- Email: [email protected]
Influence of alkaline phosphatase on phase state of the SM monolayers at the air-water interface
Colloids and Surfaces A: Physicochem. Eng. Aspects 489 (2016) 136–141
Contents lists available at ScienceDirect
Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa
Influence of alkaline phosphatase on phase state of the SM monolayers at the air-water interface Juan Wang, Runguang Sun ∗ Laboratory of Biophysics and Biomedicine, College of Physics and Information Technology, Shaanxi Normal University, Xi’an 710062, Shaanxi, China
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
• The different amount of alkaline phosphatase have an influence on the phase transition of SM monolayer at the air–water interface. • The interfacial mixing ratio of protein/lipid has been calculated by the mass conservation plots. • The images of AFM and SEM shown the microstructure of the monolayer at 5 and7.8 mN/m.
a r t i c l e
i n f o
Article history: Received 16 July 2015 Received in revised form 19 October 2015 Accepted 23 October 2015 Available online 28 October 2015 Keywords: Alkaline phosphatase Compression modulus The interfacial mixing ratio Atomic force microscopy Phase transition
a b s t r a c t The Langmuir monolayer is a most suitable model membrane system to study the interfacial interaction between protein and lipid. The surface pressure-area per molecule isotherms of the SM monolayer in the presence of different amount of alkaline phosphatase have been studied in this work. The compression modulus and the interfacial mixing ratio of protein/lipid have been calculated. The different amount of alkaline phosphatase has an influence on the phase transition of SM monolayer at the air–water interface. A coexist state of liquid expanded and liquid condensed is due to the presence of alkaline phosphatase. The process of phase transition from liquid expanded state to coexist state is longer in the presence of more amount of protein. The emergence of coexistence phase state reflects the interaction of the protein and the SM monolayer. The interfacial mixing ratio at the liquid expanded state is higher than that of the coexist state of liquid expanded and liquid condensed. The images of SEM and AFM also shown the microstructure of SM monolayers at two phase states in the presence of alkaline phosphatase. © 2015 Elsevier B.V. All rights reserved.
1. Introduction
∗ Corresponding author. E-mail address: [email protected] (R. Sun). http://dx.doi.org/10.1016/j.colsurfa.2015.10.040 0927-7757/© 2015 Elsevier B.V. All rights reserved.
Membrane models are often used to study biophysical and biochemical phenomena involved in the interaction between molecules and cellular membranes [1,2]. Lipid monolayer is a most
J. Wang, R. Sun / Colloids and Surfaces A: Physicochem. Eng. Aspects 489 (2016) 136–141
2. Materials and methods Sphingomyelin, alkaline phosphatase from bovine intestinal mucosa, Tris, Chloroform, methanol, ethanol absolute, potassium chloride, sodium chloride, hydrochloric acid were obtained from Sigma Chemical Co. (St.Louis, MO). All experiments were carried out at 25 ± 1 ◦ C and all solutions were prepared using ultrapure water. 2.1. Preparation 5 mM Tris–HCl buffer (pH 7.5, containing 150 mmol NaCl, 2 mmol MgCl2 and 0.4 mol KCl) was used as a subphase solution under the air–water interface. Alkaline phosphatase was dissolved in Tri–HCl buffer with the concentration of 0.05 mol/ml. Sphingomyelin(SM) solution was prepared in chloroform/methanol (proportion 3:1 in volume). The concentration was 0.5 mol/ml.
50
Surface pressure (mN/m)
commonly used system [3,4]. The Langmuir technique is based on the property of amphiphilic molecules to form a monolayer film when spread at the air-water interface and subsequently subjected to compression [5]. The surface pressure isotherm studies can provide the information on the phase transition of monolayers. And the interfacial interaction ratio of protein/lipid can be calculated by data of the surface pressure-area per molecule curves. The enzyme of alkaline phosphatase has a role in the biomineralization process [6]. Alkaline phosphatases from several sources have been intensively studied at the air–water interface [7,8]. The orientation and distribution of the enzyme on the lipid monolayer were studied frequently, while papers about the influence of the amount of alkaline phosphatase to the lipid monolayer are rarely reported. Alkaline phosphatase, as a medical check index, is used to help with the disease diagnosis of bone, liver and gallbladder system [9,10]. High content of alkaline phosphatase is relative to the diseases. So, the influence of different amount of the enzyme to the lipid monolayer model is worth being studied. Sphingomyelin (SM), containing saturated fatty acids, was used to as model membrane in this work. The effect of different amount of alkaline phosphatase on the phase of SM monolayer at the air–water interface was investigated.
137
SM
40
SM+0.1 nmol AP SM+0.3 nmol AP
30
SM+0.5 nmol AP SM+0.7 nmol AP
20
SM+0.9 nmol AP 10 0
50
100
150
200 2
Area per molecule (Å ) Fig. 1. The surface pressure-area per molecule isotherms of SM monolayers in the presence of different amount of alkaline phosphatase.
paper [11–13]. Phospholipids were spread at the air–water interface and different amount of alkaline phosphatase were injected under the interface. After 20–min for solvent evaporation, the monolayer was compressed to obtain a surface pressure-area per molecule ( − A) isotherm. Monolayer compression was performed at a compression rate of 0.5 cm2 s−1 . 2.3. SEM and AFM images Transfer the Langmuir monolayers onto the fresh mica at a certain surface pressure, forming Z-Langmuir film. The dipping rate for transfer was 5 mm/s. Observe their microstructure characterization by scanning electron microscope (Hitachi, Japan) and atomic force microscopy (Shimadzu, Japan). SEM samples were coated with gold before examination. AFM images can be obtained in the contacting mode using a silicon nitride pyramidal tip mounted on a 100 um long cantilever with a force constant of 0.1 N/m. 3. Results and discussion 3.1. phase transitions
2.2. Langmuir setup The SM monolayer at the air-buffer interface was run using a Teflon Langmuir trough (KSV, Finland). The surface pressure was measured by the Wilhelmy method, using a very thin plate of filter
For the pure SM monolayer, the isotherm has no clear inflection point. But after the injection of alkaline phosphatase under the air–water interface, a flat region is shown (Fig. 1). The flat region changes longer with the increase of the amount of alkaline phos-
100 80
A
SM + 0.1 nmol AP
SM + 0.5 nmol AP
SM + 0.9 nmol AP
40
40
M2
M0
0
10
M2
20
20
M1
B
SM + 0.7 nmol AP
60
SM + 0.3 nmol AP 60
0
80
compression modulus -1 ( Cs , mN/m )
compression modulus -1 ( Cs , mN/m )
SM
7 / 11 20
30
40
surface pressure ( π, mN/m )
50
0
0
M1
10
20
30
40
surface pressure ( π, mN/m )
Fig. 2. The compression modulus-surface pressure curves of SM monolayers in the presence of different amount of alkaline phosphatase.
50
J. Wang, R. Sun / Colloids and Surfaces A: Physicochem. Eng. Aspects 489 (2016) 136–141
10
10
A
6
π -1 Cs
4
6 4
2 0
27
40
38
21
36 18
0
0.9
0.7
0.5
0.3
B
24
2
0.1
π -1 Cs
π (mN/m )
M1
42
M2
8
Cs-1 (mN/m )
π (mN/m )
8
30
Cs-1 (mN/m )
138
0.1
0.3
0.5
0.7
0.9
amount of AP (nmol)
amount of AP (nmol) 95
π (mN/m )
( Cs-1)max (mN/m )
C
36 32
76
28
π -1 (Cs )max
24 20
0.0 0.1
0.3
0.5
0.7
57 0.9
amount of AP (nmol)
8 / 11
Fig. 3. The surface pressure and compression modulus of the SM monolayer at the phase transition points, M1 (A) and M2 (B). In the figure (C), the maximums of compression modulus of the SM monolayer in the presence of different amount of alkaline phosphatase are shown.
phatase. The presence of flat regions suggest the process of phase transition. The information about phase transition of the monolayer can be obtained from the surface compressional modulus-surface pressure curves. The surface compressional modulus (Cs−1 ) is a significant parameter to analyze the compressibility of the monolayer [14]. The Cs−1 can be calculated out according to the surface pressure-area per molecule isotherm and the formula as follows [15,16]:
Cs−1 = −A ∂⁄∂A
(1) T
where A is the area per molecule, is the surface pressure and T is the constant temperature. A larger Cs−1 value indicates a less compressible membrane. From the Figure 2, there is a minimum (M0 , Fig. 2A) on the compression modulus-surface pressure curve of the pure SM monolayer. The point, M0 , suggests a phase transition from the liquid expanded state to the liquid condensed state. At 37 mN/m, the value of Cs−1 reaches a maximum which indicates the monolayer is incompressible and may collapse at higher surface pressure. While after the addition of alkaline phosphatase, the two points of phase transition, M1 and M2 , are observed on the compression modulussurface pressure curves (Fig. 2B). The point at M1 suggests that the phase of monolayer transfers from the liquid expanded state to the coexist state of liquid expanded and liquid condensed. Then the point at M2 indicates a phase transition from the coexist state of liquid expanded and liquid condensed to the liquid condensed state.
At the point of M0 , the surface pressure is 13.46 mN/m and the value of corresponding Cs−1 is 35.11 mN/m. The surface pressures and Cs−1 values at the points of M1 (A) and M2 (B) are shown in Fig. 3. The phase transition at M1 occurs at 7.8m N/m approximately, while the Cs−1 value at M1 point decreases with the increase of amount of alkaline phosphatase. Notably, the point of M2 occurs at the surface pressure of 20.5 mN/m. The corresponding Cs−1 value at M2 decreases first and then increases as the increase of amount of the protein, and it disappears when the amount of alkaline phosphatase is above 0.5 nmol. The minimum of the Cs−1 value of M2 is 37.03 mN/m, higher than that of M0 in the pure SM monolayer. At the point of phase transition, the surface pressures almost remain the same value while the corresponding Cs−1 values change obviously with the increases of the AP amount. In addition, after adding the different amount of AP protein, the (Cs−1 )max values of the SM monolayer are greater than that without AP under the air–water interface (Fig. 3C). However, the (Cs−1 )max values are shown a trend of decrease with the increase of the amount of AP. Notably, the values of (Cs−1 )max have a little change when the amount of AP increases from 0.3-0.7 nmol. Meanwhile, alkaline phosphatase induces the surface pressure corresponding to the (Cs−1 )max value of monolayers decreased. 3.2. The interfacial mixing protein/lipid ratio Mass conservation of protein in the system: n0P = r · nL + nsP
(2)
J. Wang, R. Sun / Colloids and Surfaces A: Physicochem. Eng. Aspects 489 (2016) 136–141
6
*
B
A 0.8
*
ΔΑ = 1.5 Å
4
0.6
3
1
*
ΔΑ = 3 Å
2
0.4
0
15 nmol SM 18 nmol SM 20 nmol SM 22 nmol SM
2
2
* 2 ΔΑ = 2.5 Å
np (nmol)
2
ΔΑ (Å /molecule)
5
1.0
139
0.2
0 0.0
0.2
0.4
0.6
0.8
0.0 14
1.0
16
18
np (nmol)
1.0
C
*
*
ΔΑ = 1 Å
np (nmol)
3 2
0
15 nmol SM 18 nmol SM 20 nmol SM 22 nmol SM
1 0 0.0
0.2
0.4
22
D
0.8
2
ΔΑ (Å /molecule)
4
20
nL (nmol)
0
0.6
0.8
2
*
ΔΑ = 1.5 Å
0.6
2
* 2 ΔΑ = 2 Å
0.4 0.2
1.0
0.0
15
18
0
21
nL (nmol)
np (nmol)
Fig. 4. Data processing at 5 mN/m (A, B) and 7.8 mN/m(C, D). The measured area increase per SM molecule versus total amount of added alkaline phosphatase for different amounts of SM in the monolayer (A, C). And mass conservation plots at constant A∗ (B, D).
L
L
22 nmol, respectively, have been run to confirm the quality of the mass conservation plots. Then at the same level of A∗ , the n0P values for the individual nL must be subject to constancy regarding the interfacial protein/lipid ratio, r, and the amount that was partitioned into the subphase, nsP . The n0P − nL linear relation stands for the mass conservation plots according to Eq.(2). So, the interfacial mixing ratio, r, and the amount of protein partitioned into the subphase,nsP , can be calculated according to the n0P − nL linear relation. According to the surface pressure-area per molecule isotherms, the values of A are negative at the given surface pressure from 7.8 mN/m to 30 mN/m, which suggests that alkaline phosphatase in the interface carry a portion of SM molecules into the subphase, and that is to say, some amounts of SM in the interface are lost. At the air–water interface, the nL value changes to be uncertain. So, the phase transition of M2 is a false point, which is caused by the redistribution of the rest of SM molecules at the interface under the continued compression. It also shows that the presence of AP only induces the phase transition of the SM monolayer from the liquid expanded state to the coexist state of liquid expanded and liquid
0.06
5 mN/m 7.8 mN/m 0.04
r
where n0P , nsP stand for the total and subphase amounts of protein, respectively; nL is the amount of lipid; and r = nP ⁄nL denotes the interfacial mixing ratio of protein–lipid interaction [17,18]. For a given pressure the increase in per molecular area, A, under equilibrium conditions were determined from various series of surface pressure-area per molecule isotherms. Here we have to take into account only differences in the area, so the effect of a thin plate of filter paper can be ignored. For a given amount of SM in the monolayer,nL , we have plotted A∗ − n0P curves, where A∗ = A⁄n . Four groups of experiments with n =15, 18, 20 and
0.02
1.2
1.6 *
2.0
2.4
2
ΔΑ (Å /molecule) Fig. 5. The interfacial mixing radio of alkaline phosphatase/SM molecules.
condensed. The emergence of coexistence phase state reflects the interaction of the protein and the SM monolayer. the monolayer is in the liqAnd from the analysis of C−1 s uid expanded state at 5 mN/m and in the coexist state of liquid expanded and liquid condensed at 7.8 mN/m. So, in order to investigate the interfacial protein/lipid mixing radio at the two state, we only calculate the relevant data at the surface pressure 5 mN/m and 7.8m N/m (Fig. 4). The A∗ − n0p curves becaome increasingly bent in the downward direction in Fig. 4A and C. For a given amount of total protein, A∗ is decreasing with the increase of SM molecules. The linear
140
J. Wang, R. Sun / Colloids and Surfaces A: Physicochem. Eng. Aspects 489 (2016) 136–141
Fig. 6. The SEM and AFM images of the SM monolayer in the presence of 0.3nmol AP at 5 mN/m (A, C) and 7.8 mN/m(B, D)(nL = 20nmol).
mass conservation plots in Fig. 4B and D distinctly reveal a substantial portion of alkaline phosphatase in the subphase under the air–water interface. For a given amount of SM in the interface, A∗ increases as the increase of amount of total protein. The interfacial mixing ratio of protein/lipid, r, has been calculated by mass conservation plots at 5 mN/m (Fig. 4B) and 7.8 mN/m (Fig. 4D). And the function of r with A∗ has been shown in Fig. 5. The r values increase with the increase of A∗ . At 5 mN/m, it is lower than that at 7.8 mN/m for a certain value of A∗ , which suggests that the interfacial mixing ratio at the liquid expanded state is higher than that of the coexist state of liquid expanded and liquid condensed. A greater of r suggests fewer amounts of SM corresponding to an alkaline phosphatase molecule.
decreases with the increase of amount of alkaline phosphatase. The appearance of coexist state is due to the interaction between alkaline phosphatase and SM monolayer. The interfacial mixing ratio in the coexist state is greater than that in liquid expanded state, which suggests that more alkaline phosphatase molecule combine with the SM monolayer in the coexist state. The greater the amount of alkaline phosphatase, the easier its combination with membrane. In some disease of bone, liver and gallbladder system, the content of alkaline phosphatase is often high. The high content of alkaline phosphatase has a pronounced effect on the phase transition of membrane. The microstructure images from SEM and AFM provide some helpful evidence of the phase transition. The emergence of the coexist state of liquid expanded and liquid condensed may have a special influence on the membrane in cells.
3.3. SEM and AFM Acknowledgement In SEM images, the liquid expanded state of SM monolayers at 5 mN/m is shown a shape like “a series of ditches” (arrow regions in Fig. 6). At 7.8 mN/m, a shape of “cotton flocculent” (triangular regions in Fig. 6B), which suggests a liquid condensed state, also appears on the SM monolayers, except for the ditch shape. The images of AFM also show a liquid expanded state at 5 mN/m, and a coexist state of liquid expanded and liquid condensed (arrow and triangular regions in Fig. 6D, respectively) at 7.8 mN/m. 4. Conclusion Alkaline phosphatase has an influence on phase transition of the SM monolayer mainly under low pressure. The greater the amount of alkaline phosphatase, the longer the process of phase transition from liquid expanded state to the coexist state of liquid expanded and liquid condensed. At the point of phase transition, that is, in the coexist state of the monolayer, the surface compressional modulus
These authors are thankful to the Foundamental Research Funds for the Central Universities (GK201504004) for supporting this work. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfa.2015.10. 040. References [1] N. Nandi, D. Vollhardt, Helfrich’s concept of intrinsic force and its molecular origin in bilayers and monolayers, Adv. Colloid Interface Sci. 208 (2014) 110–120. [2] D. Needham, D.H. Kim, PEG-covered lipid surfaces:bilayers and monolayers, Colloids Surf. B 18 (2000) 183–195.
J. Wang, R. Sun / Colloids and Surfaces A: Physicochem. Eng. Aspects 489 (2016) 136–141 [3] N.F. Crawford, M. Micic, J. Orbulescu, D. Weissbart, R.M. Leblanc, Surface chemistry and spectroscopy of the —galactosidase Langmuir monolayer, J. Colloid Interface Sci. 453 (2015) 202–208. [4] J. Li, R. Sun, C. Hao, G. He, L. Zhang, J. Wang, The behavior of the adsorption of cytochrome C on lipid monolayers:a study by the Langmuir-Blodgett technique and theoretical analysis, Biophys. Chem. 205 (2015) 33–40. [5] G. Roberts, Langmuir-Blodgett Films, Plenum Press, New York, 1990. [6] P.A. Henthorn, Alkaline phosphatase, in: J.P. Bilezikian, L.G. Raisz, G.A. Rodan (Eds.), Principles of Bone Biology, Academic Press, New York, 1996, p. 197. [7] A. Petrigliano, A. Tronin, C. Nicolini, Deposition and enzymatic activity of Langmuir-Blodgett films of alkaline phosphatase, Thin Solid Films 284–285 (1996) 752–756. [8] L. Caseli, D.C. Masui, R.P.M. Furriel, F.A. Leone, M.E.D. Zaniquelli, Incorporation conditions guiding the aggregation of a glycosylphosphatidyl inositol (GPI)-anchored protein in Langmuir monolayers, Colloids Surf. B 46 (2005) 248–254. [9] T.J. Zhu, T.H. Zhu, X.X. Fan, The significance of alkaline phosphatase in liver diseases diagnosis, J.Qiqihar Med. Coll. 16 (3) (1995) 219 (in Chinese). [10] X.J. Wang, X.F. Yuan, S.J. Li, Alkaline phosphatase conjugated serum detection and analysis on the clinical value of diagnosis of diseases, Med. Innov. China 12 (16) (2015) 146 (in Chinese).
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[11] L. Caseli, F.N. Crespilho, T.M. Nobre, M.E.D. Zaniquelli, V. Zucolotto, ONO Jr, Using. phospholipid Langmuir and Langmuir-Blodgett films as matrix forurease immobilization, J. Colloid Interface Sci. 319 (2008) 100. [12] J.B. Peng, G.T. Barnes, I.R. Gentle, The structures of Langmuir-Blodgett films of fatty acids and their salts, Adv. Colloid Interface Sci. 91 (2001) 163. [13] G. Schwarz, J. Zhang, Chain length dependence of lipid partitioning between the air/water interface and its subphase thermodynamic and structural implications, Chem. Phys. Lipids 110 (2001) 35. [14] C.C. Hao, R.G. Sun, J. Zhang, Y.G. Chang, C.L. Niu, Behavior of sulfatide/cholesterol mixed. monolayers at the air/water interface, Colloids and Surfaces B: Biointerfaces 69 (2009) 201. [15] F.N. Zakanda, P. Laurent, M. Paquot, G.M. Lelo, M. Deleu, Alkylbetainate chlorides:Synth-esis and behavior of monolayers at the air–water interface, Thin Solid Films 520 (2011) 344. [16] A.K. Panda, K. Vasilev, S. Orgeig, C.A. Prestidge, Thermodynamic and structural studies of mixed monolayers:mutual mixing of DPPC and DPPG-with DTAP at the air–water interface, Mater. Sci. Eng. C 30 (2010) 542. [17] G. Schwarz, S.E. Taylor, Polymorphism and interactions of a viral fusion peptide in a compressed lipid monolayer, Biophys. J. 76 (1999) 3167–3175. [18] L. Zhang, R. Sun, C. Hao, J. Yang, Effect of myelin basic protein on membrane lipid monolayers composed of DPPC and DPPE molecules, Sci. Sinica Chim. 44 (2014) 1562–1569, in Chinese.
Contents lists available at ScienceDirect
Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa
Influence of alkaline phosphatase on phase state of the SM monolayers at the air-water interface Juan Wang, Runguang Sun ∗ Laboratory of Biophysics and Biomedicine, College of Physics and Information Technology, Shaanxi Normal University, Xi’an 710062, Shaanxi, China
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
• The different amount of alkaline phosphatase have an influence on the phase transition of SM monolayer at the air–water interface. • The interfacial mixing ratio of protein/lipid has been calculated by the mass conservation plots. • The images of AFM and SEM shown the microstructure of the monolayer at 5 and7.8 mN/m.
a r t i c l e
i n f o
Article history: Received 16 July 2015 Received in revised form 19 October 2015 Accepted 23 October 2015 Available online 28 October 2015 Keywords: Alkaline phosphatase Compression modulus The interfacial mixing ratio Atomic force microscopy Phase transition
a b s t r a c t The Langmuir monolayer is a most suitable model membrane system to study the interfacial interaction between protein and lipid. The surface pressure-area per molecule isotherms of the SM monolayer in the presence of different amount of alkaline phosphatase have been studied in this work. The compression modulus and the interfacial mixing ratio of protein/lipid have been calculated. The different amount of alkaline phosphatase has an influence on the phase transition of SM monolayer at the air–water interface. A coexist state of liquid expanded and liquid condensed is due to the presence of alkaline phosphatase. The process of phase transition from liquid expanded state to coexist state is longer in the presence of more amount of protein. The emergence of coexistence phase state reflects the interaction of the protein and the SM monolayer. The interfacial mixing ratio at the liquid expanded state is higher than that of the coexist state of liquid expanded and liquid condensed. The images of SEM and AFM also shown the microstructure of SM monolayers at two phase states in the presence of alkaline phosphatase. © 2015 Elsevier B.V. All rights reserved.
1. Introduction
∗ Corresponding author. E-mail address: [email protected] (R. Sun). http://dx.doi.org/10.1016/j.colsurfa.2015.10.040 0927-7757/© 2015 Elsevier B.V. All rights reserved.
Membrane models are often used to study biophysical and biochemical phenomena involved in the interaction between molecules and cellular membranes [1,2]. Lipid monolayer is a most
J. Wang, R. Sun / Colloids and Surfaces A: Physicochem. Eng. Aspects 489 (2016) 136–141
2. Materials and methods Sphingomyelin, alkaline phosphatase from bovine intestinal mucosa, Tris, Chloroform, methanol, ethanol absolute, potassium chloride, sodium chloride, hydrochloric acid were obtained from Sigma Chemical Co. (St.Louis, MO). All experiments were carried out at 25 ± 1 ◦ C and all solutions were prepared using ultrapure water. 2.1. Preparation 5 mM Tris–HCl buffer (pH 7.5, containing 150 mmol NaCl, 2 mmol MgCl2 and 0.4 mol KCl) was used as a subphase solution under the air–water interface. Alkaline phosphatase was dissolved in Tri–HCl buffer with the concentration of 0.05 mol/ml. Sphingomyelin(SM) solution was prepared in chloroform/methanol (proportion 3:1 in volume). The concentration was 0.5 mol/ml.
50
Surface pressure (mN/m)
commonly used system [3,4]. The Langmuir technique is based on the property of amphiphilic molecules to form a monolayer film when spread at the air-water interface and subsequently subjected to compression [5]. The surface pressure isotherm studies can provide the information on the phase transition of monolayers. And the interfacial interaction ratio of protein/lipid can be calculated by data of the surface pressure-area per molecule curves. The enzyme of alkaline phosphatase has a role in the biomineralization process [6]. Alkaline phosphatases from several sources have been intensively studied at the air–water interface [7,8]. The orientation and distribution of the enzyme on the lipid monolayer were studied frequently, while papers about the influence of the amount of alkaline phosphatase to the lipid monolayer are rarely reported. Alkaline phosphatase, as a medical check index, is used to help with the disease diagnosis of bone, liver and gallbladder system [9,10]. High content of alkaline phosphatase is relative to the diseases. So, the influence of different amount of the enzyme to the lipid monolayer model is worth being studied. Sphingomyelin (SM), containing saturated fatty acids, was used to as model membrane in this work. The effect of different amount of alkaline phosphatase on the phase of SM monolayer at the air–water interface was investigated.
137
SM
40
SM+0.1 nmol AP SM+0.3 nmol AP
30
SM+0.5 nmol AP SM+0.7 nmol AP
20
SM+0.9 nmol AP 10 0
50
100
150
200 2
Area per molecule (Å ) Fig. 1. The surface pressure-area per molecule isotherms of SM monolayers in the presence of different amount of alkaline phosphatase.
paper [11–13]. Phospholipids were spread at the air–water interface and different amount of alkaline phosphatase were injected under the interface. After 20–min for solvent evaporation, the monolayer was compressed to obtain a surface pressure-area per molecule ( − A) isotherm. Monolayer compression was performed at a compression rate of 0.5 cm2 s−1 . 2.3. SEM and AFM images Transfer the Langmuir monolayers onto the fresh mica at a certain surface pressure, forming Z-Langmuir film. The dipping rate for transfer was 5 mm/s. Observe their microstructure characterization by scanning electron microscope (Hitachi, Japan) and atomic force microscopy (Shimadzu, Japan). SEM samples were coated with gold before examination. AFM images can be obtained in the contacting mode using a silicon nitride pyramidal tip mounted on a 100 um long cantilever with a force constant of 0.1 N/m. 3. Results and discussion 3.1. phase transitions
2.2. Langmuir setup The SM monolayer at the air-buffer interface was run using a Teflon Langmuir trough (KSV, Finland). The surface pressure was measured by the Wilhelmy method, using a very thin plate of filter
For the pure SM monolayer, the isotherm has no clear inflection point. But after the injection of alkaline phosphatase under the air–water interface, a flat region is shown (Fig. 1). The flat region changes longer with the increase of the amount of alkaline phos-
100 80
A
SM + 0.1 nmol AP
SM + 0.5 nmol AP
SM + 0.9 nmol AP
40
40
M2
M0
0
10
M2
20
20
M1
B
SM + 0.7 nmol AP
60
SM + 0.3 nmol AP 60
0
80
compression modulus -1 ( Cs , mN/m )
compression modulus -1 ( Cs , mN/m )
SM
7 / 11 20
30
40
surface pressure ( π, mN/m )
50
0
0
M1
10
20
30
40
surface pressure ( π, mN/m )
Fig. 2. The compression modulus-surface pressure curves of SM monolayers in the presence of different amount of alkaline phosphatase.
50
J. Wang, R. Sun / Colloids and Surfaces A: Physicochem. Eng. Aspects 489 (2016) 136–141
10
10
A
6
π -1 Cs
4
6 4
2 0
27
40
38
21
36 18
0
0.9
0.7
0.5
0.3
B
24
2
0.1
π -1 Cs
π (mN/m )
M1
42
M2
8
Cs-1 (mN/m )
π (mN/m )
8
30
Cs-1 (mN/m )
138
0.1
0.3
0.5
0.7
0.9
amount of AP (nmol)
amount of AP (nmol) 95
π (mN/m )
( Cs-1)max (mN/m )
C
36 32
76
28
π -1 (Cs )max
24 20
0.0 0.1
0.3
0.5
0.7
57 0.9
amount of AP (nmol)
8 / 11
Fig. 3. The surface pressure and compression modulus of the SM monolayer at the phase transition points, M1 (A) and M2 (B). In the figure (C), the maximums of compression modulus of the SM monolayer in the presence of different amount of alkaline phosphatase are shown.
phatase. The presence of flat regions suggest the process of phase transition. The information about phase transition of the monolayer can be obtained from the surface compressional modulus-surface pressure curves. The surface compressional modulus (Cs−1 ) is a significant parameter to analyze the compressibility of the monolayer [14]. The Cs−1 can be calculated out according to the surface pressure-area per molecule isotherm and the formula as follows [15,16]:
Cs−1 = −A ∂⁄∂A
(1) T
where A is the area per molecule, is the surface pressure and T is the constant temperature. A larger Cs−1 value indicates a less compressible membrane. From the Figure 2, there is a minimum (M0 , Fig. 2A) on the compression modulus-surface pressure curve of the pure SM monolayer. The point, M0 , suggests a phase transition from the liquid expanded state to the liquid condensed state. At 37 mN/m, the value of Cs−1 reaches a maximum which indicates the monolayer is incompressible and may collapse at higher surface pressure. While after the addition of alkaline phosphatase, the two points of phase transition, M1 and M2 , are observed on the compression modulussurface pressure curves (Fig. 2B). The point at M1 suggests that the phase of monolayer transfers from the liquid expanded state to the coexist state of liquid expanded and liquid condensed. Then the point at M2 indicates a phase transition from the coexist state of liquid expanded and liquid condensed to the liquid condensed state.
At the point of M0 , the surface pressure is 13.46 mN/m and the value of corresponding Cs−1 is 35.11 mN/m. The surface pressures and Cs−1 values at the points of M1 (A) and M2 (B) are shown in Fig. 3. The phase transition at M1 occurs at 7.8m N/m approximately, while the Cs−1 value at M1 point decreases with the increase of amount of alkaline phosphatase. Notably, the point of M2 occurs at the surface pressure of 20.5 mN/m. The corresponding Cs−1 value at M2 decreases first and then increases as the increase of amount of the protein, and it disappears when the amount of alkaline phosphatase is above 0.5 nmol. The minimum of the Cs−1 value of M2 is 37.03 mN/m, higher than that of M0 in the pure SM monolayer. At the point of phase transition, the surface pressures almost remain the same value while the corresponding Cs−1 values change obviously with the increases of the AP amount. In addition, after adding the different amount of AP protein, the (Cs−1 )max values of the SM monolayer are greater than that without AP under the air–water interface (Fig. 3C). However, the (Cs−1 )max values are shown a trend of decrease with the increase of the amount of AP. Notably, the values of (Cs−1 )max have a little change when the amount of AP increases from 0.3-0.7 nmol. Meanwhile, alkaline phosphatase induces the surface pressure corresponding to the (Cs−1 )max value of monolayers decreased. 3.2. The interfacial mixing protein/lipid ratio Mass conservation of protein in the system: n0P = r · nL + nsP
(2)
J. Wang, R. Sun / Colloids and Surfaces A: Physicochem. Eng. Aspects 489 (2016) 136–141
6
*
B
A 0.8
*
ΔΑ = 1.5 Å
4
0.6
3
1
*
ΔΑ = 3 Å
2
0.4
0
15 nmol SM 18 nmol SM 20 nmol SM 22 nmol SM
2
2
* 2 ΔΑ = 2.5 Å
np (nmol)
2
ΔΑ (Å /molecule)
5
1.0
139
0.2
0 0.0
0.2
0.4
0.6
0.8
0.0 14
1.0
16
18
np (nmol)
1.0
C
*
*
ΔΑ = 1 Å
np (nmol)
3 2
0
15 nmol SM 18 nmol SM 20 nmol SM 22 nmol SM
1 0 0.0
0.2
0.4
22
D
0.8
2
ΔΑ (Å /molecule)
4
20
nL (nmol)
0
0.6
0.8
2
*
ΔΑ = 1.5 Å
0.6
2
* 2 ΔΑ = 2 Å
0.4 0.2
1.0
0.0
15
18
0
21
nL (nmol)
np (nmol)
Fig. 4. Data processing at 5 mN/m (A, B) and 7.8 mN/m(C, D). The measured area increase per SM molecule versus total amount of added alkaline phosphatase for different amounts of SM in the monolayer (A, C). And mass conservation plots at constant A∗ (B, D).
L
L
22 nmol, respectively, have been run to confirm the quality of the mass conservation plots. Then at the same level of A∗ , the n0P values for the individual nL must be subject to constancy regarding the interfacial protein/lipid ratio, r, and the amount that was partitioned into the subphase, nsP . The n0P − nL linear relation stands for the mass conservation plots according to Eq.(2). So, the interfacial mixing ratio, r, and the amount of protein partitioned into the subphase,nsP , can be calculated according to the n0P − nL linear relation. According to the surface pressure-area per molecule isotherms, the values of A are negative at the given surface pressure from 7.8 mN/m to 30 mN/m, which suggests that alkaline phosphatase in the interface carry a portion of SM molecules into the subphase, and that is to say, some amounts of SM in the interface are lost. At the air–water interface, the nL value changes to be uncertain. So, the phase transition of M2 is a false point, which is caused by the redistribution of the rest of SM molecules at the interface under the continued compression. It also shows that the presence of AP only induces the phase transition of the SM monolayer from the liquid expanded state to the coexist state of liquid expanded and liquid
0.06
5 mN/m 7.8 mN/m 0.04
r
where n0P , nsP stand for the total and subphase amounts of protein, respectively; nL is the amount of lipid; and r = nP ⁄nL denotes the interfacial mixing ratio of protein–lipid interaction [17,18]. For a given pressure the increase in per molecular area, A, under equilibrium conditions were determined from various series of surface pressure-area per molecule isotherms. Here we have to take into account only differences in the area, so the effect of a thin plate of filter paper can be ignored. For a given amount of SM in the monolayer,nL , we have plotted A∗ − n0P curves, where A∗ = A⁄n . Four groups of experiments with n =15, 18, 20 and
0.02
1.2
1.6 *
2.0
2.4
2
ΔΑ (Å /molecule) Fig. 5. The interfacial mixing radio of alkaline phosphatase/SM molecules.
condensed. The emergence of coexistence phase state reflects the interaction of the protein and the SM monolayer. the monolayer is in the liqAnd from the analysis of C−1 s uid expanded state at 5 mN/m and in the coexist state of liquid expanded and liquid condensed at 7.8 mN/m. So, in order to investigate the interfacial protein/lipid mixing radio at the two state, we only calculate the relevant data at the surface pressure 5 mN/m and 7.8m N/m (Fig. 4). The A∗ − n0p curves becaome increasingly bent in the downward direction in Fig. 4A and C. For a given amount of total protein, A∗ is decreasing with the increase of SM molecules. The linear
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J. Wang, R. Sun / Colloids and Surfaces A: Physicochem. Eng. Aspects 489 (2016) 136–141
Fig. 6. The SEM and AFM images of the SM monolayer in the presence of 0.3nmol AP at 5 mN/m (A, C) and 7.8 mN/m(B, D)(nL = 20nmol).
mass conservation plots in Fig. 4B and D distinctly reveal a substantial portion of alkaline phosphatase in the subphase under the air–water interface. For a given amount of SM in the interface, A∗ increases as the increase of amount of total protein. The interfacial mixing ratio of protein/lipid, r, has been calculated by mass conservation plots at 5 mN/m (Fig. 4B) and 7.8 mN/m (Fig. 4D). And the function of r with A∗ has been shown in Fig. 5. The r values increase with the increase of A∗ . At 5 mN/m, it is lower than that at 7.8 mN/m for a certain value of A∗ , which suggests that the interfacial mixing ratio at the liquid expanded state is higher than that of the coexist state of liquid expanded and liquid condensed. A greater of r suggests fewer amounts of SM corresponding to an alkaline phosphatase molecule.
decreases with the increase of amount of alkaline phosphatase. The appearance of coexist state is due to the interaction between alkaline phosphatase and SM monolayer. The interfacial mixing ratio in the coexist state is greater than that in liquid expanded state, which suggests that more alkaline phosphatase molecule combine with the SM monolayer in the coexist state. The greater the amount of alkaline phosphatase, the easier its combination with membrane. In some disease of bone, liver and gallbladder system, the content of alkaline phosphatase is often high. The high content of alkaline phosphatase has a pronounced effect on the phase transition of membrane. The microstructure images from SEM and AFM provide some helpful evidence of the phase transition. The emergence of the coexist state of liquid expanded and liquid condensed may have a special influence on the membrane in cells.
3.3. SEM and AFM Acknowledgement In SEM images, the liquid expanded state of SM monolayers at 5 mN/m is shown a shape like “a series of ditches” (arrow regions in Fig. 6). At 7.8 mN/m, a shape of “cotton flocculent” (triangular regions in Fig. 6B), which suggests a liquid condensed state, also appears on the SM monolayers, except for the ditch shape. The images of AFM also show a liquid expanded state at 5 mN/m, and a coexist state of liquid expanded and liquid condensed (arrow and triangular regions in Fig. 6D, respectively) at 7.8 mN/m. 4. Conclusion Alkaline phosphatase has an influence on phase transition of the SM monolayer mainly under low pressure. The greater the amount of alkaline phosphatase, the longer the process of phase transition from liquid expanded state to the coexist state of liquid expanded and liquid condensed. At the point of phase transition, that is, in the coexist state of the monolayer, the surface compressional modulus
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