ABSTRACT
Rhodanese, a ubiquitous enzyme, catalyzes the detoxification of cyanide by sulphuration reaction. Cyanide is a highly toxic substance and an ineffective detoxification of this substance may lead to inhibition of respiration, production of ATP and its dependent processes, liver damage and necrosis in aquatic organisms. Considering the habitat of this fish which is a reservoir, it is exposed to toxic substances and waste products such as cyanide, ammonia etc. If the fishes can survive despite being exposed to cyanide, then we can assume that the fish has cyanide-detoxifying mechanism and enzyme.
In this research, rhodanese was purified and characterized from the liver of Sarotherodon galilaeus using ammonium sulphate precipitation and Reactive blue-2-agarose affinity chromatography.
The specific activity of the enzyme was found to be 121RU per milligram of protein. The optimum pH of the enzyme was found at pH 8.5 while the optimum temperature was 40°C. The Km values of the substrates; KCN and Na2S2O3 were 40mM and 11.76mM respectively. Studies on the effect of cations on the enzyme showed that the activity of the enzyme was not affected by Mn2+and Ni2+., however, 0.01mM concentration of Mg2+, Ca2+, and Zn2+ inhibited the enzyme considerably.
The results of this research showed that there is activity of the enzyme; rhodanese in the liver of the fish and it implies that the organism has a cyanide-detoxifying mechanism. Thus, a prolonged exposure to cyanide will overwhelm the detoxifying mechanism eventually leading to liver damage and inhibition of respiration. Also, at very high temperature (above 70°C) and pH, the enzyme is inactivated.
CHAPTER ONE
1.0 INTRODUCTION AND LITERATURE REVIEW
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 (Smith and Urbanska, 1986). 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. The enzyme is a single polypeptide chain of 289 amino acids with molecular weight of up to 37,000dalton Sorbo (1953a). The physiological role of rhodanese in animal tissues and perhaps in plants is controversial; particularly its function in the detoxification of acute cyanide exposure (Delvin et al., 1989; Sylvester and Sander, 1990). Rhodanese is a sulphur transferase that catalyses, in vitro, the formation of thiocyanate from cyanide and thiosulphate or other suitable sulphur donors (Westley, 1980; Nagahara et al., 1996).
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 play various roles in sulphur metabolism.
Rhodanese was first crystallized and partially characterized from bovine liver by Sorbo (1953a) and subsequent studies with bovine liver and kidney enzymes by a number of investigators have provided information with regard to the mechanism of reaction and the chemical and physiological nature of the enzyme (Westley and Green, 1959, Westley, 1968). The enzyme has been purified and characterized from a number of animal tissues (Sorbo, 1953b, Agboola, and Okonji, 2004). Some of the metabolic functions of the enzyme carried out is the transfer of sulphur from thiosulphate to a nucleophilic acceptor such as reduced lipoate (Volini et al., 1966, Tomati et al., 1974,), reactivation of ferredoxin from apoferredoxin (Tomati et al., 1974) and supply of sulphide centre for the electron transport chain in mammalian tissues (White et al., 1981).
Rhodanese in its phosphorylated and dephosphorylated forms has been reported to function as a converter enzyme that interact with mitochondrial membrane bound iron–sulphur centers of the mitochondrial electron transport chain where it modulate the rate of respiration (Ogata and Volina, 1990). There is an indication of a possible role of rhodanese in providing labile sulphide necessary for the synthesis of ferredoxin in the chloroplast of spinach, parsley, cabbage, and red turnips (Tomati, 1972). It also catalyses the formation of iron-sulphur centers in Escherichia coli, and a physiological role of the enzyme in aerobic metabolism in this organism was suggested (Keith and Volina, 1987). Rhodanese was also reported to reconstitute spinach ferredoxin (Pagani et al., 1984); restore durum wheat leaves cyanide inactivated NADH: nitrate reductase activity and if added before cyanide treatment, it protects the enzyme (Tomati et al., 1976). It also restores, partially, the activity of NADH dehydogenase (Pagani and Galante, 1983). It was also found to increase the activity of malate dehydrogenase (Agro et al., 1976). Restoration of MgATP and chelator inactivated nitrogenase of Klebsiella pneumoniae has been reported (Pagani et al., 1987). A possible role of the enzyme in modulating S-amino levulinate synthetase activity has also been reported (Vazquez et al., 1987). In Thiobacillus intermedius, the mechanism of oxidation of thiosulphate to sulphate seems to involve the action of rhodanese (Charles, 1969). At pH 8.8 beef liver rhodanese however, catalyses the reduction of thiosulphate to sulphite (Koj, 1968). Its activity was also related to the oxidation of thiosulphate and elemental sulphur to sulphate by the fungus, Rhizopus oryzae (Ray et al., 1990)
1.1 OCCURRENCE AND DISTRIBUTION
The enzyme rhodanese (thiosulfate: cyanide sulfurtransferase) is an ubiquitous enzyme with activity in all living organisms, from bacteria to man. (Nagahara et al.,1999; Aminlariet al.,2007). Various tissues from different species of animals have been assayed for the presence of rhodanese, an enzyme generally considered to be responsible for the detoxification of cyanide. Rhodanese is widely distributed in both adults and young animals and is not restricted to them alone but also to aphylum arthropoda which encounter exogenous cyanide through feeding on cyanogenic plants or leaves in cyanide polluted environment (Gopal et al., 2005). This indicates that cyanide detoxification is likely to be the primary role of rhodanese in animals (Beesley et al., 1985).
The detoxification of HCN as thiocyanate was first observed by S. Lang in 1894, and the enzymatic aspects were first studied in 1933 by K. Lang who gave the name rhodanese to the enzyme concerned. Since thiocyanate is some hundred times or more less toxic than HCN, the rhodanese reaction is a true detoxification.
It appears that the concentration or activity of rhodanese in the tissues of animals varies directly with the normal nitriloside content of the general dietary characterizing each species. The livers of rats, rabbits and cows appear to be more active than those of monkeys, men, dogs, and cats
in descending order. Rhodanese activity is widely distributed in living forms as nitrilosides. This has been found in diverse forms of fish, squids, insects and plants (Anosike and Ugochukwu, 1981). The enzyme has been isolated in crystalline form by Sorbo and a substantial literature on it has developed (Beesley et al., 1985). The action of rhodanese is highly specific. It is limited not merely to nitriles but only to those nitrilosides which surrender free HCN ions upon hydrolysis (Wood et al., 1975). The administration of rhodanese has been found to protect experimental animals from doses of cyanide or its salts ten times or more in excess of normally lethal doses. The concentration of rhodanese in tissue is generally proportional to that of beta-glucosidase and always functionally in excess of the latter (Beesley et al., 1985).
Rhodanese may also appear in the absence of beta-glucosidase as in the case of the brain just as beta-glucosidase may appear in conjunction with cancer or trophoblast cells in the absence of Rhodanese. The high sensitivity of cerebral tissue to hypoxia would tend in the course of natural selection to provide a high Rhodanese activity against adventitious HCN and to exclude any enzymatic means by which the cyanide ion could be hydrolyzed in this area. The rationale for the occurrence of a high beta-glucosidase concentration in the absence of rhodanese in the case of trophoblast is associated with the role the trophoblast plays in hemopoiesis [blood formation], especially as it concerns the nitrilization of hydrocobalamin to active vitamin B-12 ((Beesley et al., 1985).
Several organs and sub-cellular organelles showed varying rhodanese activity.The Liver mitochondrial shows the highest specific activity of Rhodanese (Nagahara et al., 1998). Rhodanese is distributed mainly in pericentral hepatocytes. In the kidney Rhodanese is localised mainly in proximal tubular epithelial cells. In the brain, rhodanese is distributed mainly in glial cells (Nagahara et al., 1998). In the heart, myocardial cells contain rhodanese predominantly in the perinuclear area while in the lung; rhodanese is distributed diffusely in bronchiolar epithelial cell (Nagahara et al., 1998). Red blood cells were found to contain abundant rhodanese (Nagahara et al., 1998).
1.2 ASSAY METHODS
Many methods have been reported for the assay of rhodanese activity in various extracts. The most prominent is the one based on the colorimetric determination of thiocyanate. In this method, thiocyanate produced by the action of the enzyme on cyanide and thiosulphate, is reacted with ferric nitrate reagent to produce a red coloured complex – ferric thiocyanate which can be estimated colorimetrically at 460 nm (Sorbo, 1955; Baskin et al., 1990; Markku et al., 1999).
This method is suitable for measuring the activity of rhodanese preparation of any degree of purity (Sorbo, 1955). It is however not suitable for estimation of the enzyme in some tissue and organisms. Scott and Wright (1980) thus reported a modified method for human hemolyzates in which a protein-precipitating agent is included. Determination of the activity of rhodanese in soil samples involves incubating the buffered substrates and toluene. The method is rapid, sensitive and precise (Tabatabai et al., 1976). The assay methods used for extracts from animal tissues are not applicable in plant homogenate (Lieberei et al., 1990) and micro-organisms (Singleton et al., 1988). This is due to concomitant occurrence of both enzymic [biological] and non enzymic [non biological] thiocyante production. The non-enzymic formation of thiocyanate strongly affects the assay procedure.
A histochemical method was described in which the reducing effect of sulphite ion resulting from the enzyme reaction was used (Tanka et al., 1983). In a similar work, Cannella et al. (1984) reported a colorimetric method based on the continuous determination of sulphite. A method for the assay of rhodanese activity which uses pH-STAT apparatus with cyanide sensitive electrode to monitor the rate of the enzyme catalyzed reaction under controlled conditions has been reported (Cannella et al., 1975). A standard kinetic analysis of bovine kidney enzyme, including pH and temperature dependence is similar to those obtained by the classic spectrophotometric method.
Chew and Boey (1972) also assayed for rhodanese activity according to a modified method of Sorbo (1953). The substrate and the crude extract were equilibrated at the reaction temperature before mixing. The pH of the mixture was 8.7. The reaction mixture was then incubated for 15 minutes and the reaction was stopped by the addition of 0.5 ml 35 % formaldehyde. 2.5 ml of ferric nitrate reagent was added to give the colour reaction and absorbance was read at 460 nm.
Similarly, Lee et al. (1995) also employed a borate buffer, pH 9.4, in a slight modification of the assay method of Sorbo (1953a) to estimate the activity of rhodanese in mouse liver. Agboola and Okonji (2004) working on fruit bat liver, used a modified method of Lee et al. (1995) to assay for the activity of rhodanese.
1.3 METHODS OF PURIFICATION AND CRYSTALLIZATION OF RHODANESE
Rhodanese has been assayed from different animals and beef liver was found to be most active (Sorbo, 1951). Different extraction procedures have been described. Sorbo (1951) working on bovine liver, disintegrated in a Turmix blendor with 2.5 volumes of water and 0.2 volumes of a 20% lead acetate solution was added. When the suspension was centrifuged, a clear supernatant was obtained. However only about 55% of the total activity in the liver was extracted, the rest seems to be bound to insoluble particle. Purification procedure was as follows: the optimum purification was obtained at pH of 3.8, at this pH fractionation with ammonium sulphate was carried out in the cold room. The enzyme precipitated between 40 and 55 % saturation of ammonium sulphate and the precipitate was dissolved in 0.05 M solution sulphate and Na2HPO4 to a concentration of 120 rhodanese unit per ml. To it was added 1.3 volume of 80% saturated ammonium sulphate solution, brought to a pH 8.1 with ammonia.
The inactive precipitate was centrifuged and discarded, and the enzyme was precipitated from the supernatant by addition of 25 g ammonium sulphate per 100 ml of original solution. The precipitate was then dissolved in water. Furthermore, the ammonium sulphate fractionation was dialyzed until it is free from salt. To the dialysed enzyme was added 0.05 volumes of 0.1 acetate buffer pH 4.63 and acetone to 35 % by volume. The inactive precipitate was removed by centrifugation and discarded and the enzyme was precipitated by raising the acetone concentration in the supernatant to 50%. The precipitate now contained rhodanese purified 50-fold from the first extract and about 150-fold from liver.
Horowitz and Detoma (1970) devised a better method isolating rhoadanese from crystalline bovine liver tissue. The following steps are used.
10 pounds of liver (previously frozen and then thawed) were homogenized in a 1-gallon waring Blendor in three batches, each with 1,600 ml of 0.1 M glycine acetate buffer, pH 5.0. The homogenate was centrifuged for 30 minutes at 14,000 × g and residue was extracted with 8 litres of the same buffer. To the extracts was added solid (NH4)2SO4 mechanically with stirring to 0.40 M and the pH was adjusted to 3.8 with cold 1 M HCl. The ammonium sulphate concentration was raised to 1.4 M, and the solution was allowed to stand for at least 3 hours to permit complete precipitation.
After the removal of the precipitation by centrifugation, the ammonium sulphate concentration was raised to 2.0 M. After at least 3 hours the precipitated rhodanese was collected and stirred for 3 hours with 200ml of 10-3 Tris chloride buffer, pH 7.6, the preparation was then centrifuged and the residue discarded. The pH of the supernatant was adjusted to 7.6 with cold 1M NH4OH. The ammonium sulphate concentration was raised to 1.8 by addition of an equal volume of 3.6 M (NH4)2 SO4. After centrifugation to remove precipitate protein, the pH of the supernatant was lowered to 6.0 with cold 1.0 M acetic acid containing ammonium sulphate 1.8 M. The precipitated protein was removed by centrifugation after one hour. The pH of supernatant was lowered to 4.5 with 1.0 M acetic acid, 1.8 M (NH4)2SO4. The precipitation was then stirred for two hours and centrifuged.
The precipitated rhodanese was suspended in 200ml of 1.8 M ammonium sulphate. The pH was raised to 4.9 by addition of 1M NH4OH and the suspension was immediately centrifuged. The precipitated rhodanese was suspended in 200 ml of 1.8 M (NH4)2SO4, the pH was raised to 4.9 by addition of 1 M NH4OH and the suspension was immediately centrifuged. The precipitated rhodanese was again suspended in 200ml of 1.8 M (NH4)2SO4, the pH was raised to 7.9 by addition of 1M NH4OH and the precipitation was stirred for 3 hours.
After removal of insoluble protein by centrifugation, rhodanese was precipitated by addition of equal of 3.25 M (NH4)2SO4 which had been adjusted to pH 7.9. The enzyme crystallized (needles or plates) upon gentle stirring.
The crystals from this suspension was washed twice by suspension in 1.8 M (NH4)2SO4 pH 7.9 and stored frozen.
However, Lee et al. (1995) worked on Rhodanese from mouse liver and was able to purify the rhodanese to homogeneity using ammonium sulphate precipitation, CMS-Sephadex ion exchange, Affil-gel blue colour chromatography, hydroxylapitate and Sephacryl S-200-HR gel filtration chromatography with a purification fold of 776.
1.4 STRUCTURAL PROPERTIES OF RHODANESE
1.4.1 MOLECULAR WEIGHT
Different molecular weight of rhodanese has been reported by different researcher. (Sorbo; 1953) reported a molecular weight of 37,000 for bovine liver rhodanese. In 1967, Volini and Westley demonstrated that the enzyme exists in the monomer-dimer equilibrium and that the monomeric subunits are identical having a molecular weight of about 18,000 daltons. A similarly molecular weight of 17,500 daltons was reported for rat liver by Ploegman et al. (1979), further work by Wager in 1979 on rat liver showed that rhodanese from the rat liver has a molecular weight of 34,800, Man and Bryant (1974) reported a molecular weight of 38,000 daltons for bovine liver rhodanese. The molecular weight of the human liver rhodanese was found to be similar to that of the bovine liver rhodanese (Jarabak and Westley, 1974). Nagahara et al. (1996) obtained a molecular weight of 34,000 daltons for the same rat liver rhodanese. In 1995, Lee et al. reported a molecular weight of 34,800 daltons for the mouse liver rhodanese. While Agboola and Okonji (2004) reported a molecular weight of 35,700 daltons for rhodanese from the fruit bat liver (Eidolon helvum, Kerr).
1.4.2 AMINO ACID COMPOSITION
Using Sequential Edman Degradation of over lapping peptides obtained by selected chemical and enzymatic cleavages of rhodanese purified from chicken liver revealed that the primary structure of the enzyme contains 289 amino acids. Two hundred and twelve (212) of these amino acids matched with those obtained from bovine liver (Weiland et al., 1991). The differences were mainly due to conservative substitutions (Kohanski et al., 1990). In 1976, Baillie and Horowitz reported that between 5 and 11 tryptophan residues are present for every 26 alanine residues in bovine liver rhodanese.
In a more intensive study, involving combination of amino acid analysis, solvent perturbation difference spectroscopy, specific residue modification and direct UV spectra analysis, it was found that the enzyme contained 10 tyrosine, 8 tryptophan and 16 phenylalanine per 26 alanine residues (Baillie and Horowitz, 1976).
The results of amino acid analyses of homogenous, reduced, carboxymethylated rhodanese A (obtained from crystalline liver) and rhodanese B (obtained from bovine liver) subjected to acid hydrolysis for 22, 48 and 72 hours are shown in Table 1. Compositions for those amino acids stable to acid hydrolysis were derived by averaging the three values for carboxymethylcysteine, serine, threonine and methionine were and corrected for decomposition by extrapolation to zero time and the value obtained in the 72-hour analysis was used for isoleucine which is liberated more slowly during the hydrolytic procedure. Tryptophan was determined spectrophotometrically by the procedure of Beaven and Holiday (Beaven et al., 1952).
TABLE 1 Amino acid compositions of reduced and carboxymethylated rhodanese A (obtained from crystalline liver) and rhodanese B (obtained from bovine liver) (Blumenthal et al., 1972).
Amino acid
Rhodanese A
Number of residue per molecule
Rhodanese B
Number of residue per molecule
Lysine
8
8
Histidine
3
3
Arginine
8
8
S-carboxymethyl Cysteine
2
2
Aspartic acid + Asparagine
13
13
Threonine
8
8
Serine
11
11
Glutamic acid + Glutamine
16
16
Proline
10
10
Glycine
13
13
Alanine
12
12
Valine
11
11
Methionine
2
2
Isoleucine
5
5
Leucine
15
15
Tyrosine
5
5
Phenylalanine
7
7
Trytophan
4
4
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