1. The document discusses superoxide dismutase (SOD), an antioxidant enzyme that catalyzes the breakdown of superoxide radicals. It describes the three main types of SOD found in humans, plants, and fungi based on their metal cofactors, as well as their cellular locations and structures. 2. SOD plays important roles in reducing oxidative stress and regulating signaling pathways involved in processes like apoptosis, aging, and disease. The expression and functions of SODs have been studied in plants for improving stress tolerance and crop yields. 3. The document outlines methods for extracting and analyzing SOD from the plant Arabidopsis and the fungus Chaetomium thermophilum using gel electrophoresis and activity staining to

1. 1
Subject: Molecular Biodiversity and DNA
Analysis
Topic: Superoxide
dismutase:
types and importance in
plants and human and
fungi. Submitted to: Dr. M. Ishfaq
Submitted by: Jannat Iftikhar
MS16-01
Department of botany
University of the Punjab
Lahore.

2. Contents
1. Reactive oxygen species (ROS)
2. Role of ROS
3. Superoxide dismutase (SOD)
4. Discovery and nomenclature
5. Types of SOD
6. SOD in human
7. SOD in plants
8. SOD in fungi
9. Evolution of SOD
10. Extraction of SOD from Arabidopsis (Plant)
11. Extraction of SOD from Cheatomium
thermohilum (Fungi)
12. Conclusion 2

3. Reactive oxygen species
(ROS)
 ROS are chemically reactive chemical
species containing oxygen.
 ROS are generated as by-products
during mitochondrial electron
transport.
 ROS are formed as necessary
intermediates of metal catalyzed
oxidation reactions.
 Types of ROS includes: superoxide;
hydrogen peroxide; hydroxyl radical;
hydroxyl ion; and nitric oxide.
3

4. Role of ROS
ROS play important
role in
 Apoptosis
 Gene expression
 Activation of cell
signaling.
 Serve as both
intra- and
intercellular
messengers.
ROS also
responsible for
 Aging
 Carcinogenic
 Cell death
4

5. Superoxide dismutase
 Superoxide dismutase are
metalloproteins found ubiquitously in
all aerobic organisms. (Fridovich & McCord,
1969)
 Superoxide dismutase (SOD)
catalyzes the conversion of two
superoxide anions into a molecule of
hydrogen peroxide (H2O2) and oxygen
(O2).
5

6. Discovery
 Irwin Fridovich and Joe M. McCord,
discovered the enzymatic activity of
copper, zinc superoxide
dismutase(SOD).
 Subsequently, Fridovich's research
group also discovered the
manganese-containing and the iron-
containing SODs from E.coli and the
mitochondrial MnSOD (SOD2), now
known to be an essential mammalian
protein. (Fridovich,1975) 6

7. 7
SOD Nomenclature (Culotta et. al.,2006)

8. Types of SODs
There are three major families of
superoxide dismutase, depending on
the metal cofactor:
 Cu/Zn (which binds both copper and
zinc), (Richardson et. al., 1975)
 Fe and Mn types (which bind either
iron or manganese), (Borgstahl et. al., 1992)
 Ni type, which binds nickel. (Barondeau
et. al., 2004)
8

9. SOD in Human
Three forms of superoxide dismutase
are present in humans.
 SOD1, located in cytoplasm
 SOD2, located in mitochondria
 SOD3 is extracellular
The genes are located on
chromosomes 21 (Levnon et. al.,1985) ,
chromosome 6 (Creagan et. al., 1973) , and
chromosome 4 (Hendrickson et. al., 1990) ,
respectively. 9

10. 10
Genomic organization of the three known members of the human SOD
enzyme family. SOD3 was placed in the middle in order to demonstrate
areas of amino acid sequence homology between SOD1 and SOD3.
SOD2 has no significant amino acid sequence homology with either
SOD1 or SOD3. The size of each exon and intron, in base pairs, is
shown in association with that fragment.

11. SOD1
 SOD1, contains copper (Cu) in its
reactive center.
 SOD1 has molecular mass of about
32,000 Da. (Chang et. al. 1988)
 It is found in the cytoplasm, nuclear
compartments, and lysosomes of
mammalian cells. (Crapo et. al.,1992)
11

12. Crystal structure of the human SOD1 enzyme
(rainbow-color N-terminus = blue, C-terminus =
red) complexed with copper (orange sphere) and
zinc (grey sphere).
SOD1, contains copper (Cu) in its reactive
center.
12

13. SOD2
 This isoform of SODs has manganese
(Mn) as a cofactor and has been
localized to mitochondria of aerobic
cells (Mn-SOD or SOD2).
 It exists as a homotetramer with an
individual subunit molecular weight of
about 23,000 Da.
13

14. SOD2, contains manganese (Mn) in its
reactive center.
Active site of human mitochondrial Mn superoxide
dismutase (SOD2)
14

15. SOD3
 SOD3 is the most recently discovered
and least characterized member of the
SOD family.
 The enzyme exists as a homotetramer of
molecular weight 135,000 Da with high
affinity for heparin.
 SOD3 was first detected in human
plasma, lymph, ascites, and
cerebrospinal fluids. (Markland, 1982)
 The expression pattern of SOD3 is highly
restricted to the specific cell type and
tissues.
15

16. SOD3, contains zinc (Zn) in its reactive
center.
Crystallographic structure of the tetrameric human
SOD3 enzyme (cartoon diagram) complexed with
copper and zinc cations (orange and grey spheres
respectively).
16

17. Role of SOD in Human
 SOD1 enzyme is an important
constituent in apoptotic signaling
and oxidative stress.
 SOD2 confer protection against cell
death.
 This protein plays an anti-apoptotic role
against oxidative stress,
ionizing radiation,
and inflammatory cytokines.
 SOD3 is thought to protect
the brain, lungs, and other tissues 17

18. Clinical Significance of SOD in
Human
SOD is involved in a number of
diseases and pathologies:
 ALS, Down’s syndrome, and
premature aging are some of the
pathological conditions that develop
due to altered SOD activity and ROS
concentration.
 SOD plays in cardiovascular and
pulmonary diseases.
18

19. SOD in Plants
There are three well-known and well-
studied classes of SOD metallic
coenzymes that exist in plants.
 Fe SOD
 Mn SOD
 Cu-Zn SOD
19

20. Fe SOD
 They are thought to be the most
ancient SOD metalloenzymes.
 They are found within both
prokaryotes and eukaryotes.
 Fe SODs are most abundantly
localized inside plant chloroplasts,
where they are indigenous.
 Fe SOD is inactivated by H2O2 and is
resistant to KCN inhibition.
20

21. Fe SOD
 There are two Fe SOD groups.
 The first group is a homodimer formed
from two identical 20 kDa subunits
proteins, with 1-2 gram atom of iron in
the active centers.
 The second Fe SOD group found in
most higher plants, is a tetramer of
four equal subunits with a molecular
weight of 80-90 kDa. . This group
contains 2-4 grams of iron atom in the
active center.
21

22.  Out of 43 families investigated, the Fe
containing superoxide dismutase was
found in three families: Ginkgoaceae,
Nymphaeaceae and Cruciferae
(Brassicaceae) (Salin and Bridges, 1981).
22

23. Mn SOD
 Second, Mn SODs consist of a
homodimer and homotetramer species
each containing a single Mn(III) atom
per subunit.
 They are found predominantly in
mitochondrion and peroxisomes.
23

24. Mn SOD
 The enzyme is not inhibited by KCN or
inactivated by H2O2.
 Plant Mn SODs have approximately
65% sequence similarity to one
another and these enzymes has also
high similarity to bacterial Mn SODs
(Bowler, 1994).
24

25. Cu-Zn SOD
 Third, Cu-Zn SODs have electrical
properties very different from those of
the other two classes.
 These are concentrated in
the chloroplast, cytosol, and in some
cases the extracellular space.
25

26. Cu-Zn SOD
 There are two different groups of this
enzyme. The first group consists of
cytoplasmic and periplasmic forms
which are homodimeric.
 Homodimer has molecular weight of
32,500.
 The second group is chloroplastic and
extracellular and are homotetrameric.
(Bordo et al., 1994)
 Cu-Zn enzyme is sensitive to cyanide.
26

27. 27
(Alscher et al., 2002.)

28. Importance of SODs for
Plants
 FeSOD is essential for chloroplast
development in Arabidopsis. (Husodo et.
al., 2008)
 affect the efficiency of microspore
embryogenesis in Triticosecale. (Dubas
et. al., 2014)
 Cu-ZnSOD improves tolerance
against cold and drought stresses.
 MnSOD involved in heat-stress
tolerance during grain filling of rice.
(Takeshi et. al., 2015)
28

29. Importance of SODs for
Plants
 Cu-Zn superoxide dismutase enhance
in-vitro shoot multiplication in
transgenic plum. (Faize et. al., 2013)
 It improves the recovery of
photosynthesis in sugarcane plants
subjected to water deficit and low
substrate temperature. (Chistina et. al.,
2013)
 It is a protective enzyme against
ozone injury in snap beans
(Phaseolus vulgaris L.) (Bennet et. al.,
1982) 29

30. SOD in Fungi
C. neoformans, only two SODs were
identified,
 one cytosolic Cu/ZnSOD (SOD1)
(Hwang et al., 2003)
 one mitochondrial MnSOD (SOD2).
(Martchenko et al., 2004)
30

31. SOD in Fungi
 Four genes encoding putative Sods
have been identified in the A.
fumigatus genome. (Lambou et. al., 2010)
 a cytoplasmic Cu/ZnSOD (AfSod1p)
 a mitochondrial MnSOD (AfSod2p),
 a cytoplasmic MnSOD (AfSod3p)
 a AfSod4 displaying a MnSOD C-
terminal domain.
31

32.  During growth, AfSOD1 and AfSOD2
were highly expressed in conidia
 AfSOD3 was only strongly expressed
in mycelium.
 AfSOD4 was weakly expressed
compared with other SODs.
32

33. Role of SOD in Fungi
 Superoxide dismutases (SODs), which
provide protection against oxidative
stress, exhibit an essential role for fungal
cell survival, especially during host
invasion.
 The CuZn superoxide dismutase from
Sclerotinia sclerotiorum is involved with
oxidative stress tolerance, virulence, and
oxalate production. (Selvakumar et. al., 2012)
 Function of SODs has been investigated
yeast pathogens Candida albicans.
(Lamarre et al., 2001)
33

34.  Cu-Zn SOD involvement in virulence
is shown in Candida albicans. (Hwang et
al., 2002)
 Mn-containing SODs were shown to
be involved in protection against
various stresses in C. albicans. (Hwang
et al., 2003).
 We can also make phylogenetic tree
based on Manganese superoxide
dismutase of pathogenic fungi.
34

35. Evolution of SODs
 The appearance of SOD enzymes
was triggered by the proliferation of
photosynthetic organisms that began
to produce oxygen about 2 billion
years ago.
 Two major kinds of superoxide
dismutase appeared in prokaryotes at
that time, copper/zinc-containing
SODs and iron/manganese-containing
SODs.
35

36. Three hypothesis explains the
presence of Cu-Zn SOD in
prokaryotes
 Evolves independently in prokaryotes
and eukaryotes.
 Originated in eukaryotes and then
gene is transferred to prokaryotes.
 Originated in prokaryotes and then
transferred to eukaryotes.
36

37. Cellular Extract Preparation for
SOD (Kuo et. al., 2013)
 This protocol is to demonstrate how to
prepare the cellular extract for the
identification and characterization of
SODs in plants.
37

38. Materials and Reagents
1. Nitroblue tetrazolium (NBT) solution
2. N,N,N’,N’-Tetramethylethylenediamine
(TEMED) (Sigma-Aldrich, catalog
number: T9281)
3. Grinding buffer
4. Riboflavin solution
5. KCN (Sigma-Aldrich, catalog number:
60178)
6. H2O2(Sigma-Aldrich, catalog number:
349887)
38

39. Equipment
1. A light box (white light)
2. Centrifuge
3. Protein gel cassette
39

40. Procedure
A. Arabidopsis cellular extract
preparation
B. SOD activity staining
C. Identification of different SOD
species
40

41. Arabidopsis Cellular Extract
Preparation
1. Arabidopsis seedlings were grown at 23°C
with 16 h of light at 60–100 μmol/m2/s.
Nine-day-old seedlings were collected and
weighted.
2. Seedlings were homogenized with ice-cold
Grinding buffer (tissue weight/buffer volume
= 1 mg/3 μl). Note that the tissue and
extract should be kept at 4°C during all
extraction processes.
3. Centrifuge at 16,000 x g at 4°C for 10 min.
4. The supernatant is the resulting cellular
extract, and the amount of protein was
quantified by Bradford method (1976).
41

42. SOD Activity Staining
 Proteins or cellular extract (15 to 25 μg)
was subjected to 10% native-PAGE at
4°C.
 Wash the gel with distilled water for 3
times.
 Incubate with NBT solution in dark with
shaking for 15 min at room temperature
(RT).
 Pour off the NBT solution, wash the gel
with distilled water for 3 times.
 Incubate with Riboflavin solution in dark
with shaking for 15 min at RT. 42

43. SOD activity staining
 Pour off the Riboflavin solution, wash
the gel with distilled water for 3 times.
 Gel was illuminated with a white-light
box for 10-15 min at RT. During
illumination, immerse gel in a thin
layer of distilled water to avoid drying
the gel.
 White SOD activity bands appear in
the blue background.
43

44. Identification of different SOD
Species
44
SOD activity verification in Arabidopsis thaliana. KCN is an
inhibitor of CuZnSOD activity, whereas H2O2 inhibits both
CuZnSOD and FeSOD activities. MnSOD activity is not inhibited
by either treatment.

45. Extraction of SOD from Fungi (C.
thermophilum) (Guo et. al., 28)
 A thermostable superoxide dismutase
(SOD) from the culture supernatant of
a thermophilic fungus Chaetomium
thermophilum strain CT2 was purified
to homogeneity by fractional
ammonium sulfate precipitation, ion-
exchange chromatography on DEAE-
sepharose, phenyl-sepharose
hydrophobic interaction
chromatography.
45

46. Reagents
 Yeast extract
 Casein
 Coomassie brilliant blue
 materials for gel electrophoresis
 DEAE-Sepharose fast flow, phenyl-
sepharose,
 sephacryl S-100-sepharose
 Standard protein makers (14.4–97.4
kDa)
46

47. Organism and growth
conditions
 Chaetomium thermophilum CT2 was
isolated and preserved on potato
dextrose agar (PDA) medium.
 C. thermophilum CT2 was grown in
shake cultures at 50 C in 20 flasks
each with 50 mL liquid medium
containing (g/L): casein, 40.0; glucose,
10.0; yeast extract, 4.0;
K2HPO4.3H2O, 1.0; MgSO4.7H2O,
0.5; dissolved in distilled and tap water
(3:1). 47

48. Preparation of the crude
enzyme
 After incubation for 8 d in liquid
medium the culture fluid was filtered
and centrifuged at 8000 xg for 15 min,
4 C, and the supernatant was used for
the purification of SOD.
48

49.  All procedures of the SOD purification
were carried out at 4 C. These buffers
were used:
(A) 50 mM Tris-HCl (pH 7.5)
(B) containing 50% saturation
ammonium sulfate.
49

50. Fractional ammonium sulfate
precipitation
 Solid ammonium sulfate was added to
the supernatant to 90% saturation.
 After 12 h, the precipitate was collected
by centrifugation (10 000 g, 15 min),
dissolved in buffer A and dialyzed
overnight against three changes of the
same buffer.
 Insoluble material was removed by
centrifugation (10 000 g, 15 min) and the
supernatant was put on a DEAE-
Sepharose column.
50

51. Ion exchange chromatography
on DEAE-Sepharose column
 Ion exchange chromatography on
DEAE-Sepharose column (1X 20 cm)
equilibrated with buffer A.
 After the column was washed with five
column volumes of buffer A, a 200 mL
linear gradient of NaCl (0–0.3 M in
buffer A) was applied at a flow rate of
45 mL/h.
51

52. Phenylsepharose hydrophobic
interaction chromatography
 The sample from the DEAE-Sepharose
column with 50% saturation ammonium
sulfate added was applied to a
phenylsepharose column (13-20 cm)
previously equilibrated with buffer B.
 After the column was washed with five
column volumes of buffer B, SOD was eluted
with a 160 mL linear gradient of ammonium
sulfate from 50–0% saturation at a flow rate
of 45 mL/h.
 Fractions with SOD activity were pooled and
concentrated for determination of purity and
properties.
52

53.  To identify the type of SOD, duplicate
gels were incubated with
 10 mM KCN,
 10 mM H2O2 and
 10 mM NaN3 during activity staining to
inactivate Cu, ZnSOD, MnSOD or
FeSOD, respectively (Asada et al 1975,
Britton et al 1978).
53

54. Conclusion
 They are very important against ROS.
 They act as first line defense against
ROS.
 There expression and regulation of
both SOD and ROS should be
controlled.
54

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62. 62