Quantification of urinary oxalate by immobilized oxalate oxidase of forage sorghum leaf

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1 Indian Journal of Biotechnology Vol 3, January 2004, pp Quantification of urinary oxalate by immobilized oxalate oxidase of forage sorghum leaf Vijay Kalra and C S Pundir* Biochemistry Research Laboratory, Department of Biosciences, M D University, Rohtak , India Received 29 July 2002; accepted 24 April 2003 A partially purified oxalate oxidase from leaves of 10-day-old seedlings of forage sorghum was immobilized covalently onto alkylamine glass beads affixed on inner base of a glass beaker. The enzyme retained 5.17% of its initial activity with a conjugation yield of 182 mg/g support. After immobilization, the enzyme showed an increase in its optimum ph and K m for oxalate but slight decrease in incubation temperature and time for maximum activity as compared to free enzyme. The glass beaker bound enzyme was employed for determination of urinary oxalate. The urinary oxalate in apparently healthy persons, as measured by the glass beaker, was found to be in the range of 12.5 to 29.7 mg/l (mean 20.9 mg/l) for females and 25.7 to 45.4 mg/l urine (mean 37.2 mg/l) for males. The glass beaker provided 70 reuses of immobilized enzyme with ease in handling. Keywords: oxalate, oxalate oxidase, immobilization, forage sorghum, urine, alkylamine glass Introduction Increase in urine oxalate is observed in patients predisposed to oxalate kidney stone formation and various forms of lipid malabsorption through intestine. Among various methods available for oxalate determination in biological materials, enzymic colorimetric method employing oxalate oxidase and peroxidase is simple, sensitive and specific 1. The use of immobilized oxalate oxidase is expected to reduce the cost of enzymic procedure as it provides the reuse of enzyme, the costliest component of the method. Earlier, the authors have reported methods for discrete analysis of oxalate in urine, plasma and food stuffs by employing a Cl - and NO - 3 insensitive oxalate oxidase purified from grain sorghum leaves and commercial horseradish peroxidase, immobilized separately onto free alkylamine glass beads 2-4. Although, these enzyme-bounded, free glass beads were reused regularly for a period of six months without any considerable loss of activity, the handling of these beads was tedious and time consuming. The beads also had a chance of being lost during the transfer of the reaction mixture and their washing for reuse. This problem was overcome in the present work by affixing the alkylamine glass beads, prior to immobilization of enzyme, on the inner base of a glass beaker with a non-reactive fixative. *Author for correspondence: Tel: ; Fax: pundircs@rediffmail.com Furthermore, the enzyme employed herein has been purified partially from a much cheaper and widely available plant source, i.e. forage sorghum leaves, which is also insensitive to Cl - - and NO 3 normally found in biological fluids. Materials and Methods Materials Zirconia coated alkylamine glass beads (pore diam 55 nm; manufactured by Corning Glass Works, NewYork) were gifted by Dr H H Weetall, Environment Protection Group, Las Vegas, USA. Glutaraldehyde (25%) was obtained from BDH Poole, England and Horseradish Peroxidase (RZ=2.0) from SISCO Research Laboratories Pvt. Ltd., Mumbai. The fixative, Lakme long stay nail enamel of silver white colour (Number 754, Dew Drops), manufactured by Flora Cosmetics Ltd., Kundain, Goa (India) and forage sorghum seeds (Sorghum vulgare var. KH105) were purchased from the local market. Growing of Forage Sorghum Plants and Collection of Leaves Plants were raised from seeds of forage sorghum in the laboratory as described by Pundir and Nath 5.The leaves from 10-day-old plants were collected and stored at 20 C until used. Extraction and Partial Purification of Oxalate Oxidase from forage Sorghum Leaves Extraction from the leaves was done as described by Satyapal and Pundir 6. The frozen leaves (300 g) were homogenized in cold distilled water in 1:3 ratio

2 KALRA & PUNDIR:URINARY OXALATE DETERMINATION BY IMMOBILIZED OXALATE OXIDASE 53 (w/v) in a chilled pestle and mortar. The homogenate was passed through double layer of muslin cloth. The filtrate was centrifuged at 15,000 rpm for 30 min at 4 C. The supernatant (850 ml) was collected and treated as crude enzyme. Solid ammonium sulphate ( g) was added to the crude enzyme to give a final saturation of 0-80%. The mixture was then stirred in cold until the ammonium sulphate was dissolved. The resulting solution was centrifuged at 10,000 rpm for 30 min at 4 C. The pellet was washed carefully with distilled water and then redissolved in 8 ml sodium phosphate buffer (0.05 M, ph 7.0) and treated as partially purified enzyme. It was stored at 4 C until used. Assay of Oxalate Oxidase The assay of oxalate oxidase was carried out in a 15 ml test tube wrapped with black paper as described by Pundir 7 with modifications. The reaction mixture containing 1.7 ml sodium succinate buffer (0.05 M, ph 4.5), 0.1 ml CuSO 4 (0.01 M) and 0.1 ml partially purified enzyme was pre-incubated at 37 C for 2 min. The reaction started by adding 0.1 ml of aqueous solution of oxalate (10 mm). After incubation at 37 C for 5 min under continuous stirring, 1 ml colour reagent was added and kept at room temperature (25±2 C) for 10 min to develop the colour. The control and blank were also run in the same manner except that the enzyme solution was replaced with heat denatured enzyme and reaction buffer, respectively. A 520 of reaction mixture was read against the control in a Spectronic-20 (Milton and Roy, USA). The content of H 2 O 2 generated in the reaction was extrapolated from the standard curve of H 2 O 2. The colour reagent was prepared by dissolving 5 mg MBTH (3-methyl-2-benzo thiazolinone hydrazine), 53 mg of DMAB (3-dimethyl amino benzoic acid) and 1 mg of horseradish peroxidase in 100 ml sodium succinate buffer (0.05 M, ph 3.8) as described by Sugiura et al 8. It was stored in amber coloured bottle at 4 C and prepared fresh every week. One unit of enzyme activity is defined as the amount of enzyme that generates 1μmol H 2 O 2 /min under standard assay conditions. The protein content in various enzyme preparations was determined by the method of Bradford 9. Immobilization of Oxalate Oxidase on Inner Base of a Glass Beaker Affixation of Glass Beads on Inner Base of Glass Beaker A 25 ml glass beaker was thoroughly washed and dried. It was then applied, uniformly on the inner base, a thin layer (1mm thickness) of fixative, i. e. Lakme nail paint of silver white colour, with the help of a brush. Powder of alkylamine glass beads (100 mg) was sprinkled on this layer uniformly by an aluminium foil. The beaker was kept for 24 hrs at room temperature (25±5 C) for affixation of the glass beads. The unbound glass beads, if any, were poured off from the beaker. Immobilization of Oxalate Oxidase on Affixed Alkylamine Glass Beads Immobilization of oxalate oxidase was carried out as described by Lynn 10 with modification. Two ml glutaraldehyde (2.5%) in sodium phosphate buffer (0.05 M, ph 7.0) was added to the beaker containing affixed glass beads. The beaker was shaken gently and allowed to stand at room temperature for 2 hrs with occasional stirring. The excess of glutaraldehyde solution was removed and the affixed glass beads were washed repeatedly with 2.0 ml distilled water until the ph of discard was 7.0 to ensure the complete removal of glutaraldehyde. Final washing of the beads was done in sodium phosphate buffer (0.05 M, ph 7.0). Two ml oxalate oxidase solution [1.5 mg/ml sodium phosphate buffer (0.05 M, ph 7.0)] was added to the glass beaker and the coupling between enzyme and activated glass beads was allowed at 4 C for 48 hrs with occasional shaking. After coupling, the unbound enzyme was decanted and tested for enzyme activity and protein. The glass beads were washed 3-4 times with distilled water until no activity of enzyme was detected in consequent washing. The amount of protein bound to glass beads was estimated by determining the loss of protein from the enzyme solution during immobilization. Assay of Glass Beaker Bound Oxalate Oxidase The assay of glass beaker bound enzyme was carried out in the same beaker and in the same manner as described for free enzyme except that 0.1 ml reaction buffer replaced the free enzyme solution. The blank/control was run using the same procedure in another 25 ml glass beaker containing affixed glutaraldehyde activated glass beads on its inner base (Fig. 1). Kinetic Properties of Glass Beaker Bound Oxalate Oxidase Various kinetic properties of glass beaker bound oxalate oxidase, viz. ph optima, incubation temperature for maximum activity, time course study and effect of substrate concentration, were determined

3 54 INDIAN J BIOTECHNOL, JANUARY 2004 Fig. 1 Assay of glass beaker bound oxalate oxidase. Beaker 1: Blank, containing activated alkylamine glass beads affixed on the base and reaction mixture; Beaker 2: Test, containing oxalate oxidase of forage sorgham leaf bound on alkylamine glass beads affixed on the base and rection mixture and K m and V max were calculated. To determine the effect of ph on immobilized enzyme, the ph of reaction buffer was varied from 4 to 7, each at a final concentration of 0.05 M, using the following buffer: ph 4-6, sodium succinate buffer and ph , sodium phosphate buffer. The activation energy (E a ) of the immobilized enzyme was calculated from Arrhenius plot; whereas, K m and V max were calculated from Lineweaver-Burk plot. Determination of urinary Oxalate by Oxalate Oxidase Bound Glass Beaker Collection and Pretreatment of Urine Sample Urine samples of 24 hrs were collected in plastic bottles, containing a few drops of concentrated HCl, from apparently healthy adults (males and females) and stored at 4 C until used. Acidified urine (1 ml) was taken in a 15 ml graduated centrifuge tube and 1 ml CaCl 2 (5g//l) solution was added to the tube. The final ph was adjusted around 7.0 by adding NaOH. Then 8.0 ml absolute ethyl alcohol was added and the tubes were immediately covered with aluminium foil and kept at room temperature for precipitation. After 24 hrs, tubes were centrifuged at 3,000 rpm for 5 min and the precipitate was dissolved in 1.0 ml HCl (0.1 N) 11. Assay of Urinary Oxalate Assay of urinary oxalate was done as described for the glass beaker bound oxalate oxidase except that oxalate was replaced by 0.1 ml of pretreated urine. The oxalate concentration in the sample was Fig. 2 Standard curve of oxalate employing oxalate oxidase of forage sorghum leaf immobilized on glass beads affixed inside a glass beaker calculated from standard curve between oxalate concentrations vs A 520 (Fig. 2). Reusability and storage of glass beaker bound oxalate oxidase To reuse the glass beaker bound enzyme, the beaker was rinsed 3-4 times with reaction buffer [sodium succinate buffer (0.05 M, ph 5.0)] prior to its use in the next assay. To store the immobilized enzyme, 2 ml reaction buffer was added to the beaker and kept at 4 C until used. Results and Discussion An oxalate oxidase was purified 4-fold from leaves of 10-day-old plants of forage sorghum by ammonium sulphate fractionation. The partially purified enzyme showed a specific activity of U/mg. The enzyme was immobilized covalently onto alkylamine glass beads affixed on the inner base of a glass beaker with a non-reactive fixative. The conjugation yield was reported to be 182 mg/g support (Table 1), which is higher than that reported for grain sorghum leaf (10.8 mg/g support) and barley root enzyme on free alkylamine glass beads (6.6 mg/g support) and barley enzyme on alkylamine glass beads affixed on the inner base of glass beaker with Aaraldite fixative 2,12,13. For determination of urinary oxalate, an oxalate oxidase purified from sorghum leaf and barley root was earlier also immobilized on alkylamine glass beads and nylon tubing, respectively 2,14.

4 [ KALRA & PUNDIR:URINARY OXALATE DETERMINATION BY IMMOBILIZED OXALATE OXIDASE 55 Kinetic Properties of Immobilized Oxalate Oxidase A comparison of kinetic properties of glass beaker bound oxalate oxidase with those of its free form is summarized in Table 2. The optimum ph of enzyme was increased from 4.5 to 5.5 after immobilization. Similar results have also been shown by earlier reports on this enzyme from other plant sources, e.g. grain sorghum leaf 2, barley root 12,13,15 and beet stem 16. The increase in optimum ph of enzyme after immobilization might be due to loss of NH 2 group of enzyme for its covalent coupling with alkylamine glass beads. The enzyme showed maximum activity at 37 C, similar to that of free enzyme but slightly lower as compared to free grain sorghum leaf enzyme (40 C) 6, and slightly higher than that for free barley root enzyme (35 C) 8 and barley root enzyme immobilized to alkylamine glass beads affixed on inner base of a glass beaker (30 C) 13. The change in optimum temperature of enzyme after immobilization has also been reported in other plant enzymes 16,17-20 that might be due to alteration in configuration of enzyme or due to steric hindrance or diffusion effects, which also protect the enzyme against heat denaturation. The rate of reaction was linear up to 5 min, which is lower than that of free enzyme (7 min) but equal to that of grain sorghum enzyme immobilized on free alkylamine glass beads (5 min) 2 and barley enzyme on affixed alkylamine glass beads (5 min) 13. K m value of oxalate oxidase increased after immobilization. Moreover, immobilized enzyme had higher K m value than that reported for grain sorghum enzyme immobilized on free alkylamine glass beads ( M) 21 and barley oxalate oxidase immobilized on affixed alkylamine glass beads ( M) 13. V max value of immobilized enzyme was reported to be 0.02 µmol/min, which was almost similar to that of free enzyme but lower than that reported for free grain sorghum enzyme (0.285 µmol/min) 6 and for the same enzyme bound to free alkylamine glass beads (0.25 µmol/min) 21, and also for the barley enzyme bound to free and affixed alkylamine glass beads (0.066 µmol/min) 12. Whenever an enzyme is coupled to a solid support, the kinetic pattern of the reaction changes considerably, leading to changes in K m and V max. The changes in kinetic parameters of immobilized enzyme are controlled by four factors: Change in enzyme configuration, steric effects, microenvironmental effects, and bulk and diffusion effects 12. E a of immobilized enzyme was calculated to be 4.50 Kcal/mol, which is higher as compared to that of free enzyme ( Kcal/mol) but lower than that reported for free barley enzyme (9.140 Kcal/mol) 8, and barley enzyme immobilized onto free alkylamine glass beads (11.46 Kcal/mol) 12 and affixed alkylamine glass beads as inner base of a beaker (6.71 Kcal/mol) 13. The increase in E a after immobilization Table 1 Immobilization of oxalate oxidase of forage sorghum leaf onto zirconia coated alkylamine glass beads (pore diam 55 nm) affixed onto inner base of a glass beaker Oxalate oxidase added to 80 mg alkylamine glass beads (mg) Oxalate oxidase coupled to 80 mg glass beads (mg) % oxalate oxidase coupled Units of oxalate oxidase added % retention of sp. activity Conjugation yield (mg/g) Table 2 A comparison of kinetic parameters of oxalate oxidase of forage sorghum leaf immobilized on alkylamine glass beads affixed on inner base of a glass beaker with those of free enzyme Parameters Free oxalate oxidase Oxalate oxidase immobilized to affixed glass beads Optimum ph Optimum temperature ( C) E a (Kcal/mole) Time for linearity (min) 7 5 K m for oxalate (M) V max (μmol H 2 O 2 /min) Stability at 4 C 50% loss in 15 days 50% loss in 12 days % inhibition by NaCl (100 mm) ND ND ND = Not Detected.

5 56 INDIAN J BIOTECHNOL, JANUARY 2004 might be due to conformational changes in enzyme or change of polarity or ionic strength in the vicinity of active site of enzyme. Earlier methods for discrete analysis of urinary oxalate were also based on the quantification of H 2 O 2 generated in urinary oxalate by immobilized enzyme, using a colour reaction consisting of phenol, 4- aminophenazene and horseradish peroxidase as chromogenic system. The present method, however, has advantage that it uses partially purified oxalate oxidase from a cheaper, widely available and fungal resistant plant source, i.e. forage sorghum leaf and provides ease in handling of glass beads bound enzyme. Earlier methods, employing oxalate oxidase bound to free alkylamine glass beads, included a tedious and time consuming handling of glass beads, along with risk of their loss during the transfer of reaction mixture from reaction flask to cuvette and their washing for reuse in next assay. The present method has no such problems as the glass beads are affixed. Furthermore, the forage sorghum enzyme is insensitive to Cl -, unlike barley, moss, beet stem and banana peel enzyme 21 and thus prior to assay requires no pretreatment of urine for removal of Cl -. The oxalate values in 24 hrs urine samples as measured by the present method were in the range of 12.5 to 29.7 mg/l (mean 20.9 mg/l) for females and 25.7 to 45.4 mg/l (mean 37.2 mg./l) for males. These values of urinary oxalate are comparable to those reported earlier, e.g to 40.5 mg /l (mean 19.5 mg/l) 2, 8 to mg/l (mean 16.6 mg/l) by free sorghum oxalate oxidase 22, 18.9 to 63.9 mg/l (mean 36.9 mg/l) in isotope dilution and direct colorimetry 23, 27.9 to 43.2 mg/l (mean 37.8 mg/l) by free moss oxalate oxidase 24, 22.5 to 45.9 mg/l (mean 34.2 mg/l) by free beet stem oxalate oxidase 25, 14.0 to 35.6 mg/l in continuous flow system using barley oxalate oxidase immobilized to nylon tubing 14, 12.2 to 28.0 mg/l (mean 19.8mg/l) in discrete analysis using alkylamine glass bound barley oxalate oxidase and arylamine glass bound horseradish peroxidase 26, 4.8 to 37.9 mg/l by immune complex of banana oxalate oxidase 27, and 6.6 to 35.4 mg/l urine by acrylamide membrane bound banana oxalate oxidase 19. 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