Protein Concentration Dependence of Palmitate Binding to β-Lactoglobulin1

Protein Concentration Dependence of Palmitate Binding to b-Lactoglobulin1 QIWU WANG, JONATHAN C. ALLEN, and HAROLD E. SWAISGOOD2 Southeast Dairy Foods Research Center, Department of Food Science, North Carolina State University, Raleigh 27695-7624

stranded antiparallel b-barrel enclosing a hydrophobic pocket (10, 12, 14), but b-LG also has an a-helix lying on the surface of the b-barrel that forms a second potential hydrophobic binding pocket (10, 12). Although the site of retinol binding has been controversial, most evidence points toward the calyx formed by the b-barrel as the hydrophobic binding pocket (1, 2, 24, 25). The identity of the binding site for palmitate has also been controversial: some data ( 1 8 ) indicate that it competes with retinol for the same site, but other data suggest that these two ligands bind at different sites (7, 8, 13). Binding of retinoids to b-LG causes a quenching of the fluorescence of Trp 19 (2, 9), which is present at the bottom of the calyx; however, the interaction between b-LG and palmitate enhances the Trp fluorescence intensity (16, 17). Consequently, this enhancement of fluorescence intensity has been used to quantitate the binding of fatty acids to b-LG. Some investigators ( 8 ) have suggested that b-LG has one high affinity binding site that is close to the contact region for dimer formation. Analysis of equilibrium binding data obtained by ultrafiltration or dialysis with [14C]palmitate has led others ( 2 1 ) to conclude that two classes of binding sites are present. The magnitude of the binding constant has also been controversial, and reported values (8, 18, 21) have ranged from 107 to 105. In this study, a wide range of b-LG concentrations was used to measure the equilibrium binding of [14C]palmitate. The results indicate that b-LG has two sites that bind palmitate and, furthermore, that the binding affinity depends on the extent of protein dimerization as determined by the concentration.

ABSTRACT The binding of palmitate to b-lactoglobulin at protein concentrations ranging from 1 to 200 mM was determined using an ultrafiltration method with [14C]palmitate. Fit of the data to theoretical models required the assumption of two independent sets of binding sites; however, binding characteristics were dependent on the protein concentration. A model assuming one set of sites on the protein monomer and another on the dimer was consistent with the data. The analysis suggests that 2 mol of palmitate are bound/mol of dimer and that the binding constant is of the order of 105 M–1; a larger number of palmitate molecules are bound per mole of monomer with a smaller binding constant of the order of 104 M–1. Apparently, formation of the dimer, by hydrophobic interactions at the monomer contact site, eliminated palmitate binding sites on the monomer but formed a higher affinity pocket for binding to the dimer. ( Key words: b-lactoglobulin, palmitate, palmitate binding) INTRODUCTION b-Lactoglobulin is classified as an octinin member of the lipocalycin family (3, 5, 10, 15) and is the major protein in the whey of ruminant and some nonruminant species (10, 18, 22). Because of its binding affinity for long-chain FFA, b-LG potentially could serve as a carrier for FFA transport or as an acceptor for their removal in biological systems. For example, it has been proposed ( 1 5 ) that b-LG functions to enhance pregastric esterase activity by its binding of inhibitory fatty acids. In common with other members of the lipocalycin family, b-LG has a central core consisting of an eight-

MATERIALS AND METHODS Materials

Received April 18, 1997. Accepted July 30, 1997. 1Paper Number FSR 97-19 of the Journal Series of the Department of Food Science, North Carolina State University, Raleigh 27695-7624. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of the products named nor criticism of similar ones not mentioned. 2Corresponding author. 1998 J Dairy Sci 81:76–81

Palmitic acid (Sigma grade, approximately 99% purity) and palmitic acid-carboxy 14C (Sigma 98% purity, 11.2 mCi/mmol) were purchased from Sigma Chemical Co. (St. Louis, MO). Solutions of the desired concentrations were prepared by weight by dissolving those substances in absolute ethanol. For 76

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PROTEIN CONCENTRATION DEPENDENCE

solutions at high concentrations, [14C]palmitic acid was diluted with unlabeled palmitic acid to give the appropriate concentration. ScintiSafe Gel was purchased from Fisher Scientific (Fair Lawn, NJ). b-Lactoglobulin was prepared by bioselective adsorption with 500 ml of N-retinyl Celite™ as previously described (24, 25, 26, 27). This preparation of b-LG was greater than 96% purity as measured by size-exclusion chromatography.

System (Beckman Instruments Inc., Palo Alto, CA). Solution samples (100 ml ) were added to 4 ml of ScintiSafe Gel for counting. For the purpose of quantitation, background counts of the phosphate buffer and counts for solutions containing known amounts of [14C]palmitate were obtained in the same manner.

Ultrafiltration Method for Measuring Binding

The association constants, k1 and k2, and the stoicheometry at each site, n1 and n2, were determined by computer fitting of the data to the two-site model (23):

Solutions of b-LG in 400 ml of 50 mM sodium phosphate, pH 7.0, at concentrations ranging from 1 to 200 mM were adjusted to concentrations of palmitate ranging from 0.1 to 200 mM by addition of the appropriate stock in ethanol. The protein and FFA concentrations were chosen to include the b-LG concentration and the ratio of fat to b-LG in skim milk. The final ethanol concentration was less than 3% and should not perturb the protein structure. The mixtures were incubated in Millipore ultrafiltration cones (Millipore Corp., Bedford, MA) for 16 h or overnight at 23°C. Following incubation, the solution was centrifuged at 7000 rpm for 10 min in a Millipore microfuge, and the amount of palmitate in the ultrafiltrate was assayed by scintillation counting or by the colorimetric method. The moles of bound palmitate were calculated from the difference in concentrations between the original solution and the ultrafiltrate and the volume of the solution. Scintillation Counting for Determination of Palmitate Concentration Radioactivity of [14C]palmitate solutions was measured with a Beckman LS 3801 Liquid Scintillation

Calculation of Equilibrium Binding Parameters

n1k1C

n2k2C

+ = y¯ I + y¯ II . . . y¯ = 1 + k1C 1 + k2C

[1]

where y¯ = moles of palmitate bound per mole of protein and C = concentration of free palmitate. At low concentrations of palmitate, the equation becomes y¯ ≈ ( n 1k1 + n2k2) C . . .

[2].

Consequently, the initial slope of the y¯ versus C plot at low concentrations can be used to check the consistency of the parameters. RESULTS AND DISCUSSION As a representative of fatty acids, the binding of palmitate by b-LG has been investigated by a number of researchers using several methods (3, 4, 8, 13, 16, 18, 21). However, results have been variable, as indicated by the representative data given in Table 1. For example, association constants that were determined from changes in Trp fluorescence concomitant with

TABLE 1. Comparison of the parameters for binding of palmitate to b-LG obtained by various methods and at various protein concentrations.1 Method

Protein concentration

Flourescence measurements2 Flourescence measurements ( 8 ) Equilibrium partitioning ( 2 1 ) Equilibrium partitioning ( 2 1 )

( mM) 20 25 200 200

Ultrafiltration (this study)

200

n1

k1

n2

1.17 ± 0.053 0.93 ± 9.11 1.32 1.0 1.0 1.03 ± 0.01

( ×10–5 M–1) 20.4 ± 23.3 100 ± 5 3.91 ± 0.38 7.0 ± 0.51 6.6 ± 0.39 2.28 ± 0.03

k2 ( ×10–4 M–1)

4.11 24 6 23.4 ± 0.02

7.6 ± 1.7 0.158 ± 0.006 0.709 ± 0.014 0.36 ± 0.05

1k and k = 1 2 2Unpublished

Association constants; n1 and n2 = sites. data obtained in our laboratory. 3Means and standard errors of the means. Journal of Dairy Science Vol. 81, No. 1, 1998

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WANG ET AL.

tate concentrations, agree well with the values calculated using Equation [2] from the parameters listed (Table 2), further indicating the excellent fit of the data to the model. The conclusion that the equilibrium binding data cannot be attributed to binding to a single set of independent sites is further supported by nonlinear nature of Scatchard plots of the data as shown in Figure 2. Because of the limitation of palmitate solubility, it was not possible to approach saturation of the binding sites; therefore, determination of n1 and n2 from Scatchard plots was not possible. Results of these studies indicate that palmitate binding is significantly affected by the protein concentration. Thus, k1 and k2 decreased 6-fold and 100-fold, respectively, as the concentration increased from 1 to 200 mM and the ratio k1/k2 increased 20-fold. Previously, literature values determined from fluorescence measurements were obtained at low protein concentrations ( 8 ) . b-Lactoglobulin undergoes well-characterized self-association reactions that are dependent upon pH and temperature (11, 22). At pH 7.5, a monomer-dimer equilibrium exists with a dissociation constant: Kd ∼ 3.5 × 10–5 M (11, 28). The relationship between palmitate binding and protein concentration is revealed more clearly by comparison of the relative values for the two terms in Equation [1] at a similar ratio of free palmitate concentration to protein concentration (Table 3). As the protein concentration increases, the total binding increases, but, more significantly, the first term of the two-term

Figure 1. Equilibrium binding of [14C]palmitate to b-LG. A. Data obtained at protein concentrations of 1.0 mM ( ⁄) and 10 mM ( o) . B. Data obtained at protein concentrations of 50 mM ( o) and 200 mM ( ◊) . Small symbols represent data points calculated by the model (Equation [1]), which was used to draw the line. y = Moles of palmitate bound per mole of protein.

palmitate binding are substantially larger than those determined by two-phase equilibrium partitioning or ultrafiltration methods. In the present study, equilibrium binding data were obtained as a function of b-LG concentrations ranging from 1.0 to 200 mM using an ultrafiltration method. These data, shown in Figure 1, were analyzed with a model assuming two independent binding sites (Equation [1]), and the results are given in Table 2. The lines drawn in Figure 1 using the values listed in Table 2 indicate the fit of this model to the data. The initial slopes of the data in Figure 1, obtained by fitting at low palmiJournal of Dairy Science Vol. 81, No. 1, 1998

Figure 2. Scatchard plot of the equilibrium binding data obtained at a protein concentration of 200 mM. Experimental data ( ◊) . Data points calculated by the model, Equation [1] ( ♦) . y = Moles of palmitate bound per mole of protein; C = concentration of free palmitate.

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PROTEIN CONCENTRATION DEPENDENCE TABLE 2. Effect of protein concentration on the binding capacity and association constants ( k1 and k2) of palmitate to b-LG.1 Protein concentration

Monomer2

( mM) 1

(%) 95

10

71

50

44

200

26

n1

n2

k1

k2 ( ×10–5

1.00 ±0.01 0.99 ±0.01 1.00 ±0.01 1.03 ±0.01

24.0 ±0.02 24.0 ±0.05 24.0 ±0.05 23.4 ±0.02

13.3 ±0.06 5.46 ±1.11 2.75 ±0.02 2.28 ±0.03

k1/k2

M–1)

4.10 ±0.04 0.45 ±0.04 0.087 ±0.002 0.036 ±0.005

3.24 12.1 31.6 63.5

n1k + n2k2 ( ×10–6) 11.4 (11.8) 3 1.62 (1.62) 0.37 (0.33) 0.32 (0.28)

1Equilibrium

binding data were fit to Equation [1], a two-site ( n 1 and n2) model. were calculated using the dimer dissociation constant reported by McKenzie (11). 3The values in parentheses were obtained from plots of the experimental data. 2Values

function increased substantially, but the second term only slightly decreased. Furthermore, when the value for the first term is plotted against the percentage dimer, a linear relationship is observed (Figure 3). These results suggest that the two sets of independent binding sites may be represented by one set of sites on the monomer and another on the dimer. Quantitatively, this can be expressed as nDkDC

y¯ = y¯ I + y¯ II = ( 1 – FM) ( 1 + kDC ) +

nMkMC ( 1 + kMC )

FM . . .

[3]

Data for palmitate binding were fit to this expression with nM = 24 (monomer) and nD = 1 (dimer) to obtain values for kM, kD, and FM (Table 4). Comparison of the fraction of monomer that is present at various protein concentrations, as obtained by fitting the data for palmitate binding to Equation [3], with that calculated from the previously reported dimer dissociation constant shows excellent agreement. This simple model does not account for possible shifts in

the monomer-dimer equilibrium that result from ligand binding ( a shift in the equilibrium as a result of ligand binding is suggested by the apparent cooperativity indicated in Figure 1A). Nevertheless, the data are fit quite well, and, with the exception of data for 1 mM b-LG, both binding constants ( k D and kM) are similar for all protein concentrations. The site or sites for binding of lipophilic ligands to b-LG have been controversial (2, 3, 7, 8, 12, 13, 14, 15, 16, 18, 24, 25). Most of the current evidence suggests that retinol binds in the calyx formed by the b-barrel (1, 2, 14), although binding in a surface pocket formed by the a-helix has also been suggested (12). Evidence for competition between retinol and palmitate binding also has been contradictory. Using enhancement of tryptophanyl fluorescence to quantitate binding, Frapin et al. ( 8 ) found no competition between retinol and palmitate. We have made a similar observations using an ultrafiltration equilibrium binding technique (unpublished observation). Furthermore, Frapin et al. ( 8 ) found that alkylated and esterified b-LG did not bind palmitate, but the modified protein exhibited enhanced affinity for retinol

TABLE 3. Analysis of the effect of protein concentration on the binding of palmitate. Protein 1.0 10 50 200

Cpalmitate1 ( mM) 0.063 0.60 3.05 6.60

Cpalmitate/Cb-LG

Dimer2

y¯ I3

y¯ II3

y¯ I/y¯

0.063 0.060 0.061 0.033

(%) 5 29 56 74

0.077 0.245 0.456 0.619

0.604 0.634 0.594 0.543

0.11 0.28 0.43 0.533

1Concentration

( C ) of free palmitate. from the protein concentration using the dimer dissociation constant, 3.5 × 10–5 M–1, reported by McKenzie (11). 3Values obtained from Equation [1]. 2Calculated

Journal of Dairy Science Vol. 81, No. 1, 1998

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WANG ET AL. TABLE 4. Fit of equilibrium binding data to distinct independent sites on monomers ( M ) and dimers ( D ) of b-LG.1

Protein

Fraction of monomer ( F M) 2

kD

kM

( mM) 1.0 10 50 200

( ×10–5 0.95 0.71 0.44 0.26

X 13.3 6.33 2.53 2.32

FM (from data fit) 3

SE 2.1 3.3 0.37 0.27

M–1) X 4.53 0.38 0.34 0.20

SE 0.22 0.05 0.01 0.01

X 0.95 0.76 0.34 0.21

SE 0.02 0.09 0.04 0.02

1Equilibrium

binding data were fit to Equation [3] with nM = 24 and nD = 1. fraction of monomer was calculated using the dimer equilibrium dissociation constant reported by McKenzie (11): 3.5 × 10–5 M–1. 3The monomer fraction obtained by fitting the palmitate binding data to Equation [3]. 2The

( 6 ) . Also, increased protein stability has been observed in the presence of palmitate (3, 19), but stability was not increased when retinol was bound (19, 20). Recently, Narayan and Berliner ( 1 3 ) observed that b-LG bound 5-doxylstearic acid (the dissociation constant was 0.8 mM) without affecting retinol binding and that palmitic acid displaced bound 5doxylstearic acid from the protein. However, Puyol et al. ( 1 8 ) observed competition between palmitate and retinol in an equilibrium binding study in which high concentrations of palmitate were used (molar ratio of palmitate to retinol ranged from 23,000 to 230,000). Our results suggest that monomeric b-LG is capable of weakly binding a large number of palmitate

molecules at the surface site that is also the site of interaction between monomers during dimer formation. This surface contains hydrophobic residues that are in the region of the a-helix and b-strands A and I (10, 12, 14). Formation of the dimer eliminates much of the surface that is responsible for palmitate binding, but a smaller, higher affinity binding site is created. Thus, two molecules of palmitate are bound per dimer, and binding affinity is an order of magnitude greater than that for the monomer. These conclusions are consistent with many of the previously reported observations. For example, porcine b-LG lacks some of the hydrophobic residues in the proposed binding region, does not form dimers, and does not bind palmitate (8, 10). Higher stability in the presence of palmitate may result from a required dissociation prior to unfolding, and bound palmitate may stabilize the dimer because of its interaction with residues in both monomers. The cause for the apparent competitive binding of retinol and palmitate observed by Puyol et al. ( 1 8 ) is not clear; however, at the high concentrations of palmitate used in that study, palmitate may weakly bind in the calyx. ACKNOWLEDGMENTS This study was supported by Dairy Management, Inc. through the Southeast Dairy Foods Research Center. REFERENCES

Figure 3. Effect of protein concentration on the binding of palmitate. yI represents the first term of Equation [1]. The percentage dimer was calculated using the equilibrium dissociation constant, 3.5 × 10–5 M, reported by McKenzie (11). y = Moles of palmitate bound per mole of protein. Journal of Dairy Science Vol. 81, No. 1, 1998

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