In the Laboratory
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The Binding Constant for Complexation of Bilirubin to Bovine Serum Albumin An Experiment for the Biophysical Chemistry Laboratory Kathryn R. Williams,* Bhavin Adhyaru, and Russell Pierce Department of Chemistry, University of Florida, Gainesville, FL 32611-7200; *
[email protected] Stephen G. Schulman Department of Medicinal Chemistry, University of Florida, Gainesville, FL 32610-0485
Growing emphasis on biochemistry tracks/options for undergraduate majors has led to the need for suitable experiments for the laboratory component in biophysical chemistry (1). Instructionally, these should focus on the application of fundamental concepts of physical chemistry to systems of biological interest, preferably using commercially available and reasonably robust chemicals. It is also desirable to include instrumentation commonly employed in biochemical research. This paper summarizes the design of an experiment on the complexation of bilirubin to bovine serum albumin using molecular fluorescence measurements. In the body, degradation of the protoporphyrin component of hemoglobin results in the formation of the intensely yellow compound bilirubin (BR). The BR is carried as a complex with serum albumin to the liver, where it is conjugated with glucuronic acid and excreted in the bile. In newborns and individuals with diseased livers, the excretion mechanism often fails to rid the body of BR, resulting in jaundice and cell malfunction (2). The condition is especially serious in newborns, who are susceptible to brain damage. The binding of BR to plasma albumin presents an essential biological reaction, which can be studied in a student laboratory using purchased bilirubin and bovine serum albumin (BSA). Description of the Experiment The complexation reaction
KB =
I BRBSA 0 I0 BR T – I BRBSA 0 I0
BSA T – I BRBSA 0 I0
(2)
In eq 2, [BRBSA]0 is the concentration of BRBSA for complete complexation and I0 is the corresponding fluorescence intensity, evaluated as I at the intersection of the two straight lines. For X ≤ X0, BR is the limiting reagent and [BRBSA]0 is equal to the total concentration of BR ([BR])T. Thus, eq 2 becomes
KB =
I0 – I
I × I0 I 0 BSA T – I BR T
(3)
Likewise, for X ≥ X0, BSA is the limiting reagent and a similar equation may be written. Students evaluate I at the stoichiometric concentration fraction from the best fit to the curved region in Figure 1 and use eq 3 to calculate KB and its 95% confidence limits. The result for the data in Figure 1 is (1.2 ± 0.9) × 107, which agrees closely with the values
BRBSA
can be investigated quantitatively by the intense fluorescence of BRBSA at 540 nm with excitation at 460 nm. The experimental design utilizes the Method of Continuous Variations, also called Job’s method, which is used primarily to determine the stoichiometry of complexation. For 1:1 complexes, the binding constant
BRBSA KB = BR BSA
(1)
can also be calculated (3). The background theory and procedure are explained in detail in the student manual.W A series of solutions is prepared with constant total BR + BSA concentration (2 µM), but with BR concentration fractions (X ) varying from 0.1 to 0.9. The fluorescence intensity (I ) is measured for each solution. As shown in Figure 1, a plot of I versus X shows a maximum at the stoichiometric concentration fraction (X0).
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Intensity
BR + BSA
Students calculate X0 and its 95% confidence limits by equating the least-squares lines for the two linear regions. Since the fluorescence intensity is directly proportional to [BRBSA], eq 1 can be rewritten as
5
0 0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
X Br Figure 1. Plot of fluorescence intensity at 540 nm (λexc = 460 nm) of BRBSA vs concentration fraction BR for a total concentration (BR + BSA) of 1.99 µM. The intersection of the two linear parts at 0.50 ± 0.06 agrees well with the theoretical value of 0.50.
JChemEd.chem.wisc.edu • Vol. 79 No. 1 January 2002 • Journal of Chemical Education
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In the Laboratory
2.2 × 107, 2.7 × 107, and 2.9 × 107 observed at pH 7.4 by previous investigators (4 ). Hazards Because of the low solubility of bilirubin in water, the stock BR solution (ca. 200 µM) is prepared by laboratory personnel in N,N-dimethylformamide (DMF). Small aliquots are combined with BSA in aqueous phosphate buffer and diluted 100-fold with the same buffer. Because of the very large dilution factor, the resulting mixtures present negligible risk. However, students should be cautioned to handle the stock solution of BR in DMF with care. Dimethylformamide has been shown to have carcinogenic and mutagenic properties. It is also a skin and eye irritant (5). Conclusion The KB determination fulfills the educational goals of the biophysical chemistry laboratory using readily available materials. Students also gain experience with fluorescence measurements, including choice of the excitation and emis-
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sion wavelengths. The procedure is easily completed in a halfday laboratory period, and the data analysis teaches a simple but meaningful application of least-squares equations. Students routinely obtain good experimental results for this important biological equilibrium. W
Supplemental Material
Detailed background material, instructions for students, and an instructor’s guide are available in this issue of JCE Online. Literature Cited 1. Howard, K. P. J. Chem. Educ. 2000, 77, 1469–1471. 2. Broderson, R. Crit. Rev. Clin. Lab. Sci. 1980, 11, 305–399. 3. Bruneau, E.; Lavabre, D.; Levy, G.; Micheau, J. C. J. Chem. Educ. 1992, 69, 883–837. 4. Faerch, T.; Jacobsen, J. Arch. Biochem. Biophys. 1975, 168, 351–357. 5. Vermont Safety Information Resources, Inc. http://siri.org/ (accessed Oct 2001).
Journal of Chemical Education • Vol. 79 No. 1 January 2002 • JChemEd.chem.wisc.edu
