Materials Science and Engineering C 33 (2013) 836–843
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Effect of bovine serum albumin on the functionality and structure of catanionic surfactant at air–buffer interface Kajari Maiti a, Subhash C. Bhattacharya a, Satya P. Moulik a, Amiya K. Panda b,⁎ a b
Centre for Surface Science, Department of Chemistry, Jadavpur University, Kolkata 700 032, India Department of Chemistry, University of North Bengal, Darjeeling 734 013, India
a r t i c l e
i n f o
Article history: Received 26 June 2012 Received in revised form 9 October 2012 Accepted 5 November 2012 Available online 13 November 2012 Keywords: Coacervate DPPC BSA π A isotherm Microscopic images
a b s t r a c t Interaction of bovine serum albumin (BSA) with the solvent spread monolayer of a catanionic surfactant, octadecyltrimethylammonium dodecylsulfate, (C18TA+DS−) at the air–buffer interface was investigated by measuring the surface pressure with time and change in surface area. Dipalmitoylphosphatidylcholine (DPPC) was used as reference. Kinetics of BSA desorption from the interface to the buffer subphase, that of C18TA+DS− and DPPC through their interaction with BSA, were also studied at different BSA concentrations (in the subphase) and surface pressures. Surface pressure (π)–area (A) isotherms (at pH = 5.4, μ = 0.01, T = 298 K) revealed that the coacervate/DPPC monolayer becomes expanded in the presence of BSA at low π while their protein bound species are released into the subphase at high π. Film morphology, studied by epifluorescence microscopy (EFM) and atomic force microscopy (AFM), reveals that the sizes of the domains of both DPPC and coacervate decrease in the presence of BSA. Presence of BSA in the coacervate and DPPC monolayer was supported from AFM data analysis. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Globular proteins find uses in medicinal and pharmaceutical research for their capability to catalyze biochemical reactions, to adsorb as well as bind specifically to other molecules and subsequently form molecular aggregates [1]. Besides, they find potential applications as biochemical sensors [2]. Serum albumin is the most abundant protein in the circulatory system and contributes 80% to colloid osmotic blood pressure [3] and also maintains the pH of blood [4]. The most outstanding property of albumin lies in its ability to bind to an incredible variety of ligands and a broad range of metabolites, drugs, amphiphiles and other organic compounds [5–10]. Interaction of ionic surfactants with BSA has been widely studied because of the relatively large number of charged amino acids on its surface (net charge=−17 at pH 7) [11,12]. According to Bos et al. [13], BSA is heart shaped, comprising nine loops held together by 17 disulfide bonds, resulting in three domains (I, II, and III) each containing two subdomains or alternatively each containing one small and two large loops. These loops are mainly composed of helical segments [14]. The adsorption behavior of different proteins in combination with surfactants has been studied in detail by Miller et al. [15–17]. Interaction of ionic surfactant with BSA leads to the formation of some amphiphilic entities with altered surface activity. In the case of non-ionic surfactants, the adsorption layer is mainly formed by competitive adsorption
⁎ Corresponding author. Tel.: +91 9433347210; fax: +91 353269901. E-mail address: [email protected] (A.K. Panda). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2012.11.009
between protein and non-ionic surfactant molecules and the interaction is only hydrophobic in nature [17]. Such complexes are typically less surface active than the pure protein. Even interactions between proteins and cationic/nonionic amphiphiles as well as DPPC [18] are available in the literature, however, the studies involving catanionic surfactants with globular proteins are not so common. It is of great theoretical and practical importance to investigate such systems especially with reference to protein separation [19,20]. When the catanionic surfactants are used, their required concentrations are usually very low such that the proteins can maintain its interfacial activity even in the presence of amphiphiles [21]. Ionic surfactants could cause major structural changes in the protein conformation with subsequent changes in protein functionality which is not desirable in many applications; the uses of catanionic surfactants are thus preferred over individual components. Recently, catanionic surfactants or coacervates have gained attention of researchers for their structural resemblance to phospholipids [22,23] and their ability to form various aggregated microstructures such as micelles, vesicles, lamellar phases, etc. [24]. The catanionic surfactants are extensively used in many fields of technology and research, including pharmaceutical preparations. DPPC is the major component of lung surfactant and serum albumins are known for surfactant dysfunction [25]. The best way to understand the functionality of an amphiphile at the air–solution interface is to study its surface pressure–area isotherms [26]. In this perspective, it is believed to be worthy to study the interaction of BSA with a lipid mimicking amphiphile, catanionic surfactant, C18TA +DS − and hence to compare the results with DPPC.
K. Maiti et al. / Materials Science and Engineering C 33 (2013) 836–843
In this paper, interaction between well characterized coacervate (C18TA+DS −) and BSA has been studied by the Langmuir monolayer technique. The results are compared with the lipid, DPPC. Surface morphology of the Langmuir–Blodgett films of the coacervate and DPPC has been studied by combined epifluorescence microscopy and atomic force microscopy (AFM). We have focused our attention on the three aspects: (i) adsorption properties of BSA molecules at the air–solution interface, (ii) nature of binding of BSA with C18TA+DS − and DPPC along with their interaction kinetics at the interface and, (iii) a comparative studies between the catanionic and the phospholipid. The results are expected to generate information and understanding on the physicochemistry of lipid–protein interaction. 2. Materials and methods 2.1. Materials Octadecyltrimethylammonium bromide or C18TAB was a product of Fluka, Switzerland (purity ≥ 97%). BSA (MW = 66430 Da, purity > 98.5%), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (purity > 98%), sodium dodecysulphate (SDS) (purity > 99%), and the fluorescent probe, ([1-palmitoyl-2-{12-[(7-nitrobenz-2-oxa-1,3-diazo4-yl)amino] dodecanoyl}-sn-glycerol-3-phosphocholine]) or NBD-PC (purity> 95%) were purchased from Sigma, USA. AR grade acetic acid, sodium acetate, NaOH (BDH, India) and borax (Merck, India) were used for the preparation of buffer solutions. The following compositions were used to get buffers at ionic strength (μ) = 0.01: 500 mL 3.08 mM sodium acetate+ 500 mL 16.92 mM acetic acid (for pH 4); 500 mL 16.41 mM sodium acetate + 500 mL 3.59 mM acetic acid (for pH 5.4); and 500 mL 4.54 mM borax + 500 mL 6.37 mM NaOH (for pH 10). Chloroform and methanol of AR grade (SRL, India) were used for preparing the DPPC and coacervate solutions. Doubly distilled water (specific conductance= 2–4 μS cm−1 and surface tension = 72 mN m −1 at 298 K) was used to make BSA and buffer solutions. 2.2. Methods 2.2.1. Preparation and isolation of coacervate Equimolar amounts of aqueous C18TAB and SDS solutions were mixed with constant stirring. The formed catanionic coacervate was isolated by repeated partitioning between chloroform-water [22]. After solvent evaporation, the coacervate was vacuum-dried for 24 h and then stored. 1H NMR, XRD, FTIR, DSC study, etc., were performed on the formed coacervate to ensure that the results matched with that of reported C18TA +DS − [22]. 2.2.2. Monolayer study A Langmuir trough (Apex Inst. Co., India) with a surface area of 300 cm 2, placed on an anti-vibration table, was used. Surface pressure was measured by the Wilhelmy plate technique with an accuracy of ±0.1 mN m −1. Buffers of different pH (μ = 0.01) were used as subphases. For measuring the surface pressure (π)–area (A) isotherm of BSA, an aqueous solution of BSA (0.5 mg mL −1) was added to the buffer subphase using a Hamilton microsyringe; after careful mixing, the BSA monolayer was formed through adsorption from the subphase onto the air–buffer interface. The π–A isotherm was recorded after 30 min. For pure C18TA +DS − and DPPC at the air–buffer interface, their chloroform–methanol solutions (3:1, v/v) were spread by a Hamilton microsyringe onto the buffer subphase and after the solvent evaporation (30 min), the compression process was initiated. For the interaction and kinetic studies (adsorption or desorption processes), in the Langmuir trough, the coacervate or DPPC (dissolved in chloroform–methanol solution) was spread over the buffer subphase. After 30 min, aqueous BSA solution was gently injected at the bottom of the trough right underneath the monolayer without disturbing the monolayer. After waiting for an hour (for
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equilibration) [23], the film compression was compressed with a barrier speed of 5 cm 2 min −1. All data presented here were averages of at least three measurements for each studied condition. 2.2.3. Epifluorescence microscopy (EFM) A Motic phase contrast microscope (model: AE31, Hong Kong) was used to take the images of NBD-PC-doped (1 mol% with respect to the amphiphile) coacervate or DPPC monolayer in the absence and presence of BSA (0.25 μg mL −1) in the subphase at different surface pressures. The fluorescent probe was excited at 470 nm with a green emission (530 nm) [22,23]. The images of the compressed films at the desired surface pressure were taken with a digital camera. 2.2.4. Atomic force microscopy (AFM) Coacervate or DPPC films in the absence and presence of BSA were transferred onto the freshly cleaved mica substrate by Langmuir– Blodgett technique [27]. Ten minutes after attainment of the desired surface pressure, the pre-immersed mica substrate was lifted with an upstroke of 1 mm min −1 with a 1:1 transfer ratio. The transferred films were scanned within 2 h with an atomic force microscope (NT-MDT, Russia) with a 25 μm × 25 μm J scanner. A silicon nitride tip with a typical force constant of 120 mN m −1 was used for scanning. AFM measurements were carried out in non-contact mode in the air. The images were then flattened and analyzed with NT-MDT Solver-pro software to obtain the corresponding topography and phase images [28]. All measurements were carried out at the ambient but controlled temperature (298 ± 1 K). 3. Results and discussion 3.1. Experimental data analysis 3.1.1. A. pH dependent π–A isotherm of BSA π–A isotherm of BSA, as shown in Fig. 1 (main diagram) at 298 K at the air–buffer interface reflects its pH-dependent behavior. Although there was no significant change in the Alim (the area after which the surface pressure starts rising; Alim values were nearly constant in the range of 82–85 nm 2 molecule −1) at a different pH, however, the slopes of the curves increased steeply with increase in subphase pH and the feature was more prominent after their crossing point (at 51.5 nm 2 molecule −1 at π = 17 mN m −1). Independence of surface pressure with the pH at 17 mN m −1 implies that the orientation of BSA molecules at the interface was independent of pH at this surface pressure. BSA, can undergo reversible conformational changes with pH [29]. Different pH-dependent isomeric forms of BSA can exist
Fig. 1. Surface pressure (π)–area (A) isotherm of BSA at different pH. [BSA]=0.1 μg mL−1. Inset: Alim–[BSA] profile at T=298 K and subphase pH=5.4.
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[30]. π–A isotherms of BSA (at pH = 4 and 5.4) have another crossing point at 7.5 mN m −1. It cannot be reasoned out at present, further research is needed.
It is assumed that all the free energy of desorption of segments are equivalent. ΔGd is thus expressed as [40]:
3.1.2. B. π–A isotherm of BSA near its IP (pH = 5.4) The π–A isotherm of BSA at different concentrations in the subphase at pH 5.4 resulted in interesting features (figures shown in supplementary section). The lift-off molecular area (Alim) of the protein in its compressed monolayer gradually decreased with increasing [BSA] up to 0.24 μg mL−1, then it became constant (inset, Fig. 1). At lower concentration, higher Alim resulted because of solubility of the globular protein in the bulk. The number of BSA molecules at the air–buffer interface became less than expected. With increasing [BSA], the interface experienced greater protein coverage making Alim to decrease and finally saturation occurred at 0.24 μg mL−1. In the inset plot, two distinct transition points at 0.11 and 0.24 μg mL −1 of BSA were observed. The first transition corresponded to an orientation change of the BSA molecules in the monolayer whereas the second stood for surface saturation of BSA at the interface for maintaining a constant interfacial population. The data revealed that the constant point corresponded to the molecular area of BSA to be 40 nm2 molecule−1. The protein adsorption studies [15] revealed that at lower surface pressures an adsorbed molecule might occupy different parts of the interface (i.e., a maximum molar surface area is occupied), whereas minimum molar surface area was achieved at large π. With increasing surface coverage (i.e., stronger competition between the adsorbed protein molecules), their molar areas become smaller until finally a minimum area was reached. This implied that the adsorption layer thickness increased with increasing protein concentration. This led to an increased coverage up to an almost complete saturation of the adsorption layer at high protein concentration. The fact that the surface pressure of concentrated protein solutions is independent of the bulk concentration can also be satisfactorily explained in the framework of a monolayer model, considering a weakening of the inter-ion interactions in concentrated monolayers [31] or the possibility of two-dimensional aggregation of adsorbed protein molecules [32]. An illustration of the idea was schematically shown by Miller et al. [15]. From the result of adsorption isotherm and adsorption kinetics of lysozyme, Hunter et al. [33] have proposed that at higher protein concentration, the conformation of the lysozyme at the air– water interface changed from side-on to end-on form. From hydrodynamic experiments [34,35] and low-angle X-ray scattering studies [36], serum albumin has been postulated to be an oblate ellipsoid with dimensions of 14× 4 nm as supported by others [37]. The nature of the π–A isotherm of BSA on buffer sub-phase also varied with [BSA] but more or less similar isotherm profiles were obtained at [BSA]≥ 0.24 μg mL−1. Increased adsorption leads to smaller degree of unfolding with advancing time [38]. The adsorption kinetics of proteins was systematically investigated by Graham and Phillips [39], and later by others [38]. It is known that depending on the concentration in the bulk and surface pressure, long chain flexible polymer molecule can adopt different configuration at the interface where some segments remain adsorbed at the interface while others extend as loops into the adjacent liquid phase [40]. It has been proposed that an equilibrium exists between the desorbed segments (Nd) and the segments at the interface (Ni). The related equilibrium constant (κe) is defined as [40]:
ΔGd ðπÞ ¼ ∫ adπ
π∞
κe ¼
Ni ΔGd ðπÞ−πa ¼ exp Ns kT Nd
ð2Þ
πe
where, πe and π∞ are the equilibrium surface pressure (taken as zero) and surface pressure at which all the molecules are completely desorbed, respectively. In practice, π∞ has been taken as the maximum surface pressure that the film attains. In the case of Langmuir monolayer of a protein, the fraction of segments desorbed from the interface, r (π) is the parameter that characterizes the stability of the monolayer and can be expressed as [41]: rðπÞ ¼
−1 Nd ΔGd −πa −1 ¼ ½κe þ 1 ¼ exp þ1 : Ns kT ðNi þ Nd Þ
ð3Þ
Using Eq. (3) one can estimate π corresponding to the maximum of desorbed segments where the protein monolayer is most compact. In Fig. 2, calculated fractions of the desorbed segments of BSA at different concentrations have been plotted as a function of π. It is observed that with the increase in [BSA], the r values increased up to ~ 0.4 (at [BSA] ≤ 0.16 μg mL − 1), after which the r–π lines merged with one another. However, all the r–π lines reached to saturation at r = 0.50 in the range of 20–30 mN m − 1. The r–π lines at different pH values (inset, Fig. 2) have similar features and have reached the same saturation value (0.50). The r values decreased with increasing pH and reached the saturation at π = 23, 26 and 31 mN m − 1 at pH 4, 5.4 and 10, respectively. The results suggested that in the range of 20–30 mN m − 1, the stability of the BSA monolayer was maximum. This particular π range has been considered as the optimum π for getting the LB deposits [42]. 3.1.3. C. Interactions of C18TA +DS − and DPPC with BSA at the air–buffer interface The results of interaction of DPPC and C18TA +DS − with BSA at the air–buffer interface are presented in Fig. 3. It is observed that with increasing [BSA] in the subphase, the Alim of C18TA +DS − increased. Due to the complexation of the cationics with BSA at the interface there occurred an expansion of the monolayer. Similar behavior was noticed also for DPPC indicating the existence of significant association between BSA and DPPC. The Alim values increased with increasing [BSA] and remained constant at [BSA] = 0.11 and 0.16 μg mL −1 for C18TA +DS − and DPPC, respectively (Fig. 3, inset). Beyond these limiting concentrations, values did not change significantly for both the systems. At pH 5.4, the nature of the π–A isotherm (beyond π =
ð1Þ
where, ΔGd is the potential barrier which opposes the desorption of the whole molecule (i.e., free energy of adsorption of a molecule), Ns is the number of segments in the macromolecule (amino acid residues which in the case of BSA is 583), a is the area per protein molecule, k is the Boltzmann constant and T is the Kelvin temperature.
Fig. 2. r–π profile at different [BSA] at 298 K. [BSA] (μg mL−1): 1, 0.010; 2, 0.10, 3, 0.13; 4, 0.17; 5, 0.24; and 6, 0.32. Inset: Effect of subphase pH on the r–π profile. [BSA] in subphase=0.1 μg mL−1.
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former can accommodate more BSA molecules resulting in higher expansion of the monolayer than the latter [43,47,49,50]. 3.3. Kinetics of interfacial desorption and penetration
Fig. 3. π–A isotherms of DPPC and C18TA+DS− in absence and presence of 0.25 μg mL−1 BSA. pH=5.4 and T=298 K. Systems: 1, pure DPPC; 2, pure C18TA+DS−; 3, DPPC–BSA and 4, C18TA+DS−–BSA. Inset: Variation of Alim of C18TA+DS− (O) and DPPC (□) with [BSA].
17 mN m −1 for both the coacervate and DPPC) was found to resemble with that of the pure BSA having concentration ≥0.24 μg mL−1. The concentration difference between the coacervate and the DPPC is a measure of the difference in the extent of interaction. Similar type of reports are also available in the literature [43,44]. Compressibility modulus (CS-1) of a monomolecular film can be expressed as: −1
Cs
∂π ¼ −A : ∂A T
ð4Þ
In CS−1 vs. π plot, the maximum value (CS −1max) indicates the state of the molecules at the air–liquid interface [45]. The CS −1max values were found to be 30.8, 55.1, 145.5, 35.0 and 30.5 mN m −1, for C18TA +DS −, BSA, DPPC, C18TA +DS −–BSA and DPPC–BSA system, respectively. Results suggest that the pure coacervate (C18TA +DS −) was in liquid-expanded state, whereas pure BSA and pure DPPC were in liquid state and liquid-condensed state, respectively [22,23]. The BSA–C18TA +DS − and BSA–DPPC systems were in the liquidexpanded state. The coacervate monolayer became compact whereas the DPPC monolayer became fluidized (transferred from liquidcondensed to liquid-expanded state). These findings supported penetration of BSA into the monolayer. Wang et al. [46] have shown that human serum albumin ([HSA] = 10 −7 M) also induces a large expansion in dimyristoylphosphatidic acid (DMPA) monolayers, whose isotherms exhibit liquid-condensed to liquid-expanded (LC to LE) phase transition.
Due to its amphiphilic nature, BSA is used in different applied processes, viz., emulsions, thin films, foams, etc. [51]. At its initial stage, adsorption at the air–solution interface is diffusion controlled but the adsorbing molecule has to overcome surface pressure as well as the electrostatic energy barriers in order to locate itself at the interface [52,53]. At the interface, after attaining a certain surface pressure, BSA molecules get desorbed. Kinetics of this desorption process was studied by monitoring the reduction in π with time at different BSA concentration in the subphase (pH 5.4, and μ= 0.01 at 298 K). Results are shown in Fig. 4. Both C18TA +DS− and DPPC can withstand a moderate to high surface pressure. The possibility of penetration of BSA into the DPPC and the coacervate (C18TA+DS−) monolayer was explored by adding BSA solution underneath the compressed C18TA+DS− (Fig. 5) and DPPC (Fig. 5) monolayer. It was observed that, upon addition of BSA into subphase of the compressed monolayer, π values decreased with time and the rate depended on both π and the [BSA] in the subphase. The desorption process of BSA from the interface followed a pseudo first order kinetics obeying the relation given bellow: κ¼
ðπ −πf Þ 2:303 log t ðπi −πf Þ t
ð5Þ
where, πt, πi, and πf are the surface pressure of the monolayer at time t, initial and final, respectively, and κ is the first-order rate constant. −πf Þ log ððππti −π vs t plots were used to get κ from the initial straight lines. fÞ The πf and κ values at π = 15 mN m −1 are shown in Table 1. Results revealed that BSA desorption rate decreased with increasing [BSA]. Essentially, the inter-protein molecular interaction slowed down the rate of desorption process. At [BSA]= 0.25 μg mL−1, the simultaneous occurrence of desorption (up to t ≈ 1000 s) and adsorption process was observed for the case of C18TA +DS− (Fig. 5). At π = 15 mN m−1, pure C18TA+DS − produced a low κ value which increased with BSA addition; so was the observation for the compressed DPPC monolayer. But the desorption rate of C18TA +DS− in the presence of BSA was greater than DPPC (κDPPC−BSA 〈κC18 TAþ DS− −BSA ) as the solubilization of DPPC from its compressed monolayer by BSA was relatively easier than solubilization of C18TA+DS − under comparable states of compression because of the presence of more hydrophilic part in the former than in the latter. The unusual behavior of the π vs t graph of C18TA +DS− at πi = 15 mN m −1 and [BSA] = 0.25 μg mL−1 (Fig. 5, main diagram) was because the coacervate monolayer was in liquid-expanded state and capable of incorporating BSA into the air–buffer interface, which
3.2. Mechanism of BSA–coacervate and BSA–lipid interactions Surface activity of BSA arises from its amphiphilic nature. The attractive coacervate–protein and lipid–protein interactions can change its molecular organization at the interface. Both the coacervate and DPPC are zwitterionic. Thus, ion–ion induced dipole interaction and hydrophobic interactions are responsible for the accumulation of BSA at the interface and subsequent penetration into the coacervate or DPPC monolayer. In order to accommodate its zwitterionic head group, the coacervate and DPPC molecules bind to the hydrophobic pockets of BSA near the charged amino acids [47]. The co-adsorption of BSA along with DPPC occurred through a non-electrostatic mechanism competing with the repulsive hydration force exerted by the phospholipids [48]. The hydrophobic interaction in BSA–coacervate and protein–lipid systems allows the protein molecules to get co-adsorbed into the monolayer resulting in an expansion. Interfacial packing of the coacervate is less compact than DPPC for which the
Fig. 4. Effect of bulk concentration of BSA on its π–time (t) profile at 298 K. Subphase −πf Þ pH = 5.4. [BSA](μg mL−1): O, 0.05; ▲, 0.10; □, 0.175 and ∇, 0.25. Inset: ln ððππti −π vs t fÞ profile for [BSA] = 0.25 μg mL−1.
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Fig. 5. Surface desorption of C18TA+DS− and DPPC (inset) in the presence of BSA at pH = 5.4 at 298 K. [BSA] (μg mL−1) : ●, 0; O, 0.05; ▲, 0.10; □, 0.175 and ∇, 0.25.
under compression (at π>10 mN m−1) desorbed into the subphase following a first order kinetics. Δπ (πi–πf) vs. t profile at different initial surface pressures of compressed C18TA+DS− and DPPC monolayer films at [BSA] =0.25 μg mL−1 (at pH 5.4 and μ=0.01) at 298 K are presented in Fig. 6. The related kinetic data are given in Table 2. With increasing state of compression, the adsorption rate for both C18TA+DS−–BSA and DPPC–BSA systems decreased. For the desorption process (from πi = 15–35 mN m−1), it was found that values of the rate constant for the first system decreased with increasing [BSA] whereas the reverse case was observed for DPPC. According to Kamilya et al. [54] the incorporation of protein (egg lecithin) into the monolayer of DPPC was more favorable in its LE (liquid-expanded) region (πi ≈6 mN m−1). 3.4. Epifluorescence microscopic (EFM) study Film morphology of C18TA +DS − and DPPC monolayer in the absence and presence of BSA was studied by epifluorescence microscopy (EFM). Results are summarized in Table 3. Pure BSA did not produce any significant image (or the image contrasts were b1 μm, lower than the resolution limit). The average circular radius of the domain of pure DPPC at the air–buffer interface increased up to π =35 mN m−1 (honeycomb-like structures appeared at this π) after which it decreased and eventually the features collapsed at 40 mN m −1 (shown in supporting information). In the case of pure C18TA +DS− (Fig. 7A), about 2.5 fold increase in the size was found for a rise of π by 5 mN m−1; afterwards it decreased and collapsed at 35 mN m −1 (shown in the supporting information). At any particular π, the domain size obtained for pure DPPC was larger than that of pure C18TA+DS−, and the collapse pressure of the former was also higher than the later. It was reported [22] that the EFM images of C18TA+DS − and DPPC at the air–water interface at 298 K at 25 mN m −1 produced circular domains of average radius 30 and 67 μm, respectively; at 35 mN m −1, Table 1 Kinetic parametersa,b for the desorption of pure BSA, coacervate–BSA and lipid–BSA complexes at different protein concentration in buffer subphase (pH = 5.4) at 298 K. [BSA]/μg mL−1
0 0.05 0.10 0.175 0.25
104 k/s−1 C18TA+DS−–BSA
DPPC–BSA
Only BSA
3.90 4.42 7.21 10.5 11.4
3.72 4.25 5.63 6.50 7.14
– 11.1 9.55 8.81 8.66
a The k value calculation at πi = 15 mN m−1 was based on data only up to 1000 s as the initial desorption process was followed by the adsorption processes after 1000 s. b The error limits of k are within ±5% and the values of linear regression factors (R) are in between 0.975 and 0.980.
Fig. 6. Surface pressure (Δπ=πi− πf) vs. t plots for C18TA+DS− (A) and DPPC (B) at different initial surface pressure (πi) of the compressed film in presence of BSA (0.25 μg mL−1). Subphase pH=5.4 at 298 K. πi (mN m−1): Δ, 5; □, 10; ●, 15; , 25; O, 30 and ▼, 35.
DPPC domains were agglomerated whereas fern-leaf like structures [22] were obtained for C18TA+DS −. The compressed DPPC films produced bean shaped domains which, with time, turned into circular bodies creating cavities or pockets in them as found from fluorescence and Brewster angle microscopy (BAM) [55]. In the presence of BSA, the domain sizes of DPPC and C18TA+DS− (Fig. 7B) were found to decrease and the collapse of the monolayer films was delayed. The domains of C18TA+DS− increased at a higher rate at π ≤ 35 mN m−1, after that the rate of increase declined and finally disappeared at 45 mN m −1. The corresponding histogram of the epifluorescence images are also shown in Fig. 7. It was found that in the absence of BSA, the domains occupied 34 and 36% of the selected region, respectively at π = 25 and 30 mN m −1 whereas in the presence of BSA, they covered approximately 14, 25, 46.5 and 60% of the selected area at π = 25, 30, 35 and 40 mN m −1, respectively. Similar results were obtained for DPPC with BSA in the subphase (see supplementary information). The DPPC domain sizes increased with increasing π and became densely packed at higher π before collapsing at 52 mN m −1. 3.5. Atomic force microscopy The AFM images of the monolayer LB films of the pure coacervate (C18TA +DS −) and the mixed BSA–C18TA +DS − at 25 mN m −1 with their corresponding histograms are illustrated in Fig. 8A and B,
Table 2 Kinetic parametersa for the desorption of coacervate–protein and lipid–protein complexes on buffer sub-phase (pH = 5.4) at 298 K. [BSA] = 0.25 μg mL−1. πi/mN m−1
5 7 10 15 20 25 30 35
104 k/s−1 C18TA+DS−–BSA
DPPC–BSA
−4.56 – −3.70 11.4 7.57 4.50 4.09 3.80
−4.38 −2.55 6.50 7.14 – 9.46 13.8 24.4
a The error limits of k are within ±5% and the values of linear regression factors (R) are in between 0.978 and 0.999.
K. Maiti et al. / Materials Science and Engineering C 33 (2013) 836–843 Table 3 Size of the domains of the monolayer films of coacervate and lipid in the presence and absence of BSA ([BSA] = 0.25 μg mL−1, pH = 5.4 at 298 K). πi/mN m−1
10 15 20 25 30 35 40 45 52
Ave. circular radius/μm Only C18TA+DS−
C18TA+DS−–BSA
Only DPPC
DPPC–BSA
– – – 28 70 Collapse – – –
– – – 6 15 30 40 Collapse –
45 55 63 69 73 76 Collapse – –
32 35 36.5 40 45.5 50.5 55 – Collapse
respectively. The changes in the film morphology are distinguishable. The height of the monolayer film of pure C18TA+DS – was reported within 2 nm [56]. Here, the domains were homogeneously distributed with a mean height of 0.5 to 1 nm (Fig. 8A) and their radii varied between 4 and 6 μm. The AFM images of DPPC are not shown as it is already available in the literature [57,58]. The added BSA in the subphase penetrates into the monolayer through hydrophobic interaction (forming dimmer or trimers in some cases). The presence of BSA molecules was evidenced in the organized domains (Fig. 8B). Larger sizes of protein molecules along with the coacervate monolayer at lower π (10 mN m−1) (given in supplementary information) suggested stacking of dimmers and/or trimers of BSA molecules [59,60]. The Alim of BSA at 0.25 μg mL−1 found in the present study was ~40 nm2 molecule−1 at 298 K nearly matched with the findings of Wright and Thompson [34]. The protein molecules were heterogeneously distributed into the coacervate-matrix. With increasing π, penetration of BSA into the monolayer film decreased (the increased compactness of the coacervate or DPPC film suppressed the adsorption
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of BSA on the film) and the height of the membrane also consequently decreased. The surface roughness (root mean squared (RMS), measured at 5×5 μm area) of pure C18TA+DS− (at π=25mN m−1) and mixed C18TA+DS− with BSA at π=20 and 25 mN m−1 were found to be 0.33, 0.51 and 0.37 nm, respectively. The corresponding coefficients of kurtosis (height distribution sharpness) were estimated to be 31.6, 13.1 and 18.0, respectively. Surface roughness values were comparable to the reports of de Souza et al. [47] for di-palmitoylphosphatidyl ethanolamine–BSA system (RMS roughness=0.36) and dimyristoylphosphatidic acid–BSA system (RMS roughness=0.38). Change in the film morphology can be attributed to the distinct interfacial packing of coacervate molecules with BSA. Addition of BSA to C18TA+DS− resulted in two size distributions, one sharp and narrow (~2 nm), and the other less sharp and broad (~14 nm). At π=10 and 20 mN m−1, there appeared two broad distributions (~2 and ~6 nm) with nearly similar but much lesser intensities (data not shown). A narrow but intense single distribution was observed at π=25 mN m−1 (Fig. 8B). The results supported penetration of BSA into the coacervate monolayer and subsequent desorption of the BSA–coacervate complexes/adduct in the compressed state. Such a behavior on the BSA–coacervate film was observed from the kinetic study of the compressed monolayer discussed earlier. 4. Conclusions The interaction studies of coacervate/DPPC with BSA near its isoelectric point (where the electrostatic repulsion is minimum) revealed incorporation of BSA into the monolayers and thereby affecting the monolayer function and structure. BSA, through a diffusion controlled process, get adsorbed at the interface and consequently enhances the surface pressure. Pure BSA film is unstable in the surface pressure range of 20–30 mN m −1. Surface area of the C18TA+DS− and DPPC molecules in the Langmuir monolayer increases and reaches a saturation level with a BSA mole fraction of about 0.03 with respect
A1
A2
B1
B2
Fig. 7. Epifluorescence images of pure C18TA+DS− (A) and C18TA+DS−–BSA (B) films ([BSA] = 0.25 μg mL−1) with their corresponding histograms at π = 25 mN m−1 at pH = 5.4 and T = 298 K. Scale bar: 100 μm. Total selected area (histogram): 1900 × 1400 μm.
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K. Maiti et al. / Materials Science and Engineering C 33 (2013) 836–843
A1
A2
B1
B2
Fig. 8. AFM images of C18TA+DS− in absence (A) and presence of BSA ([BSA] = 0.25 μgmL−1) (B) at different π with their corresponding histogram at 298 K. Total selected area (histogram): 2 × 2 μm.
to the amphiphiles. At the interface, the constituents associate to produce an intermolecular cohesive film with some degree of elasticity. When BSA interacts with C18TA+DS− and DPPC, the maximum expansion occurs at [BSA] ≥0.16 and 0.11 μg mL−1, respectively. The associative interaction is more effective with DPPC than with C18TA+DS−. At π > 15 mN m−1, lipids (DPPC) or coacervate (C18TA+DS−) inhibits foaming by displacing protein molecules from the air–buffer interface and by disrupting the protein film [61]. The coacervate monolayer is found to be better permeative to BSA than DPPC. At lower π, the interaction of coacervate or DPPC with BSA leads to adsorption of the BSA molecule to the interface. At higher π, BSA deactivates the coacervate and lipid monolayer. A weak hydrophobic interaction occurs between BSA and catanionic surfactants which is significantly different from the strong interaction of anionic or cationic surfactants with BSA. It is revealed that the hydrophobic moieties of catanionic surfactants bind the apolar amino acid, whereas the polar head groups of the coacervate interacted with the peptide bond, and additionally with one or more polar amino acid residues and the coacervate mimics lipid-like behavior. This feature of catanionic surfactant has the prospect for use in the field of drug design. Acknowledgments K.M. is thankful to CSIR, Govt. of India, for a Senior Research Fellowship. S.P.M. thanks Indian National Science Academy, Government of India, for an honorary scientist position. Financial assistance from the Department of Science and Technology, Govt. of India, is acknowledged. Also the help from Mr. N. S. Das and Dr. K. K. Chatterjee of Department of Nanoscience and Technology, Department of Physics, JU is thankfully acknowledged.
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Effect of bovine serum albumin on the functionality and structure of catanionic surfactant at air–buffer interface Kajari Maiti a, Subhash C. Bhattacharya a, Satya P. Moulik a, Amiya K. Panda b,⁎ a b
Centre for Surface Science, Department of Chemistry, Jadavpur University, Kolkata 700 032, India Department of Chemistry, University of North Bengal, Darjeeling 734 013, India
a r t i c l e
i n f o
Article history: Received 26 June 2012 Received in revised form 9 October 2012 Accepted 5 November 2012 Available online 13 November 2012 Keywords: Coacervate DPPC BSA π A isotherm Microscopic images
a b s t r a c t Interaction of bovine serum albumin (BSA) with the solvent spread monolayer of a catanionic surfactant, octadecyltrimethylammonium dodecylsulfate, (C18TA+DS−) at the air–buffer interface was investigated by measuring the surface pressure with time and change in surface area. Dipalmitoylphosphatidylcholine (DPPC) was used as reference. Kinetics of BSA desorption from the interface to the buffer subphase, that of C18TA+DS− and DPPC through their interaction with BSA, were also studied at different BSA concentrations (in the subphase) and surface pressures. Surface pressure (π)–area (A) isotherms (at pH = 5.4, μ = 0.01, T = 298 K) revealed that the coacervate/DPPC monolayer becomes expanded in the presence of BSA at low π while their protein bound species are released into the subphase at high π. Film morphology, studied by epifluorescence microscopy (EFM) and atomic force microscopy (AFM), reveals that the sizes of the domains of both DPPC and coacervate decrease in the presence of BSA. Presence of BSA in the coacervate and DPPC monolayer was supported from AFM data analysis. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Globular proteins find uses in medicinal and pharmaceutical research for their capability to catalyze biochemical reactions, to adsorb as well as bind specifically to other molecules and subsequently form molecular aggregates [1]. Besides, they find potential applications as biochemical sensors [2]. Serum albumin is the most abundant protein in the circulatory system and contributes 80% to colloid osmotic blood pressure [3] and also maintains the pH of blood [4]. The most outstanding property of albumin lies in its ability to bind to an incredible variety of ligands and a broad range of metabolites, drugs, amphiphiles and other organic compounds [5–10]. Interaction of ionic surfactants with BSA has been widely studied because of the relatively large number of charged amino acids on its surface (net charge=−17 at pH 7) [11,12]. According to Bos et al. [13], BSA is heart shaped, comprising nine loops held together by 17 disulfide bonds, resulting in three domains (I, II, and III) each containing two subdomains or alternatively each containing one small and two large loops. These loops are mainly composed of helical segments [14]. The adsorption behavior of different proteins in combination with surfactants has been studied in detail by Miller et al. [15–17]. Interaction of ionic surfactant with BSA leads to the formation of some amphiphilic entities with altered surface activity. In the case of non-ionic surfactants, the adsorption layer is mainly formed by competitive adsorption
⁎ Corresponding author. Tel.: +91 9433347210; fax: +91 353269901. E-mail address: [email protected] (A.K. Panda). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2012.11.009
between protein and non-ionic surfactant molecules and the interaction is only hydrophobic in nature [17]. Such complexes are typically less surface active than the pure protein. Even interactions between proteins and cationic/nonionic amphiphiles as well as DPPC [18] are available in the literature, however, the studies involving catanionic surfactants with globular proteins are not so common. It is of great theoretical and practical importance to investigate such systems especially with reference to protein separation [19,20]. When the catanionic surfactants are used, their required concentrations are usually very low such that the proteins can maintain its interfacial activity even in the presence of amphiphiles [21]. Ionic surfactants could cause major structural changes in the protein conformation with subsequent changes in protein functionality which is not desirable in many applications; the uses of catanionic surfactants are thus preferred over individual components. Recently, catanionic surfactants or coacervates have gained attention of researchers for their structural resemblance to phospholipids [22,23] and their ability to form various aggregated microstructures such as micelles, vesicles, lamellar phases, etc. [24]. The catanionic surfactants are extensively used in many fields of technology and research, including pharmaceutical preparations. DPPC is the major component of lung surfactant and serum albumins are known for surfactant dysfunction [25]. The best way to understand the functionality of an amphiphile at the air–solution interface is to study its surface pressure–area isotherms [26]. In this perspective, it is believed to be worthy to study the interaction of BSA with a lipid mimicking amphiphile, catanionic surfactant, C18TA +DS − and hence to compare the results with DPPC.
K. Maiti et al. / Materials Science and Engineering C 33 (2013) 836–843
In this paper, interaction between well characterized coacervate (C18TA+DS −) and BSA has been studied by the Langmuir monolayer technique. The results are compared with the lipid, DPPC. Surface morphology of the Langmuir–Blodgett films of the coacervate and DPPC has been studied by combined epifluorescence microscopy and atomic force microscopy (AFM). We have focused our attention on the three aspects: (i) adsorption properties of BSA molecules at the air–solution interface, (ii) nature of binding of BSA with C18TA+DS − and DPPC along with their interaction kinetics at the interface and, (iii) a comparative studies between the catanionic and the phospholipid. The results are expected to generate information and understanding on the physicochemistry of lipid–protein interaction. 2. Materials and methods 2.1. Materials Octadecyltrimethylammonium bromide or C18TAB was a product of Fluka, Switzerland (purity ≥ 97%). BSA (MW = 66430 Da, purity > 98.5%), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (purity > 98%), sodium dodecysulphate (SDS) (purity > 99%), and the fluorescent probe, ([1-palmitoyl-2-{12-[(7-nitrobenz-2-oxa-1,3-diazo4-yl)amino] dodecanoyl}-sn-glycerol-3-phosphocholine]) or NBD-PC (purity> 95%) were purchased from Sigma, USA. AR grade acetic acid, sodium acetate, NaOH (BDH, India) and borax (Merck, India) were used for the preparation of buffer solutions. The following compositions were used to get buffers at ionic strength (μ) = 0.01: 500 mL 3.08 mM sodium acetate+ 500 mL 16.92 mM acetic acid (for pH 4); 500 mL 16.41 mM sodium acetate + 500 mL 3.59 mM acetic acid (for pH 5.4); and 500 mL 4.54 mM borax + 500 mL 6.37 mM NaOH (for pH 10). Chloroform and methanol of AR grade (SRL, India) were used for preparing the DPPC and coacervate solutions. Doubly distilled water (specific conductance= 2–4 μS cm−1 and surface tension = 72 mN m −1 at 298 K) was used to make BSA and buffer solutions. 2.2. Methods 2.2.1. Preparation and isolation of coacervate Equimolar amounts of aqueous C18TAB and SDS solutions were mixed with constant stirring. The formed catanionic coacervate was isolated by repeated partitioning between chloroform-water [22]. After solvent evaporation, the coacervate was vacuum-dried for 24 h and then stored. 1H NMR, XRD, FTIR, DSC study, etc., were performed on the formed coacervate to ensure that the results matched with that of reported C18TA +DS − [22]. 2.2.2. Monolayer study A Langmuir trough (Apex Inst. Co., India) with a surface area of 300 cm 2, placed on an anti-vibration table, was used. Surface pressure was measured by the Wilhelmy plate technique with an accuracy of ±0.1 mN m −1. Buffers of different pH (μ = 0.01) were used as subphases. For measuring the surface pressure (π)–area (A) isotherm of BSA, an aqueous solution of BSA (0.5 mg mL −1) was added to the buffer subphase using a Hamilton microsyringe; after careful mixing, the BSA monolayer was formed through adsorption from the subphase onto the air–buffer interface. The π–A isotherm was recorded after 30 min. For pure C18TA +DS − and DPPC at the air–buffer interface, their chloroform–methanol solutions (3:1, v/v) were spread by a Hamilton microsyringe onto the buffer subphase and after the solvent evaporation (30 min), the compression process was initiated. For the interaction and kinetic studies (adsorption or desorption processes), in the Langmuir trough, the coacervate or DPPC (dissolved in chloroform–methanol solution) was spread over the buffer subphase. After 30 min, aqueous BSA solution was gently injected at the bottom of the trough right underneath the monolayer without disturbing the monolayer. After waiting for an hour (for
837
equilibration) [23], the film compression was compressed with a barrier speed of 5 cm 2 min −1. All data presented here were averages of at least three measurements for each studied condition. 2.2.3. Epifluorescence microscopy (EFM) A Motic phase contrast microscope (model: AE31, Hong Kong) was used to take the images of NBD-PC-doped (1 mol% with respect to the amphiphile) coacervate or DPPC monolayer in the absence and presence of BSA (0.25 μg mL −1) in the subphase at different surface pressures. The fluorescent probe was excited at 470 nm with a green emission (530 nm) [22,23]. The images of the compressed films at the desired surface pressure were taken with a digital camera. 2.2.4. Atomic force microscopy (AFM) Coacervate or DPPC films in the absence and presence of BSA were transferred onto the freshly cleaved mica substrate by Langmuir– Blodgett technique [27]. Ten minutes after attainment of the desired surface pressure, the pre-immersed mica substrate was lifted with an upstroke of 1 mm min −1 with a 1:1 transfer ratio. The transferred films were scanned within 2 h with an atomic force microscope (NT-MDT, Russia) with a 25 μm × 25 μm J scanner. A silicon nitride tip with a typical force constant of 120 mN m −1 was used for scanning. AFM measurements were carried out in non-contact mode in the air. The images were then flattened and analyzed with NT-MDT Solver-pro software to obtain the corresponding topography and phase images [28]. All measurements were carried out at the ambient but controlled temperature (298 ± 1 K). 3. Results and discussion 3.1. Experimental data analysis 3.1.1. A. pH dependent π–A isotherm of BSA π–A isotherm of BSA, as shown in Fig. 1 (main diagram) at 298 K at the air–buffer interface reflects its pH-dependent behavior. Although there was no significant change in the Alim (the area after which the surface pressure starts rising; Alim values were nearly constant in the range of 82–85 nm 2 molecule −1) at a different pH, however, the slopes of the curves increased steeply with increase in subphase pH and the feature was more prominent after their crossing point (at 51.5 nm 2 molecule −1 at π = 17 mN m −1). Independence of surface pressure with the pH at 17 mN m −1 implies that the orientation of BSA molecules at the interface was independent of pH at this surface pressure. BSA, can undergo reversible conformational changes with pH [29]. Different pH-dependent isomeric forms of BSA can exist
Fig. 1. Surface pressure (π)–area (A) isotherm of BSA at different pH. [BSA]=0.1 μg mL−1. Inset: Alim–[BSA] profile at T=298 K and subphase pH=5.4.
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[30]. π–A isotherms of BSA (at pH = 4 and 5.4) have another crossing point at 7.5 mN m −1. It cannot be reasoned out at present, further research is needed.
It is assumed that all the free energy of desorption of segments are equivalent. ΔGd is thus expressed as [40]:
3.1.2. B. π–A isotherm of BSA near its IP (pH = 5.4) The π–A isotherm of BSA at different concentrations in the subphase at pH 5.4 resulted in interesting features (figures shown in supplementary section). The lift-off molecular area (Alim) of the protein in its compressed monolayer gradually decreased with increasing [BSA] up to 0.24 μg mL−1, then it became constant (inset, Fig. 1). At lower concentration, higher Alim resulted because of solubility of the globular protein in the bulk. The number of BSA molecules at the air–buffer interface became less than expected. With increasing [BSA], the interface experienced greater protein coverage making Alim to decrease and finally saturation occurred at 0.24 μg mL−1. In the inset plot, two distinct transition points at 0.11 and 0.24 μg mL −1 of BSA were observed. The first transition corresponded to an orientation change of the BSA molecules in the monolayer whereas the second stood for surface saturation of BSA at the interface for maintaining a constant interfacial population. The data revealed that the constant point corresponded to the molecular area of BSA to be 40 nm2 molecule−1. The protein adsorption studies [15] revealed that at lower surface pressures an adsorbed molecule might occupy different parts of the interface (i.e., a maximum molar surface area is occupied), whereas minimum molar surface area was achieved at large π. With increasing surface coverage (i.e., stronger competition between the adsorbed protein molecules), their molar areas become smaller until finally a minimum area was reached. This implied that the adsorption layer thickness increased with increasing protein concentration. This led to an increased coverage up to an almost complete saturation of the adsorption layer at high protein concentration. The fact that the surface pressure of concentrated protein solutions is independent of the bulk concentration can also be satisfactorily explained in the framework of a monolayer model, considering a weakening of the inter-ion interactions in concentrated monolayers [31] or the possibility of two-dimensional aggregation of adsorbed protein molecules [32]. An illustration of the idea was schematically shown by Miller et al. [15]. From the result of adsorption isotherm and adsorption kinetics of lysozyme, Hunter et al. [33] have proposed that at higher protein concentration, the conformation of the lysozyme at the air– water interface changed from side-on to end-on form. From hydrodynamic experiments [34,35] and low-angle X-ray scattering studies [36], serum albumin has been postulated to be an oblate ellipsoid with dimensions of 14× 4 nm as supported by others [37]. The nature of the π–A isotherm of BSA on buffer sub-phase also varied with [BSA] but more or less similar isotherm profiles were obtained at [BSA]≥ 0.24 μg mL−1. Increased adsorption leads to smaller degree of unfolding with advancing time [38]. The adsorption kinetics of proteins was systematically investigated by Graham and Phillips [39], and later by others [38]. It is known that depending on the concentration in the bulk and surface pressure, long chain flexible polymer molecule can adopt different configuration at the interface where some segments remain adsorbed at the interface while others extend as loops into the adjacent liquid phase [40]. It has been proposed that an equilibrium exists between the desorbed segments (Nd) and the segments at the interface (Ni). The related equilibrium constant (κe) is defined as [40]:
ΔGd ðπÞ ¼ ∫ adπ
π∞
κe ¼
Ni ΔGd ðπÞ−πa ¼ exp Ns kT Nd
ð2Þ
πe
where, πe and π∞ are the equilibrium surface pressure (taken as zero) and surface pressure at which all the molecules are completely desorbed, respectively. In practice, π∞ has been taken as the maximum surface pressure that the film attains. In the case of Langmuir monolayer of a protein, the fraction of segments desorbed from the interface, r (π) is the parameter that characterizes the stability of the monolayer and can be expressed as [41]: rðπÞ ¼
−1 Nd ΔGd −πa −1 ¼ ½κe þ 1 ¼ exp þ1 : Ns kT ðNi þ Nd Þ
ð3Þ
Using Eq. (3) one can estimate π corresponding to the maximum of desorbed segments where the protein monolayer is most compact. In Fig. 2, calculated fractions of the desorbed segments of BSA at different concentrations have been plotted as a function of π. It is observed that with the increase in [BSA], the r values increased up to ~ 0.4 (at [BSA] ≤ 0.16 μg mL − 1), after which the r–π lines merged with one another. However, all the r–π lines reached to saturation at r = 0.50 in the range of 20–30 mN m − 1. The r–π lines at different pH values (inset, Fig. 2) have similar features and have reached the same saturation value (0.50). The r values decreased with increasing pH and reached the saturation at π = 23, 26 and 31 mN m − 1 at pH 4, 5.4 and 10, respectively. The results suggested that in the range of 20–30 mN m − 1, the stability of the BSA monolayer was maximum. This particular π range has been considered as the optimum π for getting the LB deposits [42]. 3.1.3. C. Interactions of C18TA +DS − and DPPC with BSA at the air–buffer interface The results of interaction of DPPC and C18TA +DS − with BSA at the air–buffer interface are presented in Fig. 3. It is observed that with increasing [BSA] in the subphase, the Alim of C18TA +DS − increased. Due to the complexation of the cationics with BSA at the interface there occurred an expansion of the monolayer. Similar behavior was noticed also for DPPC indicating the existence of significant association between BSA and DPPC. The Alim values increased with increasing [BSA] and remained constant at [BSA] = 0.11 and 0.16 μg mL −1 for C18TA +DS − and DPPC, respectively (Fig. 3, inset). Beyond these limiting concentrations, values did not change significantly for both the systems. At pH 5.4, the nature of the π–A isotherm (beyond π =
ð1Þ
where, ΔGd is the potential barrier which opposes the desorption of the whole molecule (i.e., free energy of adsorption of a molecule), Ns is the number of segments in the macromolecule (amino acid residues which in the case of BSA is 583), a is the area per protein molecule, k is the Boltzmann constant and T is the Kelvin temperature.
Fig. 2. r–π profile at different [BSA] at 298 K. [BSA] (μg mL−1): 1, 0.010; 2, 0.10, 3, 0.13; 4, 0.17; 5, 0.24; and 6, 0.32. Inset: Effect of subphase pH on the r–π profile. [BSA] in subphase=0.1 μg mL−1.
K. Maiti et al. / Materials Science and Engineering C 33 (2013) 836–843
839
former can accommodate more BSA molecules resulting in higher expansion of the monolayer than the latter [43,47,49,50]. 3.3. Kinetics of interfacial desorption and penetration
Fig. 3. π–A isotherms of DPPC and C18TA+DS− in absence and presence of 0.25 μg mL−1 BSA. pH=5.4 and T=298 K. Systems: 1, pure DPPC; 2, pure C18TA+DS−; 3, DPPC–BSA and 4, C18TA+DS−–BSA. Inset: Variation of Alim of C18TA+DS− (O) and DPPC (□) with [BSA].
17 mN m −1 for both the coacervate and DPPC) was found to resemble with that of the pure BSA having concentration ≥0.24 μg mL−1. The concentration difference between the coacervate and the DPPC is a measure of the difference in the extent of interaction. Similar type of reports are also available in the literature [43,44]. Compressibility modulus (CS-1) of a monomolecular film can be expressed as: −1
Cs
∂π ¼ −A : ∂A T
ð4Þ
In CS−1 vs. π plot, the maximum value (CS −1max) indicates the state of the molecules at the air–liquid interface [45]. The CS −1max values were found to be 30.8, 55.1, 145.5, 35.0 and 30.5 mN m −1, for C18TA +DS −, BSA, DPPC, C18TA +DS −–BSA and DPPC–BSA system, respectively. Results suggest that the pure coacervate (C18TA +DS −) was in liquid-expanded state, whereas pure BSA and pure DPPC were in liquid state and liquid-condensed state, respectively [22,23]. The BSA–C18TA +DS − and BSA–DPPC systems were in the liquidexpanded state. The coacervate monolayer became compact whereas the DPPC monolayer became fluidized (transferred from liquidcondensed to liquid-expanded state). These findings supported penetration of BSA into the monolayer. Wang et al. [46] have shown that human serum albumin ([HSA] = 10 −7 M) also induces a large expansion in dimyristoylphosphatidic acid (DMPA) monolayers, whose isotherms exhibit liquid-condensed to liquid-expanded (LC to LE) phase transition.
Due to its amphiphilic nature, BSA is used in different applied processes, viz., emulsions, thin films, foams, etc. [51]. At its initial stage, adsorption at the air–solution interface is diffusion controlled but the adsorbing molecule has to overcome surface pressure as well as the electrostatic energy barriers in order to locate itself at the interface [52,53]. At the interface, after attaining a certain surface pressure, BSA molecules get desorbed. Kinetics of this desorption process was studied by monitoring the reduction in π with time at different BSA concentration in the subphase (pH 5.4, and μ= 0.01 at 298 K). Results are shown in Fig. 4. Both C18TA +DS− and DPPC can withstand a moderate to high surface pressure. The possibility of penetration of BSA into the DPPC and the coacervate (C18TA+DS−) monolayer was explored by adding BSA solution underneath the compressed C18TA+DS− (Fig. 5) and DPPC (Fig. 5) monolayer. It was observed that, upon addition of BSA into subphase of the compressed monolayer, π values decreased with time and the rate depended on both π and the [BSA] in the subphase. The desorption process of BSA from the interface followed a pseudo first order kinetics obeying the relation given bellow: κ¼
ðπ −πf Þ 2:303 log t ðπi −πf Þ t
ð5Þ
where, πt, πi, and πf are the surface pressure of the monolayer at time t, initial and final, respectively, and κ is the first-order rate constant. −πf Þ log ððππti −π vs t plots were used to get κ from the initial straight lines. fÞ The πf and κ values at π = 15 mN m −1 are shown in Table 1. Results revealed that BSA desorption rate decreased with increasing [BSA]. Essentially, the inter-protein molecular interaction slowed down the rate of desorption process. At [BSA]= 0.25 μg mL−1, the simultaneous occurrence of desorption (up to t ≈ 1000 s) and adsorption process was observed for the case of C18TA +DS− (Fig. 5). At π = 15 mN m−1, pure C18TA+DS − produced a low κ value which increased with BSA addition; so was the observation for the compressed DPPC monolayer. But the desorption rate of C18TA +DS− in the presence of BSA was greater than DPPC (κDPPC−BSA 〈κC18 TAþ DS− −BSA ) as the solubilization of DPPC from its compressed monolayer by BSA was relatively easier than solubilization of C18TA+DS − under comparable states of compression because of the presence of more hydrophilic part in the former than in the latter. The unusual behavior of the π vs t graph of C18TA +DS− at πi = 15 mN m −1 and [BSA] = 0.25 μg mL−1 (Fig. 5, main diagram) was because the coacervate monolayer was in liquid-expanded state and capable of incorporating BSA into the air–buffer interface, which
3.2. Mechanism of BSA–coacervate and BSA–lipid interactions Surface activity of BSA arises from its amphiphilic nature. The attractive coacervate–protein and lipid–protein interactions can change its molecular organization at the interface. Both the coacervate and DPPC are zwitterionic. Thus, ion–ion induced dipole interaction and hydrophobic interactions are responsible for the accumulation of BSA at the interface and subsequent penetration into the coacervate or DPPC monolayer. In order to accommodate its zwitterionic head group, the coacervate and DPPC molecules bind to the hydrophobic pockets of BSA near the charged amino acids [47]. The co-adsorption of BSA along with DPPC occurred through a non-electrostatic mechanism competing with the repulsive hydration force exerted by the phospholipids [48]. The hydrophobic interaction in BSA–coacervate and protein–lipid systems allows the protein molecules to get co-adsorbed into the monolayer resulting in an expansion. Interfacial packing of the coacervate is less compact than DPPC for which the
Fig. 4. Effect of bulk concentration of BSA on its π–time (t) profile at 298 K. Subphase −πf Þ pH = 5.4. [BSA](μg mL−1): O, 0.05; ▲, 0.10; □, 0.175 and ∇, 0.25. Inset: ln ððππti −π vs t fÞ profile for [BSA] = 0.25 μg mL−1.
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Fig. 5. Surface desorption of C18TA+DS− and DPPC (inset) in the presence of BSA at pH = 5.4 at 298 K. [BSA] (μg mL−1) : ●, 0; O, 0.05; ▲, 0.10; □, 0.175 and ∇, 0.25.
under compression (at π>10 mN m−1) desorbed into the subphase following a first order kinetics. Δπ (πi–πf) vs. t profile at different initial surface pressures of compressed C18TA+DS− and DPPC monolayer films at [BSA] =0.25 μg mL−1 (at pH 5.4 and μ=0.01) at 298 K are presented in Fig. 6. The related kinetic data are given in Table 2. With increasing state of compression, the adsorption rate for both C18TA+DS−–BSA and DPPC–BSA systems decreased. For the desorption process (from πi = 15–35 mN m−1), it was found that values of the rate constant for the first system decreased with increasing [BSA] whereas the reverse case was observed for DPPC. According to Kamilya et al. [54] the incorporation of protein (egg lecithin) into the monolayer of DPPC was more favorable in its LE (liquid-expanded) region (πi ≈6 mN m−1). 3.4. Epifluorescence microscopic (EFM) study Film morphology of C18TA +DS − and DPPC monolayer in the absence and presence of BSA was studied by epifluorescence microscopy (EFM). Results are summarized in Table 3. Pure BSA did not produce any significant image (or the image contrasts were b1 μm, lower than the resolution limit). The average circular radius of the domain of pure DPPC at the air–buffer interface increased up to π =35 mN m−1 (honeycomb-like structures appeared at this π) after which it decreased and eventually the features collapsed at 40 mN m −1 (shown in supporting information). In the case of pure C18TA +DS− (Fig. 7A), about 2.5 fold increase in the size was found for a rise of π by 5 mN m−1; afterwards it decreased and collapsed at 35 mN m −1 (shown in the supporting information). At any particular π, the domain size obtained for pure DPPC was larger than that of pure C18TA+DS−, and the collapse pressure of the former was also higher than the later. It was reported [22] that the EFM images of C18TA+DS − and DPPC at the air–water interface at 298 K at 25 mN m −1 produced circular domains of average radius 30 and 67 μm, respectively; at 35 mN m −1, Table 1 Kinetic parametersa,b for the desorption of pure BSA, coacervate–BSA and lipid–BSA complexes at different protein concentration in buffer subphase (pH = 5.4) at 298 K. [BSA]/μg mL−1
0 0.05 0.10 0.175 0.25
104 k/s−1 C18TA+DS−–BSA
DPPC–BSA
Only BSA
3.90 4.42 7.21 10.5 11.4
3.72 4.25 5.63 6.50 7.14
– 11.1 9.55 8.81 8.66
a The k value calculation at πi = 15 mN m−1 was based on data only up to 1000 s as the initial desorption process was followed by the adsorption processes after 1000 s. b The error limits of k are within ±5% and the values of linear regression factors (R) are in between 0.975 and 0.980.
Fig. 6. Surface pressure (Δπ=πi− πf) vs. t plots for C18TA+DS− (A) and DPPC (B) at different initial surface pressure (πi) of the compressed film in presence of BSA (0.25 μg mL−1). Subphase pH=5.4 at 298 K. πi (mN m−1): Δ, 5; □, 10; ●, 15; , 25; O, 30 and ▼, 35.
DPPC domains were agglomerated whereas fern-leaf like structures [22] were obtained for C18TA+DS −. The compressed DPPC films produced bean shaped domains which, with time, turned into circular bodies creating cavities or pockets in them as found from fluorescence and Brewster angle microscopy (BAM) [55]. In the presence of BSA, the domain sizes of DPPC and C18TA+DS− (Fig. 7B) were found to decrease and the collapse of the monolayer films was delayed. The domains of C18TA+DS− increased at a higher rate at π ≤ 35 mN m−1, after that the rate of increase declined and finally disappeared at 45 mN m −1. The corresponding histogram of the epifluorescence images are also shown in Fig. 7. It was found that in the absence of BSA, the domains occupied 34 and 36% of the selected region, respectively at π = 25 and 30 mN m −1 whereas in the presence of BSA, they covered approximately 14, 25, 46.5 and 60% of the selected area at π = 25, 30, 35 and 40 mN m −1, respectively. Similar results were obtained for DPPC with BSA in the subphase (see supplementary information). The DPPC domain sizes increased with increasing π and became densely packed at higher π before collapsing at 52 mN m −1. 3.5. Atomic force microscopy The AFM images of the monolayer LB films of the pure coacervate (C18TA +DS −) and the mixed BSA–C18TA +DS − at 25 mN m −1 with their corresponding histograms are illustrated in Fig. 8A and B,
Table 2 Kinetic parametersa for the desorption of coacervate–protein and lipid–protein complexes on buffer sub-phase (pH = 5.4) at 298 K. [BSA] = 0.25 μg mL−1. πi/mN m−1
5 7 10 15 20 25 30 35
104 k/s−1 C18TA+DS−–BSA
DPPC–BSA
−4.56 – −3.70 11.4 7.57 4.50 4.09 3.80
−4.38 −2.55 6.50 7.14 – 9.46 13.8 24.4
a The error limits of k are within ±5% and the values of linear regression factors (R) are in between 0.978 and 0.999.
K. Maiti et al. / Materials Science and Engineering C 33 (2013) 836–843 Table 3 Size of the domains of the monolayer films of coacervate and lipid in the presence and absence of BSA ([BSA] = 0.25 μg mL−1, pH = 5.4 at 298 K). πi/mN m−1
10 15 20 25 30 35 40 45 52
Ave. circular radius/μm Only C18TA+DS−
C18TA+DS−–BSA
Only DPPC
DPPC–BSA
– – – 28 70 Collapse – – –
– – – 6 15 30 40 Collapse –
45 55 63 69 73 76 Collapse – –
32 35 36.5 40 45.5 50.5 55 – Collapse
respectively. The changes in the film morphology are distinguishable. The height of the monolayer film of pure C18TA+DS – was reported within 2 nm [56]. Here, the domains were homogeneously distributed with a mean height of 0.5 to 1 nm (Fig. 8A) and their radii varied between 4 and 6 μm. The AFM images of DPPC are not shown as it is already available in the literature [57,58]. The added BSA in the subphase penetrates into the monolayer through hydrophobic interaction (forming dimmer or trimers in some cases). The presence of BSA molecules was evidenced in the organized domains (Fig. 8B). Larger sizes of protein molecules along with the coacervate monolayer at lower π (10 mN m−1) (given in supplementary information) suggested stacking of dimmers and/or trimers of BSA molecules [59,60]. The Alim of BSA at 0.25 μg mL−1 found in the present study was ~40 nm2 molecule−1 at 298 K nearly matched with the findings of Wright and Thompson [34]. The protein molecules were heterogeneously distributed into the coacervate-matrix. With increasing π, penetration of BSA into the monolayer film decreased (the increased compactness of the coacervate or DPPC film suppressed the adsorption
841
of BSA on the film) and the height of the membrane also consequently decreased. The surface roughness (root mean squared (RMS), measured at 5×5 μm area) of pure C18TA+DS− (at π=25mN m−1) and mixed C18TA+DS− with BSA at π=20 and 25 mN m−1 were found to be 0.33, 0.51 and 0.37 nm, respectively. The corresponding coefficients of kurtosis (height distribution sharpness) were estimated to be 31.6, 13.1 and 18.0, respectively. Surface roughness values were comparable to the reports of de Souza et al. [47] for di-palmitoylphosphatidyl ethanolamine–BSA system (RMS roughness=0.36) and dimyristoylphosphatidic acid–BSA system (RMS roughness=0.38). Change in the film morphology can be attributed to the distinct interfacial packing of coacervate molecules with BSA. Addition of BSA to C18TA+DS− resulted in two size distributions, one sharp and narrow (~2 nm), and the other less sharp and broad (~14 nm). At π=10 and 20 mN m−1, there appeared two broad distributions (~2 and ~6 nm) with nearly similar but much lesser intensities (data not shown). A narrow but intense single distribution was observed at π=25 mN m−1 (Fig. 8B). The results supported penetration of BSA into the coacervate monolayer and subsequent desorption of the BSA–coacervate complexes/adduct in the compressed state. Such a behavior on the BSA–coacervate film was observed from the kinetic study of the compressed monolayer discussed earlier. 4. Conclusions The interaction studies of coacervate/DPPC with BSA near its isoelectric point (where the electrostatic repulsion is minimum) revealed incorporation of BSA into the monolayers and thereby affecting the monolayer function and structure. BSA, through a diffusion controlled process, get adsorbed at the interface and consequently enhances the surface pressure. Pure BSA film is unstable in the surface pressure range of 20–30 mN m −1. Surface area of the C18TA+DS− and DPPC molecules in the Langmuir monolayer increases and reaches a saturation level with a BSA mole fraction of about 0.03 with respect
A1
A2
B1
B2
Fig. 7. Epifluorescence images of pure C18TA+DS− (A) and C18TA+DS−–BSA (B) films ([BSA] = 0.25 μg mL−1) with their corresponding histograms at π = 25 mN m−1 at pH = 5.4 and T = 298 K. Scale bar: 100 μm. Total selected area (histogram): 1900 × 1400 μm.
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A1
A2
B1
B2
Fig. 8. AFM images of C18TA+DS− in absence (A) and presence of BSA ([BSA] = 0.25 μgmL−1) (B) at different π with their corresponding histogram at 298 K. Total selected area (histogram): 2 × 2 μm.
to the amphiphiles. At the interface, the constituents associate to produce an intermolecular cohesive film with some degree of elasticity. When BSA interacts with C18TA+DS− and DPPC, the maximum expansion occurs at [BSA] ≥0.16 and 0.11 μg mL−1, respectively. The associative interaction is more effective with DPPC than with C18TA+DS−. At π > 15 mN m−1, lipids (DPPC) or coacervate (C18TA+DS−) inhibits foaming by displacing protein molecules from the air–buffer interface and by disrupting the protein film [61]. The coacervate monolayer is found to be better permeative to BSA than DPPC. At lower π, the interaction of coacervate or DPPC with BSA leads to adsorption of the BSA molecule to the interface. At higher π, BSA deactivates the coacervate and lipid monolayer. A weak hydrophobic interaction occurs between BSA and catanionic surfactants which is significantly different from the strong interaction of anionic or cationic surfactants with BSA. It is revealed that the hydrophobic moieties of catanionic surfactants bind the apolar amino acid, whereas the polar head groups of the coacervate interacted with the peptide bond, and additionally with one or more polar amino acid residues and the coacervate mimics lipid-like behavior. This feature of catanionic surfactant has the prospect for use in the field of drug design. Acknowledgments K.M. is thankful to CSIR, Govt. of India, for a Senior Research Fellowship. S.P.M. thanks Indian National Science Academy, Government of India, for an honorary scientist position. Financial assistance from the Department of Science and Technology, Govt. of India, is acknowledged. Also the help from Mr. N. S. Das and Dr. K. K. Chatterjee of Department of Nanoscience and Technology, Department of Physics, JU is thankfully acknowledged.
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