Lysozyme (LZM) from chicken egg white, crystallized, was supplied from Sigma (purity 95%) and stored according to the supplier information. The protein spreading solution was a mixture of concentration of 3.2 mg/mL in 10 mM phosphate buffered saline (PBS; 0.9% NaCl, pH 7.4), and the PBS was always made fresh and was used within 30 min of making the solution. 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) from bovine heart was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Spreading solutions of DPPC and DPPE were prepared in chloroform/methanol 3:1 (v/v) mixture at a concentration of 1 mg/mL. The ultra pure water used as subphase was distilled and purified with a Millipore purification system (electrical resistivity ≥ 18.2 MΩ·cm).

Surface pressure–area (π–A) isotherms were obtained by means of a computer-controlled commercial device (Minitrough, KSV, Helsinki, Finland). Symmetric compression was achieved with two moving barriers at a constant rate of 10 mm/min. The surface pressure (π) of the monolayer was measured using the Wilhelmy method, using a Wilhelmy platinum plate, with an accuracy of 0.1 mN/m and a dynamic surface pressure range 0–150 mN/m. The value was monitored with a Wilhelmy article probe hanging from a high-precision microbalance connected to a computer, and is defined as^[26]

In order to keep the area of the monolayer, pressure which changes with time has been recorded so as to obtain π–T isotherms when the lipid monolayer is compressed to a certain surface pressure. Surface pressure was set to near 10, 15, or 20 mN/m (π₀) by reducing the surface area. Once the film was set to near the desired pressure, the area was kept constant, lysozyme was injected into the subphase outside the barriers, and π was monitored until it became constant. This was deemed to be the equilibrium pressure (π_e). Compression of the DPPC monolayer occurred until the target surface pressure of 15 mN/m was reached. Monolayers have been transferred to the surface of mica substrate (transfer ratio is 1) with a method of vertical pulling transferring. Each experiment was repeated at least three times. The behavior of protein molecules on the surface of the lipid monolayer and the rearrangement of the structure can be obtained by^[20]

Figure 1 is a typical π–T adsorption curve. The first area represents the initial adsorption. π increase indicates that more molecules are adsorbed to the same interface membrane. The second regions indicate that there is a new protein molecule adsorbed to the interface, the combination, expansion, and rearrangement. The third region is due to the expansion and rearrangement of the protein molecules on the interface, and gradually reaches the saturation stable state.^[27]

Fig. 1.

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	Fig. 1. Ideal π–T curve of lysozyme interaction with lipid layer.

AFM images were acquired in air at room temperature using a SPM-9500-J3 AFM (Shimadzu Corporation, Japan), which provided both a topographical image and a phase contrast one. The sample imaging has been carried out in the contact mode through a Micro-V-shaped Cantilever probe (Olympus Corporation, Japan) with cantilever spring constant about 0.06 N/m. A 55 μm × 55 μm tube-type piezoelectric scanner and a Si₃N₄ probe were used and images in height mode were collected simultaneously with 512×512 points with scan rate of 1.0 Hz per line.

DPPC and DPPE are components of biological membranes, which have different head groups.^[28] In this paper, we change the concentration of lysozyme to study the different effects of protein on DPPC and DPPE monolayer.

The surface pressure–area isotherms for a series of mixtures of LZM with (a) DPPC and (b) DPPE at temperature of 23±1 °C are shown in Fig. 2. The DPPC isotherm displayed the characteristic plateau attributed to the first-order liquid expanded (LE) to liquid condensed (LC) phase transition at 4–7  mN/m,^[20,27] which is consistent with the reported literature.^[5] The subsequent sharp increase of surface pressure corresponds to the formation of a compact LC phase, and with further compression, a solid phase is formed.^[29] Figure 1(a) shows that, even at very low concentration, the presence of LZM molecules strongly affects the shape of curves. As the LZM concentration increases, the curves are gradually shifted toward higher area values, and the typical plateau shown by the DPPC isotherm gradually disappears. This demonstrates that LZM molecules were adsorbed onto the DPPC monolayer and strongly affect the arrangement. Lysozyme increases the orderliness of the DPPC monolayer and reduces the lipid membrane surface tension,^[28] the lateral movement of the monolayer is influenced by LZM, which further influences the phase transformation process. For lysozyme and DPPE systems, as shown in Fig. 1(b), with the increase of subphase concentration of lysozyme, the π–A curves of the monolayer are similarly shifted toward higher area values. This indicates that LZM was inserted into DPPE molecules by adsorption on membrane lipid monolayers to change the π–A curve shape.

Fig. 2.

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	Fig. 2. π-area isotherms of different subphase concentration of lysozyme interaction with (a) DPPC and (b) DPPE monolayer. (i) Pure DPPC; (ii), (vii) 0.10 mM; (iii), (viii) 0.21 mM; (iv), (ix) 0.63 mM; (v), (x) 1.04 mM; (vi) pure DPPE. ΔA = Aξ −A0, where A0 stands for mean molecular area of pure lipid monolayer and Aξ is the mean molecular area of the mixed monolayer.

In order to study the adsorption process of different subphase concentrations of lysozyme into the lipid monolayer, under different pressures, we calculate the change of mean molecular area of the two kinds of system, denoted as ΔA. When the concentration of lysozyme is 0.10 mM, the surface pressure is 5 mN/m, ΔA of DPPC and DPPE is 19.92 Å² and 9.25 Å². Variation of DPPC was significantly higher than that of DPPE. This is because when the pressure is 5 mN/m, DPPC is in LE–LC phase coexistence, and protein molecules are easily embedded into the membrane. When the surface pressure is 5 mN/m, 15 mN/m, and 25 mN/m, the curves variation of DPPE (ΔA) is greater than the change of DPPC monolayer in the amount of LZM (0.21 mM), which indicates that the effects of lysozyme molecules on DPPE is larger and there are more lysozyme molecules adsorbed on the DPPE monolayer. The reason may be that DPPE molecules are arranged loosely below the pressure of 25 mN/m, so lysozyme is more easily embedded into the monolayer on the surface. On the contrary, at the same surface pressure, the curves variation of DPPC (ΔA) is greater than the change of DPPE monolayer in the amount of LZM (0.63 mM and 1.04 mM). However, the corresponding (ΔA) of the DPPC and DPPE were negative when the pressure is 35 mN/m. This may be induced by the squeezing out of LZM from the lipid monolayer. When adding a certain amount of lysozyme, the ΔA of two systems increases with the decrease of surface pressure. The experimental results showed that the low concentration of lysozyme in subphase solution tends to adsorb or insert into the DPPE monolayer and the high concentration of lysozyme solution is more likely to be adsorbed or inserted into the DPPC monolayer. It could be expected that the interactions of the head groups could influence penetration. DPPC and DPPE are different in size and orientation of the head groups. More space is required for the larger and more hydrated PC head group than for the PE head group.

The elastic compressibility is a useful parameter for assessing elastic properties of Langmuir monolayers, which was calculated from the π–A isotherm using Eq. (1).^[30–33] The result is presented in Fig. 3. A is the area in the trough, and π is the surface pressure.

Fig. 3.

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	Fig. 3. calculated from π–A isotherm of different subphase concentrations of lysozyme interaction with (a) DPPC and (b) DPPE monolayers.

As can be seen from Fig. 3(a), with the increase of the content of lysozyme in the subphase, the maximum elastic modulus of DPPC monolayer gradually decreased. The maximum elastic modulus of pure DPPC is 245 mN/m and the maximum elastic modulus at 0.10 mM is about 175 mN/m. The maximum value of elastic modulus decreases rapidly when the subphase concentration is greater than 0.10 mM. The subphase concentration is 0.63 mM and 1.04 mM, and the maximum value of two lipids with the same elastic modulus is 51 mN/m. For the DPPE system, as shown in Fig. 3(b), the concentration of lysozyme is 0 mM, 0.10 mM, 0.21 mM, 0.63 mM, 1.04 mM, corresponding to elastic modulus maximum value of 232 mN/m, 181 mN/m, 74 mN/m, 65 mN/m, 57 mN/m. We can conclude that with the increase of the content of lysozyme in subphase, the maximum elastic modulus decreases. The elastic modulus can characterize monolayer compressibility. These phenomena indicate that the compressibility of the pure lipid is smaller than that of the mixed monolayer film. A certain amount of the lysozyme increases the monolayer compressibility and this may be due to lysozyme embedded into the lipid membrane, stabilizing it.

As soon as the predetermined π_o had been reached with particular phospholipids on the surface, the barriers were stopped and a certain amount of lysozyme was injected into the subphase, forming a concentration of 0.10 mM, 0.21 mM, 0.63 mM, 1.04 mM of the subphase solution. Figure 4 shows the π–T curve of LZM adsorption onto phospholipid when initial pressure is 15 mN/m. We can see that interaction of the lysozyme molecule with lipid undergoes three important stages: initial adsorption, adsorption rearrangement and equilibrium state expansion and rearrangement.^[13] In addition, with the increase of concentration of lysozyme, the equilibrium pressure of DPPC showed increasing trend, suggesting that with the increase of LZM, more and more LZM are adsorbed or inserted into the monolayer. In contrast, for the DPPE system, the equilibrium pressure decreases with the increase of the amount after the addition of LZM. This may be induced by the squeezing out of LZM from the DPPE monolayer due to the stronger interaction. This is consistent with the π–A isotherms analysis results.

Fig. 4.

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	Fig. 4. π–T curves of different subphase concentration of lysozyme interaction with (a) DPPC and (b) DPPE monolayer at the initial surface pressure of 15 mN/m.

Parameters are obtained according to the π–T curve in order to study the ability of lysozyme molecules better embedded in lipid monolayer, as shown in Tables 1 and 2. When the concentration of lysozyme was 0.21 mM, the surface pressure DPPC and DPPE adsorption curve in 2500 s and 500 s, respectively, changes, showing that from now on, lysozyme begins to adsorb on the lipid monolayer,^[34] interpreted as representing initial adsorption. With the increase of time, the surface pressure increases continuously, until 89951 s and 2593 s, to reach the equilibrium stage, the equilibrium pressure is 15.4 mN/m and 21.1 mN/m, respectively. This shows that more lysozyme molecules are embedded into DPPE monolayer at this concentration. In addition, the equilibrium time of interaction between lysozyme and DPPE is less than the time of the interaction with DPPC. The shapes of all the curves are very similar to that with the increase of adsorption time, in which the slope of adsorption curves at first increase and then decrease. That is in the process of lysozyme molecules with constant in a steady stream of adsorption on the membrane, until the pressure reached a relatively steady state (equilibrium pressure), protein at the interface of self-assembling and arranging.^[5] The interaction of protein and lipid is mainly through physical adsorption, namely van der Waals forces. Protein and lipid interactions generally do not form chemical bonds. Physical adsorption is reversible, so we can finally see, protein did not have unlimited adsorption on the membrane, but eventually reached a dynamic equilibrium.

Table 1.

Table 1.

Table 1. Parameter values of π–T curves of LZM/DPPC. . Concentration of lysozyme/mM Parameters T/s πo/mN·m−1 πe/mN·m−1 0.21 89951 15.0 15.4 0.63 11494 15.0 16.0 1.04 17185 15.0 18.1

Table 1. Parameter values of π–T curves of LZM/DPPC. .

Table 2.

Table 2.

Table 2. Parameter values of π–T curves of LZM/DPPE. . Concentration of lysozyme/mM Parameters T/s πo/mN·m−1 πe/mN·m−1 0.21 2593 15.0 21.1 0.63 3257 15.0 16.7 1.04 3562 15.0 14.4

Table 2. Parameter values of π–T curves of LZM/DPPE. .

When the lysozyme concentration was 0.21 mM, the lysozyme molecules were adsorbed onto the DPPC lipid monolayer (see Fig. 5(b)). With the increase of concentration of lysozyme, the aggregation of lysozyme molecules is increasingly large. When the concentration was 0.63 mM, clear lipid membranes can be seen and membrane arrangement is still more regular (Fig. 5(c)). This shows that the subphase of lysozyme molecules adsorbed on the surface of the DPPC and a small part of the lysozyme molecules embedded into the lipid monolayer, and with the increase in the amount of lysozyme, lysozyme molecules on the surface of the membrane were self-assembled. Lysozyme has little effect on the structure of the lipid monolayer. For the DPPE monolayer, when the lysozyme concentration is 0.21 mM, the same as for DPPC, lysozyme is evenly dispersed in the phospholipid. However, density is higher compared to the DPPC, as shown in Fig. 5(f). With the increase of the amount of lysozyme, lysozyme aggregation and its lipid molecules around the center to attract to the surrounding, eventually causes the DPPE film morphology to have a greater impact. LZM interaction with DPPC and DPPE is obviously different. This may be possibly due to the different head groups. The head group of DPPC and DPPE are similar in charge and differ in size and orientation of the head groups. More space is required for the larger and more PC head group than for the PE head group.^[20,26] Previous studies have shown that as lysozyme adsorbs to a PC film, it causes a stretching of the O–P–O diester bond, but at the same time lowers the chain-disorder transition temperature by a few degrees. Penetration is difficult once adsorption has occurred because even though there are polar interactions between lysozyme and the acidic phospholipids, it is excluded from interacting with the hydrophobic portion of the lipid below the chain transition temperature.^[20] Our experiments were all carried out below the transition temperature for PC (∼ 41 °C). We suggest this may cause a stretching of the O–P–O diester bond. Then penetration is more difficult for lysozyme to adsorb to the DPPC than DPPE monolayer. In addition, with surface pressure at 15 mN/m, DPPC is in the liquid condensed phase and molecules are arranged closely whereas DPPE is in liquid expanded phase and molecules are arranged loosely, so lysozyme is easily embedded into the lipid membrane, and the membrane LZM molecular quantity increases.

Fig. 5.

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	Fig. 5. The AFM images of DPPC and DPPE monolayer at different concentrations LZM amount : (a) DPPC; (e) DPPE; (b), (f) 0.21 mM; (c), (g) 0.63 mM; (d), (h) 1.04 mM; scale bar: 5 μm.

A more detailed observation of the effect of lysozyme on the DPPE monolayer is shown in Fig. 7. There are a large number of lysozyme molecules adsorbed on the DPPE monolayer, varying in size and shape. In this paper, the information of the diameter and height of lysozyme are statistical. The results show that the height and diameter of lysozyme are in the range of 3.7 nm–13.9 nm and 47.5 nm–66.8 nm respectively. The three-dimensional structure of the lysozyme molecule is nearly elliptical, and its size is 4.5 nm×3 nm×3 nm, which was obtained by x-ray analysis method in 1965.^[34] Through observation and analysis, lysozyme forms a cone polymer after adsorption on a layer. Lysozyme not only changed the conformation of lipid monolayer, but also self-assembly. This is consistent with the π–T isotherms analysis results.

Fig. 6.

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	Fig. 6. The AFM images of DPPE monolayer at 0.21 mM of LZM concentrations. Scanning range: (a) 5 μm×5 μm; (b) 2.5 μm×2.5 μm; (c) 1.25 μm×1.25 μm; (d) three-dimensional graph of panel (c).

Fig. 7.

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	Fig. 7. Interaction mechanism model of lysozyme molecule with DPPC and DPPE monolayer.

Through the analysis of the lysozyme molecule aggregation mechanism may be related to amyloid fibril nucleation stage. Similar models suggest that the proteins and peptides monomer under certain conditions form the core fiber, and the fiber core and the monomer binding, Ever fount monomer and nuclear binding form fibrils further integrated fiber.^[36] This model consists of two stages: nucleation stage and elongation stage.^[37] The extension of the protein fiber can be interrupted and then form a new kernel, so as to shorten the formation time of skin amyloid fibers. In this process, lysozyme molecules are able to aggregate into its nucleus, inevitably accompanied by thermodynamic changes of the molecular aggregation of lysozyme. This may be induced by lipid,^[38] but when lysozyme molecules are adsorbed to the lipids, lipid arrangement because of protein insertion into it. Previous literature reported that lysozyme can form amyloid fibrils under certain limiting conditions.^[36,38] The formation of lysozyme molecules was similar to the first stage of amyloid fibrils. Therefore, the lysozyme molecules can form oligomers under the induction of lipids.

Possible interfacial structural changes of the mixed lysozyme/DPPC and lysozyme/DPPE films on compression are schematically depicted in Fig. 7. For DPPC at the surface pressure of 15 mN/m, the monolayer was in the liquid condensed phase, lipid molecules were arranged closely, a small amount of lysozyme molecules embedded into the membrane, and lysozyme molecules will form irregular multimers. However, DPPE at this surface pressure which is in liquid expanded phase, has molecules arranged loosely, so lysozyme molecules easily embedded into the lipid membrane, and the number of LZM molecules on lipid membranes increased, forming conical multimeric.