Indian Journal of Biochemistry & Biophysics

Vol. 48, February 2011, pp. 42-46

Purification and partial characterization of oxalate oxidase from leaves of forage

Sorghum (Sorghum vulgare var. KH-105) seedlings
Rajender Kumar1, Vinita Hooda2 and C S Pundir1*
1
Biochemistry Research Laboratory, Dept. of Biochemistry, 2Department of Botany, M.D. University, Rohtak-124001 (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 2.0338 Kcal/mol. The rate of reaction was linear up to 7 min.
Km value for oxalate was 0.22 mM. The enzyme was stimulated by Cu2+ and inhibited by EDTA, NaCN,
diethyldithiocarbamate, Na2SO4, 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-H2SO4 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-modelling1. Breakdown of oxalate in higher
plants is of great importance, since it could lead to the
availability of free Ca2+. Two types of oxalate
degrading enzymes are known in higher plants:
oxalate decarboxylase (E.C. 4.1.1.2) which catalyzes
ATP and Co-A dependent breakdown of oxalate with
release of CO2 and formic acid and oxalate oxidase
(E.C. 1.2.3.4) which catalyzes the oxidative
breakdown of oxalate into H2O2, and CO22. The
former enzyme has been studied in pea and other
plants3. The later enzyme has generated more interest,
as it releases H2O2, 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 diseases4.
Oxalate oxidase has been found in higher plants in
two forms: membrane-bound and soluble. Membrane-
bound enzyme has been purified from beet stems5 and
Amaranthus spinosus leaves6 and soluble enzyme
from roots of barley seedlings7-9 and kernel10, banana
——————
*Corresponding author
E-mail: pundircs@rediffmail.com
Fax: (95) (1262) 294640
fruit peel11, grain Sorghum leaves12 and wheat
seedlings13. 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 laboratory14-19. 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 efficiency20.
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’-bismethylene-
acrylamide, tris base, ammoniumpersulphate, glycine,
 KUMAR et al.: OXALATE OXIDASE FROM FORAGE SORGHUM
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 laboratory21 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
protein14. The crude enzyme (15000 × g supernatant)
was purified in cold (4-8°C) by 0-80% (NH4)2SO4
fractionation, gel filtration on Sephadex G-200 and
ion-exchange chromatography on DEAE-Sephacel12.
The ultrafiltration of purified enzyme was carried out
by Amicon concentrator at 4°C. The activity and
protein content of purified enzyme were measured22.
Assay of oxalate oxidase
Assay of oxalate oxidase was carried out in a 15 ml
test tube wrapped with black paper14. The reaction
mixture containing 1.8 ml 0.05 M sodium succinate
buffer (pH 4.0), 0.1 ml CuSO4 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. A520
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 H2O2 generated in the assay was calculated from
the standard curve between H2O2 concentrations
vs. A520. One unit of enzyme was defined as the
amount of enzyme required to catalyze the generation
of 1.0 nmol of H2O2 from oxalate per min under
standard assay conditions.
The protein in various enzyme preparations was
determined using the method of Lowry et al22.
43
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 Davis23.
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 4.0-6.0: sodium succinate. To
determine the optimum temperature of the enzyme,
the reaction mixture was incubated at different
temperatures ranging from 25-50°C at 5°C intervals.
Energy of activation (Ea) 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. Km 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 Cu2+ 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-H2SO4 reaction24.
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 NaNO2
(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
44 INDIAN J. BIOCHEM . BIOPHYS., VOL. 48, FEBRUARY 2011
CuSO4 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. A520 was read. The oxalate
concentration in urine was extrapolated from standard
curve between oxalate concentration ranging from
0.01 to 0.2 mM and A520 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]
Table 1—Purification of oxalate oxidase from leaves of 10-days old forage Sorghum seedling plants
Purification step Total volume Protein
(Units/ml)
Crude enzyme (15000 × g) supernatant 1000 5.39
(NH4)2SO4 Precipitate (0-80%) 35 12.5
DEAE-Sephacel fraction 60 1.01
Sephadex G-200 fraction 100 0.15
Ultra-filtered enzyme 15 0.33
One enzyme unit was defined as amount of enzyme required to generate 1 nmol H2O2/min/ml under standard assay.
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 (Ea) of the
purified enzyme as calculated from Arrhenius plot
was 2.033 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 seedlings7 and grain Sorghum leaf12.
Km 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 Cu2+-specific chelator) caused strong inhibition of
the enzyme, indicating the requirement of Cu2+ for the
enzyme, similar to that from grain Sorghum leaf12.
Among the various metals tested each at a final
concentration of 1 mM, only CuSO4 stimulated the
enzyme. Cu+2 binds with two adjacent –SH group(s)
of peptide backbone of the enzyme to form a cupro-
sulfhydryl complex which facilitates binding of
Activity Specific activity Purification fold Yield
(Units/ml) (%)
90 16.7 1 100
771 61.68 3.69 30.0
375 371.28 22.23 25.0
180 1200 71.85 20.0
600 1818.18 108.87 10.0
 KUMAR et al.: OXALATE OXIDASE FROM FORAGE SORGHUM 45

Table 2—Properties of oxalate oxidase purified from leaves of forage Table 3—Effect of metal chelators and metal salts on forage Sorghum
Sorghum and its comparison with those from grain Sorghum leaf leaf oxalate oxidase
and barley seedlings Compound added Relative activity
Kinetic parameter Forage Grain Sorghum Barley (Final conc. 0.1 mM) (%)
Sorghum leaf leaf12 seedlings9
None 100.0
Mr (kDa) 124 120 125 EDTA 61.5
Subunit (No) 2 2 5 Diethyldithiocarbamate 30.7
Optimum pH 4.5 5.0 3.8 CaCl2 92.3
KCl 100.0
Optimum temp (°C) 37 40 37 MgSO4 138.0
Energy of activation 2.0338 4.4 NA CuSO4 176.0
(Ea) (kcal/mol) ZnSO4 61.3
Time for linearity 7 2 5 FeSO4 92.3
(min) Na2SO4 48.4
Km for oxalate (mM) 0.22 0.078 0.27 H3PO4 76.9
NaCl (0.1, 1, 10, 100, 200 mM) 100, 69.2, 61.3, 48.4, 30.4
Inhibition by NaCl ND ND 14
(1 mM) (%) Standard assay conditions were used, except for the addition as
indicated above
Stimulation by Cu2+ 76 200 10
(0.5 mM) (%) Table 4—Effect of coenzymes, Cu2+ and flavins on forage Sorghum
Stimulation by FAD 55 31 ND leaf oxalate oxidase
(0.5 mM) in presence Compounds added Relative activity
of Cu2+ (1 mm) (%) (Final conc. 0.1 mM) (%)
Nature Glycoprotein Cu2+ requiring Mn2+ None 100.0
glycoprotein requiring Riboflavin 133.3
glycoprotein NAD+ 116.6
ND = Not detected; NA = Not available FMN 145.0
FAD 155.5
oxalate to the enzyme, similar to grain Sorghum root Cu2+ 147.0
oxalate oxidase25. However, further studies are Riboflavin + Cu2+ 152.0
required to elaborate the stimulatory role of Cu+2 in this NAD+ + Cu2+ 147.0
FAD + Cu2+ 188.0
enzyme. Na2SO4 caused 52% inhibition of the enzyme,
FMN + Cu2+ 165.0
while rest of the metal salts had practically no effect.
Standard assay conditions were used, except that the dialyzed
However, NaCl at high concentration caused inhibition enzyme was used and the addition of compound as indicated above.
of enzyme, which increased with its concentration
The UV and visible spectra of purified enzyme did
(Table 3), similar to barley seedling enzyme7.
not show peak at 370 nm and 450 nm, a characteristic
Among the co-enzymes (riboflavin, FMN, FAD,
of flavoprotein. The purified enzyme was also not
NAD+) tested each at a concentration of 0.1 mM,
yellow in color. Hence this enzyme can not be
flavins caused slight stimulation of the enzyme, while classified as a flavoprotein. Nevertheless, the enzyme
NAD+ had no effect similar to that from grain solution gave brown color after heating it with
Sorghum leaf12. Although our observations suggest orcinol-H2SO4 reagent, indicating glycoprotein nature.
that forage Sorghum enzyme is not a flavoprotein, Earlier, barley root enzyme was also classified as a
slight stimulation of the enzyme by flavins might be glycoprotein9,26.
due to their protective effect against NaCl inhibition. The urinary oxalate measured in healthy male
Earlier, flavins have been reported to protect barley adults by forage Sorghum enzyme ranged from 18 to
enzyme from inhibition by sodium fluoride7. The 32 mg/L with a mean ± S.E. of 22.6 ± 0.47 (n = 12)
stimulating effect of Cu2+ on dialyzed enzyme was which is comparable to earlier reported values
increased in presence of flavins which was an additive employing oxalate oxidase from mosses (28-43 mg/L,
effect of two stimulants together and in the following mean 37.7)27, barley (10-40.5 mg/L, mean 20.9)28,
order FAD>FMN>riboflavin (Table 4). beet stem (22.9-46.1 mg/L, mean 33.8)5, banana peel
Among the other compounds tested each at a final (4.80-37.5 mg/L, mean 17.2)11, grain Sorghum
concentration of 0.1 mM, NaCN inhibited the enzyme (9-23 mg/L, mean 16.7)12 and alkylamine glass beads
activity completely, while sodium dithionate and bound forage Sorghum (25.7-45.4 mg/L, mean 37.2)15.
potassium ferricyanide caused 85% and 24% The method has the advantage that it does not require
inhibition of the enzyme reaction respectively. pre-treatment of urine for removal of chloride ion as
46 INDIAN J. BIOCHEM . BIOPHYS., VOL. 48, FEBRUARY 2011
forage Sorghum enzyme is insensitive to chloride ion
(at low concentration) unlike barley enzyme7.
Moreover the source of present enzyme is cheaper and
widely available compared to the source of earlier
reported chloride-insensitive grain Sorghum enzyme12.
In conclusion, an oxalate oxidase was purified and
characterized from forage Sorghum leaves. The
enzyme had two identical subunits, stimulated by
Cu2+ and was a glycoprotein. The enzyme is a better
analyte for urinary oxalate determination over earlier
reported enzymes.
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