Isolation and Characterization of Cytochrome C-556 from Halothiobacillus neapolitanus. PRANATHI CHEEMAKURTHI and NEWTON P.HILLIARD Department of Physical Sciences, Eastern New Mexico University, Portales, NM-88130.

1
Isolation and Characterization of Cytochrome
C-556 from Halothiobacillus neapolitanus.PRANATHI
CHEEMAKURTHI and NEWTON P.HILLIARDDepartment of
Physical Sciences, Eastern New Mexico University,
Portales, NM-88130.
Methodology Cultivation and Harvesting of
Bacterial cells Halothiobacillus neapolitanus
ATCC 23641 was grown in large scale in a 14L
vessel, New Brunswick Scientific Bio Flo 110,
with the thiosulfate as the energy source. The
conditions maintained in the vessel are aeration
with 30LPM of 5 CO2, temperature 30C, rotor
speed 180rpm, and a continuous supply of medium
with 0.4 dilution rates. The pH of the vessel is
maintained at 6.951 using sodium carbonate
(8w/v). The cells were collected for every 24
hrs in a 50L carboy and stored at 4C. These
harvested cells were concentrated using Millipore
Corporation Pellicon2 tangential flow high
performance ultra filtration unit (HPUF) with a
0.5 m2 0.22 micron GVPP membrane (50L to 2L). The
resultant cells were centrifuged by using Beckman
model J2-21 at 19,000 rpm for 30 minutes, and
obtained a pellet of cells which was stored at
-20C. Lysis of cells and protein
extraction The pellets stored at -20C were
thawed to room temperature and were lysed by
French Pressure Cell Press at 20,000 psi.
Repeating the procedure for three times results
in lysis of all cells. These lysed cells were
centrifuged at 20,000 rpm for one hour. The
supernatant liquid contains the soluble protein
and this is used for the further procedure. Ion
exchange chromatography for isolation of
proteinsThe soluble protein extract from the
lysed cells was poured on to a 517cm
diethyl-amino ethyl (DEAE) SephaCelTM column
(equilibrated with 20mM phosphate buffer, pH 7).
The unbound proteins were eluted out of the
column. These eluted proteins were again poured
on to a 58 cm Blue-Agarose column equilibrated
with phosphate buffer. Again the unbound proteins
were collected which eluted with the buffer. This
pass was then taken in to dialysis tubing and
concentrated using PEG 20,000 for 24 hrs. This
concentrated fraction was then dialyzed against
20mM MES at pH 6. Then it was poured on to a
511 cm DEAE-Sepharose TM column equilibrated
with 20mM MES at pH 6. The unbound matter is
eluted with the buffer. The bound matter was
eluted using, 0.2 M salt gradient with MES which
was then equilibrated with 20mM TRIS at pH 8.
Then it was applied on to a column of 210 cm
DEAE-Sepharose. Three fractions were eluted with
0.4 M salt gradient, the first of these fractions
contained cytochromeC-556. Characterization of
cytochromeC-556 UV/Visible spectroscopy
Absorbance spectra in the region of 280-700 nm
were taken for both the oxidized and reduced
forms of cytochrome C-556. Reduction of the
protein was done with sodium dithionite. Molecular
weight determination SDS-PAGE, polyarylamide
gel electrophoresis was used to determine the
molecular weight. 10 denaturing polyacrylamide
gel was prepared by standard method. The gel was
run according to the procedure described by
Laemmni (1970) with the purified sample and sigma
molecular weight marker loaded on it. The gel was
then stained with the Coomasie blue and the band
was compared with the sigma marker to determine
the molecular weight.
ABSTRACT Being a chemolithoautotropic bacteria,
Halothiobacillus neapolitanus, will obtain its
energy needed for growth and biosynthetic
reactions by the oxidation of sulfur. This sulfur
oxidation has economic and environmental
importance as it can be used for effective
removal of reduced sulfur compounds in various
microbial leaching processes. This sulfur
oxidation pathway contains many enzymes, and the
knowledge about this is incomplete. The present
purpose is to isolate and characterize the
cytochrome C-556 in the sulfur oxidation pathway
of H. neapolitanus by using Ion exchange column
chromatography. SDS-PAGE results indicate a
single size peptide with an apparent molecular
mass of 55kDa. Electronic absorption spectra of
the reduced form shows room temperature splitting
of a-bands with maxima at 548 and 556nm
indicating the possibility of multiple haem
environments. Additional maxima occur at 522 and
413nm.
Table 2. Diagrammatic flow chart of Isolation and
purification of cytochrome C-556.

• Results
• UV/Visible spectra of the reduced sample showed
  room temperature splitting of a-bands with maxima
  at 548 and 556 nm with the additional maxima at
  521 and 413 nm.
• SDS-PAGE results showed that the cytochrome has a
  single size of subunit with apparent molecular
  weight of 55,000 Daltons.

Conclusion From the spectral and SDS-PAGE
results it was concluded that the cytochrome
C-556 was a single size peptide of high molecular
weight with an apparent molecular weight of
55,000 Daltons and with multiple haem
environment, inferred by the room temperature
splitting of spectra
Acknowledgement This publication was made
possible by NIH Grant Number RR-16480 from the
BRIN program of the National Center for Research
Resources. Its contents are solely the
responsibility of the authors and do not
necessarily represent the official views of NIH.
I would also like to thank Dr. Newton P. Hilliard
for his guidance and support.
Figure 1 1-Dimensional SDS-PAGE showing the
apparent molecular weight of protein.
Introduction Thiobacilli are a group of bacteria
that oxidize the sulfur compounds. This oxidation
of sulfur compounds catalyzed by Thiobacilli has
both negative and positive influences on the
environment and economy applications. The
applications of these bacteria include
desulfurization of gases, waste water treatment,
metal leaching and coal desulfurization. Adverse
effects are due to the sulfuric acid produced by
the bacteria and these are the principle
causative agents for acid mine drainage, the
major source of ground water pollution in many
regions of the United States. Because of their
applications these bacteria are industrially
important. For the large scale production of
these bacteria for industrial uses, a complete
knowledge of the sulfur oxidation pathway and the
stoichiometry of the oxidation reactions are
necessary. But this knowledge of sulfur oxidation
path way is incomplete in many species of
thiobacilli. The present research, isolation and
characterization of the cytochrome C-556 from
Halothiobacillus neapolitanus is a part of group
research to complete the oxidation pathway in
H.neapolitanus. The advantages of choosing these
bacteria are the possibility of producing cells
in adequate quantities, easy genetic analysis and
acid tolerance of bacteria.
205 KDa
116 KDa
97 KDa
84 KDa
66 KDa
55 KDa
45 KDa
36 KDa

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Figure 2 Absorbance spectrum of oxidized
cytochrome C-556.
Figure 3 Absorbance spectrum of reduced
cytochrome C-556.
Table 1.
Sulfur Oxidation Pathways