Law of Thermodynamics
• 1st – energy is conserved
• 2nd - Increased disorder
• 3rd – at absolute zero, entropy is zero
Reactions
• Exergonic reaction – release of free
energy. Spontaneous
• Endergonic – require free energy from
outside
Metabolism
• The “key” to survival
• The total of all the life activities required to
sustain life.
• Each process is chemical (metabolic) in
nature
– Anabolism is a constructive activity
– Catabolism is a destructive process
Homeostasis
• The “key” to the quality of life
• Life functions are carried out in an
integrated way that results in the
maintenance of a stable internal
environment (internal dynamic equilibrium)
• This maintenance is known as
homeostasis
Water
• Polar. Solvent.
• Density – highest at 4oC. Leads to fall & spring
overturn.
• High heat of vaporization
– Water absorbs heat in phase change
• High heat capacity (specific heat)- can absorb
lots of heat with only minimal changes in
temperature
– Hydrogen bonds
– Dissipates heat when broken
– Prevents sudden temperature change
Lab 1 – Diffusion and Osmosis
Dialysis bag of sucrose (solution) was
placed in distilled water. Water moved into
the bag. There was a higher water
potential outside the bag. Water moves
toward a more negative water potential.
The bag is hypertonic to the distilled water.
The water is hypotonic to the sugar solution
in the bag.
Lab 1 Cont.
• Adding sugar to the distilled water
decreases the water potential there.
• Water potential is represented by Ψ “psi”
• Ψ = Ψp + Ψpi symbol
• At equilibrium the pressure (turgor) of the
cell wall will balance out the negative
potential of the solutes out of the cell.
pH etc…
• pH scale is used to show relative amounts of H+
and OH- ions
• pH of 7 = neutral (equal amounts of H+ and OHions)
• Acid (pH 0-7) – a compound that dissolves in
water to yield H+; a proton donor
• Base (pH 7-14) – a compound that dissolves in
water to yield OH-; a proton acceptor
• pH = 1/ log[H+] = -log[H+]
Organic Compounds
• Contain both the elements carbon and
hydrogen
Macromolecules
• Carbohydrates (sugars and starches)
• Lipids (fats, oils, and waxes)
• Proteins (functional and structural)
• Nucleic Acids (RNA and DNA)
Carbohydrates
• Consist of C, H, and O (2:1 ratio)
• Monomer – monosaccharide
• Pentose sugar has 5 carbons
Lipids, Protein and Nucleic Acid
• Lipids
– Saturated fatty acid – no double bonds
– “saturated with hydrogen!”
• Protein
- Monomers are amino acids
• Nucelic Acid
• Monomers are nucelotides
• (sugar, phosphate, and nitrogenous base)
Proteins
• Monomer – amino acids
– 20 commonly found
– 4 levels of structure
• Primary structure
• Number, type, and sequence of amino acids
• Secondary Structure
• Due to hydrogen bonding
• A twist: alpha helix, or pleat: beta pleat
• Tertiary Structure
• 3D folding pattern due to interactions between the amino acids
and their various charges
• Quaternary
• The way two or more folded subunits fit together (hemoglobin
structure)
Enzymes
•
•
•
•
•
“Organic Catalysts”
Protein nature – all enzymes are either all protein or protein with non-protein
parts known as coenzymes. Coenzymes are often vitamins.
Active site – Enzyme molecules are usually much larger than the molecule
they interact with.
“Enzyme substrate complex” with induced fit.
Factors influencing enzyme action –
– Temperature:
• In general as temperature increases, action increases
• Most efficient temperature is optimum temperature
• At high temperature enzyme denatures
– Relative amounts of enzyme and substrate:
• Rate of enzyme action varies with the amount of available substrate molecules.
• When an excess of substrate is added to a system with a fixed concentration of
enzymes, the rate of enzyme action tends to increase to a point then remain fixed as
long as the enzyme concentration remains constant.
– pH:
• Ranges and optimums differ
Enzyme Catalysis Lab #2
•
•
In this lab you used the enzyme catalase
to convert hydrogen peroxide (H202) to
water and oxygen gas.
The slope of the graph line in the early,
constant period is called the initial velocity.
It is the same for an enzyme and
substrate when conditions are the same.
It is used to compare one reaction with
another. It is constant because at the
beginning of the reaction, the number of
substrate molecules is usually large
compared to the number of enzymes.
Increasing the substrate concentration will
increase productive collisions.
Rate of reaction – pick any two points on
the straight line portion. Divide the
difference in product by the difference in
time.
uMoles2 – uMoles1 30 -20
t2 - t1
180 -120
10
60
Rate of Enzyme Catalysis
Product (in moles)
•
35
30
25
20
15
10
5
0
1
2
0.17 umoles/sec
3
4
Time (minutes)
5
6
7
Mitosis and Meiosis Lab
•
•
•
•
•
•
•
•
•
•
•
You calculated the relative duration of the phases of mitosis.
Prophase was found to have the longest relative length (50 + %)
Telophase was the shortest (~12%)
You looked at meiosis and mitosis in the formation of ascospores within the asci of
Sordaria fimicola.
Meiosis reduces the chromosome number. It starts out 2n and ends up n. (n is the
number of different chromosomes you have. In humans your n number is 23 and you
have two of each (2n) for 46 total.)
2n is called diploid
n is haploid
Sordaria is usually haploid. It is diploid only when the mycelia of 2 strains fuse to form
a diploid nucleus. It must undergo meiosis to return to its haploid state.
Meiosis (chromosomes double then divide, leaving one n)
…is followed by
Mitosis – two of each of the 4 cells from meiosis. Resulting in 8 haploid ascospores in
a sac called an ascus (plural asci). Many asci are in a fruiting body called a
perithecium.
Synapsis (coming together) and crossing over frequency can be calculated because
the alleles are independently expressed in the haploid state.
DNA
• In Progress
Central Dogma: DNA-RNA-Protein
• Replication: DNA to DNA (“semi-conservative”)
• Transcription: DNA to RNA
– Primary Transcript-result of translation
• Must be processed
– Introns-Clipped out (think “intervening”)
– Exons-stuck together
• Translation: RNA to Protein
– 3 nucleotides = 1 amino acid
DNA Replication
• 1. Start at origins
• 2. DNA Polymerase adds nucleotides
• 3. Nucleotides are only added to the 3’ end.
“Grows” in the 5’ to 3’ direction.
• 4. Have a leading strand and a lagging strand.
• 5. DNA ligase joins the lagging or “Okazaki”
fragments together.
• 6. Chains must be started with an RNA primer
added by the enzyme primase.
Lab 6 Molecular Biology Part I
• There are 3 ways to move genes between
bacteria:
– Conjugation (mating)
– Transduction (virus transfer the genetic
material)
– Transformation (direct uptake of DNA by cells)
• We did a lab on transformation.
Lab 6 Molecular Biology Part I
Transformation with pGlo
• We made the cells
“competent”- able to take up
environmental DNA.
• We mixed the competent
E.coli with plasmids.
• The plasmids we used were
already made and carried
Bacteria
Big ring of DNAone circular
chromosome
The little ring
of DNA is a
plasmid.
– 1. a gene for antibiotic
resistance.
– 2. pGlo gene
We could tell if our transformation worked because we grew the bacteria on
plates full of ampicillin. They could only grow there if they contained the plasmid
with the antibiotic gene (therefore the plasmid). If the sugar arabinose was
present it turned on the gene which made the glow in the dark protein.
Positive Control LB+ Negative Control LB/Amp- (+ or – the plasmid)
Lab 6 Molecular Biology Part II
Restriction Enzyme Cleavage
• DNA can be cut with restriction enzymes. These
have been isolated from bacteria and protect
them from viral invasion.
– They recognize palindromes. Many leave “sticky
ends.” These allow different pieces of DNA cut with
the same restriction enzyme to combine due to base
pairing.
• In this lab DNA from the bacteriophage (virus)
lambda was cut with three different restriction
enzymes. The results were electrophoresed (is
that a word?)
Eco RI
HIND III
No Enzyme
Number of Base Pairs (log)
Lab 6 Molecular Biology Part II
Restriction Enzyme Cleavage
Plot
the
known,
say
HIND
III
Distance Cm
If one of the enzymes produced pieces of known
size (Like HIND III- data was given), the size of
pieces cut with a different restriction enzyme can
be determined. To find the size of pieces cut with
ECO RI measure the bands and find the base
pairs from the intersecting line (See the red
line).
Respiration
•
Glycolysis-No oxygen needed
1.
2.
Glucose is broken into 2 pyruvate molecules.
Nets 2 ATP molecules by phosphorylation .
2 molecules of NAD+ are reduced to NADH
In fermentation pyruvate is converted to lactate,
ethanol, etc.
Occurs in the cell cytoplasm.
3.
4.
Respiration (Cellular)
1.
2.
Occurs in mitochondria
Pyruvate (See last slide) goes into the Krebs cycle
(AKA the Citric Acid Cycle).
In a series of steps enzymes transfer electrons to
coenzyme acceptors NAD+ and FAD, CO2 released.
Electron Transport Chain
3.
4.
•
•
•
•
The electron energy from NADH and FADH2 is retrieved and
stored in ATP.
ETC is made of a series of pigment containing cytochrome
compounds that serve as electron carriers.
Cytochromes are embedded in the inner membrane of the
mitochondria.
Making ATP from ADP this way is oxidative Phosphorylation.
Respiration (Cellular)
ATP Ledger
Glycolysis
Krebs
Chemiosmosis
3 per NADH
4 ATP’s -2 ATP
2 ATP’s
34 ATP’s
2 from glycolysis*
2 from Acetyl CoA
6 from Krebs cycle
2 per FADH2 2 from Krebs cycle
*
2 ATP needed to move NADH from glycolysis across the
mitochondrial membrane.
=
=
2
2
=
=
=
=
4
6
18
4
36
Respiration (Cellular)
Mitchell Hypothesis
• Chemiosmotic coupling hypothesis:
Movement of electrons through the ETC is
accompanied by a protein pumping mechanism
that sets up an energy gradient consisting of
hydrogen ions (protons) across the inner
mitochondrial membrane. These are pumped
from the inner mitochondrial matrix to the outer
compartment. As they flow back through the
molecule known as ATP Synthase, free energy
is released and conserved as ATP.
Lab 5
Cell Respiration
•
•
•
•
In this lab you compared germinating with non-germinating peas and their
rate of repiration. You also looked at the effect of temperature.
As plants respire they use up oxygen. You measured how fast they used it
up.
Non-germinating seeds are alive but dormant.
To take CO2 out of the equation a CO2 absorbent was used (KOH)
Germinating
25º
Non-germinating
O2
10º
Consumed
Colder-slower respiration
ml
25º
10º
5
Time (minutes)
Warmer-faster respiration
Germinating- faster respiration
Dormant- Slower
Photosynthesis
•
Light hits chlorophyll (strikes the antenna complexes on thylakoids in the chloroplast-photons
are funneled to the reaction center of the photosystem).
1.
2.
First occurs in the P680 reaction center in Photosystem II.
Water is cleaved to replace the excited electrons which leave the reaction center. Oxygen is
released.
The electrons move down the ETC. ATP is made.
Last acceptor is P700 reaction center in Photosystem I.
Photons boost electrons again. 2e- are energized and reduce NADH+ forming NADPH. NADP+
is reduced to NADPH. (OIL RIG)
3.
4.
5.
Steps 1-5 is the light reaction.
ATP and NADPH will be used in the dark reaction.
Photophosphorylation is the term for making ATP from the movement of electron excited by light, as
they move down the electron transport chain.
Remember there is also Substrate level phosphorylation and oxidative phosphorylation. They all
involve adding a phosphate. Substrate is a direct transfer, oxidation is when the energy comes
from breaking down food.
Photosynthesis
Light-Independent: AKA Calvin-Benson cycle: AKA
Dark Reaction (in the stroma)
1.
2.
3.
4.
CO2 is converted to carbohydrate.
CO2 is fixed as it reacts with ribulose biphosphate(
RuBP).
The above is catalyzed by the enzyme ribulose.
RuBP is regenerated so the cycle may continue.
Photorespiration-an inefficient form of the dark reaction; O2
os fixed instead of CO2. No carbohydrates are
produced.
C3- typical plant
C4- thrive in arid conditions (less photorespiration due to
special leaf anatomy)
CAM-also thrive in arid conditions