Purification and partial characterization of oxalate oxidase from leaves of forage Sorghum (Sorghum vulgare var. KH-105) seedlings

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1 Indian Journal of Biochemistry & Biophysics Vol. 48, February 2011, pp Purification and partial characterization of oxalate oxidase from leaves of forage Sorghum (Sorghum vulgare var. KH-105) seedlings Rajender Kumar 1, Vinita Hooda 2 and C S Pundir 1* 1 Biochemistry Research Laboratory, Dept. of Biochemistry, 2 Department of Botany, M.D. University, Rohtak (Haryana), India Received 22 September 2010;revised 06 December 2010 An oxalate oxidase was purified to apparent homogeneity from the leaves of 10-days old seedlings of forage Sorghum (Sorghum vulgare var. KH-105). The enzyme had a Mr of 124 kda with two identical subunits, an optimum ph of 4.5, optimum temperature of 37 C and activation energy (Ea) of Kcal/mol. The rate of reaction was linear up to 7 min. K m value for oxalate was 0.22 mm. The enzyme was stimulated by Cu 2+ and inhibited by EDTA, NaCN, diethyldithiocarbamate, Na 2 SO 4, but unaffected by NaCl at 0.1 mm concentration. Although the enzyme was stimulated by flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), UV and visible spectra of the enzyme did not match with that of a flavoprotein. The positive reaction of the enzyme with orcinol-h 2 SO 4 reagent indicated its glycoprotein nature. The superiority of the purified enzyme over earlier reported oxalate oxidases for determination of urinary oxalate has been demonstrated. Keywords: Oxalate, Sorghum vulgare, Oxalate oxidase, Forage Sorghum, Purification, Glycoprotein Oxalate has implications in cellular biochemistry and tissue re-modelling 1. Breakdown of oxalate in higher plants is of great importance, since it could lead to the availability of free Ca 2+. Two types of oxalate degrading enzymes are known in higher plants: oxalate decarboxylase (E.C ) which catalyzes ATP and Co-A dependent breakdown of oxalate with release of CO 2 and formic acid and oxalate oxidase (E.C ) which catalyzes the oxidative breakdown of oxalate into H 2 O 2, and CO 2 2. The former enzyme has been studied in pea and other plants 3. The later enzyme has generated more interest, as it releases H 2 O 2, which is required for the key cross-linking reaction of cell wall and cellular regulation. The enzyme has also attracted the attention of several workers, since it has been introduced as an analytical reagent for colorimetric determination of urinary oxalate, required in the diagnosis and medical management of urinary calculi and various intestinal diseases 4. Oxalate oxidase has been found in higher plants in two forms: membrane-bound and soluble. Membranebound enzyme has been purified from beet stems 5 and Amaranthus spinosus leaves 6 and soluble enzyme from roots of barley seedlings 7-9 and kernel 10, banana *Corresponding author pundircs@rediffmail.com Fax: (95) (1262) fruit peel 11, grain Sorghum leaves 12 and wheat seedlings 13. Earlier, presence of a soluble oxalate oxidase in the leaves of forage Sorghum seedlings and its superiority along with grain Sorghum enzyme over other plant enzymes in urinary oxalate determination has been reported from this laboratory However, this enzyme has not yet been purified and characterized from forage Sorghum, which is preferred agronomically over grain Sorghum because of its suitability for dry land and better water use efficiency 20. In the present report, we describe the purification and partial characterization of an oxalate oxidase from the leaves of forage Sorghum (Sorghum vulgare var. KH-105) seedlings. Materials and Methods 4-Aminophenazone, DEAE-Sephacel, bovine serum albumin (BSA) and oxalate from Sigma Chemical Co., USA, Sephadex G-200 (from Amarsham Pharmacia, Biotech. Sweden) and diethyldithiocarbamate, (DEIDA), α,α -dipyridyl, EDTA, copper sulphate, Coomassie brilliant blue, solid phenol, succinic acid, ammonium sulphate (enzyme grade) Folin-Ciocalteu (F.C.) reagent, riboflavin, nicotinamide adenine dinucleotide (NAD + ), flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), acrylamide, N N -bismethyleneacrylamide, tris base, ammoniumpersulphate, glycine,

2 KUMAR et al.: OXALATE OXIDASE FROM FORAGE SORGHUM 43 bromophenol blue, methanol, glacial acetic acid, N,N,N,N -tetramethylethylenediamine (TEMED), silver nitrate, sodiumdodecyl sulphate (SDS), β mercaptoethanol, glycerol and horseradish peroxidase (RZ = 3.0) from SISCO Research Laboratories Pvt. Ltd., Mumbai were used. All other chemicals were of analytic reagent grade. Collection of plant material and extraction and purification of oxalate oxidase The seeds of forage Sorghum (Sorghum vulgare var. KH-105) were purchased from the local market and ten-days old seedlings of forage Sorghum were raised in the laboratory 21 and their leaves were collected and stored immediately at 20 C until use. The frozen leaves were homogenized with cold distilled water in 1:3 ratio (w/v) in a chilled mortar and pestle. The homogenate was centrifuged at l5000 g for 30 min at 4 C and the supernatant was collected and tested for oxalate oxidase activity and protein 14. The crude enzyme (15000 g supernatant) was purified in cold (4-8 C) by 0-80% (NH 4 ) 2 SO 4 fractionation, gel filtration on Sephadex G-200 and ion-exchange chromatography on DEAE-Sephacel 12. The ultrafiltration of purified enzyme was carried out by Amicon concentrator at 4 C. The activity and protein content of purified enzyme were measured 22. Assay of oxalate oxidase Assay of oxalate oxidase was carried out in a 15 ml test tube wrapped with black paper 14. The reaction mixture containing 1.8 ml 0.05 M sodium succinate buffer (ph 4.0), 0.1 ml CuSO 4 solution (10-2 M) and 0.1 ml crude enzyme was preincubated at 37 C for 2 min. The reaction was started by adding 0.1 ml of oxalate (10-2 M). After incubating at 37 C for 10 min, 1.0 ml colour reagent was added and kept at room temperature (25 C) for 15 min to develop colour. A 520 was read in Spectronic-20 (Thermo Scientific, USA) against blank. The colour reagent consisted of 50 mg 4-aminophenazone, 100 mg solid phenol and 1 mg horseradish peroxidase per 100 ml of 0.4 M sodium phosphate buffer (ph 7.0) stored in amber coloured bottle at 4 C and prepared fresh every week. Amount of H 2 O 2 generated in the assay was calculated from the standard curve between H 2 O 2 concentrations vs. A 520. One unit of enzyme was defined as the amount of enzyme required to catalyze the generation of 1.0 nmol of H 2 O 2 from oxalate per min under standard assay conditions. The protein in various enzyme preparations was determined using the method of Lowry et al 22. Native and SDS-PAGE Slab gel-page and SDS-PAGE of purified oxalate oxidase was carried out in 7% polyacrylamide gel using the method of Davis 23. Kinetic properties of oxalate oxidase The following kinetic properties of purified oxalate oxidase were studied: To determine the optimum ph of the enzyme, the ph of the reaction buffer was varied from ph 3.0 to 6.0 using the following buffers, each at a final concentration of 0.05 M; ph 3.0 to 3.5: glycine-hcl, ph : sodium succinate. To determine the optimum temperature of the enzyme, the reaction mixture was incubated at different temperatures ranging from C at 5 C intervals. Energy of activation (E a ) of the enzyme was calculated from the Arrhenius plot. Incubation time for maximum activity of enzyme was determined by incubating the reaction mixture from 2 to 12 min at an interval of 2 min. To determine the effect of oxalate, different concentrations of oxalate ranging from 0.01 to 10.0 mm were used in the assay. K m for oxalate was calculated from Lineweaver-Burk plot. Effect of various metals, metal chelators, coenzymes, flavins and other compounds on the enzyme activity was studied at final concentration of 0.1 mm. NaCl effect was also studied at 1, 10, 100 and 200 mm. To study the effect of Cu 2+ in the presence of flavins, the enzyme was dialyzed overnight at 4 C against the buffer (0.02 M sodium phosphate, ph 7.0). The effect of copper sulphate was studied on dialyzed enzyme both alone at a final concentration of 1 mm and also in presence of FMN/FAD (0.1 mm). Glycoprotein nature of the enzyme was studied by orcinol-h 2 SO 4 reaction 24. Measurement of UV and visible spectra The UV and visible spectra of purified enzyme in 0.02 M potassium phosphate buffer (ph 7.0) was recorded at room temperature (30 C) between the wavelength 250 to 600 nm in a UV and visible spectrophotometer (make Hitachi, Japan). Determination of urinary oxalate The first morning urine sample was collected from apparently healthy male adults and stored at 4 C until use. The urine was diluted in 1:1 ratio with distilled water and its ph was adjusted to 7.0 by NaOH or HCl. To 1.0 ml diluted urine 0.1 ml buffered NaNO 2 (35 mg/10 ml 0.02 M sodium phosphate buffer ph 7.0) was added to avoid the possible ascorbate interference. The reaction mixture containing 1.8 ml 0.05 M sodium succinate buffer (ph 4.5), 0.1 ml

3 44 INDIAN J. BIOCHEM. BIOPHYS., VOL. 48, FEBRUARY 2011 CuSO 4 solution (10-2 M) and 0.1 ml purified enzyme was preincubated at 37 C for 2 min. The reaction was started by adding 0.1 ml of pre-treated urine. After incubating at 37 C for 7 min, 1.0 ml colour reagent was added and kept at room temperature for 15 min to develop colour. A 520 was read. The oxalate concentration in urine was extrapolated from standard curve between oxalate concentration ranging from 0.01 to 0.2 mm and A 520 prepared under the standard assay conditions. Results and Discussion A soluble oxalate oxidase found in the leaves of 10 days old seedling plants of forage Sorghum (Sorghum vulgare var KH-105) was purified up to apparent homogeneity, as judged in simple PAGE using Coomassie brilliant blue staining (Fig. 1). Table 1 summarizes the results of enzyme purification scheme. An overall 109-fold purification of the enzyme was achieved with 10% yield, which was better than that from grain sorghum leaf (8.8%) 12, barley seedlings (6.8%) 7, but lower than that from Amaranthus leaf (15.7%) 6. The enzyme had a Mr of 124 kda as determined by Sephadex G-200 gel Fig. 1 Native-polyacrylamide gel electrophoresis (PAGE) (a) and SDS-PAGE (b) of oxalate oxidase from forage Sorghum leaves [In both Fig. 1 (a) & (b) lane 1 represents high molecular (in kda) protein markers and lane 2 the purified forage Sorghum oxalate oxidase] filtration method which was almost similar to that of grain Sorghum leaf enzyme (120.2 kda) 12 and barley root enzyme (125 kda) 9. The SDS-PAGE of the purified enzyme revealed that it had two identical subunits, each of 62 kda (Fig. 1), similar to that of grain Sorghum leaf (two subunits, each of 62 kda) 12, but different from that of barley root (five identical subunits of Mr 26 kda) 9 and wheat seedling enzyme (five subunits each of 32.6 kda) 13. Table 2 summarizes kinetic properties of forage Sorghum oxalate oxidase and compares it with that from grain Sorghum and barley seedlings. The enzyme showed maximum activity at ph 4.5. Incubation temperature for maximum activity of enzyme was 37 C. Energy of activation (E a ) of the purified enzyme as calculated from Arrhenius plot was kcal/mole, which was lower than that from grain Sorghum leaf (4.4 kcal/mole) 12, and Amaranthus leaf (15.2 kcal/mole) 6. The rate of enzyme reaction of purified enzyme was linear up to 7 min, after which it was almost constant. The effects of varying concentration of oxalate (0.01 to 10.0 mm) on initial velocity of the enzyme reaction was hyperbolic from 0.01 to 4 mm after which the reaction rate showed a rapid decline, indicating the substrate inhibition at high concentration or product inhibition, similar to that from barley seedlings 7 and grain Sorghum leaf 12. K m for oxalate, as calculated from Lineweaver-Burk plot was 0.22 mm, which was lower than that from Sorghum leaf (0.78 mm) 12 and barley seedlings (0.42 mm) 7, but close to the enzyme from wheat seedling (0.21 mm) 13. The effect of various metal chelators and metals on the enzyme is shown in Table 3. EDTA and DEIDA (a Cu 2+ -specific chelator) caused strong inhibition of the enzyme, indicating the requirement of Cu 2+ for the enzyme, similar to that from grain Sorghum leaf 12. Among the various metals tested each at a final concentration of 1 mm, only CuSO 4 stimulated the enzyme. Cu +2 binds with two adjacent SH group(s) of peptide backbone of the enzyme to form a cuprosulfhydryl complex which facilitates binding of Table 1 Purification of oxalate oxidase from leaves of 10-days old forage Sorghum seedling plants Purification step Total volume Protein (Units/ml) Activity (Units/ml) Specific activity Purification fold Crude enzyme (15000 g) supernatant (NH 4 ) 2 SO 4 Precipitate (0-80%) DEAE-Sephacel fraction Sephadex G-200 fraction Ultra-filtered enzyme One enzyme unit was defined as amount of enzyme required to generate 1 nmol H 2 O 2 /min/ml under standard assay. Yield

4 KUMAR et al.: OXALATE OXIDASE FROM FORAGE SORGHUM 45 Table 2 Properties of oxalate oxidase purified from leaves of forage Sorghum and its comparison with those from grain Sorghum leaf and barley seedlings Kinetic parameter Forage Sorghum leaf Grain Sorghum leaf 12 Barley seedlings 9 Mr (kda) Subunit (No) Optimum ph Optimum temp ( C) Energy of activation NA (E a ) (kcal/mol) Time for linearity (min) K m for oxalate (mm) Inhibition by NaCl ND ND 14 (1 mm) Stimulation by Cu (0.5 mm) Stimulation by FAD (0.5 mm) in presence of Cu 2+ (1 mm) ND Nature Glycoprotein Cu 2+ requiring glycoprotein ND = Not detected; NA = Not available Mn 2+ requiring glycoprotein oxalate to the enzyme, similar to grain Sorghum root oxalate oxidase 25. However, further studies are required to elaborate the stimulatory role of Cu +2 in this enzyme. Na 2 SO 4 caused 52% inhibition of the enzyme, while rest of the metal salts had practically no effect. However, NaCl at high concentration caused inhibition of enzyme, which increased with its concentration (Table 3), similar to barley seedling enzyme 7. Among the co-enzymes (riboflavin, FMN, FAD, NAD + ) tested each at a concentration of 0.1 mm, flavins caused slight stimulation of the enzyme, while NAD + had no effect similar to that from grain Sorghum leaf 12. Although our observations suggest that forage Sorghum enzyme is not a flavoprotein, slight stimulation of the enzyme by flavins might be due to their protective effect against NaCl inhibition. Earlier, flavins have been reported to protect barley enzyme from inhibition by sodium fluoride 7. The stimulating effect of Cu 2+ on dialyzed enzyme was increased in presence of flavins which was an additive effect of two stimulants together and in the following order FAD>FMN>riboflavin (Table 4). Among the other compounds tested each at a final concentration of 0.1 mm, NaCN inhibited the enzyme activity completely, while sodium dithionate and potassium ferricyanide caused 85% and 24% inhibition of the enzyme reaction respectively. Table 3 Effect of metal chelators and metal salts on forage Sorghum leaf oxalate oxidase Compound added (Final conc. 0.1 mm) Relative activity None EDTA 61.5 Diethyldithiocarbamate 30.7 CaCl KCl MgSO CuSO ZnSO FeSO Na 2 SO H 3 PO NaCl (0.1, 1, 10, 100, 200 mm) 100, 69.2, 61.3, 48.4, 30.4 Standard assay conditions were used, except for the addition as indicated above Table 4 Effect of coenzymes, Cu 2+ and flavins on forage Sorghum leaf oxalate oxidase Compounds added (Final conc. 0.1 mm) Relative activity None Riboflavin NAD FMN FAD Cu Riboflavin + Cu NAD + + Cu FAD + Cu FMN + Cu Standard assay conditions were used, except that the dialyzed enzyme was used and the addition of compound as indicated above. The UV and visible spectra of purified enzyme did not show peak at 370 nm and 450 nm, a characteristic of flavoprotein. The purified enzyme was also not yellow in color. Hence this enzyme can not be classified as a flavoprotein. Nevertheless, the enzyme solution gave brown color after heating it with orcinol-h 2 SO 4 reagent, indicating glycoprotein nature. Earlier, barley root enzyme was also classified as a glycoprotein 9,26. The urinary oxalate measured in healthy male adults by forage Sorghum enzyme ranged from 18 to 32 mg/l with a mean ± S.E. of 22.6 ± 0.47 (n = 12) which is comparable to earlier reported values employing oxalate oxidase from mosses (28-43 mg/l, mean 37.7) 27, barley ( mg/l, mean 20.9) 28, beet stem ( mg/l, mean 33.8) 5, banana peel ( mg/l, mean 17.2) 11, grain Sorghum (9-23 mg/l, mean 16.7) 12 and alkylamine glass beads bound forage Sorghum ( mg/l, mean 37.2) 15. The method has the advantage that it does not require pre-treatment of urine for removal of chloride ion as

5 46 INDIAN J. BIOCHEM. BIOPHYS., VOL. 48, FEBRUARY 2011 forage Sorghum enzyme is insensitive to chloride ion (at low concentration) unlike barley enzyme 7. Moreover the source of present enzyme is cheaper and widely available compared to the source of earlier reported chloride-insensitive grain Sorghum enzyme 12. In conclusion, an oxalate oxidase was purified and characterized from forage Sorghum leaves. The enzyme had two identical subunits, stimulated by Cu 2+ and was a glycoprotein. The enzyme is a better analyte for urinary oxalate determination over earlier reported enzymes. References 1 Oke O L (1969) In: Oxalic Acid in Plants and Nutrition. World Review of Nutrition Dietetics, Xth edn., pp , Karger, Basel/New York 2 Hodgkinson A (1977) In: Oxalic Acid in Biology and Medicine, pp , Academic Press, New York 3 Giovanelli J & Tobin N F (1963) Plant Physiol 39, Sharma S Nath R & Thind S K (1993) Scanning Micros 7, Obzansky D M & Richardson K E (1983) Clin Chem 29, Goyal L, Thakur M & Pundir C S (1999) Anal Lett 32, Chiriboga J (1966) Arch Biochem Biophy 116, Whittaker M M & Whittaker J W (2002) J Biol Inorg Chem 7, Kotsira V P & Clonis Y D (1997) Arch Biochem Biophys 340, Kanauchi M, Milet J & Bamforth C W (2009) J Inst Brew 115, Raghavan K G & Devasagayam T P A (1985) Clin Chem 31, Satyapal & Pundir C S (1993) Biochem Biophys Acta 1161, Hu Y & Guo Z (2009) Acta Physiol Plantarum 31, Pundir C S (1991) Experentia 47, Kalra V & Pundir C S (2004) Indian J Biotechnol 3, Kumari M & Pundir C S (2004) Indian J Biochem Biophys 41, Pundir C S, Chauhan N S & Bhambi M (2008) Anal Biochem 374, Pundir C S, Bhambi M & Chauhan N S (2009) Talanta 77, Pundir C S, Kuchhal N K, Thakur M & Satyapal (1998) Indian J Biochem Biophys 35, Paul W U (1988) Agron. J. 80 (2), Pundir C S & Nath R (1984) Phytochemistry 23, Lowry O H, Rosebrough N J Farr A L & Randal R J (1951) J Biol Chem 93, Davis V J & Ann N Y (1964) Ann N Y Acad Sci 121, White C A & Kennedy J F (1986) In: Oligosaccharides in Carbohydrate Analysis, A Practical Approach (Chaplin & Kennedy J R L, eds), pp , IRL Press, Oxford Pundir C S & Kuchhal N K (1989) Phytochemistry 28, Whittaker M M, Pan H Y, Yuki E T & Whittaker J W (2007) J Biol Chem 282, Laker M R, Hofman A F & Meeuse B J D (1980) Clin Chem 26, Wilson D M & Liedtke R R (1991) Clin Chem 37,