Binding of Doxyl Stearic Spin Labels to Human Serum Albumin: An

Aug 25, 2014 - Ana D. Popović-Bijelić,. †. Miloš D. Mojović,. †. Snežana V. Šušnjar,. ‡ and Goran G. Bačić*. ,†. †. Faculty of Physical Chemistry, Uni...
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Binding of Doxyl Stearic Spin Labels to Human Serum Albumin: An EPR Study Aleksandra A. Pavićević,† Ana D. Popović-Bijelić,† Miloš D. Mojović,† Snežana V. Šušnjar,‡ and Goran G. Bačić*,† †

Faculty of Physical Chemistry, University of Belgrade, Studentski trg 12-16, P.O. Box 47, 11158 Belgrade, Serbia National Cancer Research Center, Pasterova 14, 11000 Belgrade, Serbia

‡

ABSTRACT: The binding of spin-labeled fatty acids (SLFAs) to the human serum albumin (HSA) examined by electron paramagnetic resonance (EPR) spectroscopy was studied to evaluate the potential of the HSA/SLFA/EPR technique as a biomarking tool for cancer. A comparative study was performed on two spin labels with nitroxide groups attached at opposite ends of the fatty acid (FA) chain, 5-doxyl stearic (5-DS) and 16-doxyl stearic (16-DS) acid. The effects of incubation time, different [SLFA]/[HSA] molar ratios, ethanol, and temperature showed that the position of the nitroxide group produces certain differences in binding between the two SLFAs. Spectra for different [SLFA]/[HSA] molar ratios were decomposed into two spectral components, which correspond to the weakly and strongly bound SLFAs. The reduction of SLFA with ascorbate showed the existence of a two component process, fast and slow, confirming the decomposition results. Warfarin has no effect on the binding of the two SLFAs, whereas ibuprofen significantly decreases the binding of 5-DS and has no effect on 16-DS. Together, the results of this study indicate that both SLFAs, 5-DS and 16-DS, should be used for the study of HSA conformational changes in blood induced by various medical conditions.

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INTRODUCTION The human serum albumin (HSA) is the most abundant blood plasma protein present in concentrations of ∼40 mg mL−1 (0.6 mM).1,2 This 66.5 kDa protein is synthesized in the liver, and from there it is secreted as a single nonglycosylated polypeptide chain.3 HSA has multiple functions under physiological conditions. It possesses a remarkable ligand-binding capacity, and can transport hundreds of endogenous and exogenous compounds, such as bilirubin, hemin, hormones, polypeptides, proteins, and free metal ions.3 Also, it binds a wide range of drugs, like aspirin, ibuprofen, diazepam, and warfarin.4 HSA represents the main carrier for fatty acids (FAs). It typically contains about 0.1−2 moles of FAs per 1 mole of albumin,2 although it may accommodate up to 6 mole under certain disease-related states.1,5 Crystallographic studies of HSA in complex with medium- and long-chain saturated FAs revealed the structure of the complex and the position of the FA binding sites.6,7 It has been shown that both, medium- and long-chain FAs, bind in seven binding sites and that mediumchain FAs bind in two additional sites. Three of the seven FA binding sites overlap with the two major binding sites, drug sites I and II (Figure 1).8,9 FA3 and FA4 overlap with drug site II, and FA7 overlaps with drug site I. The FA binding sites are located in different domains of the protein but have certain common features. The hydrophobic tail of the FA is located in a deep hydrophobic pocket, and the carboxyl group is liganded by two or three basic or polar residues.2,6 The highest affinity binding sites for this ligand on the protein were identified to be FA2, FA4, and FA5.9 © 2014 American Chemical Society

Figure 1. Crystal structure of the human serum albumin cocrystallized with seven stearic acid molecules (PDB 1e7i).7

Using electron paramagnetic (spin) resonance (EPR or ESR) spectroscopy with spin labels has a long history in studying FA binding to albumin. Such spin-labeled fatty acids (SLFAs) contain a doxyl group attached to one of the carbon atoms of the FA chain, which can be detected by EPR.10−20 Various aspects of the binding of different SLFAs were studied using a variety of EPR techniques, and valuable information has been gained, although we are still far from a general consensus view. Recently, it has been proposed that HSA/SLFA/EPR technique can be used clinically as a potential biomarker for certain diseases, most notably cancer. Namely, tumor cells release a Received: July 10, 2014 Revised: August 24, 2014 Published: August 25, 2014 10898

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for the study of the effect of ethanol). Subsequently, 60 μL of HSA of required concentration in 0.9% NaCl, pH 7.4 phosphate buffer, was added and gently mixed. Samples were incubated for 30 min at 22 °C prior to EPR measurements. For examining the effects of ethanol on the binding of SLFA to HSA, samples contained 0.1 mM HSA (in 0.9% NaCl, pH 7.4 phosphate buffer), 0.6 mM SLFA, and 0.5 M ethanol. Samples used for measuring the effect of temperature on the binding of SLFAs to HSA contained 0.1 mM HSA (in 0.9% NaCl, pH 7.4 phosphate buffer) and 0.2 mM SLFA, and were incubated for 30 min at three temperatures. The competition in the binding of 5-DS and 16-DS to HSA was investigated using ratios of [5DS]/[16-DS]/[HSA] of 1:1:1, 1:2:1, 2:1:1, and 2:2:1. All samples contained 0.1 mM HSA (in 0.9% NaCl, pH 7.4 phosphate buffer). Samples used to evaluate the effect of ibuprofen/warfarin on the binding of SLFAs to HSA contained 0.1 mM HSA (in 0.9% NaCl, pH 7.4 phosphate buffer), 0.1 mM ibuprofen or warfarin (dissolved in deionized water, 0.9% NaCl), and varying concentrations (0.05, 0.1, 0.2, 0.3, and 0.6 mM) of SLFAs. Samples were incubated for 30 min at 22 °C prior to EPR measurements. Samples used for measuring the reduction of the bound SLFA to HSA by sodium ascorbate contained 0.1 mM HSA (in 0.9% NaCl, pH 7.4 phosphate buffer) and 0.1 mM SLFA incubated for 30 min at 22 °C. Equal volumes of the SLFA/HSA complex and 2 mM sodium ascorbate were rapidly mixed, and EPR measurements were performed immediately. The kinetics of the reduction was monitored for 20 min. EPR Measurements. The X-band (9.5 GHz) EPR spectra were recorded at room temperature on a Bruker Elexsys-II EPR spectrometer under the following conditions: microwave power 10 mW, modulation amplitude 2 G, modulation frequency 100 kHz, and conversion time 240 ms. Samples were drawn into 10 cm long gas-permeable Teflon tubes (Zeus industries, Raritan, NJ) and folded into 2.5 cm long segments to improve the signal-to-noise ratio.31 Spectra were recorded and analyzed using Bruker Xepr software. Data Analysis. Experimental EPR spectra of 5-DS and 16DS complexes with HSA were simulated using the spectral analysis package EPRSIM-C, which is a five-component model that takes into account isotropic tumbling, isotropic spinexchange label−label and label-broadening agent, and anisotropic tumbling with full and partial averaging of all rotations.32

host of various compounds (proteins, peptide fragments, etc.) into the blood, which can relay very useful disease-related information.21,22 Binding of these metabolites to HSA results in the modification of its structure and function, which in turn leads to an altered HSA binding capacity for FAs that can be studied by EPR using nitroxide-labeled stearic FAs. Current literature data on cancer detection with EPR suggests that 16-doxyl stearic acid (16-DS) should be used for the study of conformational changes of HSA, due to its structural congruence with HSA and the position of the nitroxide group at the terminal part of the FA chain, also claiming that 16-DS is highly specific for HSA.23−27 However, many studies have shown that when FAs, labeled with the doxyl group located at different positions on the carbon chain, are bound to albumin, the doxyl group of the 16-DS is located in a more polar and less fixed environment than that of the 5-doxyl stearic acid (5-DS).15,16 There is nothing special in the binding of 16-DS to HSA; quite to the contrary, it may have some disadvantages over using other SLFAs, e.g., 5-DS. Namely, studies of incorporation of 16-DS to artificial and natural membranes clearly indicated that 16-DS may “bend-back” to the aqueous surface, so that the polar nitroxide group on the C16 atom is not located inside the membrane.28−30 This suggests that the same bending effect may occur during the binding of 16-DS to HSA subdomains so that the polar label on C-16 is out. Indeed, the study of the accessibility of different SLFAs bound to bovine serum albumin (BSA) has shown that 16-DS is more accessible to water-soluble ferricyanide than 5-DS, suggesting that it protrudes from the protein’s hydrophobic pocket.17 All data given above raise a serious question if indeed 16-DS is the best probe for evaluating disease-induced HSA conformational changes. It is hardly advantageous to have a reporter of conformational changes being loosely bound to the structure whose changes it should report. Therefore, the aim of this study was to compare the binding to HSA of two SLFAs, 5DS and 16-DS, which have doxyl groups near the opposite ends of the FA chain. Specifically, the effects of incubation time, different [SLFA]/[HSA] molar ratios in the temperature range 22−37 °C, and ethanol on binding of the SLFAs to HSA were investigated. Competition in binding of 5-DS and 16-DS to HSA was also studied at different [5-DS]/[16-DS]/[HSA] molar ratios. Furthermore, computer simulations of spectra were used to assess binding sites of both SLFAs to HSA. The kinetics of the reduction of SLFAs with sodium ascorbate were determined, in order to gain further insight into the location of the nitroxide group of the bound SLFAs with respect to the protein surface. Finally, the binding of both labels was studied in the presence of drug sites I and II specific ligands, warfarin and ibuprofen. The aim of this study is to gain more knowledge on the binding of SLFAs to HSA but also to lay a solid foundation upon which clinical studies on the use of the EPR/ HSA/SLFA technique as biomarkers should be performed.

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RESULTS AND DISCUSSION Typical EPR spectra of two SLFAs (5-DS and 16-DS) bound to HSA are shown in Figure 2. The dashed line connects certain spectral features of bound 5-DS and 16-DS (sharp peaks) with a spectral feature of free SLFA in the solution, indicating that the solutions of SLFAs and HSA, with [SLFA]/[HSA] ratio of 2:1, also contain a certain small amount of free SLFA. Hence, in Figure 2 it is indicated that the amount of the SLFA bound to HSA can be estimated based on the intensity of the low-field peak (Ilf) in the EPR spectrum while the amount of the free (unbound) SLFA is determined based on the intensity of the high-field peak (Ihf).11 The value of the 2All′ for 5-DS is higher than that for 16-DS, which is in agreement with previous works.11,14,17,18 From values of spectral parameters, one can in principle calculate the rotational correlation time (τc), which is a measure of the mobility of bound SLFA, i.e., the strength of binding. Our results show that values for τc (calculated using the formula from ref 16) are 26 ns for 5-DS and 9.5 ns for 16-DS. Faster

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EXPERIMENTAL SECTION Chemicals. Human serum albumin (HSA) and spin-labeled fatty acids (SLFAs, 5- and 16-doxyl stearates) were obtained from Sigma-Aldrich. Ibuprofen was obtained from SigmaAldrich, and warfarin was obtained from Fluka (St. Louis, MO, USA). Sample Preparation. One microliter of the stock solution of SLFAs in ethanol was allowed to dry on the walls of Eppendorf sample tubes (exceptions are the samples prepared 10899

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Figure 3. Effect of the [SLFA]/[HSA] molar ratio on the binding of 5DS (■) and 16-DS (●) to HSA as measured by the intensity of the low-field peak, Ilf (a.u.). All samples contained 0.1 mM HSA and varying concentrations of SLFAs, and they were incubated for 30 min at 22 °C and pH 7.4.

binding over 5-DS. Namely, the intensity of peaks depends on the amount of the compound but also on the width, which in turn depends on its motion. 16-DS shows faster motion than 5DS (9.5 ns vs 26 ns); hence, the Ilf for 16-DS is artificially higher than that for 5-DS. The absolute amount of SLFA bound to HSA can be, in principle, determined by double integration of spectra, and then the values are compared to the values of standards. However, this is not a very accurate procedure, and we were more interested in changes of the relative intensities of the low-field EPR peak with the changing of the [SLFA]/ [HSA] ratio. The binding of both SLFAs does not show saturation in the studied range of [SLFA]/[HSA] ratios. One would expect that the width (W1/2) of Ilf changes with the [SLFA]/[HSA] ratio because more WB component with a narrower line is present and WB/SB can be deduced from that. We did not observe that with confidence, indicating that this is not a sensitive procedure. Hence, simulations were performed to determine contributions of strongly and weakly bound SLFA (see Table 1).

Figure 2. EPR spectra of 5-DS (a) and 16-DS (b) bound to HSA. Both samples contained 0.1 mM HSA (in 0.9% NaCl pH 7.4 phosphate buffer), and the [SLFA]/[HSA] ratio was 2:1. EPR spectrum of free 5-DS (0.02 mM) in phosphate buffer (c). The intensities of the low-field and the high-field peaks are marked as Ilf, and Ihf, respectively. 2All′ marks the outer (maximal) hyperfine splitting. W1/2 denotes width of the peak at half-maximum.

rotation for 16-DS also has been found by other authors.10,11,15,16 One can then conclude that 5-DS is more tightly bound than 16-DS, but that would be a premature conclusion because there is more than one binding site on HSA for SLFAs15,19,20,25 and the spectra in Figure 2 are an average of the spectra from different sites with different binding characteristics. A more detailed analysis of these spectral components (weakly bound (WB), fast rotating and strongly bound (SB), slowly rotating) is given later (see Figure 6). Effect of Incubation Time and [SLFA]/[HSA] Molar Ratio. Different experimental procedures have been performed to achieve the maximum binding of 5-DS and 16-DS to HSA,18,25 and results obtained for optimal incubation time are not consistent. Therefore, the binding was assessed at 22 °C and pH 7.4 via the height of the low-field peak in the EPR spectrum of the bound SLFA. Full binding occurs at 30 min incubation time for both labels (data not shown); hence, all incubations in all further experiments were carried out for this period of time. It is worth noting that the binding of 16-DS was slower, especially at higher ratios of [SLFA]/[HSA]. Binding of SLFA to HSA. Determination of bound and free SLFA was performed at different [SLFA]/[HSA] ratios (0.5:1− 6:1). The amount of the bound SLFA linearly increases when the [SLFA]/[HSA] molar ratio is increased (Figure 3). Note that the binding is expressed in arbitrary units of the intensity of Ilf and that higher values for 16-DS do not signify increased

Table 1. Contribution of the Strongly (SB) and Weakly (WB) Bound EPR spectral Components to the Overall Simulated Spectra of 5-DS and 16-DS Bound to HSA at Different [SLFA]/[HSA] Molar Ratios (The Goodness of All Simulation Fits Was 90%) to each simulated spectrum arises from two components associated with anisotropic tumbling. The simulated EPR spectra of these two components for both SLFAs bound to HSA at 22 °C and pH 7.4 are shown in Figure 6. On the basis of their spectral widths (2All′), they were assigned to the weakly bound (WB) and strongly bound (SB) SLFAs within the HSA molecule, where 2All′ for SB is larger than that for WB for each SLFA. The widths of the simulated spectra of the 5-DS/HSA complex, and its corresponding WB and SB components, are larger than those for 16-DS, indicating again that 5-DS is more immobilized than 16-DS when bound to HSA. The 2All′ for the WB component of 16-DS is considerably smaller than that for the rest of the sites, indicating again that at least some portion of 16-DS is protruding to the surface of HSA due to the relatively hydrophilic nature of its tail. The contributions from both spectral components for various [SLFA]/[HSA] molar ratios are given in Table 1. The SB/WB 10901

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Figure 7. EPR spectra of individual spin labels ([SLFA]/[HSA] = 4) on the left. Sum of two spectra 5-DS/HSA and 16-DS/HSA (top right). Spectrum of SLFAs mixture ([5-DS]/[16-DS]/[HSA] = 2:2:1) bound to HSA (bottom right).

Figure 6. Experimental and simulated EPR spectra of (a) 5-DS and (b) 16-DS bound to the HSA and their respective main components representing the weakly (WB) and strongly (SB) bound SLFA. The simulation was performed for the experimental EPR spectra measured at 22 °C and pH 7.4 (the samples contained 0.1 mM HSA and 0.2 mM SLFA).

Table 2. 2All′ Values of 5-DS and 16-DS Bound to HSA Individually and in a Mixture at Different [SLFA]/[HSA] Ratiosa

ratio decreased as the [SLFA]/[HSA] molar ratio was increased, indicating that the SLFAs first occupy the higher affinity, SB, sites. The values in Table 1 are in good correlation with those of Gurachevsky et al.,18 where a similar simulation has been used. For example, at 20 °C with 16-DS/HSA = 1.8:1, they obtained 0.86 for SB and 0.14 for WB. Competition between 5-DS and 16-DS. To elucidate whether 5-DS and 16-DS bind to the same sites in HSA, mixtures of both 5-DS and 16-DS were incubated with HSA. A study of the competition between 5-DS and 16-DS in binding to HSA was performed by analyzing spectra of mixtures containing [5-DS]/[16-DS]/[HSA] ratios of 1:1:1, 1:2:1, 2:1:1, and 2:2:1. The spectra in Figure 7 demonstrate that, for the [SLFA]/[HSA] ratio 4:1 (i.e., [5-DS]/[16-DS]/[HSA] ratio of 2:2:1), the amount of unbound SLFA is increased in the mixture compared to the samples containing only 5-DS or 16DS. This difference also can be clearly observed when comparing the mixture spectra with the sum of individual spectra. The spectral shape was observed and values of 2All′ were measured (Table 2) to find out which of the two SLFAs contributes more to the unbound SLFA in the mixture. The spectra of SLFA mixtures bound to HSA with equimolar amounts of 5-DS and 16-DS have values of 2All′ closer to that of 16-DS. The same holds for the [5-DS]/[16-DS]/[HSA] ratio of 1:2:1, where there is more 16-DS; however, for the [5DS]/[16-DS]/[HSA] ratio of 2:1:1, when there is more 5-DS, the 2All′ value is higher than that for 16-DS alone and lower than that for 5-DS. The possibility that 5-DS and 16-DS compete for the same binding sites at BSA has been reported previously,11 but using individual titration rather than competitive binding. Taking into account the much closer resemblance of overall spectral shapes of SLFA mixtures with HSA to spectra of 16-DS/HSA than to spectra of 5-DS/HSA, together with the values of 2All′, it may be suggested that 16-DS

a

[5-DS]/[HSA]

2All′ (G)

1:1 2:1 4:1 [16-DS]/[HSA]

64.1 63.6 63.7 2All′ (G)

1:1 2:1 4:1 [5-DS]/[16-DS]/[HSA]

59.5 59.4 59.2 2All′ (G)

1:1:1 1:2:1 2:1:1 2:2:1

61.0 61.7 62.2 60.6

The HSA concentration was 0.1 mM in all samples.

binds with greater affinity to HSA than 5-DS when they are introduced together. Such results are in good accordance with the published affinity constants.15 It has been proposed earlier that when any two of the SLFAs (5-DS, 10-DS, and 12-DS), regardless of whether they are the same or different, are bound to HSA, they form an antiparallel arrangement.36 This concept of antiparallel binding implies that the chains of two SLFAs have parallel directions, but their carboxylic groups lay at the opposite sides. In such an arrangement, doxyl groups of 5-DS and 16-DS approach each other closely, leading to the decrease in binding affinity, due to the steric interferences. Such an explanation is in good agreement with the data, indicating that the doxyl group of 16-DS protrudes the surface of HSA and that the same moiety of 5-DS is located in the interior of HSA.17,25,37 In spite of the conclusion that when mixed together, 16-DS binds to HSA with greater affinity than 5-DS, the latter may still be a better candidate for the HSA/SLFA/ EPR method for cancer diagnosis, because there is a smaller amount of unbound 5-DS than 16-DS when they are 10902

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analyzed. In liposomes where only one location of the SLFA within the bilayer is possible, the reduction follows the singlecomponent first-order kinetics.38,39 Taken together all data indicate that at least two different binding sites exist within HSA for each SLFA. The observed difference in the reduction rates suggests that each SLFA is bound to at least two different sites on HSA (arbitrarily connected to the SB and WB sites) being differently accessible to water (ascorbate). The faster kinetics, observed during the first 10 min, correspond to the reduction of SLFAs bound predominantly to sites more accessible to water but with the contribution of the less accessible sites. It is evident from both Figure 8 and Table 3 that the fastreducing component of the 16-DS is reduced much faster than that for 5-DS. Having in mind that the rate of reduction is similar for all SLFAs in the buffer, this demonstrates that the nitroxide group of 16-DS is more accessible to water than that of 5-DS, suggesting that it protrudes from HSA, as proposed earlier.16,17 The reduction of SLFA in a slowly reduced site is slower for 5-DS than for 16-DS, indicating that this moiety could be buried more deeply into the hydrophobic core of HSA than 16-DS. After 10−15 min, all SLFAs readily exposed to ascorbate are almost completely reduced. The contributions of the SB and WB EPR spectral components were determined at 0 and 15 min of the reduction reaction. Table 4 shows that the SB/WB

introduced separately, which is consistent with previous results.36 Reduction of Spin Labels with Ascorbate. An alternative method of assessing the state of SLFA bound in a predominantly hydrophobic environment has been developed38 and subsequently applied for assessing SLFAs within liposomes39 or bound to the serum albumin.13,14 In this method the accessibility of water to spin labels is tested by using ascorbate, which reduces SLFA to the EPR silent hydroxylamines, and the rate of reduction is monitored by observing the time dependence of the disappearance of the SLFA EPR signal. Reduction kinetics for both SLFAs are given in Figure 8, where

Table 4. Contribution of the Strongly (SB) and Weakly (WB) Bound EPR Spectral Components to the Overall Simulated Spectra of 5-DS and 16-DS Bound to HSA at 0 and 15 min during the Ascorbate Reduction Reaction (The Goodness of All Simulation Fits Was