylglycerol. Three water molecules release in the process.
⎯ During this ester bond formation, three water molecules are released. The three fatty acids in the
triacylglycerol may be similar or dissimilar.
⎯ We also call fats triacylglycerols or triglycerides because of their chemical structure. Some fatty
acids have common names that specify their origin.
⎯ For example, palmitic acid, a saturated fatty acid, is derived from the palm tree. Arachidic acid is
derived from Arachis hypogea, the scientific name for groundnuts or peanuts.
⎯ Fatty acids may be saturated or unsaturated. In a fatty acid chain, if there are only single bonds
between neighboring carbons in the hydrocarbon chain, the fatty acid is saturated.
⎯ Saturated fatty acids are saturated with hydrogen. In other words, the number of hydrogen atoms
attached to the carbon skeleton is maximized. Stearic acid is an example of a saturated fatty acid
(Figure 3.14).

Figure 3.14 Stearic acid is a common saturated fatty acid.

⎯ When the hydrocarbon chain contains a double bond, the fatty acid is unsaturated. Oleic acid is an
example of an unsaturated fatty acid (Figure 3.15).

Figure 3.15 Oleic acid is a common unsaturated fatty acid.

⎯ Most unsaturated fats are liquid at room temperature. We call these oils. If there is one double bond
in the molecule, then it is a monounsaturated fat (e.g., olive oil), and if there is more than one
double bond, then it is a polyunsaturated fat (e.g., canola oil).
⎯ When a fatty acid has no double bonds, it is a saturated fatty acid because it is not possible to add
more hydrogen to the chain's carbon atoms.
⎯ A fat may contain similar or different fatty acids attached to glycerol. Long straight fatty acids
with single bonds generally pack tightly and are solid at room temperature.
⎯ Animal fats with stearic acid and palmitic acid (common in meat) and the fat with butyric acid
(common in butter) are examples of saturated fats. Mammals store fats in specialized cells, or
adipocytes, where fat globules occupy most of the cell’s volume.
⎯ Plants store fat or oil in many seeds and use them as a source of energy during seedling development.
⎯ Unsaturated fats or oils are usually of plant origin and contain cis unsaturated fatty
acids. Cis and trans indicate the configuration of the molecule around the double bond.
⎯ If hydrogens are present in the same plane, it is a cis fat. If the hydrogen atoms are on two
different planes, it is a trans fat. The cis double bond causes a bend or a “kink” that prevents the
fatty acids from packing tightly, keeping them liquid at room temperature (Figure 3.16).
 ⎯ Olive oil, corn oil, canola oil, and cod liver oil are examples of unsaturated fats. Unsaturated fats
help to lower blood cholesterol levels, whereas saturated fats contribute to plaque formation in the
arteries.

Figure 3.16 Saturated fatty acids have hydrocarbon chains connected by single bonds only. Unsaturated fatty
acids have one or more double bonds. Each double bond may be in a cis or trans configuration. In
the cis configuration, both hydrogens are on the same side of the hydrocarbon chain. In
the trans configuration, the hydrogens are on opposite sides. A cis double bond causes a kink in the chain.

Trans Fats
⎯ The food industry artificially hydrogenates oils to make them semi-solid and of a consistency
desirable for many processed food products.
⎯ Simply speaking, hydrogen gas is bubbled through oils to solidify them. During this hydrogenation
process, double bonds of the cis- conformation in the hydrocarbon chain may convert to double bonds
in the trans- conformation.
⎯ Margarine, some types of peanut butter, and shortening are examples of artificially hydrogenated
trans fats.
⎯ Recent studies have shown that an increase in trans fats in the human diet may lead to higher levels
of low-density lipoproteins (LDL), or “bad” cholesterol, which in turn may lead to plaque deposition
in the arteries, resulting in heart disease.
⎯ Many fast-food restaurants have recently banned using trans fats, and food labels are required to
display the trans-fat content.

Omega Fatty Acids

⎯ Essential fatty acids are those that the human body requires but does not synthesize. Consequently,
they have to be supplemented through ingestion via the diet.
⎯ Omega-3 fatty acids (like those in Figure 3.17) fall into this category and are one of only two known
for humans (the other is omega-6 fatty acid). These are polyunsaturated fatty acids and are omega-3
because a double bond connects the third carbon from the hydrocarbon chain's end to its neighboring
carbon.

Figure 3.17 Alpha-linolenic acid is an example of an omega-3 fatty acid. It has three cis double bonds and,
as a result, a curved shape. For clarity, the diagram does not show the carbons. Each singly bonded carbon
has two hydrogens associated with it, which the diagram also does not show.
 ⎯ The farthest carbon away from the carboxyl group is numbered as the omega (ω) carbon, and if the
double bond is between the third and fourth carbon from that end, it is an omega-3 fatty acid.
⎯ Nutritionally important because the body does not make them, omega-3 fatty acids include alpha-
linoleic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), all of which are
polyunsaturated. Salmon, trout, and tuna are good sources of omega-3 fatty acids.
⎯ Research indicates that omega-3 fatty acids reduce the risk of sudden death from heart attacks, lower
triglycerides in the blood, decrease blood pressure, and prevent thrombosis by inhibiting blood
clotting. They also reduce inflammation and may help lower the risk of some cancers in animals.
⎯ Like carbohydrates, fats have received considerable bad publicity. It is true that eating an excess
of fried foods and other “fatty” foods leads to weight gain.
⎯ However, fats do have important functions. Many vitamins are fat soluble, and fats serve as a long-
term storage form of fatty acids: a source of energy. They also provide insulation for the body.
⎯ Therefore, we should consume “healthy” fats in moderate amounts on a regular basis.

Waxes
⎯ Covers some aquatic birds' feathers and some plants' leaf surfaces. Because of waxes' hydrophobic
nature, they prevent water from sticking on the surface (Figure 3.18). Long fatty acid chains
esterified to long-chain alcohols comprise waxes.

Figure 3.18 Lipids comprise waxy coverings on some leaves.

Phospholipids
⎯ Are major plasma membrane constituents that comprise cells' outermost layer.
⎯ Like fats, they are comprised of fatty acid chains attached to a glycerol or sphingosine backbone.
⎯ However, instead of three fatty acids attached as in triglycerides, there are two fatty acids forming
diacylglycerol, and a modified phosphate group occupies the glycerol backbone's third carbon (Figure
3.19).
⎯ A phosphate group alone attached to a diacylglycerol does not qualify as a phospholipid. It is
phosphatidate (diacylglycerol 3-phosphate), the precursor of phospholipids. An alcohol modifies the
phosphate group.
⎯ Phosphatidylcholine and phosphatidylserine are two important phospholipids that are in plasma
membranes.

Figure 3.19 A phospholipid is a molecule with two fatty acids and a modified phosphate group attached to a
glycerol backbone. Adding a charged or polar chemical group may modify the phosphate.
⎯ A phospholipid is an amphipathic molecule, meaning it has a hydrophobic and a hydrophilic part.
⎯ The fatty acid chains are hydrophobic and cannot interact with water; whereas, the phosphate-
containing group is hydrophilic and interacts with water (Figure 3.20).
Figure 3.20 The phospholipid bilayer is the major component of all cellular membranes. The hydrophilic head
groups of the phospholipids faces the aqueous solution. The hydrophobic tails are sequestered in the middle
of the bilayer.
⎯ The head is the hydrophilic part, and the tail contains the hydrophobic fatty acids.
⎯ In a membrane, a bilayer of phospholipids forms the structure's matrix, phospholipids' fatty acid
tails face inside, away from water; whereas, the phosphate group faces the outside, aqueous side
(Figure 3.20).
⎯ Phospholipids are responsible for the plasma membrane's dynamic nature. If a drop of phospholipids is
placed in water, it spontaneously forms a structure that scientists call a micelle, where the
hydrophilic phosphate heads face the outside and the fatty acids face the structure's interior.

Steroids
⎯ Unlike the phospholipids and fats that we discussed earlier, steroids have a fused ring structure.
⎯ Although they do not resemble the other lipids, scientists group them with them because they are also
hydrophobic and insoluble in water.
⎯ All steroids have four linked carbon rings and several of them, like cholesterol, have a short tail
(Figure 3.21). Many steroids also have the –OH functional group, which puts them in the alcohol
classification (sterols).

Figure 3.21 Four fused hydrocarbon rings comprise steroids such as cholesterol and cortisol.
⎯ Cholesterol is the most common steroid. The liver synthesizes cholesterol and is the precursor to
many steroid hormones such as testosterone and estradiol, which gonads and endocrine glands secrete.
⎯ It is also the precursor to Vitamin D. Cholesterol is also the precursor of bile salts, which help
emulsifying fats and their subsequent absorption by cells.
⎯ Although lay people often speak negatively about cholesterol, it is necessary for the body's proper
functioning.
⎯ Sterols (cholesterol in animal cells, phytosterol in plants) are components of the plasma membrane of
cells and are found within the phospholipid bilayer.
⎯ Reference: https://openstax.org/books/biology-2e/pages/3-3-lipids

Virtual laboratory Experiments

= Lipids are a heterogeneous class of naturally occurring organic substances grouped together not by the
presence of a distinguishing functional group or structural feature but rather based on common solubility
properties.
= Lipids are plant and animal products that are all insoluble in water and highly soluble in one or more
organic solvents, such as ether, chloroform, benzene, and acetone.
= The most abundant class of lipids and major components of depot or storage lipids in plant and animal
cells are the triglycerides.
= Triglycerides that are generally liquid at room temperature are called oils and those that are generally
solid are called fats.
= Lipids also include waxes which are esters of fatty acids and long chain monohydric alcohols; phosphorous
containing compounds such as phosphatides, sphingomyelin, and cerebrosides which are readily isolated from
the nervous tissues; and steroids such as cholesterol and hormones.
= The degree of unsaturation in a lipid is measured by its iodine number defined as the number of grams of
iodine that would add to the double bonds present in 100 grams of the lipid.
= Animal fats have low iodine numbers while vegetable oils have higher values.
Source: Biochemistry Lab Manual, De La Salle Lipa
⎯ Qualitative Analysis of Fats and Oils
- Fats and oils are concerted source of energy. Certain percentage of body weight of human being is
fat and 20-35% of calories should come from fat.
- Fats in the diet are essential for good health and are needed for the growth of the body and the
processing of vitamins. They make up part of all cells and help to maintain the body temperature.
They form fatty tissue around delicate organs to protect them from injury.
- Chemically, fats and oils are triesters of glycerol and higher fatty acids. They are of animal or
plant origin. Desi ghee is animal ghee while vanaspati ghee is vegetable ghee.
- Fats are solids while oils are liquids at ordinary temperature.
- Fats and oils may be saturated or unsaturated.
= Saturated fat - contain only single bonds within the carbon chain. Saturated fats are of animal
origin and are usually present in solid form. It increases the blood cholesterol level. Some examples
are meat fat, butter etc. Coconut oil and palm oil also contain saturated fat.
= Unsaturated fats - contain double bonds within the carbon chain. Unsaturated fat is found in fish
like salmon and tuna, nuts, seeds etc.
- Some Important Tests for the Detection of Oils and Fats
= Solubility test - Oils and fats are soluble in organic solvents like, chloroform, alcohol etc. but
are insoluble in water.
= Translucent Spot test - Fats and oils have higher boiling points so at room temperature they cannot
absorb enough heat to evaporate. When fat or oil is place on a sheet of paper, it diffracts light.
The diffracted light can pass from one side of the paper to another side and produces a translucent
spot.
= Acrolein test - Acrolein test is used to detect the presence of glycerol or fat. When fat is
treated strongly in the presence of a dehydrating agent like potassium bisulphate (KHSO4), the
glycerol portion of the molecule is dehydrated to form an unsaturated aldehyde, acrolein that has a
pungent irritating odour.
= Baudouin test - This test is used to detect the presence of seasame oil. Seasame oil gives a
characteristic rose red colour with concentrated hydrochloric acid and furfural solution. Vanaspati
ghee contains 5% seasame oil while pure desi ghee does not contain seasame oil. So this test can be
applied to find out whether the given sample of desi ghee contains vanaspati ghee or not.
= Huble's test - This test is used to detect the degree of unsaturation in oil or fat. Huble’s
reagent reacts with alcoholic solution of iodine that contains some mercuric chloride. During the
reaction, the violet colour of iodine fades away if the oil or fat is unsaturated. If the oil or fat
is saturated, the violet colour of iodine does not fade away.
Reference: https://amrita.olabs.edu.in/?brch=8&cnt=1&sim=210&sub=73
Virtual Laboratory: https://www.youtube.com/watch?v=l2QOi9mZoFc
⎯ Estimation of Iodine Value in Fats and Oils
- The iodine value is a measure of the relative degree of unsaturation in oil components, as
determined by the uptake of halogen.
- Iodine value (IV) is an index of the unsaturation, which is the most important analytical
characteristic of oil (Otunola et al., 2009).
- It has been found that there is a relative loss of the C18:2 fatty acid and a decrease in the
iodine value of oil after heating due to more intensive thermo-oxidative transformations that occur
as compared to heated oil containing food (Tynek et al., 2001).
- Because the melting point and oxidative stability are related to the degree of unsaturation, IV
provides an estimation of these quality factors.
- The greater the iodine value, the more unsaturation and the higher the susceptibility to oxidation.
Peanut oil (IV 82–107) is more saturated than corn (IV 103–128), cottonseed (IV 99–113), or linseed
(IV 155–205) oils; however, it is considerably less saturated than coconut (IV 7.7–10.5), palm (IV
44–54) or butter (IV 25–42) oils.
- This parameter is extremely important in the palm oil industry, where it is used to follow the
fractionation process.
 - The protocol for iodine value determination usually comprises a titration procedure, such as the
Wijs method [61].
- In this procedure, iodine chloride is used for double-bond saturation analysis, and the content of
consumed iodine is measured by titration with 0.1 mol L− 1 sodium thiosulfate solution.
Reference: https://onlinelibrary.wiley.com/doi/pdf/10.1016/0307-4412(94)90174-0
Virtual Laboratory: https://www.youtube.com/watch?v=_5ObG6fIAdQ
⎯ Estimation of Saponification Value in Fats and Oils
- Saponification value number represents the number of milligrams of potassium hydroxide required to
saponify 1g of fat under the conditions specified.
- It is a measure of the average molecular weight (or chain length) of all the fatty acids present.
- As most of the mass of a fat/tri-ester is in the 3 fatty acids, the saponification value allows for
comparison of the average fatty acid chain length.
- The long chain fatty acids found in fats have a low saponification value because they have a
relatively fewer number of carboxylic functional groups per unit mass of the fat as compared to short
chain fatty acids.
- If more moles of base are required to saponify N grams of fat, then there are more moles of the fat
and the chain lengths are relatively small, given the following relation:
Number of moles = mass of oil / average molecular mass
- The calculated molar mass is not applicable to fats and oils containing high amounts of
unsaponifiable material, free fatty acids (>0.1%), or mono- and diacylglycerols (>0.1%).
- Handmade soap makers who aim for bar soap use NaOH (sodium hydroxide, lye). Because saponification
values are listed in KOH (potassium hydroxide) the value must be converted from potassium to sodium
to make bar soap; potassium soaps make a paste, gel or liquid soap.
- To convert KOH values to NaOH values, divide the KOH values by the ratio of the molecular weights
of KOH and NaOH (1.403).
- Standard methods for analysis are for example: ASTM D5558 for vegetable and animal fats, ASTM D 94
(for petroleum) and DIN 51559.
Virtual Laboratory: https://www.youtube.com/watch?v=ersHDsiAVko

BIOCHEMISTRY-LABORATORY
NUCLEIC ACIDS
Nucleic Acids: DNA and RNA
⎯ Nucleic acids are informational molecules made up of polymers of nucleotides linked together by
phosphodiester bonds.
⎯ There are two types of nucleic acids: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
⎯ These nucleic acids are similar in that the repeating unit of both is the nucleotide. However, there
are some functional as well as structural differences between these two types of nucleic acids.
⎯ Functionally, DNA is the macromolecule which stores the genetic information of individuals.
⎯ RNA transmits this information to make proteins, the molecule responsible for visible or observable
traits.
⎯ DNA is the organism's genotype. This molecule then dictates the type and structure of proteins, the
organism's phenotype.
⎯ Structurally, the nucleotide that is the building block of nucleic acids consists of a base, sugar,
and phosphate group.
⎯ For both DNA and RNA, characteristic structural differences of the bases and sugars exist.
⎯ The bases are of two types, namely 1) purine bases which contain the purine ring, and 2) pyrimidine
bases which contain the pyrimidine ring. The purine bases for both DNA and RNA are adenine (A) and
guanine (G).
⎯ Both also contain the pyrimidine bases, cytosine (C), but these polynucleotides differ in the other
pyrimidine base such that only DNA contains thymine (T) while uracil (U) is found only in RNAs.

Nucleic Acids
⎯ Are the most important macromolecules for the continuity of life. They carry the cell's genetic
blueprint and carry instructions for its functioning.

DNA and RNA

⎯ The two main types of nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
⎯ DNA is the genetic material in all living organisms, ranging from single-celled bacteria to
multicellular mammals.
 ⎯ It is in the nucleus of eukaryotes and in the organelles, chloroplasts, and mitochondria. In
prokaryotes, the DNA is not enclosed in a membranous envelope.
⎯ The cell's entire genetic content is its genome, and the study of genomes is genomics. In eukaryotic
cells but not in prokaryotes, DNA forms a complex with histone proteins to form chromatin, the
substance of eukaryotic chromosomes.
⎯ A chromosome may contain tens of thousands of genes. Many genes contain the information to make
protein products.
⎯ Other genes code for RNA products. DNA controls all of the cellular activities by turning the genes
“on” or “off.”
⎯ The other type of nucleic acid, RNA, is mostly involved in protein synthesis. The DNA molecules never
leave the nucleus but instead use an intermediary to communicate with the rest of the cell.
⎯ This intermediary is the messenger RNA (mRNA). Other types of RNA—like rRNA, tRNA, and microRNA—are
involved in protein synthesis and its regulation.
⎯ DNA and RNA are comprised of monomers that scientists call nucleotides.
⎯ The nucleotides combine with each other to form a polynucleotide, DNA or RNA.
⎯ Three components comprise each nucleotide: a nitrogenous base, a pentose (five-carbon) sugar, and a
phosphate group (Figure 3.31).
⎯ Each nitrogenous base in a nucleotide is attached to a sugar molecule, which is attached to one or
more phosphate groups.

Figure 3.31 Three components comprise a nucleotide: a nitrogenous base, a pentose sugar, and one or more
phosphate groups. Carbon residues in the pentose are numbered 1′ through 5′ (the prime distinguishes these
residues from those in the base, which are numbered without using a prime notation). The base is attached
to the ribose's 1′ position, and the phosphate is attached to the 5′ position. When a polynucleotide forms,
the incoming nucleotide's 5′ phosphate attaches to the 3′ hydroxyl group at the end of the growing chain.
Two types of pentose are in nucleotides, deoxyribose (found in DNA) and ribose (found in RNA). Deoxyribose
is similar in structure to ribose, but it has an H instead of an OH at the 2′ position. We can divide bases
into two categories: purines and pyrimidines. Purines have a double ring structure, and pyrimidines have a
single ring.
⎯ The nitrogenous bases, important components of nucleotides, are organic molecules and are so named
because they contain carbon and nitrogen.
⎯ They are bases because they contain an amino group that has the potential of binding an extra
hydrogen, and thus decreasing the hydrogen ion concentration in its environment, making it more
basic.
⎯ Each nucleotide in DNA contains one of four possible nitrogenous bases: adenine (A), guanine (G)
cytosine (C), and thymine (T).
⎯ Scientists classify adenine and guanine as purines. The purine's primary structure is two carbon-
nitrogen rings.
⎯ Scientists classify cytosine, thymine, and uracil as pyrimidines which have a single carbon-nitrogen
ring as their primary structure (Figure 3.31). Each of these basic carbon-nitrogen rings has
different functional groups attached to it.
⎯ In molecular biology shorthand, we know the nitrogenous bases by their symbols A, T, G, C, and U. DNA
contains A, T, G, and C; whereas, RNA contains A, U, G, and C.
 ⎯ The pentose sugar in DNA is deoxyribose, and in RNA, the sugar is ribose (Figure 3.31). The
difference between the sugars is the presence of the hydroxyl group on the ribose's second carbon and
hydrogen on the deoxyribose's second carbon.
⎯ The carbon atoms of the sugar molecule are numbered as 1′, 2′, 3′, 4′, and 5′ (1′ is read as “one
prime”). The phosphate residue attaches to the hydroxyl group of the 5′ carbon of one sugar and the
hydroxyl group of the 3′ carbon of the sugar of the next nucleotide, which forms a 5′–
3′ phosphodiester linkage.
⎯ A simple dehydration reaction like the other linkages connecting monomers in macromolecules does not
form the phosphodiester linkage.
⎯ Its formation involves removing two phosphate groups. A polynucleotide may have thousands of such
phosphodiester linkages.

DNA Double-Helix Structure

⎯ DNA has a double-helix structure (Figure 3.32). The sugar and phosphate lie on the outside of the
helix, forming the DNA's backbone.
⎯ The nitrogenous bases are stacked in the interior, like a pair of staircase steps. Hydrogen bonds
bind the pairs to each other.
⎯ Every base pair in the double helix is separated from the next base pair by 0.34 nm.
⎯ The helix's two strands run in opposite directions, meaning that the 5′ carbon end of one strand will
face the 3′ carbon end of its matching strand. (Scientists call this an antiparallel orientation and
is important to DNA replication and in many nucleic acid interactions.)

Figure 3.32 Native DNA is an antiparallel double helix. The phosphate backbone (indicated by the curvy
lines) is on the outside, and the bases are on the inside. Each base from one strand interacts via hydrogen
bonding with a base from the opposing strand.
⎯ Only certain types of base pairing are allowed. For example, a certain purine can only pair with a
certain pyrimidine.
⎯ This means A can pair with T, and G can pair with C, as Figure 3.33 shows. This is the base
complementary rule.
⎯ In other words, the DNA strands are complementary to each other.
⎯ If the sequence of one strand is AATTGGCC, the complementary strand would have the sequence TTAACCGG.
⎯ During DNA replication, each strand copies itself, resulting in a daughter DNA double helix
containing one parental DNA strand and a newly synthesized strand.

Figure 3.33 In a double stranded DNA molecule, the two strands run antiparallel to one another so that one
strand runs 5′ to 3′ and the other 3′ to 5′. The phosphate backbone is located on the outside, and the
bases are in the middle. Adenine forms hydrogen bonds (or base pairs) with thymine, and guanine base pairs
with cytosine.
RNA
⎯ Ribonucleic acid, or RNA, is mainly involved in the process of protein synthesis under the direction
of DNA.
⎯ RNA is usually single-stranded and is comprised of ribonucleotides that are linked by phosphodiester
bonds.
⎯ A ribonucleotide in the RNA chain contains ribose (the pentose sugar), one of the four nitrogenous
bases (A, U, G, and C), and the phosphate group.
 ⎯ There are four major types of RNA: messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA),
and microRNA (miRNA).
⎯ The first, mRNA, carries the message from DNA, which controls all the cellular activities in a cell.
⎯ If a cell requires synthesizing a certain protein, the gene for this product turns “on” and the
messenger RNA synthesizes in the nucleus.
⎯ The RNA base sequence is complementary to the DNA's coding sequence from which it has been copied.
However, in RNA, the base T is absent, and U is present instead.
⎯ If the DNA strand has a sequence AATTGCGC, the sequence of the complementary RNA is UUAACGCG. In the
cytoplasm, the mRNA interacts with ribosomes and other cellular machinery (Figure 3.34).

Figure 3.34 A ribosome has two parts: a large subunit and a small subunit. The mRNA sits in between the two
subunits. A tRNA molecule recognizes a codon on the mRNA, binds to it by complementary base pairing, and
adds the correct amino acid to the growing peptide chain.
⎯ The mRNA is read in sets of three bases known as codons. Each codon codes for a single amino acid.
⎯ In this way, the mRNA is read, and the protein product is made. Ribosomal RNA (rRNA) is a major
constituent of ribosomes on which the mRNA binds.
⎯ The rRNA ensures the proper alignment of the mRNA and the Ribosomes.
⎯ The ribosome's rRNA also has an enzymatic activity (peptidyl transferase) and catalyzes peptide bond
formation between two aligned amino acids.

Transfer RNA (tRNA)

⎯ Is one of the smallest of the four types of RNA, usually 70–90 nucleotides long. It carries the
correct amino acid to the protein synthesis site.
⎯ It is the base pairing between the tRNA and mRNA that allows for the correct amino acid to insert
itself in the polypeptide chain.
⎯ MicroRNAs are the smallest RNA molecules and their role involves regulating gene expression by
interfering with the expression of certain mRNA messages. Table 3.2 summarizes DNA and RNA features.

⎯ Even though the RNA is single stranded, most RNA types show extensive intramolecular base pairing
between complementary sequences, creating a predictable three-dimensional structure essential for
their function.
⎯ As you have learned, information flow in an organism takes place from DNA to RNA to protein. DNA
dictates the structure of mRNA in a process that scientists call transcription, and RNA dictates the
protein's structure in a process scientist call translation.
⎯ This is the Central Dogma of Life, which holds true for all organisms; however, exceptions to the
rule occur in connection with viral infections.
Reference: https://openstax.org/books/biology-2e/pages/3-5-nucleic-acids

Virtual laboratory Experiments

⎯ DNA Extraction Experiment
= DNA extraction
- Is the technique used to isolate DNA in a biological sample.
As shown in this photo, DNA, a long stringy molecule, can be lifted out of a solution using a glass rod or
wooden stick which it naturally wraps around when turned.
 = Importance
- The extraction of DNA is pivotal to biotechnology. It is the starting point for numerous
applications, ranging from fundamental research to routine diagnostic and therapeutic decision-
making.
- Extraction and purification are also essential to determining the unique characteristics of DNA,
including its size, shape, and function.
= Discovery
- DNA was first isolated by the Swiss physician, Friedrich Miescher, in 1869 while working in the
laboratory of the biochemist Felix Hoppe-Seyler.
- This he did as part of a project to determine the chemical composition of cells which he saw as the
means to unravelling the fundamental principles of the life of cells. Initially, he began this
research by using lymphocytes drawn from lymph nodes but was unable to get sufficient quantities for
analysis so switched to using leucocytes, white blood cells, which he gathered from pus found on
fresh surgical bandages collected from a nearby surgical clinic.
- In the course of his work on leucocytes he noticed the precipitation of a substance when acid was
added and that this dissolved following the addition of alkali. Mierscher decided to call the new
substance 'nuclein' by virtue of its presence in the nuclei of the cell.
- Upon further analysis, Miescher noted that the chemical composition of the substance differed from
proteins and other known molecules. He speculated that it played a central role in cells and was
involved in the division of cells.
- Following this, Miescher developed a method for isolating nuclein from salmon sperm. Many advances
have been made to the methods for extracting and purifying DNA since Miescher's time.
- From the 1950s, routine laboratory extraction of DNA became reliant on the use of density gradient
centrifuges.
- Until very recently most methods for extracting DNA remained complex, labor-intensive and time-
consuming. They also provided only small quantities of DNA.
- Today, there are many specialized extraction methods. These are generally either solution-based or
column-based.
- The extraction of DNA has become much easier with the emergence of commercial kits and the
automation of the process. Such changes have both sped up production and increased the yield of DNA.
= Application
- The ability to extract DNA is of primary importance to studying the genetic causes of disease and
for the development of diagnostics and drugs.
- It is also essential for carrying out forensic science, sequencing genomes, detecting bacteria and
viruses in the environment and for determining paternity.
Reference: https://www.whatisbiotechnology.org/index.php/science/summary/extraction/dna-extraction-
isolates-dna-from-biological-material
- Now that you have learned concepts about DNA extraction, it is now time for you to click on the
link below. This will show you a virtual laboratory simulation on how DNA extraction is done. Happy
viewing and learning!
Reference: https://learn.genetics.utah.edu/content/labs/extraction/
- It is now your turn to extract or isolate DNA from a relatively simple and accessible plant
material, a banana! You can do this simple experiment at home using simple materials.
Biochem Exer 9_Isolation of Banana DNA (PDF)
⎯ DNA Sequencing Experiment
= What is DNA sequencing?
- DNA sequencing determines the order of the four chemical building blocks - called "bases" - that
make up the DNA molecule.
- The sequence tells scientists the kind of genetic information that is carried in a particular DNA
segment.
- For example, scientists can use sequence information to determine which stretches of DNA contain
genes and which stretches carry regulatory instructions, turning genes on or off.
- In addition, and importantly, sequence data can highlight changes in a gene that may cause disease.
 - In the DNA double helix, the four chemical bases always bond with the same partner to form "base
pairs." Adenine (A) always pairs with thymine (T); cytosine (C) always pairs with guanine (G).
- This pairing is the basis for the mechanism by which DNA molecules are copied when cells divide,
and the pairing also underlies the methods by which most DNA sequencing experiments are done.
- The human genome contains about 3 billion base pairs that spell out the instructions for making and
maintaining a human being.
= How new is DNA sequencing?
- Since the completion of the Human Genome Project, technological improvements and automation have
increased speed and lowered costs to the point where individual genes can be sequenced routinely, and
some labs can sequence well over 100,000 billion bases per year, and an entire genome can be
sequenced for just a few thousand dollars.
= What new sequencing methods have been developed?
- Since the completion of the Human Genome Project, technological improvements and automation have
increased speed and lowered costs to the point where individual genes can be sequenced routinely, and
some labs can sequence well over 100,000 billion bases per year, and an entire genome can be
sequenced for just a few thousand dollars.
= Are newer sequencing technologies being developed?
- One new sequencing technology involves watching DNA polymerase molecules as they copy DNA - the
same molecules that make new copies of DNA in our cells - with a very fast movie camera and
microscope, and incorporating different colors of bright dyes, one each for the letters A, T, C and
G.
- This method provides different and very valuable information than what's provided by the instrument
systems that are in most common use.
- Another new technology in development entails the use of nanopores to sequence DNA. Nanopore-based
DNA sequencing involves threading single DNA strands through extremely tiny pores in a membrane. DNA
bases are read one at a time as they squeeze through the nanopore.
- The bases are identified by measuring differences in their effect on ions and electrical current
flowing through the pore. Using nanopores to sequence DNA offers many potential advantages over
current methods.
- The goal is for sequencing to cost less and be done faster. Unlike sequencing methods currently in
use, nanopore DNA sequencing means researchers can study the same molecule over and over again.
= What do improvements in DNA sequencing mean for human health?
- Researchers now can compare large stretches of DNA - 1 million bases or more - from different
individuals quickly and cheaply.
- Such comparisons can yield an enormous amount of information about the role of inheritance in
susceptibility to disease and in response to environmental influences.
- In addition, the ability to sequence the genome more rapidly and cost-effectively creates vast
potential for diagnostics and therapies.
- Although routine DNA sequencing in the doctor's office is still many years away, some large medical
centers have begun to use sequencing to detect and treat some diseases.
- In cancer, for example, physicians are increasingly able to use sequence data to identify the
cancer a patient has. This enables the physician to make better choices for treatments.
- Researchers in the NHGRI-supported Undiagnosed Diseases Program use DNA sequencing to try to
identify the genetic causes of rare diseases. Other researchers are studying its use in screening
newborns for disease and disease risk.
- Moreover, The Cancer Genome Atlas project, which is supported by NHGRI and the National Cancer
Institute, is using DNA sequencing to unravel the genomic details of some 30 cancer types.
- Another National Institutes of Health program examines how gene activity is controlled in different
tissues and the role of gene regulation in disease.
- Ongoing and planned large-scale projects use DNA sequencing to examine the development of common
and complex diseases, such as heart disease and diabetes, and in inherited diseases that cause
physical malformations, developmental delay, and metabolic diseases.
- Comparing the genome sequences of different types of animals and organisms, such as chimpanzees and
yeast, can also provide insights into the biology of development and evolution.
Reference: https://www.genome.gov/about-genomics/fact-sheets/DNA-Sequencing-Fact-Sheet
- A more interesting and engaging discussion on the principles and application of DNA sequencing can
also be seen in this link below.
Reference: https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-sequencing-pcr-
electrophoresis/a/dna-sequencing
Virtual Laboratory: http://www.bch.cuhk.edu.hk/vlab2
⎯ Gel Electrophoresis Experiment
- A very interesting and engaging discussion of some preliminary principles of gel electrophoresis
can be read in the useful link below.
= Key points
- Gel electrophoresis is a technique used to separate DNA fragments according to their size.
- DNA samples are loaded into wells (indentations) at one end of a gel, and an electric current is
applied to pull them through the gel.
- DNA fragments are negatively charged, so they move towards the positive electrode. Because all DNA
fragments have the same amount of charge per mass, small fragments move through the gel faster than
large ones.
- When a gel is stained with a DNA-binding dye, the DNA fragments can be seen as bands, each
representing a group of same-sized DNA fragments.
= Introduction
- Suppose you have just done a PCR reaction, making many copies of a target DNA region. Or perhaps
you’ve done some DNA cloning, trying to "paste" a gene into a circular DNA plasmid.
- Now, you want to check and see whether your PCR worked, or whether your plasmid has the right gene
in it. - What technique can you use to visualize (directly observe) the fragments of DNA? Gel
electrophoresis
- Gel electrophoresis is a technique used to separate DNA fragments (or other macromolecules, such as
RNA and proteins) based on their size and charge.
- Electrophoresis involves running a current through a gel containing the molecules of interest.
Based on their size and charge, the molecules will travel through the gel in different directions or
at different speeds, allowing them to be separated from one another.
- The suffix phoresis means "migration" or "movement." The prefix electro tells us that we are using
electricity to make molecules migrate.
- All DNA molecules have the same amount of charge per mass. Because of this, gel electrophoresis of
DNA fragments separates them based on size only. Using electrophoresis, we can see how many different
DNA fragments are present in a sample and how large they are relative to one another. We can also
determine the absolute size of a piece of DNA by examining it next to a standard "yardstick" made up
of DNA fragments of known sizes.
= What is a gel?
- As the name suggests, gel electrophoresis involves a gel: a slab of Jello-like material. Gels for
DNA separation are often made from a polysaccharide called agarose, which comes as dry, powdered
flakes. When the agarose is heated in a buffer (water with some salts in it) and allowed to cool, it
will form a solid, slightly squishy gel. At the molecular level, the gel is a matrix of agarose
molecules that are held together by hydrogen bonds and form tiny pores.
- At one end, the gel has pocket-like indentations called wells, which are where the DNA samples will
be placed:

- Before the DNA samples are added, the gel must be placed in a gel box. One end of the box is hooked
to a positive electrode, while the other end is hooked to a negative electrode. The main body of the
box, where the gel is placed, is filled with a salt-containing buffer solution that can conduct
current. Although you may not be able to see in the image above, the buffer fills the gel box to a
level where it just barely covers the gel.
- The end of the gel with the wells is positioned towards the negative electrode. The end without
wells (towards which the DNA fragments will migrate) is positioned towards the positive electrode.
= How do DNA fragments move through the gel?
- Once the gel is in the box, each of the DNA samples we want to examine (for instance, each PCR
reaction or each restriction-digested plasmid) is carefully transferred into one of the wells. One
well is reserved for a DNA ladder, a standard reference that contains DNA fragments of known lengths.
Commercial DNA ladders come in different size ranges, so we would want to pick one with good
"coverage" of the size range of our expected fragments.
- Next, the power to the gel box is turned on, and current begins to flow through the gel. The DNA
molecules have a negative charge because of the phosphate groups in their sugar-phosphate backbone,
so they start moving through the matrix of the gel towards the positive pole. When the power is
turned on and current is passing through the gel, the gel is said to be running.
- A typical voltage for running an agarose DNA gel would be in the range of 80 – 120V. A higher
voltage will make the gel run faster but may also melt it if it runs for a long period of time. A
lower voltage will make the gel run more slowly (which can be convenient if you want it to finish up
at a particular time, say, after you get back from lunch).
 DNA samples are loaded into wells at negative electrode end of gel.
Power is turned on and DNA fragments migrate through gel (towards the positive electrode).
After the gel has run, the fragments are separated by size. The largest fragments are near the top of
the gel (negative electrode, where they began), and the smallest fragments are near the bottom
(positive electrode).
- As the gel runs, shorter pieces of DNA will travel through the pores of the gel matrix faster than
longer ones. After the gel has run for a while, the shortest pieces of DNA will be close to the
positive end of the gel, while the longest pieces of DNA will remain near the wells. Very short
pieces of DNA may have run right off the end of the gel if we left it on for too long.
= Visualizing the DNA fragments
- Once the fragments have been separated, we can examine the gel and see what sizes of bands are
found on it. When a gel is stained with a DNA-binding dye and placed under UV light, the DNA
fragments will glow, allowing us to see the DNA present at different locations along the length of
the gel.

- The bp next to each number in the ladder indicates how many base pairs long the DNA fragment is.
- A well-defined “line” of DNA on a gel is called a band. Each band contains many DNA fragments of
the same size that have all traveled as a group to the same position. A single DNA fragment (or even
a small group of DNA fragments) would not be visible by itself on a gel.
- By comparing the bands in a sample to the DNA ladder, we can determine their approximate sizes. For
instance, the bright band on the gel above is roughly 700 base pairs (bp) in size.
Reference: https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-sequencing-pcr-
electrophoresis/a/gel-electrophoresis
- Now, you can click on the link below to lead you to the virtual lab experiment simulation on the
process of Gel Electrophoresis.
Virtual Laboratory: https://learn.genetics.utah.edu/content/labs/gel/
https://www.labxchange.org/library/items/lb:LabXchange:9548bee3:lx_simulation:1