Indian Journal of Experimental Biology Vol. 48, January 2010, pp. 83-86
Screening and partial immunochemical characterization of sulfite oxidase from plant source Ausaf Ahmad1* & Sarfraz Ahmad2 1
Department of Biochemistry, Faculty of Science, Jamia Hamdard (Hamdard University), Hamdard Nagar New Delhi 110 062, India 2 Florida Hospital Medical Center, Cancer Institute and University of Central Florida, College of Medicine, 2501 N. Orange Ave., Orlando, FL 32804, USA Received 25 November 2008; revised 4 September 2009
Sulfite oxidase [SO; EC 1.8.3.1] catalyses the physiologically vital oxidation of sulfite to sulfate, the terminal reaction in degradation of sulfur containing amino acids, cysteine and methionine. Sulfite oxidase from vertebrate sources is among the best studied molybdenum enzymes. Existence of SO in plants has been established recently by identification of a cDNA from Arabidopsis thaliana encoding a functional SO. The present study was undertaken to identify herbaceous and woody plants (viz., Azardirachta indica L., Cassia fistula L., Saraca indica L., Spinacea oleracea L., and Syzyzium cumini L.), a relatively less explored source, having significant SO activity and to characterize some of its immuno-biochemical properties. The Syzyzium cumini was chosen to characterize SO as it showed maximum enzyme activity in the crude extract as compared to other plants. Absorption spectra of SO revealed two peaks at 235 and 277 nm, but no distinct peak in the visible region could be observed. Crude extract of all the plants were taken into considerations for immuno-biochemical studies. Despite of significant protein structure-functional similarities between plant and animal SO, no cross-reactivity could be established between the two sources of SO. These data suggested that plants SO, however, differed with regards to their immunobiochemical properties. Keywords: Sulfite oxidase, Plant sources, Molybdoenzyme, Molybdenum, Sulfite, Oxidase, Immuno-biochemical Properties
Occurrence of sulfite oxidase (SO; EC 1.8.3.1) in plants has been controversial for a long time because this enzyme catalyzed a reaction that counteracts sulfate assimilation. Plant sulfite oxidase (PSO) has a sulfite-detoxifying function. Sulfite is a toxic ____________ *Address for correspondence- Department of Biochemistry & Biophysics, University of Rochester Medical Center, 601 Elmwood Ave., Box 712, Rochester, NY 14642, USA Telephone - +1-585-275-3329; Fax - +1-585-275-6007 Email-
[email protected]
metabolite that has to be removed in order to protect the cells against a surplus of sulfite, which is derived from SO2 gas in the atmosphere1,2. It is assumed that SO could possibly serves as “safety valve” for detoxifying excess amount of sulfite and protecting the cell from sulfitolysis3. Existence of SO in plants was finally established by Eilers et al4 through the identification of a cDNA from Arabidopsis thaliana encoding a functional PSO. Among animals, it is a well studied enzyme involved in degradation of sulfur-amino acids. SO from animal sources (animal SOs) contains a molybdenum cofactor (Moco) and a heme domain5, whereas PSO lacks the heme domain4,6. Thus, among eukaryotes, PSO is the simplest Moco enzyme possessing only one redox center. Unlike animal SOs that is localized in mitochondria4, PSO is a peroxisomal enzyme4,7. The present communication describes the screening of five different herbaceous and woody plants for the presence of SO, and to investigate any potential immunological relationship with the animal SO. Additionally, the enzyme from crude extract of Syzyzium cumini was characterized with respect to its spectroscopic properties. Materials — Fresh leaves from five selected plants (viz., Azardirachta indica L., Cassia fistula L., Saraca indica L., Spinacea oleracea L., and Syzyzium cumini L.) were obtained from the campus of the Hamdard University, New Delhi. Goat liver was obtained freshly from a local slaughter house and rabbit was provided by the Animal House Facility of Hamdard Univeristy, New Delhi. Polyvinylidene difluoride (PVDF) membrane was purchased from BioRad Laboratories (Hercules, CA). The materials used for the dot blot analysis were obtained from Sigma Chemical Co (St. Louis, MO). All other chemicals/reagents used in this study were of the analytical grade. Homogenization and extraction — Leaves of plants were collected, washed with distilled water and crude extracts were prepared as described earlier4, containing 0.1 mM EDTA and 25 mM Tris-Cl, pH 8.0 (Tris buffer); 0.1 mM, phenylmethylsulfonylfluoride (PMSF); 2%, polyvinylpyrrolidone (w/v); and 2.5 mM, sodium molybdate and subsequently used for screening of SO enzyme activity.
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Enzyme activity measurement — Enzyme activity of SO in crude extracts obtained from the leaves was measured according to the method described by Cohen and Fridovich8. One unit of enzyme per milliliter was defined as the amount of enzyme that catalyze the transformation of one nmole of potassium ferricyanide into potassium ferrocyanide per min at 25°C. Quantification of protein — Total protein concentration of the crude extracts from different plant leaves were determined by the classical method of Lowry et al9. Absorption spectra — Absorption spectrum of SO from crude preparation of S. cumini was recorded at 25°C on a Perkin-Elmer Lambda Bio 20 double-beam spectrophotometer between the ranges of 200 to 600 nm in Tris-buffer (as noted above). Immunodiffusion — Goat liver SO was purified by the method as described by Ahmad et al10. The polyclonal antibodies against the purified goat liver SO were raised in rabbit utilizing the procedure as described previously11. Ouchterlony double immunodiffusion was carried out to find out whether there is any homologies between SO from goat liver and the plant leaves’ crude preparation explored in this study. The polyclonal antibodies against purified goat SO raised in rabbit were placed in center well of petri-plate coated with 1% melted agar in phosphate buffer saline (PBS). The crude preparations of different plants were placed in peripheral wells. The in-house purified goat liver SO was placed in one of the peripheral wells, which served as a positive control. The plate was incubated in humid chamber for 24 h. Finally, the plate was stained with Coomassie brilliant blue R-250 stain [0.1% Coomassie brilliant blue R-250, 50% methanol (v/v)/10% glacial acetic acid (v/v)] for 30 min followed by de-staining of the plate with a de-staining solution [35% methanol (v/v)/5% glacial acetic acid (v/v)].
Saraca indica L., Spinacea oleracea L., and Syzyzium cumini L.; Table 1). The leaves collected from S. cumini showed the highest level of enzymatic/specific activities for SO (25.6 EU/ml and 203.2 EU/mg protein, respectively) compared to the other four plant leaves (Table 1). Since S. cumini plant leaves showed highest SO activity, so this plant was selected as a major source in order to further characterize the plant enzyme with respect to its spectroscopic properties. The absorption spectrum obtained from the partially purified SO in the leaves of S. cumini has been shown in Figure 1. The spectrum was obtained using Tris-buffer at 25°C in the wavelength range of 200 to 600 nm. In the spectrum there are two maxima in the UV region (one at 235 nm and the other at 277 nm). However, no distinct absorption band was observed in the visible region (Fig. 1). Table 1—Data on the protein content and enzyme activities of sulfite oxidase in the crude extracts of leaves obtained from various plants Plants
Azardirachta indica L. Cassia fistula L. Saraca indica L. Spinacea oleracea L. Syzygium cumini L.
Total protein (mg/mL)
Enzyme activity (EU/mL)
Specific activity (EU/mg Protein)
0.54
17.24
32.20
1.35
21.80
16.20
0.11
15.66
145.70
4.90
3.02
00.62
0.13
25.60
203.20
Dot-blot analysis — To confirm the results obtained with the immunodiffusion technique, the SO immunoreactivity was further checked by, dot-blot analysis as described earlier12 and the membrane was developed by using an enhanced chemiluminesence method as described elsewhere13. The total protein content and SO enzyme activity levels were determined in the leaves of five domestic plants (Azardirachta indica L., Cassia fistula L.,
Fig. 1—Absorption spectrum of partially purified sulfite oxidase from Syzygium cumini in 25 mM Tris-Cl buffer, pH 8.0 at 25°C.
NOTES
Utilizing immunodiffusion technique, precipitin arc was obtained only with the goat liver sulfite oxidase (i.e., the positive control), and no precipitin arc could be found with any of the extracts of plant leaves used. Similarly, by employing the dot-blot analysis, again it was only the purified goat liver SO, which showed positive signal after the addition of substrate (Fig. 2). Molybdenum-containing enzymes are found throughout the biological system, which catalyze critical reactions in the metabolism of purines and aldehydes, as well as nitrogen- and sulfur-containing compounds. SO is ubiquitous among animals. However, its occurrence in plant kingdom has been somewhat controversial because the enzyme catalyzed a reaction that counteracts sulfate assimilation. Animal SO catalyzes the terminal step in oxidative degradation of cysteine, methionine8, whereas PSO has a sulfite-detoxifying function. Sulfite is a toxic metabolite that has to be removed in order to protect the cell against a surplus of sulfite derived from SO2 gas in the atmosphere1,2 or during the decomposition of sulfur-containing amino acids15. In the present study, SO was observed in five domestic plants and S. cumini showed highest specific activity in the crude leaf extracts. The amounts of protein levels present in the extracts of plants, except S. oleracea, were found to be relatively low. This suggested that PSO had high turnover number. Enzyme present in the crude extract of leaves although showed maximum absorption band at
Fig. 2—Dot-blot analysis of sulfite oxidase obtained from goat liver and plant sources to compare the immuno-reactivity responses. Different concentrations of sample applied to the membrane. [Lane 1 – Positive control (goat liver SO); and Lane 2-6 – crude extract from five different plant leaves as described in the text].
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277 nm, but did not show any absorption band in the visible region (around 400 nm), a characteristic of animal SO. This observation suggested that PSO did not have cytochrome c to accept electron at the terminal step of SO catalyzed reaction in animals. This is consistent with a couple of previous observations4,16. However, Hemann et al.17 obtained absorbance maxima at 360 and 480 nm from A. thaliana SO by using a modified procedure which removes the apo-enzyme lacking the molybdenum center and they have attributed 360 and 480 nm maxima with dithiolene-to-molybdenum charge transfer transition and cysteine-to-molybdenum charge transition, respectively. In plants, the function of SO electron acceptor could be taken over by some other entity. Earlier, it has been believed that plant peroxisomes are likely candidates to fulfill the role of physiological electron acceptor for PSO18, but recently Hänsch et al.19 have identified oxygen as the terminal electron acceptor for PSO and hydrogen peroxide as the product of this reaction in addition to sulfate. The latter findings might also explain the peroxisomal localization of plant sulfite oxidase. SO gene is present in higher and lower plants, and the protein encoded is highly conserved because the antibodies directed against A. thaliana SO detect proteins of the correct size in a wide range of plants. Cloning and characterization of plant SO was possible by using sequence homologies to the mammalian counterpart (as there are 46% sequence similarities between A. thaliana and chicken liver SO). Recently, the crystal structure of A. thaliana PSO has been determined at 2.6 Å by Schrader et al.20 that reveals a remarkable structural conservation between plant and animal enzymes. Attempt to investigate any possible similarities between goat liver SO and PSO from five plants sources failed to indicate any crossreactivity as determined by the Ouchterlony double immunodiffusion and dot-blot analyses (Fig. 2). In fact, the goat liver SO have 45, 68 and 87% homologies with A. thaliana, chicken liver SO and human liver SO, respectively (Ahmad A, unpublished data). These results obviously point to the fact that the animal and plant SOs are immunologically different from each other despite carrying some structurefunctional similarities in the protein molecule. AA is grateful to the Hamdard National Foundation, New Delhi, for providing a research fellowship grant to carry out some of the studies reported in this communication.
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