Papel de las actividades superóxido dismutasa y catalasa en la ...

Journal of Fish Diseases 2006, 29, 355–364
PDíaz-Rosales et al. Superoxide dismutase and catalase in P. damselae ssp. piscicida
number of bacteria needed to kill 50% of the
inoculated fish (Reed & Müench 1938). Strain
Lg h41/01 with an LD 50 ¼ 2.8 · 10 4 cfu g )1 fish
was considered virulent for sole and strain EPOY-
8803-II with LD 50 > 7.7 · 10 6 cfu g )1 fish was
considered non-virulent.
Bacterial growth conditions
Bacteria were stored at )80 °C in tryptic soy broth
(TSB; Oxoid Ltd., Basingstoke, UK) containing
2% NaCl and 20% glycerol. Bacteria were cultured
on tryptic soy agar (TSA; Oxoid) containing 2%
NaCl and incubated at 22 °C for 48 h. One colony
was used to inoculate 5 mL TSBs and incubated for
18 h at 22 °C with shaking. Aliquots (25 lL) of
these cultures were used to inoculate 250 mL TSBs
which was incubated at 22 °C with shaking. The
incubation time varied depending on the culture
condition and strain to be assayed.
Different growth conditions were assayed to
determine the potential induction of SOD and
catalase activities. Thus, 250-mL culture flasks were
supplemented with an iron chelant, dipyridyl
(100 lm), FeCl 3 Æ6H 2 O (100 lm) or MnSO 4 Æ2H 2 O
(250 lm) to determine the influence of iron and
manganese availability on enzymatic activity. In
order to induce oxidative stress, methyl viologen
(0.2 mm), which generates superoxide radicals, was
added to mid-exponential cultures, which were then
incubated for 8 h before centrifugation. The
potential induction of enzymatic activities by
hydrogen peroxide was tested in cultures after the
addition of two pulses of hydrogen peroxide, one of
20 lm in the mid-exponential phase, and another of
2mm in the early stationary phase. Cells were
harvested after 1-h incubation. The influence of the
growth phase was investigated with bacteria harvested
from mid-exponential (OD 600 ¼ 0.4–0.6) and
early stationary (OD 600 ¼ 1–1.2) phase cultures.
Preparation of crude extracts
Bacteria were harvested from cultures grown as
described above by centrifugation at 2000 g for
20 min at 4 °C and washing twice in 25 mm
potassium phosphate buffer containing 1 mm disodium
ethylene diamine tetraacetic acid (EDTA;
Sigma-Aldrich, St. Louis, MO, USA), pH 7.2 and
0.5 mm phenyl methylsulphonyl fluoride (Sigma)
followed by re-suspension in 1 mL of the same
buffer. Suspensions were sonicated on ice for 120 s
(four pulses of 30 s with 15 s cooling between
bursts). Lysates were clarified twice by centrifugation
at 10 000 g for 20 min at 4 °C. Supernatants
were assayed for the detection of SOD and catalase
and quantification of enzymatic activity on acrylamide
gels. Total protein concentration was determined
by the method of Bradford (1976) using
bovine serum albumin as standard.
Polyacrylamide gel electrophoresis
Electrophoresis was performed in non-denaturing
discontinuous polyacrylamide mini-gels using the
Bio-Rad Mini Protean II System (Bio-Rad Laboratories,
Richmond, CA, USA) with a 10% acrylamide/bis
separating gel (1.5 m Tris–HCl, pH 8.8)
and a 4% acrylamide/bis stacking gel (0.5 m Tris–
HCl, pH 8.3). The extracts in the sample buffer
were applied to the gel at a concentration of
20–24 lg protein per lane. Gels were then stained
for SOD or catalase and peroxidase activities.
Detection and quantification of SOD activity
Superoxide dismutase activity was visualized on gels
by nitroblue tetrazolium (NBT; Sigma) negative
staining (Beauchamp & Fridovich 1971). Briefly,
gels were washed in distilled water, soaked in a
solution of 2.45 mm NBT for 20 min, followed by
10-min incubation in darkness in a solution
containing 50 mm potassium phosphate buffer
(pH 7.2), 0.028 mm riboflavin (Sigma) and
28 mm tetramethylethylenediamine (TEMED;
Sigma). Gels were illuminated on a light box to
develop a dark background with achromatic bands
corresponding to SOD activity, due to inhibition of
the photochemical reduction of NBT to formazan
blue.
The method employed to quantify SOD activity
is based on the ability of SOD to inhibit the
reduction of NBT by superoxide (Winterbourn,
Hawkins, Brian & Correll 1975; Worthington
Enzyme Manual 1993). One unit is defined as the
amount of enzyme causing half the maximum
inhibition of NBT reduction. Different volumes of
extracts were added to cuvettes containing 0.2 mL
of a solution of 0.1 m EDTA, 0.3 mm sodium
cyanide (NaCN; Sigma) and 0.1 mL of 1.5 mm
NBT. Then, 0.05 mL of 0.12 mm riboflavin was
added at zero time and at timed intervals. All
cuvettes were incubated in a light box for 12 min
and absorbance at 560 nm was read at timed
Ó 2006
Blackwell Publishing Ltd
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