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Research Article
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A Method to Study the Effects of Chemical and Biological
Reduction of Molybdate to Molybdenum Blue in Bacteria |
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Yunus Shukor,
Burhanuddin Shamsuddin,
Othman Mohamad,
Khalid Ithnin,
Nor Aripin Shamaan
and
Mohd. Arif Syed
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ABSTRACT
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In this research, we modify a previously developed assay
for the quantification molybdenum blue to determine whether inhibitors
to molybdate reduction in bacteria inhibits cellular reduction or inhibit
the chemical formation of one of the intermediate of molybdenum blue;
phosphomolybdate. We manage to prove that inhibition of molybdate reduction
by phosphate and arsenate is at the level of phosphomolybdate and not
cellular. We also prove that mercury is a physiological inhibitor to molybdate
reduction. We suggest the use of this method to assess the effect of inhibitors
and activators to molybdate reduction in bacteria.
Key words: Molybdate reduction, E. cloacae
strain 48, molybdenum blue, inhibitors
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How
to cite this article:
Yunus Shukor, Burhanuddin Shamsuddin, Othman Mohamad, Khalid Ithnin, Nor Aripin Shamaan and Mohd. Arif Syed, 2008. A Method to Study the Effects of Chemical and Biological
Reduction of Molybdate to Molybdenum Blue in Bacteria. Pakistan Journal of Biological Sciences, 11: 672-675. DOI: 10.3923/pjbs.2008.672.675 URL: https://scialert.net/abstract/?doi=pjbs.2008.672.675
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INTRODUCTION Molybdate reduction to molybdenum
blue is a phenomenon that is more than one hundred years old. According
to Levine (1925), the phenomenon was first reported in E. coli
by Capaldi and Proskauer (1896). Since then, other reports on molybdate
reduction by other bacterium has been reported in approximately in a ten-year
interval (Jan, 1939; Marchal and Girard, 1948; Woolfolk and Whiteley,
1962; Bautista and Alexander, 1972). After a silence of nearly 13 years,
microbial molybdate reduction resurfaced again in a report on its reduction
by E. coli K12 (Campbell et al., 1985). Sugio et al.
(1987, 1988) reported the reduction of molybdate into molybdenum blue
by Thiobacillus ferreoxidans and identified the sulfur:ferric ion
oxidoreductase (SFORase) as the enzyme responsible for the reduction.
Further studies by Yong et al. (1997) however, showed that molybdate
reduction in T. ferreoxidans is chemically mediated by ferrous
iron added into the media; a known fact of molybdate chemistry (Lee, 1977;
Sidgwick, 1984). The efforts of Sugio et al. (1988) were followed
by Ghani et al. (1993), who reported that another heterotrophic
bacterium, Enterobacter cloacae strain 48 (EC 48) was able to reduce
molybdate to molybdenum blue. A new mechanism of molybdate reduction in
EC 48 was proposed involving phosphomolybdate as an intermediate between
molybdate and molybdenum blue (Shukor et al., 2000). It was also
shown that unlike Thiobacillus ferreoxidans where the reduction
of molybdate to molybdenum blue is probably due to ferrous iron, in EC
48 it is predominantly enzymatic (Shukor et al., 2002) using the
modified method of Munch and Ottow (1983). Using phosphomolybdate as a
substrate, the Mo-reducing enzyme was partially purified and characterized
(Shukor et al., 2003).
Previously, we have devised a standard curve to quantify
molybdenum blue from EC 48 using 12 MP reduced by ascorbate to molybdenum
blue (Shukor et al., 2000). This method could be a testing tool
to simulate the effect of various interfering substances (inhibitors or
activators) on the production of molybdenum blue from E. cloacae
strain 48 and for other biological reduction of molybdate to molybdenum
blue. This would prevent misleading analysis of the effect of activators
and inhibitors to the activity of the molybdenum-reducing microbes in
the future. MATERIALS AND METHODS
Enterobacter cloacae Strain 48 was originally isolated
from Chengkau, Malaysia (Ghani et al., 1993) and was grown on agar plate
and in low phosphate (2.9 mM phosphate) media (pH 7.0) containing glucose
(1%), (NH4)2SO4 (0.3%), MgSO4.7H2O
(0.05%), NaCl (0.5%), yeast extract (0.0.5%), Na2MoO4.2H2O
(0.242%) and Na2HPO4 (0.05%). All chemicals are
of analytical grade. Molybdenum blue is produced in this media but not
at high phosphate media (100 mM phosphate). The only difference between
the high and low phosphate media is the phosphate concentration.
Preparation of ascorbate-reduced molybdenum blue: Briefly, 12-phosphorous
molybdate or phosphomolybdate (BDH) was prepared in distilled water as
a 5 mM stock solution and the pH adjusted to pH 5.0 with HCl. Ascorbic
acid was prepared as a 25% solution in distilled water and kept at 4°C
for a maximum of one week. One hundred microlitres from the 12 MP stock
solutions was added to 100 µL ascorbic acid. Suitable volumes of chemicals
(phosphate, mercury and arsenate) from stock solutions were added and
the final volume adjusted to one milliliter with distilled water. After
12 h of incubation, the absorbance was read at the wavelength of 865 nm.
Preparation of cellular- reduced molybdenum blue: EC 48 culture
grown in 250 mL high phosphate media (100 mM phosphate) was used in the
inhibitor studies for the cellular reduction of phosphomolybdate since
it contained high molybdenum-reducing activity although the media did
not turn blue. Cells were harvested after 24 h through centrifugation
at 10, 000 g. The pellet was washed with distilled water and resuspended
in 100 mL low phosphate media minus yeast extract and 10 mL aliquots was
added into sterile bijou bottles. Incubation period was for 24 h. Phosphate
(Na2HPO4.2H2O) was added into the bottles
from a 1 M stock solution (pH 7.0) at a final concentration from 10 to
200 mM. Heavy metals such as mercury (HgCl2.2H2O,
JT Baker, Phillipsburg, USA) and arsenate (AsHNa2O4.7H2O,
Fluka, USA) were added into reaction vessels in the same manner as phosphate
above. After incubation period has elapsed, the wavelength at OD 865 nm
was read. Concentration of inhibitor causing 50% reduction in activity
(IC50) was calculated using a one-phase exponential decay model
for non-linear regression analysis performed using GraphPad Prism version
4.00 for Windows, GraphPad Software, San Diego California USA, http://www.graphpad.com.
The experiments above were carried out in triplicate.
RESULTS
AND DISCUSSION
When phosphate was added into EC 48 media, molybdenum blue
production from EC 48 decreased significantly. A similar profile was obtained
with the ascorbate reduced 12 MP (Fig. 1). The inhibitory
effect on the molybdenum reduction in EC 48 is probably due to either
the 12 MP complex not forming since molybdate is
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Fig. 1: |
Profile of ascorbate (chemical)
reduction of 12-MP (•) and EC 48 (o) reduction
of molybdate in the presence of phosphate. The error bars represent
mean±standard error of the mean for three replicates |
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Fig. 2: |
Profile of (chemical) reduction
of 12 MP (•) and EC 48 (o) of molybdate in
the presence of arsenate. The error bars represent mean±standard
error of the mean for three replicates |
not converted to the polyions form at neutral pH (pH 7)
or (and) through the disruption of the phosphomolybdate complex at high
concentration of phosphate ions. Both conditions could occur simultaneously
as observed by Glenn and Crane (1956) who reported that molybdenum blue
is unstable in neutral pH and high concentration of phosphate (100 mM).
Campbell et al. (1985) also reported that cellular reduction of
phosphomolybdate by E. coli K-12 requires an acidic pH (pH 5-6)
and high concentration of phosphate exceeding 50 mM is inhibitory to molybdate
reduction.
Figure 2 shows that arsenate appears to
inhibit Mo-reducing enzyme activity. Arsenate is an inhibitor to oxidative
and substrate level phosphorylation
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Fig. 3: |
Profile of (chemical) reduction
of 12 MP (•) and EC 48 (o) reduction of molybdate
in the presence of mercury. The error bars represent mean±standard
error of the mean for three replicates |
(Dawson et al., 1969) and thus it is tempting to
conclude that arsenate is a true cellular Mo-reducing enzyme inhibitor
with a calculated IC50 value of 4.67 mM (R2 = 0.99).
However, arsenate also inhibits chemical molybdate reduction by ascorbate
with a calculated IC50 value of 6.27 mM (R2 = 0.96).
Arsenate is similar to phosphate in terms of forming oxyanions and it
was reported that arsenate can replace phosphate ions in the phosphomolybdate
complex and would interfere with the phosphate determination method by
negatively affecting the Mo-blue synthesis (Clesceri et al., 1989).
Thus it can be suggested that the mechanism of inhibition of molybdate
reduction by arsenate is at the level of the phosphomolybdate complex
by interrupting phosphomolybdate formation or destabilising the complex,
both causing a reduction of phosphomolybdate species available for reduction
by bacteria as well as chemical reducing agents. Thus care must be exercised
in interpreting inhibition results concerning Mo-reducing enzyme.
Mercury significantly reduces bacterial Mo-reducing capability
(50%) at the concentration of 0.1 mM (Fig. 3). The calculated
IC50 value for mercury is 0.065 mM (R2 = 0.98).
Mercury does not affect the ascorbic acid reduction of phosphomolybdate
into Mo-blue as arsenate and phosphate did when it was subjected to the
experiment of the effect of interfering agents as secondary reactions.
This suggests that the inhibition seen with mercury is on the Mo-reducing
enzyme. Thus, amongst all of inhibitors tested, only mercury gives a true
enzyme inhibition results. The mechanism of mercury inhibition on Mo-reducing
enzyme is probably via its binding to the sulfhydryl active sites, inactivating
the enzymes (Dawson et al., 1969).
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