P1252108596wYmkL

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Lab 2 6 Extraction and characterization of
lactate dehydrogenase (LDH) Bovine and chicken
tissues were homogenized, and LDH was extracted
through centrifugation Protein concentrations
of the samples were determined, and LDH enzyme
activity was tested Polyacrylamide gel
electrophoresis was run to further characterize
the enzyme molecular size and).
2
Tissue Homogenates Liver 1- 4
Muscle 1-3
Pure LDH 5ug 2 ug 0.5 ug
Discontinuous nondenatured gel with enzymatic
staining.
3
Tissue Homogenates Liver 1- 4
Muscle 1-3
Pure LDH 10 ug
MW (KD)
200
4 mm
97 66
10mm
(16 mm) (27 mm)
45 31
(M4) (25 mm)
(20 mm)
SDS PAGE with Coomasee staining
(nonspecific protein staining)
4
Migration distance of LDH 25 mm. Whats its
molecular size?
5

(For protein concentration determination you also
need to plot a similar curve)
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• How to write a lab report?
• Introduction
• (background, hypothesis or objectives)
• What do you know about LDH
• The purpose of this study is to characterize
  LDH and
• Methods and Materials - Use past tense!!
• Results
• Numbers, graphs, tables and some words to
  describe them.
• Discussion and conclusion
• The significance of this study
• Conclusion the biological, chemical and
  physical
• characteristics of LDH are

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Photosynthetic electron transport

• Phototrophs
• Absorb solar radiation and divert the energy
  through the electron transport chain.
• Produce their own carbohydrates from CO2 and H2O

9

• Categories of organisms according to the
• carbon source and energy source
• Carbon source
• Autotrophs CO2
• Heterotrophs Organic molecules
• from other organisms
• Energy source
• Phototrophs Light
• Chemotrophs Oxidation of organic or
• inorganic compounds

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Photoautotrophs CO2 as carbon source light
as energy source. Chemoheterotrophs Organic
molecules as carbon source Energy from
oxidation of organic or inorganic
compounds. Often, same organic compound will
satisfy all requirements.
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Photosynthesis

•   Unlike in heterotrophs, photosynthesis is the
• energy production process by reductive
• carboxylation of inorganic substrates.
•  
• It involves fixing of CO2 and reduction of
  carboxylic acid.
• It has two steps light reaction and dark
• reaction.
•  

12

• Two steps of photosynthesis

• Light reactions - photo phase
• Absorption of light energy by chlorophyll or
• other pigments
• Dark reactions - synthesis phase
• Carbon metabolism to make carbohydrates.
• Light is not directly required.
• The two parts can be combined as one net
• reaction CO2 2H2A ? CH2On 2A
•   (H2A electron donor H2S, H2, NH3 and H2O)

13
Fig. 17.16
In light reaction, H2O loses electrons to become
O2.
The electrons and H are picked up by NADP, ?
NADPH Solar energy is stored in the form of ATP
that is used to for glucose synthesis in the dark
reaction.
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Tomorrows lab starts at 1 pm.
16

• Light reactions - photo phase
• Light energy is absorbed by chlorophyll and
• other pigments
• Dark reactions - synthesis phase
• Carbon metabolism to make carbohydrates.
• Light is not directly required.
• Net reaction of photosynthesis
• CO2 2H2A ? CH2On 2A
•  H2A, electron donor.
• For phototrophic bacteria, H2A can be H2S, H2
• or NH3. For plants the electron donor is always
• H2O.

17
Visible light (wavelength 350 - 800 nm) is the
energy source for photosynthesis. Light
quantities are defined as photons represented by
hv.
18
Chloroplast
The apparatus for light absorption and
carbon fixing in eukaryotic photosynthetic cells.
19

• Stroma
• Gel-like matrix within the inner compartment. It
  contains the enzymes for the dark reactions.

• Thylakoids
• Membranes folded into sacs
• Contain light receiving pigments, electron
• carriers and ATP synthase.
• They are arranged into stacks called grana
• (granum).

20
  The first step of photosynthesis is light
absorption by pigments located in thylakoid
membrane.   Chlorophylls, the most important
type of pigment contains four pyrrol rings and
one Mg in the center.
This structure is similar to that of the heme
group in hemoglobin.
21
Chlorophyll a
Mg porphyrin
phytol side chain
22

• Different types of light absorbing pigments
• Green plants
• Chlorophylls a and b.
• Bacteria
• Bacteriochlorophyll.

• Accessory pigments
• Carotenes and phycobilins - absorb light
• outside the range of chlorophyll.

23
Chlorophylls play major roll in photosynthesis
and are found in most green plants. Absorption
is strong in the regions of 430 (blue) and 630
700 (red). (They reflect green
light). Chlorophyll family includes a, b and
bacterio- chlorophill.
24
Chlorophyll a
saturated bond in bacteriochlorophyll
in bacteriochlorophyll
I
II
N
N
Mg
N
N
III
IV
O
H
C
H
O
H
phytol side chain
O
C
O
25
Besides chlorophyll, most photosynthetic cells
contain secondary pigments, which absorb the
wavelength that are not efficiently absorbed by
chlorophylls (470-630nm).
Two types of secondary pigments 1). Carotenoids
including beta-carotene and
xanthophill 2). Phycobilins They absorb light
and transfer the energy to Chlorophyll.
26
Photosynthetic Light Reactions The directions of
electron flow are opposite in chloroplasts and
mitochondria
In mitochondria, electrons flow from NADH to H2O
a thermodynamically downhill reaction.
Whereas in chloroplasts, H2O becomes electron
donor, the receptor is NADP, a coenzyme. The
reaction is energy consuming and the energy is
from light.
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Light reactions

• 2 H2O 2 NADP
• 2 H O2 2 NADPH

light
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Photosystems
h?
First light is absorbed by light harvesting
complexes or photosystems.

• Each photosystem contains one primary acceptor -
  chlorophyl
• and a set of accessory
• pigments helping funneling additional light.

29

• Photosystem I - P700
• Chlorophyll a and accessory pigments
• absorb light in 600-700 nm range

• Photosystem II - P680
• Chlorophyll a, b and accessory pigments
• Absorb light with a maximum at 680 nm
• All plants and bacteria have system I.
• Higher plants that can evolve O2 contain
• both I and II.

30
Photosystem I
Reduction potential, V
31
The electron flow in photosynthesis involves
energy level change in electron carrier
molecules. Electrons have two energy levels G
and S1, which involve different orbital.
Electrons can be excited by light energy and
change from a ground state orbital in G to the
excited state orbital in S1 (Fig. 17.19).
Electrons with high energy level can flow
downhill to the next carrier or the ultimate
receiver, NADP.
32
Light is absorbed by P700, an electron is
activated to a higher energy level, P700 becomes
P700, an excited molecule, The activated
electron then moves downhill via electron
transport chain to NADP that is then reduced to
NADPH.
33
In green plants, the photosystems I and II are
linked. The empty electron hole, P700 in
photosystem I is filled by another electron
transport chain driven by photosystem II.
34
Linkage of photosystems I and II

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  When light hits P680 in photosystem II, an
electron will also be activated and go down
another electron transport chain (pheophytin
?plastocyanin). Plastocyanin then delivers
the electron to P700, where it can be excited
by light.  
36
The P680 formed will then be filled with
electrons from H2O. One water molecule can
produce 4H, 4e, and one O2. This is how O2
is produced by plant metabolism. The electron
flow is Z scheme.  
37
Photosystem II
38
In chloroplasts, the electron transport from
H2O to NADP involves both photosystems I and
II, which are linked with electron transport
chain.   Linkage of Photosynthesis I and II Z
Scheme
39
Photosystems I and II

• Net reaction
• 2 H2O 2 NADP
• O2 2 NADPH 2 H
• Eight photons are required to deliver four
• electrons that are necessary to produce two
• NADPH, a coenzyme required for dark reaction.

8 h?
40
Photophosphorylation

• The process that converts light energy to
• ATP, the energy form that can be used for
• biosynthesis.
• Very similar to oxidative phosphorylation.

When the cell obtained enough NADPH, it uses the
solar energy to produce ATP.   The electron
pathway for ATP production is cyclic and only
involves photosystem I.
41
Cyclic photophosphorylation

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Photophosphorylation
The electron excited in P700 is passed to Ao ?
A1 ? Fe-S complex ? Ferredoxin. From ferredoxin,
it is shunted to cytochrome bf complex, (instead
to NADP) then from cytochrome bf complex to
plastocyanin. In the mean time, H are pumped
from stroma into intermembrane space of the
chloroplast and ATP is synthesized from ADP.

43
Photophosphorylation
proton pump
thylakoid membrane
44
When electrons are transfered from H2O to NADP,
H are pumped across the inner membrane. H
gradient drives the synthesis of ATP Protein
complexes CF0 and CF1 are the ATP synthases of
chloroplasts. Free Pi is needed for ATP to be
produced. The low energy electron can return
to P680 and to be excited by light.
45

• The photophosphorylation pathway is
• a cyclic process in photosystem I.
• No H2O is consumed and no NADPH or O2
• is produced.
• ADP is phosphorylated.

Four characteristic of cycle electron pathway,
p540
46
  There is a balance between the cyclic and
noncyclic pathways, depending on the cellular
concentration of ATP and NADPH, which are both
needed for dark reactions.  
47
Reductive carboxylation   In plants and some
bacteria, the cellular structures can be
synthesized from inorganic materials such as
CO2. The process is called Calvin cycle (or
carbon cycle) which involves formation of new
C-C bonds that links carbon units to longer
chain of carbohydrates. It also involves
reduction of CO2 to form CH2O (carbohydrates).
48
Synthesis of carbohydrates

• The Calvin Cycle
• The dark reactions - fixation of CO2.
• Each cycle contains four stages and fix
• one carbon at a time.
• Six cycles are needed for one glucose to
• be synthesized.

49

• Overall reaction for one glucose
• 6 CO2 12 NADPH 12H 18 ATP 12 H2O
• C6H12O6 12 NADP 18 ADP 18 Pi

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Light reactions

• 2 H2O 2 NADP
• 2 H O2 2 NADPH

light
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Photosystems II

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Photosystem I
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Reduction potential, V

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For the big picture, study Fig. 17.25
55
Photosystems I and II

• Net reaction
• 2 H2O 2 NADP
• O2 2 NADPH 2 H
• Eight photons are required to deliver four
  electrons
• to produce two NADPH.

8 h?
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Cyclic photophosphorylation

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58
The driving force of ATP synthesis is proton
motive force
proton pump
thylakoid membrane
59
  There is a balance between the cyclic and
noncyclic pathways, depending on the cellular
concentration of ATP and NADPH, which are both
needed for dark reactions.  
60
Reductive carboxylation   Inorganic materials
such as CO2 can be used for the synthesis of
cellular structure. In Calvin cycle (or carbon
cycle), CO2 is reduced to form CH2O
(carbohydrates). Also, new C-C bonds are formed,
which links carbon units to longer chain of
carbohydrates.
61

• Overall reaction for one glucose
• 6 CO2 12 NADPH 12H 18 ATP 12 H2O
• C6H12O6 12 NADP 18 ADP 18 Pi

62
Calvin cycle

• Stage 1
• Addition of CO2 to an acceptor molecule,
  ribulose-1,5-bisphosphate .
• Catalyzed by Ribulose-1,5-bisphosphate
  carboxylase /oxygenase (rubisco).
• The 6C intermediate product is then cleaved into
  two molecules of 3-phosphoglycerate.
•  
•  

63

• Stage 1

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3-phosphoglycerate
O
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ribulose-1,5- bisphosphate
?-keto acid intermediate
64

• Stage 2
• Entry of 3-PG into the mainstream of metabolism
• 3-phosphoglycerate is first phosphorylated into
• 1,3 bisphosphoglycerate that is then reduced
• to glyceraldehydes 3-P.

ATP
ADP
O
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C
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3-PG 1,3-BPG G3-P
65
Glyceraldehydes 3-P can then be used for the
synthesis of glucose. From G3-P to glucose is
the same as gluconeogenesis in animal cells,
except that NADPH has been used here.

 
66

• Stage 3
• Synthesis of carbohydrates from G3-P

Starch Cellulose Sucrose
ADP
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Also study Table 17.4
Dihydroxyacetone 3-phosphate (DHAP)
G 3-P
67

• Stage 4 
• Regeneration of Ribulose 1,5-Bisphosphate
  (RuBP)
• For one glucose to be produced, 6 carbons
• are needed. One is from CO2, five are from
• RuBP.
• CO2 is from the environment, RuBP has to be
• synthesized by the cell.
• For one molecule of glucose to be produced
• the cell needs to consume 6 CO2 and run 6
• carbon cycles.

68

• Only one of each six cycles results in
  carbo-hydrate production.
• The other five are used to regenerate the RuBP.
• The first step is the conversion of G3-P to DHAP

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isomerase
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3-phospho- glycerate
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(E4P, erythrose 4-P, X5P, xylulose 5-P S7P,
sedoheptulose 7-P)
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E4P, erythrose 4-P, X5P, xylulose 5-P S7P,
sedoheptulose 7-P Read the hand out.
71
Out of six molecules of G3P and DHAP, only one
can be used to form F1,6-BP, which leads to one
glucose to be made. Five of the G3P and DHAP
molecules have to run a series of reactions to
regenerate RuBP.
72
  Do plants use O2? Yes.
73
Photorespiration

• Rubisco can also act as an oxygenase by
  substituting O2 for CO2.

Phospholycolate
C
O
C
rubisco
O
CHOH

CHOH
O
CO2
C
CHOH
Ribulose 1,5-BP (RuBP)
3-phosphoglycerate
74

• This appears to be a wasteful counter productive
  pathway - oxygen is consumed, CO2 is produced.
• Useful organic material, RuBP is broken down to
  CO2

The rate of carboxylase reaction is 4 times that
of the oxygenase reaction, under normal
condition. (Rubisco has higher affinity for
CO2).
75
In hot and dry weather, pores on leaves are
closed to eliminate evaporation. CO2 can not
get in, O2 can not get out. Photorespiration
becomes predominant.
In C3 plants
76

• Some tropical plants (sugar cane, corn, sorghum
  ...)
• have evolved a way to reduce photorespiration
• and boost photosynthesis during hot and dry
• weather.
• An optional pathway for carbon fixation is
  called
• Hatch-Slack pathway.

77
Hatch-Slack pathway in C4 plants


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O2 production is eliminated.