TITLE: Isolation and Some Properties of Partially Purified Rhodanese from the
Hepatopancreas of Freshwater Prawn (Macrobrachium rosenbergii De Man)
Running Title: Rhodanese from Freshwater Prawn.
ABSTRACT
Rhodanese (cyanide: thiosulphate sulphur transferase; E.C.2.8.1.1) was isolated using
ammonium sulphate fractionation and ion exchange chromatography in hepatopancreas
of giant freshwater prawn ((Macrobrachium rosenbergii De Man). The enzyme had a
specific activity of 7.5 rhodanese unit per milligram (RU/mg). The Km values for KCN
and Na2S2O3 were 10 mM and 2.56 mM respectively. An optimum pH and temperature
were 6.5 and 50oC for the enzyme activity. Inhibition studies on the enzyme showed that
the activity of the enzyme was inhibited by Ba2+ and Zn2+ but not affected by Mn2+, Co2+,
Sn2+, Ni2+, and NH42+.
KEY WORDS: Rhodanese, freshwater prawn, detoxification, cyanide toxicity.
Table of Content
Page
Title Page
1
Abstract
2
Introduction
4
Materials and Methods
6
1
Materials
6
Enzyme Extraction and Purification
6
Enzyme assay and protein concentration determination
7
Kinetic studies
7
Effect of pH
8
Effect of temperature
8
Effect of cation
8
Results
9
Purification of rhodanese
9
Kinetic parameters
9
Effects of pH and temperature on enzyme activity
9
Effect of cations
9
Discussion
10
References
12
Figures
15
Tables
21
INTRODUCTION
Rhodanese (Cyanide: thiosulphate sulphur transferase; E.C.2.8.1.1) is a
multifunctional, mitochondrial sulphur transferase that catalyses the detoxification of
cyanide by sulphuration in a double displacement (ping pong) mechanistic reaction
2
(Smith and Urbanska 1986). It catalyses, in vitro, the formation of thiocyanate from
cyanide and thiosulphate or other suitable sulphur donors.
It is widely distributed occurring in varieties of plants and animals, where its activity is
modulated by a number of factors including differences in species, organs, sex, age and
diet. A well characterized rhodanese from bovine liver was found to be a single
polypeptide chain of molecular weight 32,900 daltons and composed of 293 amino acids
(Russell et al. 1978; Ploegman et al. 1978).
It is generally believed that the major function of rhodanese is cyanide
detoxification (Smith and Urbanska 1986; Buzaleh et al. 1990). This function is more
prominent in mammals where highly cytotoxic cyanide is converted to a less toxic
thiosulphate and excreted through the kidney (Bourdoux et al. 1980; Cagianut et al. 1984;
Keith et al. 1989). In plants, a close relationship exists between rhodanese activity and
cyanogenesis, which suggest that the enzyme provides a mechanism for cyanide
detoxification in cyanogenic plants (Smith and Urbanska 1986). Rhodanese may play
various roles in sulphur metabolism (Volini et al. 1966; Tomati et al. 1974; White et al.
1981; Keith and Volini 1987; Nagahara et al. 1998).
The enzyme has been purified and characterized from a number of animal tissues
(Ploegman et al. 1979; Sylvester and Saunders 1990; Lee et al. 1995; Nagahara et al.
1998; Ali et al. 2001; Agboola and Okonji 2004).
It is widely distributed in liver tissues of different animals (Sorbo 1953 a; Jarabak
and Westley 1974; Blumenthal et al. 1978; Lee et al. 1995; Nagahara et al. 1996;
3
Agboola and Okonji 2004), such as beef liver, horse liver, guinea pig liver, human liver
and pig liver. Rhodanese activity has also been established in leaves, the peel and the
tuberous part of Manihot esculenta Crantxz (Anosike and Ugochukwu 1981).
Freshwater prawns belong to the genus Macrobrachium. The species
Macrobrachium rosenbergii de Man, occur throughout the tropics and subtropics on all
continents except Europe. Freshwater prawn is omnivorous and coprophagous, and they
are capable of digesting a wide range of foods of both plant and animal origin (Fujimura,
and Okamoto 1972; Armstrong et al. 1976; Aquacop 1977; Tonguthai 1997).
Asejire dam is a man made dam located at Asejire, a small farm settlement along
the boarder of Oyo and Osun State, South West, Nigeria. The dam serves as the main
source of pipe borne water supply to the towns and cities around. Effluent from industries
around the area flows into the dam, making the water polluted with compounds such as
metals, cyanide and other toxic compounds (Ayoade et al. 2006). It is therefore assumed
that the existence and survival of this animal in such aquatic environment will depend on
the ability to sufficiently detoxify cyanide. This paper reports the presence of rhodanese
in prawn and also the results of the investigation of its catalytic properties.
MATERIALS AND METHODS
MATERIALS: Life Giant freshwater prawns were purchased from commercial fish
farmers at Asejire Lake in Oyo State of Nigeria. Reagents which were of analytical grade
were used and were obtained from either Sigma or BDH. They include sodium
thiosulphate (pentahydrate), potassium cyanide, boric acid, sodium borate, formaldehyde,
4
ferric nitrate, nitric acid, sodium chloride, ammonium sulphate (enzyme grade). CM
Sepharose CL- 6B and Bio-gel P4 were obtained from Pharmacia Fine Chemical,
Uppsala, Sweden.
ENZYME EXTRACTION AND PURIFICATION
The giant freshwater prawns were bought alive from Asejire dam and transported
in an ice parked bucket. The hepatopancreas of the prawns were quickly excised and kept
in the freezer. All buffers contained 10 mM sodium thiosulphate to stabilize the enzyme
The frozen hepatopancreas was allowed to thaw at room temperature and
weighed. The hepatopancreas was homogenized using a Warring Blender in 2 volumes
of 0.1 M acetate-glycine buffer, pH 7.8 according to the method of Lee et al. (1995). The
homogenate was stirred and centrifuged for 15 min at 10,000 rpm at 60 C. The
supernatant was brought to 65 % ammonium sulphate concentration, stirred continuously
for one hour and left overnight. The resulting precipitate was dialyzed against 50 mM
citrate buffer, pH 5.0. The dialysate was centrifuged at 15,000 rpm for 15 min at 60 C.
The supernatant was collected and then used for the ion exchange step.
CM-Sepharose 6B gel (cation exchanger) was provided preswollen. The gel was
washed several times with citrate buffer, pH 5.0. It was then packed into a 2.5 X 40 cm
column. The gel was equilibrated with citrate buffer and the dialyzed sample was layered
on it. Fractions of 3 ml were collected from the column at the rate of 26 ml per hour. The
protein concentration of the fractions collected was monitored spectrophotometrically at
280 nm. The fractions were also assayed for enzyme activity. The fractions with high
enzyme activity were pooled and preserved in 65 % ammonium sulphate solution.
ENZYME ASSAY AND PROTEIN CONCENTRATION DETERMINATION
5
Rhodanese activity was measured during purification and routinely according to
the method of Agboola and Okonji (2004). The reaction mixture contained, in final
concentration, 25 mM borate buffer, pH 9.4, 50 mM KCN, 50 mM Na2S2O3 and 20 µl of
the enzyme in 1 ml cuvette of 1 cm path length. The mixtures were incubated for 1 min at
room temperature. The reactions were terminated by the addition of 0.5 ml of 15 %
formaldehyde. The termination step was completed by the addition of 1.5 ml of ferric
nitrate solution [10 g Fe (NO3)2 .9H2O and 20 ml HNO3 (sp. g 1.40) and distilled water to
100 ml] i.e. Sorbo reagent (Sorbo 1953 a). Absorbance was read at 460nm. The activity
of the enzyme is expressed in Rhodanese unit (RU). One Rhodanese unit is taken as the
amount of enzyme which under the given conditions will produce an optical density
reading of 1.08 at 460 nm (Sorbo 1951).
Bradford method (1976) was used routinely to measure the protein concentration
of the enzyme using bovine serum albumin (BSA) as a standard.
KINETIC STUDIES
The enzyme in the ammonium sulphate precipitate was first desalted on a BioGel-P-4 column using 10 mM phosphate buffer (pH 7.0) The Km and Vmax, were then
determined according to the method of Lee et al. (1995) in a typical rhodanese assay
described earlier. The parameters (Km, Vmax, and Kcat) for the two substrates were
determined by varying the concentration of KCN (between 10 mM and 70 mM) at
varying fixed concentrations of Na2S2O3 (between 5 mM and 40 mM) and also varying
the concentration of Na2S2O3 (between 50 mM and 600 mM) at varying fixed
concentrations of KCN (between 5 mM and 40 mM). The ammonium sulphate precipitate
of the enzyme was first desalted on a Bio-gel P4 column using a 10 mM phosphate
6
buffer, pH 7.0 containing 10 mM sodium thiosulphate . The kinetic parameters were then
determined from the double reciprocal plots using the method of Siegel (1976).
EFFECT of pH
The effect of pH on rhodanese activity was determined by assaying the enzyme
using 0.2 M citrate between pH 4.0-6.5 and 0.2 M phosphate buffers at pH 7.0-8.0. 1ml
of the reaction mixture contained 0.1 M of the required buffer, 0.05 M of KCN, 0.05 M
Na2S2O3 and 0.02 ml of the enzyme solution.
EFFECT of TEMPERATURE
To determine the effect of temperature on the activity of rhodanese, the enzyme
was assayed at temperatures between 250 C and 600 C. The assay mixture was incubated
at the indicated temperatures for 10 min followed by the addition of the enzyme.
EFFECT of CATIONS
The effect of the following salts nickel chloride, ammonium chloride, tin chloride,
manganese chloride, cobalt chloride, barium chloride and zinc chloride on the activity of
rhodanese was investigated. Assays were carried out under standard conditions at two
different concentrations (0.5 mM and 1.0 mM) of each metal
RESULTS
PURIFICATION of RHODANESE
The partially purified enzyme specific activity was 7.5 rhodanese unit per mg.
The elution profile after CM-Sepharose ion exchange chromatography is shown in Figure
1. Only one activity peak was obtained.
7
KINETIC PARAMETERS
The kinetic parameters were determined by double reciprocal plots and secondary
plot. Figures 2 and 3 shows the various plots for thiosulphate and KCN respectively
while Table 2 shows the various values obtained for the kinetic parameters of the
substrates.
EFFECTS of pH AND TEMPERATURE ON ENZYME ACTIVITY
There was an increase in the enzyme activity between pH 4.5 and 6.0. An
optimum pH was observed at pH 6.5 while the activity of the enzyme decreased between
pH 7.0 and 8.0 (Figure 4). An optimum temperature of 500 C (Figure 5) was obtained for
rhodanese from the hepatopancreas of freshwater prawn.
EFFECTS of CATIONS
The results of the effect of various cations on the activity of rhodanese from the
hepatopancreas of the giant freshwater prawn revealed that Mn2+, Co2+, Sn2+ and NH4+
had little or no effect on the activity of the enzyme. However, both concentrations (0.5
mM and 1.0 mM) of Ba2+, Zn2+ and Ni2+ inhibited the enzyme considerably as seen in
Table 3.
DISCUSSION
Several animals are able to eat cyanogenic plants due primarily to inherent
cyanide detoxifying mechanism in these organisms. The giant fresh water prawn is
omnivorous and capable of digesting a wide range of food both of plant and animal origin
(Fujimura and Okamoto 1972; Armstrong et al. 1976; Aquacop 1977; Tonguthai 1997).
8
The Asejire lake is man made constructed on river Osun (one of the series of the rivers
which drain into coastal lagoons and creeks bordering the Atlantic Ocean) (Ayoade et al.
2006). The industries around the lake discharges varieties of pollutants in their waste
water including heavy metals, organic toxins, resin pellets, oils, cyanide containing
compounds etc (Lameed and Obadara 2006).
Rhodanese (thiosulphate: cyanide sulphur transferase) represent one of the chief
enzymes of cyanide detoxication (Sorbo 1951, 1953a, and b; Ploegman et al. 1979;
Westley 1980; Sylvester and Saunders 1990; Lee et al. 1995; Nagahara et al. 1998; Ali et
al. 2001). The enzyme was partially purified to near homogeneity by ammonium sulphate
precipitation and ion exchange chromatography on CM Sepharose CL 6B.
This work shows the existence of rhodanese in the hepatopancreas of giant fresh
water prawn (Macrobrachium rosenbergii de Man). The specific activity of freshwater
rhodanese (7.25 RU/mg) was found to be lower than the results reported by Sorbo
(1953a) [256 RU/mg for bovine liver enzyme] and Agboola and Okonji (2004) [137
RU/mg for fruit bat liver enzyme]. Lee et al. (1995) reported a value of 1,076 RU/mg for
mitochondrial enzyme from mouse liver. A value of 1240 RU/mg was obtained for
human liver (Jarabak and Westley 1974).
The results of the kinetic parameters are similar to those from other animal
sources. It was noted that the Km value of KCN (10.0 mM) for freshwater prawn
rhodanese was lower than that of the bovine liver rhodanese (19.0 mM) while the
Na2S2O3 value of 4.4 mM is lower than that of rat liver enzyme i.e. 2.56 mM. This shows
that the affinity of freshwater prawn rhodanese for thiosulphate is higher than that of rat
liver enzyme. This suggests the role of the enzyme in detoxification of cyanide in the
9
animal, considering the industrial activities that goes on around the environment (Asejire
Lake) of the freshwater prawn.
The optimum pH of 6.5 was obtained for fresh water prawn. This result is similar
to the report of Lameed and Obadara (2006) on the pH of Asejire Lake. They found that
the water had pH of 5.7, 6.3 and 6.5 at three different points around Asejire area an
indication of acidic water and might be toxic to life. The pH value obtained for
freshwater prawn, therefore, could be physiological for optimal activity of rhodanese and
possibly other enzymes of the animal. Whereas a pH of 8.3 was reported by Lang (1933a)
when working with rabbit liver enzyme. Chew and Boey (1972) reported pH value of
10.2– 11.0 with tapioca leaves. Lee et al. (1995) reported a pH of 9.4 for mouse liver
rhodanese and whose activity was almost abolished at pH 6.0 and they also showed that
the thiocyanate production increased over alkaline pH up to 9.5. An alkaline enzyme was
also reported by Agboola and Okonji (2004) for fruit bat liver with a pH of 9.0. We
obtained a temperature optimum of 50 ºC which is in good agreement with that from
bovine liver rhodanese of 50 ºC as reported by Sorbo (1953b). While the work of Chew
and Boey (1972) on tapioca leaves showed a maximum temperature between 57 and 59
ºC. A very low optimum temperature of 20 ºC was reported for mouse liver rhodanese
Lee et al. (1995).
The presence of heavy metals in the aquatic environment of the freshwater prawn
was investigated by Lameed and Obadara (2006). Their report shows the presence of
some toxic heavy metals which include cadmium, lead, nickel, and mercury at the Asejire
Lake. Some of these metals were found to inhibit rhodanese from the hepatopancreas of
freshwater prawn. Metal ions showing inhibition are those that have strong affinity for
10
ligands such as phosphate, cysteinyl and histidyl side chain of protein (Vallee and Ulmer
1972; Stokinger 1984). Inhibition of the enzyme in giant fresh water prawn by Zn2+ and
Ba2+ was found to be more than 70% while there was a 50% inhibition by Ni2+. This
result implies the interaction of these metal ions with the sulphydryl groups of the
enzyme catalytic sites (Ulmer and Vallee 1972; Lee et al. 1995; Nagahara et al. 1999).
Lee et al. (1995) had reported that Zn
2+
is a potent inhibitor of rhodanese. A similar
result was obtained by Agboola and Okonji (2004) with fruit bat liver.
REFERENCES
Agboola FK, Okonji RE (2004). Presence of rhodanese in the cytosolic fraction of the
fruit bat (Eidolon helvum) liver. J. Biochem. Mol. Biol. 37 (33): 275-281.
Anosike EO, Ugochuckwu EN (1981). Characterization of rhodanese from Cassava
leaves and tubers. J. Exp. Bot. 32: 1021-1027.
11
Aquacop (1977). Macrobrachium rosenbergii de Man Culture in Polynesia. In Progress
in Developing a Mass Intensive Larval Rearing Technique in Clear Water. Proceedings
of the World Mariculture Society 8: 311-326.
Armstrong DA, Stephenson MJ, Knight AW (1976). Acute toxicity of nitrite to larvae of
the giant Malaysian prawn, Macrobrachium rosenbergii. Aquacul. 9: 39-46.
Ayoade AA, Fagade SO, Adebisi AA (2006). Dynamics of limnological features of two
man-made lakes in relation to fish production. Afric. J. Biotech. 5 (10): 1013-1021.
Bourdoux P, Manita M, Hanson A, Ermans AM (1980). Cassava toxicity: the role of
linamarin. In: A.M. Ermans, N.M. Mbulamoko, F. Delange, R. Ahluwalia (eds) Role of
Cassava in the Etiology of Endemic Goiter and Cretinism IDRC. Ottawa, Canada, pp.
133-152.
Bradford KM (1976). A rapid and sensitive method for the quantitation of micrograme
quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:
248-254.
Buzaleh AM, Vazquez ES, Battle AMD (1990). The effect of cyanide intoxication on
hepatic rhodanese kinetics. Gen. Pharmacol. 21(2): 219 - 222.
Cagianut B, Rhyner K, Furrer W, Schnebli H (1981). Thiosulphate–sulfurtransferase
(rhodanese) deficiency in Leber’s hereditary optic atrophy (Letter). Lancet II, 981–982.
Chew MY, Boey CG (1972). Rhodanese of tapioca leaf. Phytochem. 11, 167-169.
Fujimura T, Okamoto H (1972). Notes on progress made in developing a mass
12
culturing technique for Macrobrachium rosenbergii in Hawaii. In: T.V.R. Pillay (ed)
Coastal Aquaculture in the Indo-Pacific Region. Fishing News (Books) Ltd., London. pp.
313-327.
Jarabak R, Westly J (1974). Human liver rhodanese: non-linear kinetic behavior in a
double displacement mechanism. Biochem. 13: 3233–3236.
Keith A, Volini M (1987). Properties of E. coli rhodanese. J. Biol. Chem. 262 (14):65956604.
Keith A, Procell LR, Kirby SD, Baskin SI, (1989). The inactivation of
rhodanese by
nitrite and inhibition by other anions in vitro. J. Biochem. Toxicol. 4: 29 –34.
Lameed GA, Obadara PO (2006). Eco-Development impact of coca-cola industries
on biodiversity resources at Asejire area Ibadan, Nigeria. J. Fish. Inter. (2-4): 55-62.
Lang K (1933a). Die rhodanese-building in tiekorper: biochemical. Ztschr. 259: 243-256.
Lee CH, Hwang JH, Lee YS, Cho KS (1995). Purification and characterization
of mouse liver rhodanese. J. Biochem. and Mol. Biol. 2228: 170-176.
Lineweaver H, Burk D (1934). The determination of enzyme dissociation constant.
J. Amer. Chem. Soc. 5: 658-666.
Nagahara N, Ito T, Minam M (1999). Mercaptopyruvate sulphuretransferase as a defence
against cyanide toxications. Molecular properties and mode of detoxification. Histol. and
Histopath. 14: 277-1286.
Price N, Stephen L (1982). Introduction to enzyme kinetics. In: Fundamentals of
Enzymology. Oxford University Press, Oxford, pp. 126-137.
13
Russell J, Weng L, Keim PS, Heinrikson RL (1978). The covalent structure of bovine
liver rhodanese. J. Biol. Chem. 253: 8102–8108.
Segel HI (1976). Biochemical calculation. In: How to Solve Mathematical Problems in
General Biochemistry. 2nd Edition, John Wiley and Sons Inc, pp. 450-471.
Smith J, Urbanska KM (1986). Rhodanese activity in Lotus corniculatus sensu lato. J.
Nat. Histol. 20 (6): 1467 –1476.
Sorbo BH (1951). On the properties of rhodanese. Acta chem scand. 5: 724-726.
Sorbo BH (1953a). Crystalline rhodanese. Acta chem scand. 7: 1129-1136.
Sorbo BH (1953b). Crystalline rhodanese. Enzyme catalysed reaction Acta chem scand
7: 1137-1145.
Stokinger HE (1981). Patty’s industrial hygiene and toxicology. Clayton, C.D. and
Clayton, F.E. (ed), John Wiley and Sons, New York, USA, pp. 1493- 2060.
Sylvester DM, Sander CC (1990). Immunohistochemical localization of rhodanese.
Histochem. J. 22: 197–200.
Tomati U, Matarese R, Federici G (1974). Ferredoxin activation by rhodanese.
Phytochem. 13: 1703-1706
Tonguthai K (1997). Diseases of the Freshwater Prawn, Macrobrachium rosenbergii,
Aquatic Animal Health Research Institute, Bangkok University. AAHRI Newsletter 4 (2)
Volini M, Westley J (1966). The mechanism of the rhodanese catalysed thiosulphate
lipoate reaction. Kinetic analysis. J. Biol. Chem. 241 (22): 5168-5176.
14
3
OD460
OD280
Pooled fraction
0.30
2
0.20
0.15
1
OD460
OD280
0.25
0.5
0.10
0.25
0.05
0
0.00
0
10
20
30
40
50
60
70
0.0
80
FRACTION NUMBER
Figure 1: The elution profile of freshwater prawn hepatopancreas after CM-Sepharose
ion-exchange chromatography. The 65% ammonium dialysate was layered on the column
(2.5 X 40 cm) which was first washed with 250 ml of citrate buffer, pH 5.0 containing 10
mM sodium thiosulphate and later eluted with 200 ml linear gradient of 0.0-0.5 M NaCl
at a flow rate of 30 ml/hr. Fractions of 5 ml was collected.
15
1.00
1/ Vm ax
0.75
0.50
0.25
0.4
-0.25
0.25
0.75
1.25
1.75
1/[S]
1/V
0.3
0.2
0.1
0.00
0.25
0.50
0.75
1.00
1/[S]
Figure 2: Effect of varying concentration of sodium thiosulphate. Lineweaver-Bulk
plots showing the effect of varying concentration of sodium thiosulphate at different
fixed concentration of potassium cyanide (KCN). The reaction mixture contained 25 mM
borate buffer, pH 9.4, sodium thiosulphate concentration between 50 mM and 600 mM,
the indicated concentration of potassium cyanide and 20 µl of enzyme solution in a final
volume of 1.0 ml.
The insert is a replot of slope (1/Vmax) versus 1/[S].
16
1/ Vm ax
0.3
1.5
0.2
0.1
-0.05 0.00
0.05
0.10
0.15
0.20
0.25
1/[S]
1/V
1.0
0.5
-0.5
0.5
1.5
2.5
3.5
4.5
1/[S]
Figure 3: Effect of varying the concentration of potassium cyanide. Lineweaver-Bulk
plots showing the effect of varying concentration of potassium cyanide at different fixed
concentration of sodium thiosulphate (Na2S2O3). The reaction mixture contained 25 mM
borate buffer, pH 9.4, potassium cyanide concentration between 10 mM and 70 mM, the
indicated concentration of sodium thiosulphate and 20 µl of enzyme solution in a final
volume of 1.0 ml. The insert is a replot of slope (1/Vmax) versus 1/[S].
17
V (activity)RU/ml/min
100
50
0
4
5
6
7
8
pH
Figure 4: Effect of pH on the activity of freshwater prawn hepatopancreas rhodanese.
The assay mixture contained 0.025 M of the appropriate buffer, 0.05 M KCN, 0.05 M
Na2S2O3 and 20 µl of enzyme solution in a final volume of 1.0 ml. Determination of pH
optimum using 10 mM citrate , pH 4.0-6.5 and 10 mM phosphate buffer, pH 7.0-8.0.
18
Activity (Ru/ml/min)
2.0
1.5
1.0
0.5
0.0
30
50
40
60
Temperature( 0C)
Figure 5: The activity-temperature profile indicating the optimum temperature
Assays were in a 25 mM borate buffer, pH 9.4. Aliquot of 25 ml (enzyme solution) was
assayed at temperatures between 25 0C and 60 0C. The assay mixture was first incubated
at the indicated temperatures for 10 min before the reaction was initiated by the addition
of the enzyme.
19
TABLE 1: Summary of the purification of rhodanese from the hepatopancreas of the
giant freshwater prawn.
FRACTION
Crude Extract
TOTAL
PROTEIN
(mg)
1899
65%
663.3
Ammonium
sulphate
precipitate
Ion-exchange
579.1
chromatography
TOTAL
ACTIVITY
(RU/)
2954.5
SPECIFIC
ACTIVITY
(RU//mg)
1.55
PURIFICATION YIELD
FOLD
(%)
1.00
100
3777.4
5.70
3.70
35
4201.3
7.25
4.70
30
Each step was carried out as described in the test. Activity was measured by estimating
the amount of thiocyanate formed. Protein concentration was determined by the method
of Bradford (1976). One Rhodanese unit is taken as the amount of enzyme which under
the given conditions will produce an optical density reading of 1.08 at 460 nm
20
TABLE 2: Kinetic parameters of KCN and Na2S2O3 as substrates of rhodanese in
the hepatopancreas of the giant freshwater prawn
SUBSTRATE
KCN
Na2S2O3
Km (mM)
10 .0
2.56
Vmax (RU/ml/)
25 .0
4.0
Kcat (s-1)
Kcat / Km (M-1 s-1 )
2,500
250.0
400
156.3
All Km and Vmax values are means of three determinations. These kinetic parameters were
evaluated from the secondary plots according to the method of Segel (1976).
21
TABLE 3: Effects of cations on the activity of rhodanese from the hepatopanceas of the
giant freshwater prawn.
% ENZYME RESIDUAL ACTIVITY
SALT
500 µM
1000 µM
NONE
100
100
ZnCl2
41
46
MnCl2
79
75
CoCl2
82
84
BaCl2
43
47
NH4Cl
81
83
NiCl2
55
56
SnCl2
78
72
Enzyme assays were carried out under standard assay condition as described in
the test. The final concentrations of each cation in the assay mixtures were 250
µM and 500 µM.
22
23