Biochemistry

1. Discussion of Quiz: True or False
• The valence electron of N is 5.
1. N2 has covalent bonds.
2. N2 has double bonds.
3. N2 is polar.
4. The observation “N2 + 3H2  2NH3” is
quantitative.
5. N2 and H2 can form hydrogen bonds.

2. Quiz
1. Draw all 9 isomers of C7H16.
2. Identify the molecule as cis-trans and/or E-Z.
3. Identify the functional group(s) in the
molecule.
CH3
CH3
H
F
O
O
OHOH
SH

3. Tips for Today’s Experiment
• Unless specified, the maximum number of drops
before recording “no change” is 10 drops.
• The recording of data is similar to the previous
experiment.
• Some test tubes will become hot due to certain
reactions. Be careful in handling those.
• If the experiment says “note any change”, most
likely (but not always) there is a change.
• Make sure that all your group mates have seen
the changes before proceeding to the next step.
• The concentrations of the chemicals are much
higher than the previous experiment. Please take
extra care in handling these chemicals.

4. Tips for Today’s Experiment
• As much as possible, do the experiments with
salicylic acid first, as the reagent quickly
crystallizes back out of the solvent.
• Because the litmus paper may interfere with the
reactions, a stirring rod and watch glass will be
provided. Do not dip the litmus paper into the
reactions.
• The colors of the original solutions must be
recorded to see if any change has really occurred.

5. Biochemistry

6. Proteins
• Amino acids are the monomers and contain an
amino group, a carboxyl group, and a side chain.
• The amino group of one amino acid reacts with
the carboxyl group of another amino acid by a
dehydration reaction, forming a peptide bond.
• Polypeptides are polymers of any combination of
the 20 amino acids.
• Proteins are three-dimensional biologically
functional molecules composed of one or more
polypeptides.

7. Functions of Proteins
• Enzymes speed up chemical reactions.
(Example: digestive enzymes)
• Defense proteins protect against disease.
(Example: antibodies)
• Storage proteins store amino acids. (Example:
casein in milk, ovalbumin in egg white)
• Transport proteins carry substances. (Example:
hemoglobin in blood)

8. Functions of Proteins
• Hormones coordinate activities. (Example:
insulin)
• Contractile and motor proteins help move the
body. (Example: actin and myosin in muscle)
• Receptors help cells respond to stimuli.
(Example: neurotransmitters in nerve cells)
• Structural proteins provide support. (Example:
keratin in hair, collagen in connective tissues,
silk in spider webs and cocoons)

9. Types of Amino Acids Based on Side
Chain
• Hydrophobic nonpolar: glycine, alanine,
valine, leucine, isoleucine, methionine,
phenylalanine, tryptophan, proline
• Hydrophilic polar: serine, threonine, cysteine,
tyrosine, asparagine, glutamine
• Acidic: aspartic acid, glutamic acid
• Basic: lysine, arginine, histidine
• Cysteine has sulfhydryl groups.

10. Levels of Structure in Proteins
• Primary structure is the linear sequence of amino
acids.
• Secondary structure is the combination of α-
helices, β-pleated sheets, triple helix, and/or β-
turns formed by hydrogen bonding in the
polypeptide backbone.
• Tertiary structure is the three-dimensional shape
formed by the combination of hydrophobic
interactions, disulfide bridges, and electrostatic
attractions in the side chains.
• Quaternary structure is the association of
multiple tertiary structures, forming a functional
protein.

11. Tertiary Structure
• Hydrophobic interactions are due to van der
Waals interactions of hydrophobic amino
acids.
• Disulfide bridges are covalent bonds formed
between sulfhydryl groups of amino acids.
• Electrostatic attractions are due to the ionic
charges of acidic and basic amino acids.

12. Question
• Determine the type of tertiary structure that
will dominate in the following polypeptides.
1. A polypeptide with many valine and leucine
amino acids
2. A polypeptide with many glutamic acid and
lysine amino acids
3. A polypeptide with many cysteine amino
acids

13. Protein Structure
• Denaturation is a permanent change in the shape
of the protein due to changes in the environment
(pH, salt concentration, temperature).
• Chaperonins or chaperone proteins are molecules
that assist in the proper folding of proteins.
• X-ray crystallography is the technique to
determine the three-dimensional structure of a
protein.
• NMR (nuclear magnetic resonance) spectroscopy
may also help in the determination of structure.
• Bioinformatics may be used to predict structure.

14. Technique: X-Ray Crystallography
• The technique is only possible for proteins
that can be crystallized.
• The technique cannot show dynamic motion
of a protein inside the cell.
1. A crystallized protein is placed in front of an
X-ray beam.
2. The protein will scatter the X-rays into an
orderly pattern, a phenomenon known as
diffraction.

15. Technique: X-Ray Crystallography
3. The diffraction pattern is recorded as spots by
a digital detector.
4. Based on the pattern and the primary
structure of the protein, a three-dimensional
model may be constructed by computer
software.
5. The computer software makes an electron-
density map of the spots using a mathematical
technique known as Fourier transform.

16. Technique: How to Grow Crystals
A. Microbatch Method
• The mother liquor contains all components
needed for crystal formation, including the buffer
and the precipitating agent.
• The precipitating agent is the material that will
cause the protein to precipitate and may be a
salt, polymer, organic solvent, or combinations of
them.
• The mother liquor and proteins are mixed at high
concentrations to cause precipitation of the
protein as a crystal.
• The method is done in oil to prevent evaporation.

17. Technique: How to Grow Crystals
B. Vapor-Diffusion Method
1. The undersaturated protein solution is mixed
with a drop of the mother liquor.
2. The solution is equilibrated (put into chemical
equilibrium) with solution containing only
mother liquor. The difference in concentration
causes water to evaporate into the mother
liquor, leaving the drop more concentrated.
3. The supersaturation of the protein and the
higher concentration of the precipitating agent
causes the protein to precipitate as a crystal.

18. Technique: NMR Spectroscopy
• The technique relies on nuclear spin angular
momentum of nuclei, which is only possible for
certain elements like 1H, 13C, 15N, 19F, and 31P.
• 1H is typically used because of its high sensitivity
and natural abundance, but proteins contain a lot
of 1H molecules, requiring two-dimensional NMR
techniques.
1. A strong, static magnetic field is applied to the
protein.
2. Nuclear spin generates a magnetic dipole that
can be either parallel (low energy) or antiparallel
(high energy).

19. Technique: NMR Spectroscopy
3. A short pulse of electromagnetic energy is
applied at right angles to the nuclei in the magnetic
field, causing some low-energy nuclei to become
high-energy nuclei.
4. The change is recorded as an absorption
spectrum, and data from several repeated
experiments are averaged to minimize errors.
5. Nuclear spins are then measured by atoms
through space (nuclear Overhauser effect
spectroscopy or NOESY) and by covalent bonds
(total correlation spectroscopy or TOCSY).
6. A computer software combines these data to
form a three-dimensional model.

20. Nucleic Acids
• Nucleotides are the monomers and are composed of
a nitrogen-containing base, a pentose sugar, and one
or more phosphate groups.
• Nucleosides occur when the phosphate groups are
removed.
• Nucleic acids or polynucleotides are the polymers
formed by phosphodiester linkages, which are
composed of a phosphate group joining the sugars of
two nucleotides.
• DNA (deoxyribonucleic acid) stores the genetic
information of an organism that is passed from
generation to generation.
• RNA (ribonucleic acid) copies the DNA and is
converted into protein.

21. Main Features of DNA and RNA
• The structure of DNA is a double helix, two
strands that spiral around an imaginary axis.
• The two strands are antiparallel, one is 5’3’,
the other is 3’5’.
• In DNA, A pairs with T, and C pairs with G
always.
• When making a copy of RNA, the same pairing
is followed, except U is used instead of T.

22. Question
• Suppose there is a strand 5’-ATCGGCTA-3’.
1. What is the sequence of the antiparallel
strand in DNA?
2. If this were copied in RNA, what will be the
sequence?

23. Question
• Suppose that a particular protein is bound to
many fatty acids. This particular protein
probably contains
A. Leucine
B. Tyrosine
C. Aspartic acid
D. Histidine

24. Technique: Spectrophotometry
• This technique may be used to quantify the
amount of biomolecule present in a sample.
1. Use one of the colorful tests that is currently
being used to identify biomolecules.
2. Perform the colorful test in a cuvette with a
positive sample.
3. Using a machine known as
spectrophotometer, determine the
wavelength at which maximum absorption
occurs.

25. Technique: Spectrophotometry
4. Perform the test using samples of known and
differing concentrations at the determined
wavelength.
5. Plot the results into a straight line, which is
known as the calibration curve.
6. Determine the concentration of the unknown
sample by measuring at the determined
wavelength and comparing with the calibration
curve.

26. The Microscope

27. Light Microscopy
• The microscope is an instrument that magnifies
objects that are too small to be seen by the eye.
• Microscopy is the science of studying objects
using a microscope.
• Light microscopy uses transmitted light to view
objects.
1. A simple light microscope such as magnifying
lens uses a single lens.
2. A compound light microscope uses a set of
lenses or lens systems to magnify objects and
different colorful stains to provide image
contrast.

28. Mechanical Parts of a Compound
Microscope
• The base is the bottom-most part that supports the
entire microscope.
• The pillar is the part above the base that supports the
entire microscope.
• The inclination joint allows for tilting of the microscope
for the convenience of the user.
• The arm is the curved or slanted part which is held
while carrying the microscope.
• The stage is the platform where object to be examined
is placed and secured by stage clips and may be
moveable.

29. Mechanical Parts of a Compound
Microscope
• The eye is the circular opening through the central part
of the stage and is used to allow light to pass up
through the stage.
• The mechanical stage control knobs are two knobs that
are usually located below the stage. One knob controls
forward/reverse movement, and the other controls
right/left movement.
• The body tube is attached to the arm and bears the
lenses.
• The draw tube is the cylindrical structure on top of the
body tube that holds the ocular.
• The nosepiece is a rotating disc where the objectives
are attached. When moving the nosepiece, the fingers
should be placed on the disc and not the objectives.

30. Mechanical Parts of a Compound
Microscope
• The dust shield lies atop the nosepiece and keeps dust
particles from settling on the objectives.
• The coarse adjustment knob is geared to the body tube
and elevates or lowers it when rotated, bringing the
object into approximate focus.
• The fine adjustment knob is a smaller knob for delicate
focusing bringing the object into perfect focus.
• The condenser adjustment knob elevates or lowers the
condenser to regulate the intensity of light.
• The iris diaphragm lever is a lever in front of the
condenser that may be moved horizontally to open and
close the diaphragm.

31. Optical Parts of a Compound
Microscope
• The mirror is located beneath the stage and has
concave and plane surfaces to gather and direct light to
illuminate the object. Some microscope models have
no mirror and have built-in lamp instead, mounted
below the sub-stage for illumination.
• The sub-stage consists of two components.
1. The iris diaphragm regulates the amount of light
necessary to obtain a clearer view of the object.
2. The condenser is a set of lenses between the mirror
and the stage that concentrates light rays on the
specimen.
• The eyepiece or ocular is a set of lens found on top of
the body tube which functions to further magnify the
image produced by the objective lenses.

32. Optical Parts of a Compound
Microscope
• The objectives are metal cylinders attached below the
nosepiece which contain specially ground and polished
lenses. There are usually three objectives in use.
1. The low power or 16mm objective (LPO) gives the lowest
magnification (10x) and is used to locate the specimen as
it possesses a relatively large field of vision.
2. The high power or 4mm objective (HPO) gives higher
magnification, usually 40x or 43x, and is used to focus on
the finer details of the specimen.
3. The oil immersion objective (OIO) gives the highest
magnification, usually 97x or 100x, and is used wet either
with cedar wood oil or synthetic immersion oil.

33. Proper Handling of a Compound
Microscope
• A microscope may be seriously damaged if dropped or
bumped against a hard object.
• The microscope should always be carried with both
hands in a straight upright position, one under the base
and the other on the arm. The eyepieces are not
attached and will fall out if the microscope is carried at
an angle or upside down.
• Place the microscope at least 5cm from the edge of the
table to prevent it from falling.
• The clips must be tightly clipped on the stage, and the
mirror must be in vertical position.
• Do not touch the mirror with your fingers. Handle it by
the frame to tip it at any desired angle.

34. Proper Handling of a Compound
Microscope
• Always keep the stage dry and clean.
• Never allow liquids, particularly acids and alcohol, to
come in contact with any part of the microscope. Do
not incline the microscope when examining fresh
mounts.
• After using the microscope, the LPO should be in
position, and the coarse adjustment knob should be
moved down until it stops.
• The student should always report immediately to the
instructor any defects (missing or damaged part) of the
microscope.

35. Some Terminologies
• The magnifying power of an objective is shown by a figure
engraved on the sleeve.
• The numerical aperture (NA) is also engraved on the sleeve,
next to the magnification (0.30 on the LPO, 0.65 on the HPO,
and 1.30 on the OIO).
• The resolution limit or resolving power is related to the NA
and is the ability to reveal closely adjacent details as separate
and distinct. It is a function of the wavelength of light and the
design of the condenser.
• The shortest wavelengths of visible light provide the
maximum resolution. The maximum resolving power of a
good medical laboratory microscope is 0.25μm (the resolving
power of the human eye is 0.25mm).

36. Some Terminologies
• The field of view is the entire area that can be seen
through the microscope.
• The larger the magnifying power of an objective lens, the
smaller the field of view is.
• The working distance of a particular objective is the
distance between the front lens of an objective and the
object on the slide when it is in focus.
• In most microscopes, when a new objective is brought into
use, the specimen should be in focus though enlarged and
blurred (parfocal) and in the same position in relation to
the field of view as in the previous objective (parcentral).

37. Some Terminologies
• An optical section consists of the field that is in
sharp focus at any plane below the surface of a
more or less transparent specimen when seen
through the microscope.
• Depth of field is the thickness of the object that is
in sharp focus.
• The magnification of an object is the degree to
which an object is enlarged or reduced using a
system of lenses.
• The linear magnification is the product of the
magnifying powers of the objective and the
ocular.

38. How to View Specimens Under a
Compound Light Microscope
• Clean the mirrors and lenses with lens paper or lint
free cloth. Avoid using tissue paper as it may scratch
the delicate lenses.
• When properly used, the microscope should cause no
eye strain.
• Try to keep both eyes open when working the
monocular microscope (microscope with only one
eyepiece), and use the dominant eye to look through
the ocular.
• If you wear glasses, it will not be necessary to use them
with the microscope, since the microscope
automatically corrects for this.

39. How to View Specimens Under a
Compound Light Microscope
• Before placing any prepared slide on the stage, make it
a habit to examine the specimen with the eye.
• Look at the label which describes the material
mounted on the center of the slide.
• If the slide is dirty, clean it by rubbing lightly with a soft
cloth or paper towel.
• Bring the objective as far away from the stage as
possible, and place a prepared slide on the stage,
centering the objective on the focus over the hole.
• Secure the slide with stage clips. Take care that the clip
is never on the cover slip; otherwise, the specimen will
be squashed.

40. How to View Specimens Under a
Compound Light Microscope
• Adjust the mirror so that the entire field is evenly
illuminated.
• The diaphragm should be opened as wide as possible in
both cases, and the condenser should be as near the stage
as it can move.
• Try readjusting the mirror and the diaphragm to get the
most effective illumination.
• When using higher magnifications, more light is needed
than when using lower magnifications.
• For most microscopic work, it is best to keep the
condenser at its highest level. Only rarely is it desirable to
lower it slightly. When the condenser is used at a lowered
position, the resolving power is greatly reduced.

41. How to View Specimens Under a
Compound Light Microscope
• The amount of light may be decreased by lowering the
condenser or closing the diaphragm.
• Start by bringing the LPO as close as possible to the slide,
looking from the side to make sure that the objective does
not touch the slide. Make sure the side with the cover slip
is up.
• Look into the eyepiece, and slowly move the object away
from the slide until the image comes into view. Focus until
the image becomes distinct.
• Use the fine adjustment knob to make smaller
adjustments and sharpen the focus.
• The image, as seen under the microscope, is inverted so
that the right side of the object is seen at the left, and the
top is seen at the bottom and vice-versa for both
circumstances.

42. How to View Specimens Under a
Compound Light Microscope
• If it is necessary to move the image in one direction
to center it, practice moving the specimen in the
opposite direction.
• Once the specimen is in focus under LPO, and the
portion to be examined further is at the center of the
field of vision, rotate the nosepiece so that the HPO
is in position.
• Again, watch from the side taking care that the
objective does not run into the slide.
• Focus, but this time, use only the fine adjustment
knob.

43. How to View Specimens Under a
Compound Light Microscope
• When you have finished viewing a slide and wish
to view another, use the following procedure:
1. Be sure the LPO is in position. Loosen stage
clips.
2. Remove the slide by slipping it horizontally
forward (never up, as it might touch the lens in
the objective).
3. Return the slide to the instructor, and take
another slide.