Gel Filtration Permits Separation of Bound and Unbound Forms of Phenol Red with Bovine Serum Albumin (BSA)

Oscar Lu* and Dave McKenzie (Bench #1 Tues/AM) Biochemistry Laboratory, 3029 Haworth Hall, Department of Biomolecular Sciences, University of Kansas
*

Corresponding author: ouellette.andrew@gmail.com,Teaching asst., BIOL 637

Received: October 26, 2010 Using gel filtration chromatography, fractions from eight different samples of eight different concentrations of bound (to bovine serum albumin) and free (unbound) phenol red dye were collected. Absorption data was collected from each of the fractions using a spectrophotometer and recorded in Excel. The data was processed via a template Excel worksheet to generate a dissociation constant value (Kd = 2.32) of phenol red and bovine serum albumin ligand-protein complex and a maximum specific binding (Bmax= 0.1334). Then using the equation (Kd=1/Ka) the formation constant (Kf) of phenol red and bovineserum albumin ligand-protein complex was calculated to be 0.4310. I. Introduction The utilization of gel filtration chromatography (a subgroup of size-exclusion chromatography) using a buffered aqueous solution as the mobile phase provides a simple and convenient way to study ligand-protein interactions. The essential ligand-protein interactions in biological systems are defined by the non-covalent bonding between small and larger molecules, and are further characterized by the affinity of two molecules for each other via the formation constant Kf. The formation constant is calculated and derived from experimental results of the spectroscopic readings of the gel filtration chromatography fractions. This experiment uses gel filtration chromatography to study the interaction between the dye phenol red and the protein bovine serum albumin as a model for ligand-protein interactions. Phenol red binds to bovine serum albumin to form a dye-protein complex. The complexs relatively larger size compared to unbound dye allows for them to be readily separated by gel filtration chromatography. The operation of the gel filtration chromatography in the context of this lab is based on a column filled with a stationary phase consisting of Sephadex G-25150 gel resin, inert particles that contain small pores of controlled size. Any analytes in the sample solution larger than the pore size will be restricted to the space between the beads. The resulting restriction in accessibility to column volume leads to the rapid elution of larger molecules through the column. The small molecules with accessible volume through their diffusion in and out of the beads will have much greater distances to travel to reach the end of the column, therefore eluting slower. In context of this experiment, the larger dye-protein complex will elute into the fractions before the smaller sized phenol red dye molecules. Equal volumes of multiple fractions are collected from the sample. The fractions are analyzed with a spectrophotometer for their absorbance at 430nm (approximately the yellow color the phenol red dye). An excel based calculation of the absorbance versus the fraction number allows for the determination of a Bmax and Kd, and thereby resulting in a Kf for the phenol red dye and bovine serum albumin defining the interaction ligand-protein interaction between the two. II. Experimental Approach Samples of eight different samples with eight different concentrations of bound and unbound dye were prepared following Table 1 below by eight different teams.
1 Dye ( L) Buffer ( L) BSA ( L) 20 580 700 2 30 570 700 3 40 560 700 4 55 545 700 5 75 525 700 6 100 500 700 7 125 475 700 8 150 450 700

Table 1.
0.10 M Sodium Acetate (at pH 4.3) was used as Buffer. The dye was 4.0% Phenol Red Dye in 0.10 M Sodium Acetate (at pH 4.3) Buffer. BSA consisted of Bovine Serum Albumin Solution at a 50 mg/mL concentration. Each of the eight combinations produced eight different concentrations of bound and unboud dye, and were each prepared in separate 1.7 mL centrifuge tubes.

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After sample preparation was completed, each team prepared a column. 30 mL was taken from a supply of well-suspended resin slurry (Sephadex G-25150 gel resin in Buffer). The well-suspended resin was quickly poured into the column to form a homogenous column of gel resin upon sedimentation. Buffer was drained until the meniscus reached the surface of the Resin bed. Then a 150 L aliquot of the sample was applied to the top of the Resin bed. The sample was allowed to penetrate the resin bed by repeating a washin process five times, each time with 150 L of Buffer. The liquid level above the resin bed was built-up to 3 centimeters above the resin bed with additions of buffer, without disturbance of the resin bed. Then a total of forty 1.75 mL fractions were collected in 1.7mL centrifuge tubes (with caps removed) while buffer was replenished as needed to maintain a liquid level 3 centimeters on top of the resin bed. The spectrophotometer was zeroed with a 2mL cuvet filed with Buffer at 430nm. Then all forty fractions were each read at 430nm in a 2mL cuvet. III. Computational Methods Absorption data for the forty fractions of each of the eight teams were copy and pasted into eight separate-but-identical Excel worksheet template. Using an absorption coefficient of 29 mM-1 cm-1, the spreadsheet converted the fraction readings to nmoles of dye per fraction and then displayed a column profile. The spreadsheet summed the peaks and calculated the mM concentration of each of the samples using 376.36 g/mole as the molecular weight of phenol red with a 1.4 correction factor applied to [LM]. Two peaks were produced: the first peak for [LM] and the second peak for [L] ([L] specifying the concentration of unbound phenol red dye and [LM] specifying bovine serum albumin bound phenol red dye). Values from [L] and [LM] were carried into the Combined Data Worksheet where the ratio of dyebound protein to initial protein (v) was calculated. Using a molecular weight of 66,430 daltons, v was plotted against [L] for saturation binding data in Prism, resulting in Kd and Bmax values. The formation constant Kf for the phenol red dye and bovine serum albumin dye-protein complex was then derived from the Kd using the equation (Kd=1/Ka). IV. Results and Analysis A. Gel Filtration Chromatography Fig. 1 shows the elution profile of a mixture of Phenol Dye Red and Bovine serum albumin, pH 4.3. There appears to be two peaks which read for Phenol Red (at 430nm): one small

peak early in the elution process, and one larger peak later in the elution process. The first small peak represents protein-bound phenol red. The second, and larger peak, represents the elution of free, or unbound, phenol red.

Fig. 1. Elution profile of bovine serum albumin and phenol red mixture, at pH 4.3.

Phenol Red Binding to BSA

0.15

0.10

0.05

0.00 0

10

[L], m M

Fig. 2. Binding curve of Phenol Red to Bovine Serum Albumin, at pH 4.3, based on eight varying concentrations of Phenol Red (with outliers removed). Binding Curve derived from Fig.3 below, where v is the ratio of dye-bound protein to initial protein, and [L] is the concentration of phenol red in mM.

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One site -Specific binding

Best-fit values Bmax Kd Std. Error Bmax Kd 0.1334 2.32 > 0.01401 0.7251 > > 0.08882 to 0.1780 0.01277 to 4.627 8.360 9.483

Data Plotted Above

[L], mM 1.424 2.000 2.413 3.832 5.979 6.888 0.105 0.102
Gray rows (>) were judged to be outliers and not included in graph.

v 0.048 0.056 0.029 0.095 0.656 0.205

95% Confidence Intervals

Bmax Kd

performed the experiment with sample 1, and excel worksheet derived values. ***The conversion of absorbance to mol was completed by taking the given absorbance of a given fraction and dividing the value by (29 times 1.75). ***Peak 1 was a summation of the mole values of absorbance from fractions 4 to 10. ***Peak 2 was a summation of the mole values of absorbance from fractions 11 to 40. ***[LM] was then obtained by taking Peak 1s value and dividing by (0.15*1.4), and [L] was obtained by taking Peak 2s value and dividing by (0.15). ***Then given an initial concentration [M]0 of 0.405 mM BSA, v is calculated by taking [LM] divided by [M]0. (For elution profile curve generated from Fig. 5.s data, see Fig. 1)

Goodness of Fit
Degrees of Freedom R square Absolute Sum of Squares Sy.x Runs test Points above curve Points below curve Number of runs P value (runs test) Deviation from Model Number of points Analyzed 2 3 3 0.5 Not Significant 5 3 0.9284 0.00021 0.008358

Gel filtration chromatography allowed for the separation of free, unbounded, phenol red and the BSA bounded phenol red dye-protein complex. The larger dye-protein complex (ligand-protein complex) eluted
Ligand-Protein Binding - Gel Filtration Columns

Fig. 3. Above is the data and statistical break down of the binding curve presented in Fig. 2. Based on the Prism produced analysis of the compilated data from the eight teams, it could be reported that Bmax was 0.1334 and Kd was 2.32. All outliers identified by the Prism computer based analysis were removed.

Kd=1/Kf
Since Kd=2.32 2.32=1/Kf Kf=0.4310
Fig. 4. Based on the given calculated data from the Excel worsheets and Prism analysis, the formation constant of the phenol red and bovine serum albumin was calculated to be 0.4310.

Sample 1 - 20 uL Frac # A430 nm moles 1 0.0023 0.0001 2 0.0045 0.0003 3 -0.0003 0.0000 4 -0.0018 -0.0001 5 0.0008 0.0000 6 0.0062 0.0004 7 0.0186 0.0011 8 0.0048 0.0003 9 0.0021 0.0001 10 0.004 0.0002 11 0.0045 0.0003 12 0.0025 0.0002 13 0.0034 0.0002 14 0.0069 0.0004 15 0.0141 0.0009 16 0.0361 0.0022 17 0.0751 0.0045 18 0.1438 0.0087 19 0.2346 0.0142 20 0.3338 0.0201 21 0.404 0.0244 22 0.4392 0.0265 23 0.4232 0.0255 24 0.3661 0.0221 25 0.2988 0.0180 26 0.2272 0.0137 27 0.1619 0.0098 28 0.1145 0.0069 29 0.0779 0.0047 30 0.0539 0.0033 31 0.0333 0.0020 32 0.0263 0.0016 33 0.0182 0.0011 34 0.0134 0.0008 35 0.0071 0.0004 36 0.0047 0.0003 37 0.003 0.0002 38 0.0027 0.0002 39 0.0039 0.0002 40 0.006 0.0004 Peak 1 = 0.0021 Peak 2 = 0.2136 [LM] = 0.0195 [L] = 1.4242 All Peak 1's increased by a factor of 1.4 to compensate for protein quenching of dye absorbance. Sm pl # 1 [L] mM 1.424 v 0.048 [LM] mM 0.020 [M]o mM 0.405 v/[L] mM-1 0.034

Fig. 5. Above are the recorded absorbance values for team one which

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Carry the values from columns [L] (x-axis) and v (y-axis) to Prism. * [L] and [LM] increased by a factor of 2.4375 to compensate for short light path.

first and showed a small peak 1, and then the unbounded phenol red eluted out afterwards thereby generating a large peak 2. Excel and Prism analysis of the spectroscopically obtained absorbance from each of the 40 fractions from all eight phenol red concentrations concluded the Bmax and Kd of the phenol red bound BSA complex to be 0.1334 and 2.32mM, respectively, therefore resulting in a formation constant (Kf) of the dye-protein complex to be 0.4310mM-1. V. Discussion Two peaks were produced from the gel filtration chromatography elution and spectroscopic analysis. The first peak was smaller than that of the second. The smaller first peak comes from the first four to ten elution collections, and is indicative of the elution of the phenol red and BSA dye-protein complex. Based on the fundamental process and mechanism of gel filtration chromatography, larger analytes (such as the dye-protein complex) are restricted to the volume between the resin beads of the gel bed, whereas smaller analytes ,that are equal to or smaller in size than the size-specific-pores of the resin beads, will have greater distance to travel due to its accessibility to the volume of the matrices of the porous beads. With less volume accessibility in the column the larger dye-protein would therefore elute out from the column before the free, unbound phenol red dye. The protein-bounded dye, in addition to being larger than the unbound dye and the first to elute from the column, also has a lower absorbance due to protein (BSA) quenching of the dye absorbance. By using absorbance values from the collected fractions, the Excel and Prism calculations and analysis were able to report a Kd of 2.32mM. The Kd is the dissociation constant of the phenol red and BSA, which characterizes the propensity for the two (the dye-protein complex) to reversibly dissociate. The inverse relationship to the dissociation constant (Kd) is the formation constant (Kf). The formation constant was found to be 0.4310mM-1: derived from (Kd=1/Kf) and calculated using the reported Kd. In contrast to the dissociation constant, the formation constant expresses the affinity of phenol red and BSA for one another to form the dye-protein complex. Mathematically speaking, the inverse relationship of Kd and Kf automatically suggests that a large Kd implies a small Kf . From a conceptual standpoint, it should make sense that the greater the affinity of two components for one another, the less likely they are to separate, and likewise, the smaller the affinity two components have for one another the more likely they are to dissociate. The conceptualization and interpretation of the data and analysis are all based on the parameters that there is

one binding site per protein in the experiment. With the possibility that there could be more than one binding site, comes the possibility of cooperative binding and/or allosteric interactions where the binding of one molecule could influence the binding of additional identical molecules. The possibility of cooperative binding could result in a curve different than that of Fig. 2 as well as a formation constant whose variation in value would be dependent on the concentration of ligand. In addition to the possibility of BSA possessing more than one binding site, which can skew the resulting formation constant, is the possibility that some of the dye-complex may have dissociated in the column. Since fewer dye-complex is eluted and more dye is eluted, the result would be an artificially low peak 1 and artificially high peak 2. This in turn would lead to an artificially low [LM] and thereby a low v. As a result the dissociation constant obtained would be smaller than what it would actually be, leading to an artificially high formation constant value. VI. Conclusions The gel filtration chromatography combined with computer calculation templates, led to a formation constant (Kf) characterizing the affinity of phenol red dye for BSA, and vice versa, with a value of 0.4310. VII. References
1

Boyer, Rodney. Biochemistry Laboratory: Modern Theory and Techniques. San Francisco: Benjamin Cummings, 2006. Print. Nelson, David L. Lehninger: Principles of Biochemistry. 5th. New York: W.H. Freeman and Company, 2008. Print. Debro, Joseph R. "The Application of Gel Filtration to the Measurement of The Binding of Phenol Red by Human Serum Proteins." Journal of Chromatography. 10. (1963): 68-72. Print.

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