Physicochemical and kinetic characteristics of rhodanesefrom the liver of African catfish Clarias gariepinus Burchellin Asejire lake

Omolara Titilayo Akinsiku ÆFemi Kayode Agboola Æ Adenike Kuku ÆAdeyinka Afolayan

Received: 9 December 2008 / Accepted: 21 April 2009 / Published online: 18 June 2009

� Springer Science+Business Media B.V. 2009

Abstract Two forms of rhodanese were purified

from the liver of Clarias gariepinus Burchell, desig-

nated catfish rhodanese I (cRHD I) and rhodanese II

(cRHD II), by ion-exchange chromatography on a CM-

Sepharose CL-6B column and gel filtration through a

Sephadex G-75 column. The apparent molecular

weight obtained for cRHD I and cRHD II was

34,500 ± 707 and 36,800 ± 283 Da, respectively.

The subunit molecular weight determined by sodium

dodecyl sulphate–polyacrylamide gel electrophoresis

was 33,200 ± 283 and 35,100 ± 141 Da for cRHD I

and cRHD II, respectively. Atomic absorption spec-

trophotometric analysis revealed that cRHD II con-

tained a high level of iron (Fe), which presumably was

responsible for the brownish colour of the preparation.

In contrast, no Fe was identified in cRHD I, and its

preparation was colourless. Further characterization of

cRHD II gave true Michaelis–Menten constant (Km)

values of 25.40 ± 1.70 and 18.60 ± 1.68 mM for

KCN and Na2S2O3, respectively, an optimum pH of

6.5 and an optimum temperature of 40�C. The

Arrhenius plot of the effects of temperature on the

reaction rate consisted of two linear segments with a

break occurring at 40�C. The apparent activation energy

values from these slopes were 7.3 and 72.9 kcal/mol.

Inhibition studies on the cRHD II enzyme showed that

the activity of the enzyme was not affected by Mn2?,

Co2?, Sn2?, Ni2? and NH4?, but Zn2? inhibited the

enzyme considerably.

Keywords Aquatic organisms � Asejire Lake �Catfish (Clarias gariepinus) � Cyanide �Cyanide detoxification � Cyanogenic plants �Fish � Rhodanese

Introduction

Rhodanese (thiosulphate: cyanide sulphurtransferase,

EC 2.8.1.1) is a sulphur transferase that catalyses, in

vitro, the formation of thiocyanate from cyanide and

thiosulphate or other suitable sulphur donors. Differ-

ent thiosulphonates are able to replace thiosulphate in

the rhodanese reaction (Sorbo 1953a), and sulphite,

sulphanate and persulphide can also serve as donor

substrates (Sorbo 1957; Villarejo and Westley 1963;

Koj 1968; Nagahara et al. 1999). In vivo, however,

the enzyme is multifunctional: it supplies sulphide for

the formation of iron–sulphur centres, maintains the

sulphane pool and participates in selenium metabolism

and thiamine biosynthesis (Smith and Urbanska 1986;

The data presented in this report is part of the research carried

out by O. T. Akinsiku in partial fulfilment for the degree of

Master of Science in Biochemistry at the Obafemi Awolowo

University, Ile-Ife, Nigeria.

O. T. Akinsiku � F. K. Agboola (&) � A. Kuku �A. Afolayan

Department of Biochemistry, Obafemi Awolowo

University, Ile-Ife, Nigeria

e-mail: fkagbo@oauife.edu.ng

123

Fish Physiol Biochem (2010) 36:573–586

DOI 10.1007/s10695-009-9328-4

Bordo and Bork 2002). It is a ubiquitous enzyme that is

active in all living organisms, from bacteria to Homo

sapiens and plants (Cosby and Summer 1945; Him-

wich and Saunders 1948; Sorbo 1951; Jarabak and

Westley 1974; Anosike and Ugochukwu 1981; Lee

et al. 1995; Nagahara et al. 1999; Agboola and Okonji

2004; Aminlari et al. 2007). Studies on its intracellular

distribution have revealed that it is present in the

nucleus, mitochondrion and cytosol, with the greatest

concentration in the mitochondrion (Sorbo 1951;

Nagahara et al. 1999; Ali et al. 2001). It is generally

believed that the major function of rhodanese is

cyanide detoxification, especially in mammals in

whom cyanide is converted to the less toxic thiocyanate

and excreted through the kidney (Westley 1980). In

plants, a close relationship exists between rhodanese

activity and cyanogenesis, which suggests that the

enzyme provides a mechanism for cyanide detoxifica-

tion in cyanogenic plants (Smith and Urbanska 1986).

Urbanization and industrial, agricultural and pop-

ulation growth have been accompanied by a tremen-

dous increase in the discharge of a wide diversity of

pollutants to receiving water bodies, causing desirable

effects on different components of the aquatic envi-

ronment (Tolba 1982). Organic pollution of inland

waters in Nigeria, in contrast to the situation in

developed countries of the world, is often the result of

extreme poverty and economic and social under-

development. Fish and marine resources in Nigeria

face total collapse or extinction due to over-fishing and

the destruction of marine life and natural habitats by

pollution of water bodies. Unregulated and excessive

use of pesticides for fishing and the deliberate disposal

and dumping of toxic and hazardous wastes into water

bodies are significant causes of massive fish kills and

the loss of aquatic life and habitats in this country.

Lake Asejire, a man-made lake constructed on the

Osun river in 1972 in Oyo State, Nigeria, has elevated

levels of contaminants due to receiving effluents from

various industries and the breakdown of ecological

balance caused by the widespread destruction of flora

and fauna diversities (Lameed and Obadara 2006).

Despite this high level of pollution, the lake still

supports aquatic life. Cyanide, produced by toxic gases

during the pyrolysis of plastic or nitrile-based polymer

fibres, by extracts of plants containing cyanogenic

glycosides (e.g. cassava) or from industrial waste (e.g.

electroplating), is one of the likely chemical pollutants

present in the water. The African catfish (Clarias

gariepinus), like many other fish, inhabits diverse

water bodies, ranging from lakes, streams, rivers,

swamps to floodplains (Bruton 1979; Clay 1979).

Some of these water bodies are situated in areas where

they are prone to contamination and pollution from

various sources. This paper reports on the purification

and partial characterization of rhodanese isolated in

the liver of Clarias gariepinus. It is assumed that

this enzyme, among other functions, plays a role in

cyanide detoxification in catfish and, consequently, to

its adaptation or survival in such polluted waters.

Materials and methods

Materials

Sodium thiosulphate (pentahydrate), potassium cya-

nide, ammonium sulphate (enzyme grade) and phenyl

methane sulphonyl fluoride (PMSF) were obtained

from BDH Chemical (Poole, UK). Coomassie Brilliant

Blue, bovine serum albumin (BSA) and the standard

proteins contained in the Sigma Molecular Weight

Markers Calibration kit for sodium dodecyl sulphate–

polyacrylamide gel electrophoresis (SDS–PAGE;

molecular weight marker range 14,000–70,000),

including ovalbumin (chicken), pepsin (porcine stom-

ach mucosa), trypsinogen, (bovine, PMSF-treated),

b-lactoglobulin (bovine), lysozyme (egg white) and

a-chymotrypsinogen (bovine pancreas), were obtained

from the Sigma Chemical Company (St. Louis, MO).

CM-Sepharose CL-6B and Sephadex G-100 columns

were obtained from Pharmacia Fine Chemical (Upp-

sala, Sweden), and Bio-Gel P4 was obtained from Bio-

Rad Laboratories (Hercules, CA). All other reagents

were of analytical grade and were obtained from either

Sigma or BDH. All buffers contained 10 mM sodium

thiosulphate to stabilize the enzyme. The catfishes

were collected from Asejire Lake, Ibadan, Nigeria.

The livers were quickly excised and stored in the

freezer until required for analysis.

Methods

Enzyme and protein assays

Rhodanese activity was assayed and estimated

according to Agboola and Okonji (2004). The protein

concentration was determined using a modified

574 Fish Physiol Biochem (2010) 36:573–586

123

method of Gornall et al. (1949) and BSA as the

standard.

Enzyme preparation

A sixty grams wet weight of tissue (obtained from ten

fish) was homogenized in three volumes of 10 mM

sodium thiosulphate containing 1 mM PMSF (Buffer

A) in a Warring Blender. The homogenate was

filtered through a double layer of cheese cloth and the

debris rehomogenized in one volume of buffer A. The

second homogenate was also filtered through cheese

cloth. The combined filtrate was centrifuged at

20,000 rpm for 15 min at 4�C using a Beckman

Optima LE-80K ultracentrifuge (Beckman Coulter,

Fullerton, CA). The supernatant was brought to 65%

ammonium sulphate saturation by the addition of

solid ammonium sulphate and left for 12 h. The

resulting precipitate was collected by centrifugation

at 15,000 rpm for 30 min at 4�C and stored in a

minimal amount of Buffer A.

A CM-Sepharose CL-6B cation-exchanger resin

was washed and equilibrated in 0.2 M sodium

acetate, pH 5.4 (Buffer B) and packed into a

2.5 9 40-cm column. The ammonium sulphate pre-

cipitate was first desalted on a Bio-Gel P4 column

and then layered on the ion exchange column. The

column was first washed with 240 ml of 30 mM

sodium acetate pH 5.4 (Buffer C) followed by elution

with a 200 ml linear gradient of 0–1.0 M KCl in

Buffer C. The fractions were monitored at 280 nm for

protein and assayed for rhodanese activity. The active

fractions in two distinct peaks were pooled and

dialyzed in 70% glycerol in Buffer C. The brown-

coloured fraction was then rechromatographed on the

ion-exchange column under the same conditions.

Sephadex G-75 resin was swollen and equilibrated

in 0.1 M phosphate buffer, pH 7.2 (Buffer D) and

then packed into a 1.5 9 70-cm column. The pre-

ceding two active fractions were run separately.

Three-millilitre fractions were collected at a flow rate

of 15 ml/h. Protein was monitored at 280 nm, and the

fractions were assayed for enzyme activity. The

pooled active fraction was dialyzed in 70% glycerol

in Buffer D. The two enzyme fractions were desig-

nated cRHD I and cRHD II. Polyacrylamide gel

electrophoresis in the absence of SDS was carried out

by the procedure described in the Pharmacia’s

manual (Polyacrylamide Gel Electrophoresis,

laboratory techniques, revised edition, Feb.1983) to

ascertain the purity of the preparation.

Characterizations

Apart from molecular weight determination and the

presence of metal ions in association with the

enzyme, only cRHD II was further characterized.

Molecular weight determinations

The SDS–PAGE was carried out to determine the subunit

molecular weight on an 10% acrylamide gel using the

Sigma Molecular Weight Markers Calibration kit

(molecular weight marker range 14,000–70,000) for the

standards. The native molecular weight was determined

by gel filtration on a Sephadex G-100 column (1.5 9

70 cm). The standard proteins were BSA (67,000;

3 mg/ml) plus a-chymotrypsinogen (25,000; 3 mg/ml)

and ovine albumin (45,000; 3 mg/ml) plus lysozyme

(15,000; 3 mg/ml). A total of 3 ml of each combination

was layered and eluted with 10 mM phosphate buffer,

pH 7.0 at a flow rate of 10 ml/h. Fractions of 3 ml were

collected and monitored for protein at 280 nm.

Determination of kinetic parameters

The concentration of KCN varied between 5 and 50 mM

at different fixed concentrations (ranging from 10 to

50 mM) of Na2S2O3 and vice versa in an assay mixture

containing 10 mM borate buffer, pH 5.4 and 0.1 ml

of the appropriately diluted cRHD II enzyme. The

reciprocal of the initial reaction velocity (1/v) against the

reciprocal of the varied substrates at the fixed concen-

tration of the other substrate was plotted. The vertical

intercepts of these double reciprocal plots (representing

1/Vmax app) and the abscissa intercept (representing

1/Km app) were then replotted, respectively, against the

reciprocal of the fixed concentration of the other

substrate (secondary replots) in accordance with a

ping-pong bi-bi reaction mechanism (Florini and Ves-

tling 1957; Cleland 1970; Segel 1975). The true Km

values of the substrates were then estimated from the

values of the slope and intercept of the secondary replots.

Effect of pH

The effect of pH on cRHD II activity was studied

using the methods of Agboola and Okonji (2004).

Fish Physiol Biochem (2010) 36:573–586 575

123

The enzyme was assayed using the following buffers:

50 mM citrate buffer (pH 4.0–6.5), 10 mM phosphate

buffer (pH 7.0–8.5) and 50 mM borate buffer (pH

9.0–11). A reaction mixture of 1 ml contained 0.5 ml

of the respective buffer, 0.2 ml of 0.25 mM KCN,

0.2 ml of 0.25 mM Na2S2O3 and 0.1 ml of the

enzyme solution.

Effect of cations

The method of Lee et al. (1995) was used to study the

effect of various metal ions on cRHD II activity. The

salts of the cations include CoCl2, MnCl2, NH4Cl,

NiCl2, SnCl2, ZnCl2 and MgCl2 at final concentra-

tions of 0.05 and 0.1 mM in enzyme assay mixture.

Effect of temperature

cRHD II was assayed at temperatures between 0� and

70�C to determine the optimum temperature of the

enzyme. The assay mixture was first incubated at the

indicated temperature for 10 min prior to initiating a

reaction by the addition of an aliquot of the enzyme

which had been previously equilibrated at the same

temperature. The residual activity was then assayed.

The heat stability of the enzyme was determined by

incubating 0.6 ml of the enzyme for 1 h at 30�, 40�, 50�,

60� and 70�C, respectively. A 0.1-ml aliquot was

withdrawn at 10-min intervals and assayed for residual

activity. The activity was expressed as a percentage of

activity of the enzyme incubated at 30�C as the control.

Ultraviolet/visible light spectrophotometric scanning

The post-gel filtration enzyme preparation was dia-

lysed against several changes of distilled water to

remove any ions contributed by the buffer. The

dialysate was scanned at wavelengths between 200

and 600 nm using a Cintra 101 Double Beam UV/

VIS Spectrophotometer (GBC Scientific Equipment

Pty Ltd, Australia).

Atomic absorption spectrophotometry

The dialysate was examined for the presence of

various divalent metal ions: Fe2?, Mg2?, Mn2?,

Ca2?, Co2?, Zn2?, Ni2? and Cu2?. These metal ions

were estimated by atomic absorption spectrophotom-

etry using an air-acetylene flame in the Alpha-4

ChemTech spectrophotometer. The sample was exten-

sively dialyzed against two change of distilled water to

remove any ions contributed by the buffer before it

was digested in a mixture of concentrated HNO3 and

HClO4 (1:1) according to the methods of Kaur et al.

(2006).

Results

Purification and molecular weight determination

The result of the purification of rhodanese from the

liver of catfish is summarized in Table 1. The elution

profile after ion-exchange chromatography on the

CM-Sepharose CL-6B column is shown in Fig. 1.

Two peaks of enzyme activity were obtained from

this analysis in the breakthrough volume without

the salt gradient elution. These two peaks were also

obtained in two subsequent repetitions of this

experiment, which lead us to consider the peaks as

two different forms of rhodanese, subsequently

designated catfish rhodanese I (cRHD I) and rhoda-

nese II (cRHD II). cRHD II was observed to be

brownish in colour, and this colour could not be

removed by the ion-exchange and gel-filtration

steps. The elution profile on the Sephadex G-75

column is shown in Fig. 2a and b. The photograph

showing the result of PAGE in the absence of SDS is

shown in Fig. 3.

The native molecular weights of the two forms of

the enzyme were estimated by gel filtration to be

34,500 ± 707 and 36,800 ± 283 Da for cRHD I and

cRHD II, respectively, while the subunit molecular

weights estimated by SDS–PAGE were 33,200 ± 283

and 35,100 ± 141 Da for cRHD I and cRHD II,

respectively.

Kinetic parameters

A typical double reciprocal plot of the change in

KCN concentrations at fixed Na2S2O3 concentrations

is shown in Fig. 4a and that of varying concentrations

of Na2S2O3 at fixed KCN concentrations is shown in

Fig. 4b. The lines are almost parallel, depicting an

enzyme ping-pong mechanism (Florini and Vestling

1957; Cleland 1970; Segel 1975). After the secondary

replots, the kinetic parameters were estimated; these

are summarized in Table 2.

576 Fish Physiol Biochem (2010) 36:573–586

123

Effect of pH and temperature

The enzyme showed a temperature optimum of

40�C (Fig. 5). The Arrhenius plot of the activity–

temperature profile consisted of two linear segments

with different slopes. The sudden break at 40�C

indicates a conformational change in the enzyme.

The two slopes of the plot yielded apparent activa-

tion energies of 7.3 and 72.9 kcal/mol, respectively.

The enzyme retained 70, 60, 35, and 20% of its

activity at 40, 50, 60 and 70�C when heated for 1 h

(Fig. 6). The rate constants of the pseudo-first order

heat denaturation processes are 2.31 9 10-2,

3.81 9 10-2, 4.07 9 10-2 and 5.06 9 10-2 min-1

with corresponding half-life values of 30, 18.19,

17.03 and 13.70 min at 40, 50, 60 and 70�C. There

was an increase in activity when the enzyme was

assayed between pH 5.0 and 6.5, which later

Table 1 Summary of the purification of catfish liver rhodanese

Fractiona Volume

(ml)

Total activityb

(RU/min)

Total proteinc

(mg)

Specific activityb

(RU/min per

milligram)

%

Yield

Purification

fold

Crude extract 240 6793 4644 1.5 100 1

65% Ammonium sulphate precipitate 80 5708 920 6.2 84 4

Ion-exchange on CM-sepharose CL-6B column

I 40 1785 92 19.4 31 13

II 31 1726 90 19.1 30 13

Dialysis and concentration in glycerol

I 35 414 10 41.4 23 28

II 28 396 9 44.0 23 29

Gel filtration on sephadex G-75 column

I 30 78 1.07 73.0 19 49

II 25 70 0.97 72.0 18 48

Each of the steps was carried out as described in the Methods sectiona cRHD I and cRHD II are represented as fractions I and II, respectivelyb Rhodanese activity was measured according to the method of Agboola and Okonji (2004). One unit of activity is represented as the

rhodanese unit (RU). One RU was taken as the amount of enzyme which gave the optical density reading at 460 nm of 1.08 in the test

system (Sorbo 1953a)c Protein concentration was determined by the Biuret method

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

1 13 25 37 49 61 73 85 97 109 121 135 147

Fraction number

0

0.5

1

1.5

2

2.5

3

3.5

4

Ab

s (4

60n

m)

cRHD I

cRDH II

1.0 M KCl

0.5 M KCl

0.00

Ab

s (2

80n

m)

Fig. 1 Ion-exchange

chromatography through a

CM-Sepharose CL-6B

column. Two activity peaks

were obtained, which were

designated cRHD I and

cRHD II, respectively. Solidline, filled circles OD280,

broken line, filled trianglesOD460. Analysis was carried

out using the 65%

ammonium sulphate

precipitate. The elution with

KCl gradient is also shown.

OD = optical density

Fish Physiol Biochem (2010) 36:573–586 577

123

decreased gradually. The pH optimum was obtained

at pH 6.5.

Effect of cations

The result of the effect of various cations on the

activity of catfish liver rhodanese is as shown in

Table 3. Mn2?, Mg2?, Ca2?, NH4?, Sn2? and Co2?

had little effect on the activity of the enzyme.

However, both concentrations (0.5 and 1.0 mM)

of Zn2? that were tested inhibited the enzyme

considerably, while Ni2? inhibited rhodanese activity

by approximately 50%.

Ultraviolet/visible light spectrophotometric

scanning

A single peak was obtained for cRHD I at about 280 nm

(Fig. 7a), while two distinct peaks were obtained for

cRHD II (Fig. 7b). The first peak was the normal protein

peak at 280 nm, and the second was seen at about

406 nm, which indicates the presence of divalent ions.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39

Fraction number

Ab

s (2

80n

m)

0

0.1

0.2

0.3

0.4

0.5

0.6

Ab

s (4

60n

m)

0

0.5

1

1.5

2

2.5

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39

Fraction number

Ab

s (2

80n

m)

0

0.1

0.2

0.3

0.4

0.5

0.6

Ab

s (4

60n

m)

cRHD I

cRHD II

a

b

Fig. 2 Gel filtration

chromatography. a Elution

profile the cRHD I fraction,

b elution profile of the

cRHD II fraction. Fractions

were run separately.Solidline, filled triangles OD460,

broken line, filled circlesOD460

578 Fish Physiol Biochem (2010) 36:573–586

123

Metal ion content

The divalent metal ion content determined by atomic

absorption spectroscopy showed that the metal ion in

cRHD I was negligible, with the exception of Fe2?

and Mg2?, which were present at 0.53 and 0.34 ppm,

respectively. However, among the metal ions tested

for in cRHD II, Fe2? showed the highest concentra-

tion (11.9 ppm), followed by Ni2? (0.9 ppm), Mg2?

(0.47 ppm), Mn2? (0.45 ppm), Zn2? (0.35 ppm),

Ca2? (0.06 ppm), Cu2? and Co2? (0.00 ppm).

Discussion

The African catfish (Clarias gariepinus) inhabits

calm waters of lakes, streams, rivers, swamps and

floodplains, many of which are subject to contami-

nation from various sources, such as effluents

from manufacturing companies. Studies on the food

composition of C. gariepinus indicate that this fish is

either an omnivore or a predator (Bruton 1979; Clay

1979). Micha (1973) found that C. gariepinus fed

mainly on aquatic insects, fish and higher plant

debris. Some of these food items contain cyanogenic

glycosides which upon hydrolysis release cyanide or

hydrocyanic acid (HCN) (Wokes and Willimott 1951;

Montgomery 1965). Cyanide is a potent cytotoxic

agent that kills the cell by inhibiting cytochrome

oxidase of the mitochondrial electron transport chain

(Warburg 1911; Keilin 1929; Nagahara et al. 1995).

Metal processing and organic chemical industries as

well as iron and steel production are major sources of

cyanide released to aquatic environment (Smith et al.

1978, 1979; ATSDR 1989). Other sources include

hydrogen cyanide used in the fumigation of ships

infested by rodents or insects and cyanide salts used

in ore-extracting processes. Moreover, in the local

traditional settings around the river Osun, from which

Asejire Lake was constructed, it is common for

cassava tubers to be steeped in the flowing river water

for processing into other food products. This is a

major source of cyanide in this water. There are

reports that fishes are the most sensitive aquatic

organisms to cyanide, and cyanide hazards to fish,

wildlife, livestock and man are well documented.

Adverse effects on swimming and reproduction have

been recorded at levels of between 5 and 7.2 lg free

cyanide per litre, with lethal effects usually occurring

at levels of 20–76 lg/l (Eisler 1991). Massive kills of

fresh water fish due to the accidental discharge of

cyanide wastes are common occurrences (Holden and

Marsden 1964; Leduc 1978; Towill et al. 1978; EPA

1980). In one case, cyanide-containing mine effluents

from a Canadian tailings pond released into a nearby

creek killed more 20,000 steelhead (Oncorhynchus

mykiss; Leduc et al. 1982). However, the fact that

various species of catfish and other animals have been

able to adapt and survive in cyanide-contaminated

habitats has lead to the hypothesis that the rhodanese

enzyme, which has been demonstrated to be the

major hepatic enzyme for cyanide detoxification in

animals, provides an inherent safety for catfish and

other aquatic animals against the toxicity of cyanide.

Thus, catfish can be used as a model of survival and

adaptation of aquatic animals in cyanide-polluted

water. Rhodanese catalyses the formation of the

easily excreted thiocyanate from cyanide and thio-

sulphate and acts as general transferase for the

CRUDE A B

Fig. 3 Polyacrylamide gel electrophoresis carried out in the

absence of SDS using the continuous phosphate buffer system.

A single band of protein was obtained after staining with

Coomassie Brilliant Blue. A cRHD I, B cRHD II

Fish Physiol Biochem (2010) 36:573–586 579

123

-0.04 0.00 0.04 0.08 0.12

0.1

0.2

Inte

rcep

t 1/[

Na2

S2O

3]

-0.05 0.00 0.05 0.10 0.15

0.1

0.2

0.3

0.4

0.5

1/[KCN] (mM-1)1/[KCN] (mM-1)

Slo

pe 1

/[N

a2S

2O3]

-0.04 0.00 0.04 0.08 0.12

0.1

1/[Na2S2O3](mM-1) 1/[Na2S2O3](mM-1)

Inte

rcep

t 1/[

KC

N]

-0.04 0.01 0.06 0.11

0.1

0.2

0.3

0.4

Slo

pe 1

/[K

CN

]

(a)

ai aii

(b)

bi bii

Fig. 4 Double reciprocal

plots and their secondary

replots for the

determination of kinetic

parameter. a The plot of the

reciprocal of the initial

reaction velocity (1/v)

against 1/[S] at different

fixed KCN and varying

concentrations of Na2S2O3.

The line of best-fit was

drawn by regression. The

initial reaction velocity, v,

was measured in rhodanese

units (RU) per millilitre per

minute, and the substrate

(given in square bracketunder the graph) was

measured in millimolar.

Large filled circle 10 mM

KCN, filled triangle 20 mM

KCN, filled square 30 mM

KCN, small filled circle40 mM KCN, open square50 mM KCN. ai A plot of

the ordinate intercept

(1/Vmax app) of a against

1/[KCN], aii a plot of the

abscissa intercept (1/Km

app) of a against 1/[KCN].

b The plot of 1/v against 1/

[S] at different fixed

Na2S2O3 and varying

concentrations of KCN.

Large filled circle 10 mM

Na2S2O3, filled triangle20 mM Na2S2O3, filledsquare 30 mM Na2S2O3,

small filled circle 40 mM

Na2S2O3, open square50 mM Na2S2O3. bi A plot

of the ordinate intercept of

b against 1/[Na2S2O3], bii a

plot of abscissa intercept of

b against 1/[Na2S2O3].

App = apparent

580 Fish Physiol Biochem (2010) 36:573–586

123

exchange of divalent sulphur in the biosphere (Ogata

et al. 1989). Other enzymes involved in cyanide

detoxification include 3-mercaptopyruvate sulphur-

transferase (3-MST), thiosulphate reductase, cysta-

thionase and albumin (enzyme-like behaviour in

cyanide detoxification) (Lieske et al. 1996).

Our research has documented the existence of two

forms of rhodanese in the liver of the catfish Clarias

gariepinus. These two forms have a similar molecular

size. In an earlier study, Blumenthal and Heinrikson

(1971) obtained two active peaks (rhodanese A and

B) when bovine liver rhodanese was purified on a

DEAE-Sephadex column in combination with PAGE.

Horowitz and DeToma (1970) introduced the ion-

exchange step to replace and improve the yield from

acidic (pH 4.5) and ammonium sulphate precipita-

tions. These researchers also found that these two

forms were identical with respect to kinetic param-

eters, molecular weight and other structural proper-

ties. Based on their data, they reasoned that

rhodanese B may have arisen during the course of

purification by the deamination of the A form. They

also established that rhodanese A and B did not arise

due to population differences of several livers. The

existence of the bovine rhodanese enzyme in a

monomeric–dimer equilibrium of molecular weight

about 18,000–36,000 had earlier been demonstrated

by Volini et al. (1967). Ogata et al. (1989) reported

that the two enzymes of Blumenthal and Heinrikson

(1971) were phospho- and dephosphorhodaneses. In

the study reported here, cRHD II had a brownish

colour that was not removed by rechromatography on

the same ion-exchanger and the subsequent gel

filtration step. However, these preparations were

adjudged to be pure by the presence of a single

protein band after electrophoresis in both the pres-

ence and absence of SDS, respectively. An investi-

gation into the presence of ions in the preparations by

spectrophotometric scanning and atomic absorption

spectrophotometry revealed that cRHD II contains

the metal ion Fe2?, as evidenced by an absorption

Table 2 Summary of kinetic parameters

Parameters KCN Na2S2O3

Km (mM) 25.4 ± 1.70 18.6 ± 1.68

Vmax (RU/ml) 8.22 ± 0.59 9.06 ± 0.51

The kinetic parameters for the two substrates were determined

by the method of Florini and Vestling (1957) and Segel (1975)

20 30 40 50 60 700.0

0.5

1.0

1.5

2.0

Temperature (°C)

Act

ivity

(R

U/m

l/min

)

Fig. 5 Effect of temperature on catfish liver rhodanese

0 10 20 30 40 50 60 700

50

100

Time (min)

% A

ctiv

ity

Fig. 6 Effect of temperature on the stability of catfish liver

rhodanese. The activity at 30�, 40�, 50�, 60� and 70�C was

expressed as a percentage of activity of the enzyme incubated

at 30�C. Open triangle 30�C, filled circle 40�C, open square50�C, filled diamond 60�C, open circle 70�C

Table 3 Effect of cations on catfish liver rhodanese activity

Salt Enzyme residual activity (%)

0.5 mM 1.0 mM

None 100 100

MgCl2 89 89

MnCl2 85 83

NH4Cl 81 79

SnCl2 79 78

CoCl2 78 78

NiCl2 54 53

ZnCl2 41 40

Fish Physiol Biochem (2010) 36:573–586 581

123

peak at 406 nm and the high level of Fe2? based on

atomic absorption spectrophotometry. This high level

of Fe2? was probably responsible for the brownish

colour. Given the presence of Fe2?, one may suggest

that cRHD II is a mitochondrial form of the enzyme,

while cRHD I is the cytosolic or soluble form. Volini

et al. (1967) reported that bovine liver rhodanese

contains zinc, with at least one zinc ion per monomer.

However, the identification of metal ions in our study

may have arisen from the isolation procedure

employed, possibly from the inconclusive separation

of the mitochondrial from the cytosolic fraction due

primarily to the limitations of our centrifugation

procedures. Both rhodanese and 3-MST are localized

in both mitochondria and the cytosol of animals,

plants and microorganisms (Cosby and Summer

1945; Himwich and Saunders 1948; Sorbo 1951;

Jarabak and Westley 1974; Taniguichi and Kimura

1974; Koj et al. 1975; Anosike and Ugochukwu 1981;

Kuo et al. 1983; Lee et al. 1995; Vazquez et al. 1997;

Nagahara et al. 1999; Agboola and Okonji 2004).

Another possibility is that it may have arisen from the

preparative procedures employed in a way similar to

the bovine rhodanese in which the predominance of A

or B depends on the mildness of the purification

medium (Blumenthal and Heinrikson 1971). A milder

means of purification involves the inclusion of Zn2?

and thiosulphate in the buffers instead of precipita-

tion by pH change in which strong acid and base were

used in the pH adjustments (Blumenthal and Hei-

nrikson 1971).

The specific activity of the rhodaneses from the

catfish liver, 73 and 72 RU/mg for cRHD I and cRHD

II, respectively, were rather low. Agboola and Okonji

(2004) obtained 136.6 RU/mg for fruit bat liver

rhodanese, Sorbo (1953a) and Himwich and Saunders

(1948) obtained 256 and 131 RU/mg, respectively,

for bovine liver rhodanese. The apparent molecular

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

190 290 390 490 590 690 790

WAVELENGTH

AB

SO

RB

AN

CE

0

0.5

1

1.5

2

2.5

WAVELENGTH

AB

SO

RB

AN

CE

190 290 390 490 590

Fig. 7 Ultraviolet/visible

light spectrophotometric

scanning of cRHD I (a) and

cRHD II (b)

582 Fish Physiol Biochem (2010) 36:573–586

123

weight obtained for cRHD I and cRHD II in our study

were 34,500 ± 707 and 36,800 ± 283 Da, respec-

tively, which is in line with reports that rhodanese has

molecular weight value approximately between

33,000 and 37,000 Da (Wang and Volini 1968;

Blumenthal and Heinrikson 1971; Nagahara et al.

1999). Various molecular weights have been obtained

for rhodanese from a variety of different sources:

bovine liver (37,000 Da; Sorbo 1953a, 1955), human

liver (37,000 Da; Jarabak and Westley 1974), mouse

liver (34,800 Da; Lee et al. 1995) and rat liver

(34,000 Da; Nagahara and Nishino 1996). The sub-

unit molecular weights determined by SDS–PAGE

were 33,200 ± 283 and 35,100 ± 141 Da for cRHD

I and cRHD II, respectively. These values are very

close to those obtained by Lee et al. (1995) and

Agboola and Okonji (2004), which were 34,000 and

35,700 Da for fruit bat and mouse rhodanese,

respectively. This result suggests that the enzyme is

a monomeric protein. While Jarabak and Westley

(1974) and Lee et al. (1995) showed that purified

mouse and human liver rhodaneses are monomeric

proteins, other researchers have shown that they are

dimers of two identical subunits of molecular weights

18,000–19,000 Da (Blumenthal and Heinrikson

1971; Volini et al. 1967). Volini et al. (1967)

reported that bovine rhodanese existed as a mono-

mer–dimeric molecule of molecular weight of

18,000–36,000. Russell et al. (1978) and Ploegman

et al. (1978), based on a three-dimensional structure

analysis, showed that bovine liver mitochondrial

rhodanese is a single polypeptide chain with a

molecular weight of 32,000 Da. Since both forms

(cRHD I and cRHD II) of rhodanese isolated in our

study had molecular weights of about 35,000, the

possibility that the two forms are monomeric and

aggregate forms of one of the other need not be

considered (Blumenthal and Heinrikson 1971). How-

ever, given the similar molecular weight for the two

forms, as also obtained for the two bovine rhodanes-

es, which also have other similar structural properties,

one can suggest that the cRHD I and cRHD II may

not be structurally different. Isoenzymes with struc-

tural similarity are not uncommon, as in venom

phospholipase A from Crotalus adamateus and

mushroom tyrosinases (Blumenthal and Heinrikson

1971).

The true Km values, as determined by the equation

of the bireactant enzyme ping-pong mechanism

(Florini and Vestling 1957; Cleland 1970; Segel

1975), for KCN and Na2S2O3 were 25.4 ± 1.70 and

18.6 ± 1.68 mM, respectively, for cRHD II. The

enzyme rhodanese catalyses the transfer of the outer

(sulphane) sulphur of thiosulphate to cyanide, thereby

forming the products thiocyanate and sulphite by way

of a double-displacement (ping-pong) mechanism in

which a covalent enzyme–sulphur (substituted

enzyme) intermediate is formed. Both of the initial

velocity patterns obtained in this experiment are

basically parallel lines (Fig. 4a, b). These patterns are

diagnostic of a double-displacement mechanism

(Cleland 1970; Segel 1975), and they resemble the

patterns observed with bovine liver rhodanese with

thiosulphate and cyanide as the varied substrates

(Schlesinger and Westley 1974) and the kinetics of

prokaryotic sulphurtransferases (Aird et al. 1987).

The ping-pong action is common in reactions

involving the transfer of a functional group between

two different molecules as in transaminases, transac-

ylases or transphosphorylases. Initial velocity studies

with bireactant mechanisms only serve to distinguish

the sequential mechanism from the ping-pong one

and to define certain kinetic constants. The parallel

lines in double-reciprocal plots in this experiment are

indicative of the ping-pong binary complex mecha-

nism. These values are compared to those reported in

earlier studies in Table 4. In Table 4, it can be seen

that the Km values of catfish liver rhodanese are

higher than those of mouse liver, bovine liver, human

liver and Acinetobacter rhodaneses, indicating that

the affinity of the catfish enzyme for these substrates

is less than that of the other enzymes and that it

would catalyze the detoxification reaction with less

efficiency. It should be noted, however, that the

results of the catfish (this work), bovine liver

(Schlesinger and Westley 1974), human liver (Jara-

bak and Westley 1974) and Acinetobacter (Aird et al.

1987) enzymes were determined from the double-

reciprocal plots and the appropriate secondary plots

and, therefore, are more comparable. This less

effective enzymatic system may be due to a lower

exposure to cyanide, in contrast to its mammalian

counterparts which are continually exposed through

their diet. However, it should be noted that cyanide

detoxification is a secondary benefit of rhodanese,

which has a number of major physiological roles,

including the supply of sulphide for the formation of

iron sulphur centres for the electron transport chain.

Fish Physiol Biochem (2010) 36:573–586 583

123

Different optimum temperatures have been

reported for rhodanese from different organisms. Ezzi

et al. (2003) obtained a wide temperature optimum of

35–55�C for rhodanese enzyme in some Trichoderma

strains. A value as high as 50� and 57–59�C was

obtained by Sorbo (1953b) and Chew and Boey

(1972) for bovine liver and tapioca leaves, respec-

tively. Himwich and Saunders (1948) also obtained an

optimum temperature of between 38� and 40�C for

bovine liver rhodanese. Agboola and Okonji (2004)

reported 35�C for the rhodanese in the cytosolic

fraction of fruit bat liver. The optimum temperature

obtained was 40�C for rhodanese from the liver of the

catfish from Asejire Lake. With the level of pollution

in this water, there will be various metabolic activities

going on in this lake which may result in the release of

heat. This ambient temperature may have conditioned

the enzyme to function at higher temperatures.

The Arrhenius plot of the effects of temperature on

the reaction rate consists of two linear segments with a

break occurring at 40�C. The apparent activation

energy values from these slopes are 7.3 and 72.9 kcal/

mol, respectively. The 7.3 kcal/mol obtained for

catfish liver rhodanese is close to the 7.5 kcal/mol

reported for bovine liver (Fruton and Simmonds 1963)

and also falls within the physiological activation

energy range (1–25 kcal/mol) for physiological pro-

cesses in living organisms (Fruton and Simmonds

1963; Whitaker 1972). The second value of 72.9

kcal/mol lies within the range (40–100 kcal/mol)

for protein denaturation (Whitaker 1972). The heat

stability experiment showed that the enzyme was

stable up to 50�C for about 30 min.

Pollutants in water include wide spectrum of

chemicals and pathogens which sometimes alters

the acidity, conductivity and temperature of the

water. The water sample collected from Asejire Lake

showed a pH of 5.7, 6.3 and 6.5 at different locations

of the Lake (Lameed and Obadara 2006). This is an

indication of acidic water. An optimum pH of 6.5 was

obtained for rhodanese from the liver of the catfish

from Asejire Lake.

Inhibition studies on the enzyme with a number of

chloride salts showed that the activity of the enzyme

was not affected by Mn2?, Co2? and Sn2? while

NH4?, Zn2? and Ni2? inhibited the enzyme consid-

erably. This is a result of the level of metal ions

present in the water as recorded by Lameed and

Obadara (2006). Metal ions showing a high potential

to inhibit enzyme activity are those that have a strong

affinity for ligands, such as phosphate, cysteinyl and

the histidyl side chains of proteins (Ulmer and Vallee

1972).

In conclusion, the physicochemical and kinetic

properties of rhodanese from the liver of African

catfish Clarias gariepinus are similar to those from

the livers of other animals. In particular, the kinetic

parameters obtained would allow the enzyme to

Table 4 Comparison of Km values of cRHD II with other rhodanese preparations

Substrate Km (mM)

Catfish

liveraFruit bat

liverbMouse

livercBovine

liverdBovine

livereHuman

liverfRat

livergAcinetobacter sulphane

sulphur transferaseh

KCN 25.4 13.36 12.5 19.0 0.06 9.5 NA 0.03

Na2S2O3 18.6 19.15 8.3 6.7 4.0 4.5 4.4 3.2

The Michaelis constant (Km) of KCN and Na2S2O3 of different mammalian liver rhodaneses in comparison with that from cat fish

liver

NA, Data not availablea This workb Agboola and Okonji (2004)c Lee et al. (1995)d Sorbo (1953a)e Schlesinger and Westley (1974)f Jarabak and Westley (1974)g Nagahara and Nishino (1996)h Aird et al. (1987)

584 Fish Physiol Biochem (2010) 36:573–586

123

function in the conversion of cyanide, from various

sources in the water, to thiocyanate and thereby

presumably in the detoxification of the poisonous

cyanide, ultimately improving the survival of the fish

in polluted water.

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