In the Laboratory
W
Removal of Zinc from Carbonic Anhydrase A Kinetics Experiment for Upper-Level Chemistry Laboratories Kathryn R. Williams* and Bhavin Adhyaru Department of Chemistry, University of Florida, Gainesville, FL 32611-7200; *
[email protected] As a continuation of efforts to design a laboratory program in physical measurements for systems of biochemical interest (1–3), this article describes a new experiment on the kinetics of deactivation of carbonic anhydrase. The experiment is presently used in the biophysical chemistry laboratory, but it can be adapted for laboratories in biochemistry or instrumental analysis. An optional procedure using 65Znlabeled carbonic anhydrase can be used as a special project or for a course in radiochemistry. Description of the Experiment Carbonic anhydrase (CA), the enzyme that catalyzes the interconversion of carbon dioxide and bicarbonate, requires one Zn(II) ion in its active site. Removal of the zinc cofactor by complexation to another ligand leaves the apoenzyme, which is totally inactive. Thus, the rate of zinc removal can be monitored by removing aliquots of the reaction mixture at suitable time intervals and assaying for the remaining enzyme activity. The usual assay substrate is p-nitrophenyl acetate, which is hydrolyzed to p-nitrophenol. The initial rate, which is proportional to the concentration of the holoenzyme, is measured by the increase in the absorbance of the product at 348 nm. A number of ligands can be used to remove the zinc, but many of the reactions are slow and unfeasible for a student laboratory. (For example, dialysis versus 1,10phenanthroline at pH 5 requires several days.; ref 4 ) However, 2,6-pyridinedicarboxylate, commonly known as dipicolinate (dipic), is extremely efficient (5), allowing complete Zn removal within one hour at room temperature. Thus, students can easily perform several kinetic runs in a four-hour laboratory period. Several groups (6–10) have studied the CAZn兾dipic exchange reaction, but the conditions of the student experiment described here most closely correspond to those of Kidani and Hirose (6, 7). When dipic is present in large excess, the reaction is pseudo-first-order with respect to CAZn:
d [apoCA ] dt
= kobs [CAZn ]
(1)
The proposed mechanism involves initial formation of a ternary CAZn dipic complex that slowly breaks apart to form the zinc–dipicolinate complex and apoCA. In the steps shown below, dipic is further abbreviated to L and the equilibrium constant for the ternary complex is denoted KEML (enzyme– metal–ligand): KEML
CAZn + L
fast
CAZnL
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(2a)
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CAZnL
ZnL + L
kd slow
fast
apoCA + ZnL
(2b)
ZnL2
(2c)
With L in large excess, the third step is very fast and does not influence the rate. The complete derivation of the rate equation from the mechanism is provided in the Supplemental Material.W The result in integrated form is:
ln
[CAZn] 0 − [apoCA ] [CAZn] 0
= ln (FCAZn ) = −
k d KEML [L ] 0 1 + KEML [L ] 0
t
(3)
In eq 3, the argument of the logarithm is equal to the fraction of CAZn remaining at time t, FCAZn. Thus, a plot of ln(FCAZn) versus time should be linear with slope (= kobs ) given by slope = −kobs = −
k d KEML [L ] 0
(4)
1 + KEML [L ] 0
The FCAZn values are given by the fractional remaining enzymatic activity. To obtain kd and KEML, both sides of eq 4 are inverted to give: 1 kobs
=
1 1 1 + k d KEML [ L ]0 kd
(5)
Students measure kobs for a series of dipicolinate concentrations and plot 1兾kobs versus 1兾[L]0. The intercept gives 1兾kd and the slope is 1兾(kdKEML ). Optional Experiment Using 65Zn-Labeled Carbonic Anhydrase Referring to eq 3, FCAZn can also be evaluated by monitoring the formation of the Zn(dipic)2 complex. This presents an instructive way to introduce students to radioisotopes by using carbonic anhydrase labeled with 65Zn and observing the 65Zn(dipic)2 product. The isotope decays by both positron emission and electron capture with a half-life of 245 days. Filtration devices designed to fit into the tops of microcentrifuge tubes are used to separate the 65Zn(dipic)2 from
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the enzyme. The 65Zn(dipic)2 in the filtrate may be determined using either a liquid scintillation counter or a NaI(Tl) system to detect the accompanying 1.15 MeV gamma. For the optional experiment, the instructor prepares the labeled carbonic anhydrase by adding 65Zn to the apoenzyme. Students handle only the small quantity needed for the experiment, typically about 1.2 × 105 cpm (less than 0.1 µCi). Hazards For the enzyme assays, 0.003 M p-nitrophenyl acetate must be freshly prepared by the students. They must also work with a fairly concentrated solution (0.40 M) of dipicolinate. According to the available MSDS, the toxicological properties of these compounds have not been studied, but both are known to cause irritation to the eyes, skin, and to the respiratory and digestive systems. However, the total quantities handled by the students are small (25 mg pnitrophenyl acetate and less than 1 mL dipicolinate). All remaining solutions (carbonic anhydrase, phosphate buffer, and HEPES buffer) present minimal hazard. For the optional experiment, students handle very small levels of 65Zn. They should wear gloves, but otherwise may work on an open benchtop covered with plastic-backed absorbent paper. At the end of the experiment, they should count swipes of the work area to detect (and subsequently remove) any contamination. The instructor works with greater quantities when preparing the labeled carbonic anhydrase, and s/he should have adequate training in use of radioactive materials. Licensing and disposal requirements will vary according to local regulations.
Results The first-order plots obtained by a typical student group using the standard procedure (enzyme activity method, described in detail in the Supplemental MaterialW) are shown in Figure 1. The optional procedure (65Zn detection) gives similar results. A plot of the reciprocal of kobs (obtained from the slopes of the lines in Figure 1) versus the reciprocal of [dipic] using the pooled results of five student groups is shown in Figure 2. The values of KEML and kd obtained from Figure 2 are 7.7 ± 1.5 M1 and 0.43 ± 0.08 min1, respectively, at 25 C and pH 7.4. Kidani and Hirose obtained 102 M1 and 0.0115 min1 working at 0 C and pH 7.0 (6). Because of the difference in reaction conditions, the student data are not directly comparable to the literature values. However, the authors, in a separate experiment, obtained a kobs of 0.0072 min1 using 0.10 M dipic at 0 C and pH 7.4, which compares favorably with the kobs of 0.0062 min1 obtained by Hidani and Hirose at the same temperature and pH 7.0 (6). (Note that this corresponds to a reaction halflife of over 90 minutes at 0 C, which is undesirably long. The conditions used in the student experiment give half-lives that range from about 2 to 20 minutes.) Conclusion The deactivation of carbonic anhydrase is a beneficial experiment for students in biophysical chemistry. They easily obtain data for five dipicolinate concentrations within a four-hour laboratory period, and the only instrumentation requirements are a constant temperature bath and a UV–vis
-5.00
30.00
0.016 M 0.032 M 0.050 M 0.10 M 0.20 M
min 1 k obs
∆A ∆t
-8.00
25.00
∆A ∆t
-7.00
t
∞
-6.00
20.00
15.00
10.00
-10.00
5.00
Ln
-9.00
-11.00 0
10
20
30
40
50
60
70
0.00 0.00
80
Figure 1. Plots of ln[(∆A/∆t )t − (∆A/∆t )∞] versus time, where (∆A/ ∆t )t and (∆A/∆t )∞ are the initial rates of the enzyme assays at time t and at infinite time, respectively. In the assays, p-nitrophenyl acetate is hydrolyzed to p-nitrophenol; ∆A/∆t corresponds to the increase in absorbance of p-nitrophenol (348 nm) averaged over the first 100 s. [dipic] = 0.016 M ( 夽), 0.032 M (䊏), 0.050 M (䉭), 0.10 M (䊊), and 0.20 M (䉬). Data were obtained at 25 ºC and pH 7.4 (0.125 M phosphate).
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20.00
30.00
40.00
1 [dipic]
Time / min
1046
10.00
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50.00
60.00
70.00
ⴚ1
M
Figure 2. Plot of 1/kobs versus 1/[dipic] at 25 ºC and pH 7.4. Pooled data from five student groups.
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spectrophotometer. For the optional experiment, a microcentrifuge and either a liquid scintillation or NaI(Tl) counter are also needed. The experiment works well with native bovine carbonic anhydrase (mixture of two isozymes), which is not expensive. Students learn several important biochemical concepts and observe the necessity of zinc(II) in the active site of the enzyme. W
Supplemental Material
Instructions for the students, including a prelab assignment, and notes for the instructor are available in this issue of JCE Online. Literature Cited 1. Williams, Kathryn R.; Adhyaru, Bhavin; Pierce, Russell E.; Schulman, Stephen G. J. Chem. Educ. 2002, 79, 115–116.
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2. Williams, Kathryn R.; Adhyaru, Bhavin; German, Igor; Alvarez, Eric. J. Chem. Educ. 2002, 79, 372–373. 3. Williams, Kathryn R.; Adhyaru, Bhavin; German, Igor; Russell, Thomas. J. Chem. Educ. 2002, 79, 1475–1476. 4. Lindskog, Sven; Malmström, Bo G. J. Biol. Chem. 1962, 237, 1129–1137. 5. Hunt, John B.; Rhee, Moo-Jhong; Storm, Carlyle B. Anal. Biochem. 1977, 79, 614–617. 6. Kidani, Yoshinori; Hirose, Junzo; Koike, Hishashi. J. Biochem. 1976, 79, 43–51. 7. Kidani, Yoshinori; Hirose, Junzo. J. Biochem. 1977, 81, 1383– 1391. 8. Pocker, Y.; Fong, Conrad T. O. Biochemistry 1980, 19, 2045– 2050. 9. Pocker, Y.; Fong, Conrad T. O. Biochemistry 1983, 22, 813– 818. 10. Hendry, P.; Lindoy, L. F.; Yellowlees, D. Inorg. Chim. ActaLett. 1982, 65, L237–L239.
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