Purification and Physicochemical Properties of Rhodanese
from Liver of Goat, Capra Aegagrus Hircus
Blessing
Ejeme Ogudugu, Nelson Adedeji Ademakinwa, Esther Nkechi Ezinma, Femi Kayode
Agboola
Blessing Ejeme
Ogudugu, Esther Nkechi Ezinma, Femi Kayode Agboola, Department of Biochemistry, Obafemi
Awolowo University, Ile-Ife, Osun State, Nigeria
Nelson
Adedeji Ademakinwa, Department
of Biochemistry, Faculty of Basic Medical Sciences. Olabisi Onabanjo
University, Ikenne, Ogun State. Nigeria
Correspondence to: Femi Kayode Agboola, Department of
Biochemistry, Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria
Email: fagboola@yahoo.com
Telephone: +2348034738078
Received: August 12,
2015
Revised: September 1, 2015
Accepted: September 5, 2015
Published online: September 22, 2015
ABSTRACT
Rhodanese from the
liver of domestic goat (Capra aegagrus hircus) was extracted, purified
and the catalytic as well as physicochemical properties determined in order to
gain an insight into how domestic goats are able to feed and thrive
successfully on cyanogenic plants such as sorghum, millet and cassava. A
domestic goat was purchased from a freehold rearing place in Ile-Ife, Osun
State, Nigeria, where it was slaughtered and the liver excised. It was rinsed
with normal saline (0.9 % NaCl, pH 7.4) to remove blood and other impurities.
Approximately 95 g of the liver was homogenized in 3 volumes of 0.1 M acetate
glycine buffer, pH 7.8 containing 1 mM ԑ-amino-n-caprioc
acid and 10 mM sodium thiosulphate in a blender, after which it was centrifuged
for 15 min at 12,000 rpm at 4¡æ. A specific activity of 1.55 micromole
thiocyanate formed per millilitre per minute (Rhodanese Unit; RU) per mg of
protein (U/mg) with a purification fold of 1.88 and 36 % yield was obtained
from the rhodanese extracted from the liver of goat rhodanese after
CM-Sephadex, Sephacryl S-400 and Reactive Blue 2- agarose column
chromatography. An apparent molecular weight of 36 kDa was obtained on
Sephacryl S-400 and a subunit molecular weight of 19 kDa was obtained from
Sodium dodecyl sulphate polyacylamide gel electrophoresis (SDS-PAGE). A Km
value of 0.034¡À0.007 mM and Vmax of 16.70¡À3.36 units/mL was obtained
for Na2S2O3 while a Km value of
0.038¡À0.004 mM and Vmax of 18.77¡À1.58 units/mL was obtained for KCN.
An optimal pH of 8.5, 9.5 and 7.0 were obtained using citrate-phosphate-borate,
Tris-HCl and citrate-phosphate buffers respectively. Goat liver rhodanese
showed an optimal temperature of 40¡æ. All the chloride salts used which
included KCl, MgCl2, CaCl2, MnCl2, AlCl3
and NH4Cl inhibited rhodanese activity, with the highest inhibition
was observed with MnCl2 and least inhibition observed with KCl. Goat
liver rhodanese was inhibited by all active site inhibitors, with the the
highest inhibition observed with arsenic acid. It can therefore be concluded
that domestic goats (Capara aegagrus hircus) are able to feed and survive on
cyanogenic plants due to the presence of the cyanide detoxyfying enzyme,
rhodanese, present in their liver at high activity with suitable kinetic
properties.
Key words: Cyanide; Rhodanese; Goat liver
Ogudugu BE,
Ademakinwa NA, Ezinma EN, Agboola FK. Purification and Physicochemical
Properties of Rhodanese from Liver of Goat, Capra Aegagrus Hircus. Journal of Biochemistry and Molecular Biology Research 2015; 1(3):
105-111 Available from: URL:
http://www.ghrnet.org/index.php/jbmbr/article/view/1350
INTRODUCTION
Cyanide is a highly toxic compound that is readily absorbed and causes
death by preventing the use of oxygen by tissues. This toxicant is widespread
in the environment and many naturally occurring substances as well as
industrial products contain cyanide[1]. More than 2,000 species of
plants are known to contain cyanogenic glycosides[2]. It has been
reported that ingestion of cyanogenic glycosides in forage crops can result in
the death of grazing animals[3]. Many studies had reported the death
of birds from cyanide poisoning through several routes, including exposure to
cyanide salts or ingestion of cyanogenic plants[4].
Living
organisms detoxify cyanide when contacted from feed, water bodies and the
environment due largely to a number of enzyme catalyzed reactions among which
are rhodanese (thiosulphate: cyanide sulphurtransferase, EC 2.8.1.1) and
3-mercaptopyruvate sulphurtransferase (EC 2.8.1.2). These enzymes are widely
distributed[5]. Of all cyanide detoxifying enzymes, rhodanese is the
most important and studied mechanisms by which cyanide ion, CN-, is removed
from the body by enzymatic conversion to less toxic thiocyanate, SCN, in the
presence of sulphur have been reported[6,7].
Domestic goats
being herbivorous animals feed majorly on plants most of which are cyanogenic.
The major diet of locally reared goats in Nigeria includes cassava, sorghum and
millet. Cassava contains the cyanogenic glycoside linamarin, that is hydrolysed
on its marceration by the enzyme linamarase to produce a sugar molecule and
acetone cyanohydrin, which is further broken down by ¦Á-hydroxynitrile lyase to
yield acetone and hydrogen cyanide.
The present
study aims to isolate, purify and determine the biochemical properties of
rhodanase from the liver of a domestic goat, Capra aegagrus hircus.
Materials
and Method
1. Chemicals
The standard proteins for SDS-PAGE were obtained from Sigma Chemical
Company (St. Louis, Mo., USA). CM-Sephadex C-25 and Sephacryl S-400 was
obtained from Pharmacia Fine Chemical, Uppsala, Sweden. All other reagents were
of analytical grades.
2. Enzyme Extraction
The goat was slaughtered and the liver was immediately excised and kept
in an ice bucket. It was stored in the freezer until required. The frozen liver
was thawed at room temperature and rinsed with cold saline (0.9 % NaCl, pH 7.4)
to remove blood and other impurities. Ninety-five grams (95 g) of the liver was
minced into smaller pieces and homogenized in three volumes of 0.1 M acetate
glycine buffer, pH 7.8 containing 1 mM ԑ-amino-n-caprioc acid and 10 mM
sodium thiosulphate (Buffer A) with a Warring Blender for about 5 min. The
homogenate was filtered through a double layer of cheese cloth, centrifuged at
12, 000 rpm for 30 min at 4¡æ using a HITACHI High Speed Refrigerated
Centrifuge. The pellet collected after the first round of centrifugation was
suspended in one volume of the same buffer and centrifuged as earlier
described. The first and second supernatant was combined, the pellets were
discarded and an aliquot of the supernatant was then assayed for rhodanese
activity and protein concentration.
3. Enzyme Assay and Protein Concentration Determination
Rhodanese activity was assayed according to Agboola and Okonji[8].
The reaction mixture consisted of 50 mM borate buffer, at pH 9.4, 200 ¦ÌL of 250
mM KCN, 200 ¦ÌL of 250 mM Na2S2O3 and 20 ¦ÌL of enzyme solution in a total volume
of 1 ml. The mixture was incubated for 1 min at room temperature and the
reaction was stopped by the addition of 500 ¦ÌL of 15 % formaldehyde followed by
the addition of 1.5 mL of Sorbo reagent [10.1 g Fe(NO3)3
9H2O and 20 ml of concentrated nitric acid in 100 mL of distilled
water]. The absorbance was read at 460 nm. One unit of activity is represented
as the Rhodanese unit (RU). One RU was taken as the amount of enzyme which
under the given condition produced an optical density reading of 1.08 at 460nm
per minute which is 10 µ-equivalent of thiocyanate[8]. The protein
concentration was determined according to Bradford[9] using Bovine
Serum Albumin (BSA) as the standard protein.
4. Protein Purification
4. 1. Ammonium Sulphate Precipitatio: The supernatant obtained from
the centrifugation step was brought to 70 % ammonium sulphate saturation (43.6
g/100 mL) by the slow addition of solid ammonium sulphate. This was kept for 1
hour with occasional stirring until all the salt had dissolved completely in
the supernatant. The mixture was left in the fridge for about 12 hr followed by
centrifugation at 15,000 rpm for 30 min at 4¡æ. The
supernatant was discarded and the precipitate was collected and resuspended in
a small amount of Buffer A.
4. 2.
Chromatography on CM-Sephadex: The dialysed extract (28 mL) was
layered on the CM-Sephadex column (1.5¡Á10 cm).
Fractions of 4 ml were collected from the column at a rate of 30 ml per hour.
Protein was monitored spectrophotometrically at 280nm. The fractions were also
assayed for rhodanese activity. The active fractions were pooled and
immediately dialysed against several changes of 50 % glycerol to store the
enzyme.
4. 3. Gel
filtration on Sephacryl S-400: Twenty milliliters (20 mL) of the post
CM-Sephadex sample was layered on Sephacryl S-400 column (1.5¡Á100 cm). The column was eluted
with 400 ml of 10 mM phosphate buffer, pH 7.2 containing 10 mM sodium
thiosulphate. Fractions of 5 mL were collected from the column at a rate of 10
mL per hour. The active fractions were pooled and immediately dialysed against
several changes of 50% glycerol to store the enzyme.
4. 4.
Reactive Blue 2-Agarose Affinity Chromatography: Affinity chromatography
was carried out on Reactive Blue-2 Agarose column (1.5¡Á10 cm). Two milliliters (2 mL) of the post gel filtration aliquot was
layered on the column. The column was then washed with 240 mL of buffer to
remove unbound protein followed by elution with a 200 mL linear gradient of
0-1.0 M KC1 in 50 mM citrate buffer, pH 5.0 containing 10 mM sodium
thiosuphate. Fractions of 2 mL were collected from the column at a rate of 42
mL per hour. Protein was monitored spectrophotometrically at 280 nm. The active
fractions were pooled and immediately dialysed.
5. Determination of Native and Subunit Molecular Weights
The SDS-PAGE was carried out on a 12% acrylamide solution to determine
the subunit molecular weight. The standard proteins were bovine albumin
(66,000), egg albumin (45,000), glyceraldehyde-3-phosphate dehydrogenase
(36,000), carbonic anhydrase (29,000), trypsinogen (24,000), trypsin inhibitor
(20,100) and ¦Á-lactalbumin (14,000). After electrophoresis, the gels were
stained in 1% Coomasie brilliant blue R-250 in a solution containing 10 %
acetic acid and 10 % methanol for about 2 h followed by destaining in the same
solution. The native molecular weight was determined using gel filtration on a
Sephacryl S-400 column (2.5¡Á90 cm). The
standard proteins were Bovine serum albumin (66,000), ovalbumin (45,000),
peroxidase (40,000) and ¦Á-chymotrypsinogen (25,000).
6. Determination of Kinetic Parameters
The kinetic parameters (Km and Vmax) of the enzyme
were determined by varying the concentrations of KCN between 0.01 M and 0.05 M
at fixed concentration of 0.2 M Na2S2O3. Also,
the concentration of Na2S2O3 was varied
between 0.01 M and 0.05 M at fixed concentration of 0.2 M KCN. The parameters
were estimated from the plots of the reciprocal of initial reaction velocity
(1/V) versus reciprocal of the varied substrates 1/[S] at each fixed
concentrations of the other substrate[10].
7. Effect of pH on the Enzyme Activity
The effect of pH on the goat liver rhodanese activity was performed by
assaying the enzyme using different buffers at the indicated pHs:
citrate-phosphate buffer (pH 3-7), 50 mM of citrate (pH 4-6), 50 mM phosphate
(7-8), 0.1 M Tris- HCl buffer (pH 7-11) and 50 mM borate (pH 7-11)[8].
8. Optimum Temperature
The enzyme was assayed at temperatures between 0¡æ and 70¡æ to investigate the effect of temperature on the
activity of the enzyme and to determine the optimum temperature of the enzyme.
The assay mixture was first incubated at the indicated temperature for 10 min
before initiating reaction by the addition of an aliquot of the enzyme which
had been equilibrated at the same temperature. The residual enzyme was then
assayed routinely.
9. Determination of Heat Stability
The heat stability of the enzyme was determined by incubating the enzyme
for 1 hr at 30¡æ, 40¡æ, 50¡æ, 60¡æ and 70¡æ respectively.
1.0 ml was withdrawn at 10 min interval and assayed for residual activity. The
activity at 30¡æ, 40¡æ, 50¡æ, 60¡æ and 70¡æ was expressed
as a percentage of activity of the enzyme incubated at 30¡æ which was the
control.
10. Effect of salt on the Enzyme Activity
The method of Lee et al[11] was used to study the
effect of various metal ions on the activity of the goat liver rhodanese. The
salts include NH4C1, MgCl2, CaCl2, MnCl2,
AlCl3 and KCl and at the final concentrations of 0.5 mM and 1.0 mM.
11. Effect of Active Site Inhibitors
The chemical nature of the active site of the enzyme was studied by
including certain sulphydryl reagents as well as certain reagents that are
inhibitors of thiol groups in the assay mixture. These reagents include
cycloheximide, iodoacetate, N-methylmaleimide, arsenic acid, and
N-iodoacetyl-N-(5-sulfo-1-naphthyl) ethylenediamine and 5,
5¡¯-dithiobis-(2-nitrobenzoic acid). The routine assay contained 0.5 mM and 1.0
mM of the reagent.
Results
The CM-Sephadex chromatographic step gave a peak of rhodanese activity
after elution with 0-1 M KCl (Figure 1). Also, one peak of rhodanese activity
was obtained when the post-CM-Sephadex sample was layered on Sephacryl S-400
(Figure 2). The Reactive Blue 2-Agarose Affinity column also gave a single peak
of enzyme activity (Figure 3).
The pure
enzyme had a specific activity of 1.55 micromole thiocyanate formed per
millilitre per minute (Rhodanese Unit; RU) per mg of protein (U/mg), a
purification fold of 1.88 and a percentage yield of 36. The summary of the
purification of goat rhodanese is shown in Table 1. Only one band was observed
after gel electrophoresis of the purified rhodanese either in the presence or
absence of sodium dodecyl sulphate (Figure 4).
Gel filtration
on Sephacryl S-400 column resulted in an apparent molecular weight of 36 kDa
(Figure 5) while the subunit molecular weight obtained on 12% acrylamide is 20
kDa (Figure 6).
The
Lineweaver-Burk plots for the determination of kinetic parameters Km
and Vmax of goat liver rhodanese were shown in Figures 7
and 8 and the result summarized in Table 2.
The optimum pH
was 8.5 (Figure 9). The optimum temperature obtained for the goat liver
rhodanese was 40¡æ (Figure 10).
The Arhenius
plot of temperature effect on rhodanese activity at pH 7.0 is biphasic (Figure
11). The apparent activation energy values from these slopes are 7.3 kcal/mol
and 72.9 kcal/mol respectively. The goat liver rhodanese was thermostable,
retaining about 90 % of its activity at 40¡æ after 60 min
(Figure 12).
The effect of
various salt ions on the activity of goat liver rhodanese was presented in
Table 3 and the effect of sulphydryl reagents and other active site inhibitors
was presented in Table 4.
Discussion
In this study, the enzyme was purified to homogeneity using 70%
ammonium sulphate precipitation, ion-exchange on CM-Sephadex, gel filtration on
Sephacryl S-400 and affinity chromatography on Reactive Blue 2-agarose column.
The purified enzyme was obtained as a well separated single band on
SDS-Polyacrylamide gel electrophoresis prepared at 12% acrylamide
concentration.
The specific
activity obtained from the purification of goat rhodanese was 1.5 RU/mg of
protein with 36 % yield and a purification fold of 1.88. Different folds of
purification have been obtained from the purification of rhodanese from various
sources such as a purification of 150 folds from beef liver[12], 776
folds from mouse liver[11], 49 and 48 folds from catfish rhodanese I
and II[13], purification of 5.6 folds from mudskipper rhodanese[14]
and purification of 1.7 folds from rainbow trout[15].
A molecular
weight of 36 kDa was obtained for goat liver rhodanese on a gel filtration
which is in agreement with the molecular weight range of this enzyme
established to be approximately between 33,000 and 37,000 Da[16-18].
Different molecular weights have been obtained for rhodanese from various
sources and they include 37.1 kDa from beef liver[12], 34.8 kDa from
mouse liver[11], 34,500¡À707 daltons, 36 kDa from fruit bat[8]
and 36,800¡À283 for catfish rhodanese I and II[13] which is
relatively similar to the molecular weight obtained for goat rhodanese from
this study. Sedimentation equilibrium studies shows that there are two forms of
bovine liver rhodanese in crystalline enzyme preparations. One form dissociates
to a species with a limiting molecular weight close to 19,000, while the second
form is nondissociable under the same experimental conditions and exhibits a
molecular weight of ~33,000. Previous evidence indicated that the bovine
rhodanese of Mr ~37,000 was a dimer of similar, possibly identical, subunits
with two catalytic sites per dimer[19]. In contrast to this
evidence, a recent report of data from amino acid sequence and x-ray
crystallographic studies has indicated that bovine liver rhodanese is a single
polypeptide chain of Mr ~33,000 containing one catalytic site[20].
So a subunit molecular weight of ~20,000 Da obtained for goat liver rhodanese
on a 12% acrylamide is similar to one form of bovine liver rhodanese that
dissociates to a species with a limiting molecular weight close to 19,000 Da
suggesting that rhodanese from goat liver is a homodimer[12].
From the study
carried out on rhodanese from goat liver, the Km for both substrates
was relatively lower compared to rhodanese from other mammalian sources such as
human liver[21], bovine liver[12], mouse liver[11],
rat liver[18], but in close range with what was obtained for bovine
liver[22], velvet monkey[23] and acinetobacter sulphane
sulphur transferase[24]. The values reported for goat liver
rhodanese is an indication that the enzyme has high affinity for the substrate
and it would catalyse the detoxification reaction of cyanide to a harmless
compound called thiocyanate more efficiently considering the level of exposure
of goat to cyanogenic glycosides present in its diet majorly cassava, sorghum
and millet. The high affinity of goat liver for cyanide could explain how the
organism is able to survive and reproduce feeding on these cyanogenic plants.
The optimum pH
values compares well with that obtained for rhodanese from other mammals such
as pH 8.3 from rabbit liver rhodanese[25], pH 8.0 and 9.0 from
bovine liver rhodanese[12] and pH 9.4 from mouse liver rhodanese[11].
In this study,
an optimum temperature of 40¡æ was obtained which compares well
with the values obtained from other studies such as 50-59¡æ and 38-400C for bovine liver[21] and a much lower optimum
temperature of 25¡æ for mouse liver[11].
These temperature values are also close to those of non-mammalian rhodanese
e.g. 50-59¡æ for tapioca leaf rhodanese[27], 35-55¡ãC in Trichoderma
strains[12], 35¡æ for rhodanese from fruit bat liver[8],
40¡æ catfish liver rhodanese[13], 25¡æ for rainbow
trout[15] and 500C from mudskipper[14]. The thermal
stability experiment showed that goat liver rhodanese was thermostable,
retaining about 90 % of its activity at 40¡æ after 60 min.
The enzyme had 52 %, 30 % and no activity at 50, 60 and 70¡æ respectively. However, after heating the enzyme for 2 hr, the enzyme
retained its full activity at 30¡æ, had 77 % activity at 40¡æ, but lost all its activity at 50, 60 and 70¡æ respectively.
Ploegman et al[20] reported that bovine liver rhodanese
consists of two equally sized, similarly folded domains stabilized by extensive
hydrophobic interactions. This enzyme appears sensitive to thermal
inactivation, a process which apparently results from thermally induced
transitions of the native structure which leads to the exposure of hydrophobic
surfaces and irreversible protein association[29].
Arrhenius plot of effects of temperature on reaction rate consists of
two linear segments with a break occurring at 45¡æ. The apparent activation energy values from these slopes are 7.3
kcal/mol and 72.9 kcal/mol respectively. The first activation energy value of
7.3 kcal/mol is closely related to the 7.5 kcal/mol reported for bovine liver
rhodanese[12] and also falls within the physiological activation
energy range (1 kcal- 25 kcal) for physiological processes in living organisms[30].
The second activation energy value of 72.9 kcal/mol lies within the range (40
kcal/mol-100 kcal/mol) for protein denaturation[31].
From the
result obtained from the study of the effect of salts especially those of
chloride on goat liver rhodanese, all the salts inhibited goat rhodanese
activity although with differing levels of inhibition. According to Stokinger[30],
metal ions showing inhibition of rhodanese activity are those that have strong
affinity for ligands such as phosphate, cysteinyl and histidyl side chain of
protein.
Inhibition of
rhodanese by 5, 5¡¯-dithiobis-2-nitrobenzoic acid (DTNB), an aromatic nitro
compound is similar to what was obtained by Aminlari[33] in his
study of the active site of rhodanese with disulphide reagents. DTNB is usually
used for probing the active site of rhodanese because it reacts with a
favourable equilibrium constant with sulphydryl groups, and especially because
it generates an intensely chromophoric product, thionitrobenzoate which can be
monitored spectrophotometrically. DTNB usually oxidizes rhodanese by causing a
formation of an intermolecular disulphide bond between its monomers. Disruption
of the conformation of the active site of rhodanese by DTNB could perhaps be
responsible for the inhibition observed in this study.
The
physicochemical and catalytic properties of rhodanese isolated from the liver
of goat are similar to what has been obtained from other mammalian sources. The
low Km observed signifies high affinity of rhodanese for its
substrates. This may support how goats are able to efficiently feed on
cyanogenic foods such as yam, cassava, millet, sorghum e.t.c. all of which are
high in hydrogen cyanide content without experiencing cyanide toxicity.
Conclusion
In conclusion, this work has established the presence of rhodanese with
properties similar to that of enzymes from vertebrates, and the biophysical and
other structural properties of this enzyme are required to assign roles and
physiological function to the enzyme.
CONFLICT OF INTERESTS
The authors have no conflicts of interest to declare.
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Peer reviewer: Leonid Breydo,
Department of Molecular Medicine, University of South Florida, Tampa, USA.
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