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. cause the ammonium ion produced is toxic, its ratehf production and removal are important for the utilization of urease i n artificial kidney devices. Thus, the kinetic HzNCONHz+ 3H20 + CO, f + 2NH40H
decomposes into ammonium and cyanate ions, which interfere with the assay. Enzyme: Horse gram seeds are ground into powder. The powder (1.0 g) is stirred with 10 mL of phosphate buffer containing 5 mM 2-mercaptoethanol for 1h. The suspension is centrifuged a t 2000 rpm for 1 5 min. The snpernatant is used as the stock enzyme solution and stored a t 3-5 "C. Phenol is corrosive and should be handled and weighed carefully.
Caution:
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 ensiformis) and soya beans (Glycine man). I t also occurs in other plant tissues and microorganisms. In this experiment we report the kinetic study of urease extracted from the seeds of Dolichos biflorus (also called Dolichos uniflorus, 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 distinguishing 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 Berthelot'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 concentration 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 intensity of color obtained, and its reproducibility (5). Experimental Reagents and Equipment Buffer: 0.1 M sodium phosphate buffer, pH 7.01, containing 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 solutions of fresh urea on the day of use because urea slowly 556
Journal of Chemical Education
Phenol-Nitroprusside: Dissolve 4.7 g of analyticalgrade reagent phenol in 100 mL of deionized water containing 6 mg of sodium nitroprusside. Store in a brown bottle a t 4 "C. The reagent is stable for 3 months. 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 arnmaniafree environment. Caution:
Hypochlorite Reagent: Dissolve 2 g of sodium hydroxide in 100 mL of deionized water containing 7.5 mL of a commercially 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 Berthelot reaction. Passing chlorine into alkaline hypochlorite solution is essential to get consistent results.) Special Equipment: visible spectrophotometer or calorimeter, 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 substrate 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 reaeent. ". ., The mlxrure was incubated at 50-6O'C for I0 min after vortexing. Itengrnt blanks tiom which enz,vmc had heen irmittcd w(!rt, pr(:p:~ndand trrarrd in a similar mannel- The abiorbancc 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 ammonium 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 l 1 :3 dilution rave a maximum nl)i~,rcontainine 0.5 ~ m o at bance of 0.7, which is used i n this experiment for calculating reaction rates. If the absorbance is greater than 0.8, the solution is diluted further with deionized water, and the absorbance is measured. Do not forget to multiply absorbance value by
Figure 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 of enzyme that would hydrolyze 1pmol of urea per minute under the reaction conditions described above. We have found this procedure very useful in detailed kiuetic studies of urease from Dolichos biflorus seeds and other leguminous seeds. Treatment of Experimental Data
For each assay determine t h e velocity, t h a t is, the amount of urea converted in 1min for each substrate concentration. Then average the data and calculate the average velocity. Plot velocity against substrate concentration. Calculate the reciprocals of velocity and substrate concentration, and plot l l u vs. 11s. Results and Discussion
A plot of velocity vs. substrate concentration is a rectangular hyperbola (Fig. 1). The mathematical equation that explains this behavior is called the Michaelis-Menten rate law, "=-
" & ,
K,+S
(1)
where u is the initial velocity; V, is the maximal velocity; and K, is the Michaelis constant. Its derivation can be found in anv text book of biochemistrv " (6). . . At a particular set of experimental conditions (pH, temperature, buffer concentration, etc.), values of K, and V,. can be experimentally evaluated to provide information about the kinetic properties ofthe enzyme under study. values of K, and v,, can be determined graphically from the Michaelis-Menten curve shown in Figure 1.In this experimentsK, and V, were found to be 6.5 mM (substrate concentration a t half of maximal velocity) and 1.5 x 104mM/min. More precise values for K, and V, may be obtained if the same experimental data are displayed graphically in
Figure 2. linear form. The linear form of the Michaelis-Menten equation can be obtained by taking the reciprocal of both sides of eq 1. 1 u
1
"m,
+-
Y=a+bx
K, (2) " m S
This double reciprocal form of the Michaelis-Menten rate law predicts that a plot of l l u vs. 11s would be linear with an ordinate intercept (y-axis)of IN,,, abscissa intercept (x-axis) of -llK,, and a slope of KJV,,,. Such a plot, the Lineweaver-Burke plot, is shown in Figure 2 for the urease-urea reaction from the experimental data. From 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 determlncd from x-axis and y-iixii intercepts ova stra~ght line, they are considered to be more rclial)le than those ohtained by approximation from the Michaelis-Menten curve (Fig. 1). The experiment described above is easy to carry out and can be completed by students in a 3-4-h laboratory period a s part of biochemist~lorganicchemistry laboratory exercise. This experiment may be considered for a biochemistry undergraduate course to illustrate the basic principles of enzyme kinetics. ~
~
k
~
~
~
l
~
d
I thank P V. Sundaram, Director, Centre for Protein Engineering and Biomedical Research, Voluntary Health Services, Madras, for providing facilities. Literature Cited sumner J. B,J,Bioi, Cham, 1926,69,435, 2. uixon, N. E.; G ~ ~ c.;B~ I~ ~R. I L.; ~~zerner,~. ~, I J~ A ~~chpm. . SW. 1915,97,4131. 3. ~ t . 1 M ~ ~ . &bat, E. A. ~ i ~ 1916.9.869. h ~ ~ ~ ~ l ~
w.;
i: ~ { 6. R=W",
~ ~ ~ ~ ~ ; ~ a ~ a lsl, ~ ~ ~ , ~ ~ J . u. Biochemistry;N ~~ ~ a fI f e - ~B ~U: ~ I ~ ~ ~ ~ 1989:~ O ~ ,169. NC,
Volume 72 Number 6 June 1995
~
"
557
.
l
~
~
