Detection of an oxalate oxidase in grain Sorghum roots

Pundir, C.S.; Kuchhal, N. K.

doi:10.1016/0031-9422(89)80251-1

Cited by 13 publications

(8 citation statements)

References 11 publications

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“…The roots were collected and oxalate oxidase was extracted and partially puri®ed as described previously. 6 The enzyme was further puri®ed by gel ®ltration on a Sephadex G-200 column (1´52 45 cm) using 20 mmol/L potassium phosphate buffer (pH 7´0), followed by passage of the active fractions through a DEAE-Sephacel column (2´52 29 cm) using a linear gradient of KCl (0±0´6 mol/L) in 20 mmol/L potassium phosphate buffer pH 7´0. Active DEAE-Sephacel fractions were precipitated with 0±70% (NH 4 ) 2 SO 4 and then further puri®ed on a Sephadex G-200 column, (1´52 30 cm) using 20 mmol/L potassium phosphate buffer (pH 7´0).…”

Section: Preparation and Puri®cation Of Oxalate Oxidasementioning

confidence: 99%

“…In our laboratory, we have puri®ed an oxalate oxidase from roots of 10-day-old seedling plants of a grain sorghum hybrid (CSH-5), which is insensitive to physiological concentrations of chloride (250 mmol/L) and nitrate (8 mmol/L) and therefore more suitable for the enzymic determination of urinary oxalate than enzymes from other sources. 6 …”

Section: From the Department Of Bio-sciences Maharshi Dayanand Univementioning

confidence: 99%

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Quantification of urinary oxalate with a chloride- and nitrate-insensitive oxalate oxidase

Kuchhal

Thakur²,

Pundir³

2000

Ann Clin Biochem

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Methods for the determination of urinary oxalate employing oxalate oxidase extracted from mosses, barley, banana peel and beet stems may suffer from interference by physiological concentrations of chloride and nitrate normally found in urine. 1±4 The normal daily excretion of chloride and nitrate into urine is 250 mmol/L and 8 mmol/L, respectively. This interference can be removed either by passing the urine through an ion-exchange column or by precipitating the oxalate from the urine and redissolving it prior to assay. 1,5 This pretreatment reduces the sensitivity and reproducibility of the determination of oxalate. In our laboratory, we have puri®ed an oxalate oxidase from roots of 10-day-old seedling plants of a grain sorghum hybrid (CSH-5), which is insensitive to physiological concentrations of chloride (250 mmol/L) and nitrate (8 mmol/L) and therefore more suitable for the enzymic determination of urinary oxalate than enzymes from other sources. 6 METHODS Preparation and puri®cation of oxalate oxidaseApart from Sephadex and Sephacel (which were obtained from Pharmacia LKB, Uppsala, Sweden) all reagents were obtained from Sigma Chemical Co (St Louis, MO, USA). Ten-day-old seedling plants of grain sorghum hybrid (CSH-5) were raised in the laboratory. The roots were collected and oxalate oxidase was extracted and partially puri®ed as described previously. 6 The enzyme was further puri®ed by gel ®ltration on a Sephadex G-200 column (1´52 45 cm) using 20 mmol/L potassium phosphate buffer (pH 7´0), followed by passage of the active fractions through a DEAE-Sephacel column (2´52 29 cm) using a linear gradient of KCl (0±0´6 mol/L) in 20 mmol/L potassium phosphate buffer pH 7´0. Active DEAE-Sephacel fractions were precipitated with 0±70% (NH 4 ) 2 SO 4 and then further puri®ed on a Sephadex G-200 column, (1´52 30 cm) using 20 mmol/L potassium phosphate buffer (pH 7´0). The active fractions of the last step were pooled and treated as puri®ed enzyme. The puri®ed enzyme exhibited a single band in polyacrylamide gel disc electrophoresis using Coomassie blue as protein stain and had a speci®c activity of 1´9 units/mg. One enzyme unit is de®ned as the amount of enzyme required to produce 1 mmol of H 2 O 2 /min/mL at pH 5´0 and 40 Ê C. The puri®ed enzyme was stable over a period of 1 year when stored at 2 20 Ê C in 20 mmol/L sodium phosphate buffer pH 7´0. Oxalate assayTo determine urinary oxalate content, 24-h urine samples were collected from apparently healthy individuals in 2-L plastic bottles containing 15 mL of concentrated HCl. The ®nal pH of the urine was maintained below pH 1´5 during and after the collection. Acidi®ed urine (1´0 mL) was diluted with potassium phosphate buffer (100 mmol/L pH 7´0) in 1:1 ratio and its ®nal pH was adjusted to between 5´0 and 7´0 by adding 5 mol/L NaOH dropwise with constant mixing. To prevent oxalogenesis from ascorbate present in the urine, 0´1 mL of buffered NaNO 2 (35 mg/10 mL in 10 mmol/L sodium phosphate buffer pH 7´0) was added to 2´0 mL of diluted urine.The colour reagent f...

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Section: Preparation and Puri®cation Of Oxalate Oxidasementioning

confidence: 99%

Section: From the Department Of Bio-sciences Maharshi Dayanand Univementioning

confidence: 99%

Quantification of urinary oxalate with a chloride- and nitrate-insensitive oxalate oxidase

Kuchhal

Thakur²,

Pundir³

2000

Ann Clin Biochem

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“…Various methods have been formulated for the assay of oxalate from different sources, for instance, gas-liquid chromatography, ion chromatography, high performance liquid chromatography, mass spectrometry and enzymatic determination methods have all been developed. Of these, the oxalate oxidase activity-based determination of oxalate has become very popular and is used widely because of its simplicity, specificity and sensitivity (Pundir et al, 1985). Oxalic acid has usually been seen as an inert end product of metabolism and only plants have been reported to be able to metabolize oxalic acid and oxalates.…”

Section: Wwwintechopencommentioning

confidence: 99%

The Roles of Germin Gene Products in Plants Under Salt Stress

Çalışkan¹

2011

Abiotic Stress Response in Plants - Physiological, Biochemical and Genetic Perspectives

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“…For ox a late anal y sis, the most used en zyme has been the ox a late oxidase (E.C. 1.2.3.4) from dif fer ent veg eta bles sources such as bar ley 3 , ba nanas 4 , amarantus 5 , sorghum 6 , beets 7 , etc. As far as we know, there are no meth ods that use the bio log i cal sup ports for en zyme im mo bi li za tion that are or not em ployed in flow in jec tion anal y sis de scribed in the lit er ature.…”

Section: Introductionmentioning

confidence: 99%

“…In re cent years a large num ber of meth ods [1][2][3][4][5][6][7] have been de vel oped to im mo bi lize en zymes on solid ma tri ces. The most pre dom i nant method is that of car rier bind ing, and many com mer cially avail able im mo bi lized en zymes are found with syn thetic ma tri ces.…”

Section: Introductionmentioning

confidence: 99%

Oxalate determination in urine using an immobilized enzyme on sorghum vulgare seeds in a flow injection conductimetric system

Neto¹,

Tubino²,

Godinho³

et al. 1997

J. Braz. Chem. Soc.

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Desenvolveu-se um método por injeção em fluxo para determinação de oxalato em urina, baseado na utilização da oxalato oxidase (E.C. 1.2.3.4) imobilizada em sementes trituradas de Sor ghum vulgare, variedade BR-303. Amostras de 200 µL contendo oxalato são introduzidas num fluxo de água deionizada que passa por um reator, em forma de coluna, preenchido com o ma te rial enzimático ativado. O dióxido de carbono produzido pela reação enzimática é conduzido, pelo fluxo, até uma cela de permeação, contendo uma membrana de PTFE, onde permeia para um outro fluxo de água deionizada. Este fluxo passa por uma cela de condutividade. A presença de dióxido de carbono provoca uma diferença na condutividade, proporcional à concentração de oxalato originalmente presente na amostra. Os resultados obtidos mostram uma faixa de linearidade en tre 0,05 e 0,50 mmol dm -3. O método proposto, quando comparado com o procedimento enzimático da Sigma, mostra uma boa correlação (Y = 0,006( ±0,016) + 0,98( ±0,019) X, r = 0,9995, Y = condutividade em µS e X = concentração em mmol dm -3 ), seletividade e sensibilidade. O novo procedimento de imobilização promove grande aumento de estabilidade da enzima permitindo a determinação de oxalato por cerca de seis meses. Cerca de 13 determinações podem ser realizadas por hora. A precisão do método proposto é bastante satisfatória (d.p.r. = ± 3,2 %).A flow-injection (FI) method was de vel oped for the de ter mi na tion of ox a late in urine. It was based on the use of ox a late oxidase (E.C. 1.2.3.4) im mo bi lized on ground seeds of the BR-303 Sor ghum vulgare va ri ety. A re ac tor was filled with this ac ti vated ma te rial, and the sam ples (200 µL) con tain ing ox a late were passed through it, car ried by a deionized wa ter flow. The car bon di ox ide pro duced by the en zyme re ac tion per me ated through a microporous PTFE mem brane, and was re ceived in a wa ter ac cep tor stream, pro mot ing con duc tiv ity changes propor tional to the ox a late con cen tra tion in the sam ple. The re sults ob tained showed a use ful lin ear range from 0.05 to 0.50 mmol dm -3 . The pro posed method, when com pared with the Sigma enzy matic pro ce dure, showed good cor re la tion (Y = 0.006( ±0.016) + 0.98( ±0.019)X; r = 0.9995, Y = con duc tiv ity in µS, and X = con cen tra tion in mmol dm -3), se lec tiv ity, and sen si tiv ity. The new im mo bi li za tion ap proach pro motes greater sta bil ity, al low ing ox a late de ter mi na tion for 6 months. About 13 de ter mi na tions can be per formed per hour. The pre ci sion of the pro posed method is about ± 3.2 % (r.s.d).

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Detection of an oxalate oxidase in grain Sorghum roots

Cited by 13 publications

References 11 publications

Quantification of urinary oxalate with a chloride- and nitrate-insensitive oxalate oxidase

Quantification of urinary oxalate with a chloride- and nitrate-insensitive oxalate oxidase

The Roles of Germin Gene Products in Plants Under Salt Stress

Oxalate determination in urine using an immobilized enzyme on sorghum vulgare seeds in a flow injection conductimetric system

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