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HIGH SCHOOL OF DUNDEE
ADVANCED HIGHER
SUMMARY NOTES
UNIT 1
CELLS AND PROTEINS

1

1
...
g
...
1, 0
...
3 and
so on
Dilutions in a log dilution series differ by a constant proportion, e
...
10-1, 10-2, 10-3
and so on

Production of a standard curve to determine an unknown
Plotting measured values for known concentrations to produce a line or curve
allows the concentration of an unknown to be determined from the standard curve

Use of buffers to control pH
Addition of acid or alkali has very small effects on the pH of a buffer, allowing the
pH of a reaction mixture to be kept constant
pH can be measured using a meter or an indicator

Method and uses of a colourimeter to quantify concentration and turbidity
Calibration with appropriate blank as a baseline
use of absorbance to determine concentration of a coloured solution using suitable
wavelength filters
use of percentage transmission to determine turbidity, such as cells in suspension

3

(c) Separation techniques

Use of centrifugation to separate substances of differing density
More dense components settle in the pellet
Less dense components remain in the supernatant

Paper and thin layer and affinity chromatography can be used for separating different
substances such as:
different amino acids
different sugars

The speed that each solute travels along the chromatogram depends on its differing
solubility in the solvent used

Principle of affinity chromatography and its use in separating proteins
A solid matrix or gel is created with specific molecules bound to the matrix or gel
Soluble, target proteins in a mixture, with a high affinity for these molecules,
become attached to them as the mixture passes down the column
Other non-target molecules with a weaker affinity are washed out

4

Principal of gel electrophoresis and its use in separating proteins and nucleic acids
Charged molecules move through an electric field applied to a gel matrix

Native gels separate proteins by their shape, size and charge
Native gels do not denature the molecule so that separation is by shape, size and
charge

SDS-PAGE separates proteins by size alone
SDS-PAGE gives all the molecules an equally negative charge and denatures
them, separating proteins by size alone

Proteins can be separated from a mixture using their isoelectric points (IEPs)
IEP is the pH at which a soluble protein has no net charge and will precipitate out of
solution

If the solution is buffered to a specific pH, only the protein(s) that have an IEP of that pH will
precipitate

Proteins can also be separated using their IEPs in electrophoresis
Soluble proteins can be separated using an electric field and a pH gradient
A protein stops migrating through the gel at its IEP in the pH gradient because it
has no net charge

5

(d) Detecting proteins using antibodies

Immunoassay techniques are used to detect and identify specific proteins
These techniques use stocks of antibodies with the same specificity, known as monoclonal
antibodies
(Knowledge of monoclonal antibody production is not required)

An antibody specific to the protein antigen is linked to a chemical “label”
The “label” is often a reporter enzyme producing a colour change, but
chemiluminescence, fluorescence and other reporters can be used
In some cases, the assay uses a specific antigen to detect the presence of
antibodies

Western blotting is a technique, used after SDS-PAGE electrophoresis
The separated proteins from the gel are transferred (blotted) onto a solid medium
The proteins can be identified using specific antibodies that have reporter enzymes attached

6

(e) Microscopy

Bright field microscopy is commonly used to observe
whole organisms
parts of organisms
thin sections of dissected tissue
individual cells

Fluorescence microscopy uses specific fluorescent labels to bind to and visualise certain
molecules or structures within cells or tissues

7

(f) Aseptic technique and cell culture

Aseptic technique eliminates unwanted microbial contaminants when culturing microorganisms or cells
Aseptic technique involves the sterilisation of equipment and culture media by heat
or chemical means and subsequent exclusion of microbial contaminants

A microbial culture can be started using an inoculum of microbial cells on an agar medium,
or in a broth with suitable nutrients
Many culture media exist that promote the growth of specific types of cells and
microbes

Animal cells are grown in medium containing growth factors from serum
Growth factors are proteins that promote cell growth and proliferation
Growth factors are essential for the culture of most animal cells

In culture, primary cell lines can divide a limited number of times, whereas tumour cells lines
can perform unlimited divisions

Plating out of a liquid microbial culture on solid media allows the number of colony-forming
units to be counted and the density of cells in the culture estimated
Serial dilution is often needed to achieve a suitable colony count

Method and use of haemocytometer to estimate cell numbers in a liquid culture

Vital staining is required to identify and count viable cells

8

2
...

The proteome is larger than the number of genes, particularly in eukaryotes, because more
than one protein can be produced from a single gene as a result of alternative RNA splicing

Not all genes are expressed as proteins in a particular cell
Genes that do not code for proteins are called non-coding RNA genes and include
those that are transcribed to produce tRNA, rRNA, and RNA molecules that control
the expression of other genes

The set of proteins expressed by a given cell type can vary over time and under different
conditions
Some factors affecting the set of proteins expressed by a given cell type are:
metabolic activity of the cell
cellular stress
the response to signalling molecules
diseased versus healthy cells

9

(b) The synthesis and transport of proteins
(i) Intracellular membranes

Eukaryotic cells have a system of internal membranes, which increases the total area of
membrane
Because of their size, eukaryotes have a relatively small surface area to volume
ratio
...


Amino acids are linked by peptide bonds to form polypeptides
(recognise the chemical structure of a peptide bond from a diagram)

Amino acids have the same basic structure, differing only in the R group present
R groups of amino acids vary in size, shape, charge, hydrogen bonding capacity
and chemical reactivity

Amino acids are classified according to their R groups:
basic (positively charged)
acidic (negatively charged)
polar
hydrophobic
(classify amino acids according to the R group present, but names and structures of
individual amino acids are not required)

The wide range of functions carried out by proteins results from the diversity of R groups

The primary structure is the sequence in which the amino acids are synthesised into the
polypeptide

Hydrogen bonding along the backbone of the protein strand results in regions of secondary
structure
alpha helices
parallel or anti-parallel beta sheets
turns

14

The polypeptide folds into a tertiary structure
This conformation is stabilised by interactions between R groups:
hydrophobic interactions
ionic bonds
London dispersion forces
Hydrogen bonds
disulphide bridges – covalent bonds between R groups containing sulphur

Quaternary structure exists in proteins with several connected polypeptide subunits
Quaternary structure describes the spatial arrangement of the subunits

A prosthetic group is a non-protein unit tightly bound to a protein necessary for its function
The ability of haemoglobin to bind oxygen is dependent upon the non-protein haem
group

Interactions of the R groups can be influenced by temperature and pH
Increasing temperature disrupts the interactions that hold the protein in shape; the
protein begins to unfold, eventually becoming denatured
The charges on acidic and basic R groups are affected by pH
As pH increases or decreases from the optimum, the normal ionic interactions
between charged groups are lost, which gradually changes the conformation of the
protein until it becomes denatured

15

(ii) Ligand binding changes the conformation of a protein

A ligand is a substance that can bind to a protein
R groups not involved in protein folding can allow binding to ligands
Binding sites will have complementary shape and chemistry to the ligand
As a ligand binds to a protein-binding site the conformation of the protein changes
This change in conformation causes a functional change in the protein

Allosteric interactions occur between spatially distinct sites
Many allosteric proteins consist of multiple subunits (have quaternary structure)
Allosteric proteins with multiple subunits show co-operativity in binding, in which changes in
binding one subunit alter the affinity of the remaining subunits
The binding of a substrate molecule to one active site of an allosteric enzyme
increases the affinity of the other active sites for the binding of subsequent
substrate molecules
This is of biological importance because the activity of allosteric enzymes can vary
greatly with small changes in substrate concentration
...
Membrane proteins
(a) Movement of molecules across membranes

The fluid mosaic model of cell membranes:
-

phospholipid bilayer which acts as a barrier to ions and most uncharged polar
molecules
integral proteins embedded in membranes
peripheral proteins attached to surface membranes

Regions of hydrophobic R groups allow strong hydrophobic interactions that hold integral
proteins within the phospholipid bilayer
Integral membrane proteins interact extensively with the hydrophobic region of
membrane phospholipids
Some integral proteins are transmembrane proteins, for example channels, transporters and
many receptors
...

Some small molecules such as oxygen and carbon dioxide pass through the bilayer
by simple diffusion

Facilitated diffusion is the passive transport of substances across the membrane through
specific transmembrane proteins

19

To perform specialised functions, different cell types have different channel and transporter
proteins
Most channel proteins in animal and plant cells are highly selective
Channels are multi-subunit proteins with the subunits arranged to form water-filled
pores that extend across the membrane

Some channel proteins are gated and change conformation to allow or prevent diffusion
Ligand-gated channels can be controlled by the binding of signal molecules
Voltage-gated channels are controlled by changes in ion concentrations

Transporter proteins bind to the specific substance to be transported and undergo a
conformational change to transfer the solute across the membrane
Transporters alternate between two conformations so that the binding site for a
solute is sequentially exposed on one side of the bilayer, then the other

Active transport uses pump proteins that transfer substances across the membrane against
their concentration gradient
Pumps that mediate active transport are transporter proteins coupled to an energy
source

Some active transport proteins hydrolyse ATP directly to provide energy for the
conformational change required to move substances across the membrane
ATPases hydrolyse ATP

20

(b) Ion transport pumps and generation of ion gradients

For a solute carrying a net charge, the concentration gradient and the electrical potential
difference combine to form the electrochemical gradient that determines the transport of the
solute
A membrane potential (an electrical potential difference) is created whether is a
difference in electrical charge on the two sides of the membrane

Ion pumps, such as the sodium-potassium pump, use energy from the hydrolysis of ATP to
establish and maintain ion gradients
The sodium potassium pump transports ions against a steep concentration gradient using
energy directly from ATP hydrolysis
It actively transports sodium ions out of the cell and potassium ions into the cell

The pump has high affinity for sodium ions inside the cell
binding of sodium ions occurs
phosphorylation of the pump by ATP
conformation of the pump changes
affinity for sodium ions decreases
sodium ions released outside of the cell

potassium ions bind to the pump outside the cell
dephosphorylation of the pump
conformation of pump changes
potassium ions taken into cell and released
affinity returns to start

For each ATP hydrolysed, three sodium ions are transported out of the cell and two
potassium ions are transported into the cell
...

(details of the apical and basal membranes are not required)

22

4
...

Binding changes the conformation of the receptor, which initiates a response within the cell

Different cell types produce specific signals which can only be detected and responded to by
cells with the specific receptor
Signalling molecules may have different effects on different target cell types due to
differences in the intracellular signalling molecules and pathways that are involved

In a multicellular organism, different cell types may show a tissue-specific response to the
same signal
...


Phosphorylation cascades allow more than one intracellular signalling pathway to be
activated
Phosphorylation cascades involve a series of events with one kinase activating the
next in the sequence and so on
...
This starts a phosphorylation cascade inside the
cell, which eventually leads to GLUT4-containing vesicles being transported to the
cell membrane

Diabetes mellitus can be caused by failure to produce insulin (type 1) or loss of receptor
function (type 2)
...

Exercise also triggers recruitment of GLUT4, so can improve uptake of glucose to fat and
muscle cells in subjects with Type 2
...


26

Depolarisation of a patch of membrane causes neighbouring regions of membrane to
depolarise and go through the same cycle, as adjacent voltage-gated sodium channels are
opened

When the action potential reaches the end of the neuron it causes vesicles containing
neurotransmitter to fuse with the membrane – this releases neurotransmitter, which
stimulates a response in a connecting cell

Restoration of the resting potential allows the inactive voltage-gated sodium channels to
return to a conformation that allows them to open again in response to depolarisation of the
membrane

Ion concentration gradients are re-established by the sodium-potassium pump, which
actively transports excess ions in and out of the cell
Following repolarisation the sodium and potassium ion concentration gradients are
reduced
...


PDE catalyses the hydrolysis of a molecule called cyclic GMP (cGMP)
Each active PDE molecule breaks down thousands of cGMP molecules per second
The reduction in cGMP concentration as a result of its hydrolysis affects the
function of ion channels in the membrane of rod cells

This results in the closure of ion channels in the membrane of rod cells, which triggers nerve
impulses in neurons in the retina
A very high degree of amplification results in rod cells being able to respond to low light
intensities

In cone cells, different forms of opsin combine with retinal to give different photoreceptor
proteins, each with a maximal sensitivity to specific wavelengths: red, greens, blue or UV

28

5
...


It consists of different protein structures including microtubules, which are found in all
eukaryotic cells
Microtubules are hollow cylinders composed of the protein tubulin
They radiate from the microtubule organising centre (MTOC) or centrosome
(knowledge of other cytoskeleton proteins is not required)

Microtubules control the movement of membrane-bound organelles and chromosomes

Cell division requires remodelling of the cytoskeleton
Formation and breakdown of microtubules involves polymerisation and depolymerisation of
tubulin
Microtubules form the spindle fibres, which are active during cell division
...
This
is followed by cytokinesis, in which the cytoplasm is separated into two daughter
cells

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