Kinetic Study of the Enzyme Urease from Dolichos biflorus
K. R. Natarajan
Center for Advancement of Biochemical Sciences, 113, Habibullah Road, Madras 600 017, India
Urease was the first enzyme to be crystallized (1) and
also the first enzvme shown to contain nickel (2).Its abilitv
to catalyze the hydrolysis of u r u to c;~rl)on-dioxidennd
nnimoni;~olivr.i . pruspects for use in art~ficialk~dnevs.Re-
. powder (1.0 g) is stirred with 10 mL of phosphate buffer
cause the ammonium ion produced is toxic, its ratehf pro-
duction and removal are important for the utilization of
urease i n artificial kidney devices. Thus, the kinetic
HzNCONHz+ 3H20 + CO, f + 2NH40H
study of the urease-catalyzed hydrolysis of urea is primary
and fundamental i n the search towards develooment of an
artificial kidney.
The main source of urease is jack beans (Canaualia ensi-
formis) and soya beans (Glycine man). I t also occurs in
other plant tissues and microorganisms. In this experi-
ment we report the kinetic study of urease extracted from
the seeds of Dolichos biflorus (also called Dolichos uni-
florus, horse gram, horse grain, madras grain, kollu), an
annual vine widely cultivated in tropical Asia and Africa.
I t is used as a fodder crop and is also grown extensively a s
a cover crop i n these regions. Seed meal that contains anti
At human blood group specificity is routinely used in dis-
tinguishing between Al and A? blood groups or to confirm
the presence of Al blood-group substance (3). Seeds of
Dolichos biflorus have been selected as a source of urease
because i t is inexpensive (the cheapest edible legume) and
readily available (found i n grocery stores i n India) with
high levels of urease.
Assay Principle
The procedure for the urease assay i s based on Ber-
thelot's reaction (4). The ammonium ion formed reacts
with phenol in the presence of hypochlorite to give the blue
dye, indophenol.
Indophenol (Blue)
Intensity of the color produced is proportional to the con-
centration of urea in the sample and is measured a t 630 nm.
The reaction requires a pH around 13 and is complete within
2 0 3 0 min a t 37 "C. The color remains constant for up to 24
h. Addition of sodium nitroprusside, which acts as a catalyst
in the reaction, increases the yield of indophenol dye, the in-
tensity of color obtained, and its reproducibility (5).
Experimental
Reagents and Equipment
Buffer: 0.1 M sodium phosphate buffer, pH 7.01, con-
taining 1mM EDTA (disodinm salt)
Substrate: P r e ~ a r ea 0.1 M urea (Simna) solution i n
phosphatv bulrer. ;\rid two drops o f ~ l 1 ~ l ' ~pnwnvirive.
:ii
L'rea solu~ionis stable when stored at 4 'C. Preuare solu-
tions of fresh urea on the day of use because urea slowly
556 Journal of Chemical Education
decomposes into ammonium and cyanate ions, which in-
terfere with the assay.
Enzyme: Horse gram seeds are ground into powder. The
containing 5 mM 2-mercaptoethanol for 1h. The suspen-
sion is centrifuged a t 2000 rpm for 1 5 min. The snper-
natant is used as the stock enzyme solution and stored a t
3-5 "C.
Caution: Phenol is corrosive and should be handled and
weighed carefully.
Phenol-Nitroprusside: Dissolve 4.7 g of analytical-
grade reagent phenol in 100 mL of deionized water con-
taining 6 mg of sodium nitroprusside. Store in a brown
bottle a t 4 "C. The reagent is stable for 3 months.
Caution: Chlorine and alkaline hypochlorite solution
should be used in a fuming hood.
Careful: All reagents should he free from ammonium ion,
and the experiment should be carried out in an arnmania-
free environment.
Hypochlorite Reagent: Dissolve 2 g of sodium hydroxide
in 100 mL of deionized water containing 7.5 mL of a com-
mercially available bleach (5% NaOCI). Pass chlorine gas
into this solution and store in a brown bottle a t 4 "C. (Use
of commercial bleach alone gave erratic results i n the Ber-
thelot reaction. Passing chlorine into alkaline hypochlorite
solution is essential to get consistent results.)
Special Equipment: visible spectrophotometer or calo-
rimeter, pH meter, and centrifuge
Enzyme Assay
Appropriate volumes of 0.1 M urea solution were added
in each tube containing 10 KL of horse gram extract and
buffer to give final concentrations of urea ranging from
0.001 M to 0.05 M i n the reaction mixture. Volume was
adjusted i n each tube to give 1 mL of reaction mixture.
There should be a 10-s staggering in the addition of sub-
strate between tubes down the line. Reaction mixture as
prepared above was incubated for exactly 5 min a t 30 OC
and terminated by the addition of 2.0 mL of phenol reagent
".
followed bv 2.0 mL of alkaline hv~ochlorite ., The
reaeent.
mlxrure was incubated at 50-6O'C for I0 min after vortex-
ing. Itengrnt blanks tiom which enz,vmc had heen irmittcd
w(!rt, pr(:p:~ndand trrarrd in a similar mannel- The abior-
bancc a1 630 rlm was mcasurcd ;~ft(!r1:3 dilution. 1 volume
of solution:2 volumes of water) with deionized water.
Ammonium ion released as a result of urea hydrolysis
was determined by referring to a previously prepared
standard curve relating absorbance a t 630 n m for ammo-
nium ions using 5 x lo4 M ammonium chloride solution
~ r e u a r e dfrom 1M NHaCl solution. Under the conditions
;,f & cxpvrimrnt, 1 n i i of ammonium chloride solution
containine 0.5 ~ m o atl 1 :3 dilution rave a maximum nl)i~,r-
bance of 0.7, which is used i n this experiment for calculat-
ing reaction rates.
If the absorbance is greater than 0.8, the solution is di-
luted further with deionized water, and the absorbance is
measured. Do not forget to multiply absorbance value by
Figure

Figure 2.
the dilution factor. The release of 2 pmol of ammonia was
considered to be equivalent to the hydrolysis of 1pmol of
urea. One unit of urease activity is defined a s the amount linear form. The linear form of the Michaelis-Menten
of enzyme that would hydrolyze 1pmol of urea per minute equation can be obtained by taking the reciprocal of both
under the reaction conditions described above. sides of eq 1.
We have found this procedure very useful in detailed ki- 1 1 K,
uetic studies of urease from Dolichos biflorus seeds and - +- (2)
u "m, " m S
other leguminous seeds. Y=a+bx
Treatment of Experimental Data
This double reciprocal form of the Michaelis-Menten
For each assay determine t h e velocity, t h a t is, the rate law predicts that a plot of l l u vs. 11s would be linear
amount of urea converted in 1min for each substrate con- with an ordinate intercept (y-axis)of IN,,, abscissa inter-
centration. Then average the data and calculate the aver- cept (x-axis) of -llK,, and a slope of KJV,,,. Such a plot,
age velocity. Plot velocity against substrate concentration. the Lineweaver-Burke plot, is shown in Figure 2 for the
Calculate the reciprocals of velocity and substrate concen- urease-urea reaction from the experimental data. From
tration, and plot l l u vs. 11s. the reciprocal plot, values of K, and V, were found to be
7.4 mM and 1.6 x lo4 mMimin. Because these results are
Results and Discussion determlncd from x-axis and y-iixii intercepts ova stra~ght
A plot of velocity vs. substrate concentration is a rectan- line, they are considered to be more rclial)le than those oh-
gular hyperbola (Fig. 1). The mathematical equation that tained by approximation from the Michaelis-Menten
explains this behavior is called the Michaelis-Menten rate curve (Fig. 1).
law, The experiment described above is easy to carry out and
"=- "
&
, can be completed by students in a 3-4-h laboratory period
(1)
K,+S a s part of biochemist~lorganicchemistry laboratory exer-
where u is the initial velocity; V, is the maximal velocity; cise. This experiment may be considered for a biochemistry
and K, is the Michaelis constant. Its derivation can be undergraduate course to illustrate the basic principles of
found in anv text book of biochemistrv enzyme kinetics.
" (6).
. .
At a particular set of experimental conditions (pH, tem- ~ ~ k ~ ~ ~ l ~ d
perature, buffer concentration, etc.), values of K, and V,.
can be experimentally evaluated to provide information I thank P V. Sundaram, Director, Centre for Protein En-
about the kinetic properties ofthe enzyme under study. gineering and Biomedical Research, Voluntary Health
values of K, and v,, can be determined Services, Madras, for providing facilities.
graphically from the Michaelis-Menten curve shown in Literature Cited
Figure 1.In this experimentsK, and V, were found to be sumner J. B,J,Bioi, Cham, 1926,69,435,
6.5 mM (substrate concentration a t half of maximal veloc- 2. uixon, N. E.; G ~ ~ c.;B~ I~ ~R. ~~zerner,~.
I L.; ~, I J~ A ~~chpm.
. SW. 1915,97,4131.
ity) and 1.5 x 104mM/min. 3. ~ t . 1 M w.;
~ ~ . &bat, E. A. ~ i ~ 1916.9.869.
h ~ ~ ~ ~ l ~
More precise values for K, and V, may be obtained if
the same experimental data are displayed graphically in
i: ~ {
6. R=W",
~ ~ ~ ~ ~ ; ~ a ~ a lsl,
J . u. Biochemistry;N ~~ ~
a fI
~ ~ ~ , ~ ~
f e - ~B ~U: ~ I ~ ~ ~ ~ O ~ ,169.
1989:~ NC,
~ " . l ~ ~

Volume 72 Number 6 June 1995 557