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Hooking horseradish peroxidase by using the affinity Langmuir-Blodgett technique for an oriented immobilization
Accepted Manuscript Title: Hooking Horseradish Peroxidase by using the affinity Langmuir-Blodgett technique for an oriented immobilization Author: Ye Peng Hu Ling-Ling Du Yu-Zhi Xu Yong-Juan Ni Hua-Gang Chen Cong Lu Xiao-Lin Huang Xiao-Jun PII: DOI: Reference:
S0169-4332(16)32699-X http://dx.doi.org/doi:10.1016/j.apsusc.2016.11.237 APSUSC 34542
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APSUSC
Received date: Revised date: Accepted date:
25-8-2016 30-10-2016 30-11-2016
Please cite this article as: Ye Peng, Hu Ling-Ling, Du Yu-Zhi, Xu Yong-Juan, Ni HuaGang, Chen Cong, Lu Xiao-Lin, Huang Xiao-Jun, Hooking Horseradish Peroxidase by using the affinity Langmuir-Blodgett technique for an oriented immobilization, Applied Surface Science http://dx.doi.org/10.1016/j.apsusc.2016.11.237 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Hooking Horseradish Peroxidase by using the affinity Langmuir-Blodgett technique for an oriented immobilization
Ye Peng,*,† Hu Ling-Ling,† Du Yu-Zhi,† Xu Yong-Juan,† Ni Hua-Gang,† Chen Cong,‡ Lu Xiao-Lin, § and Huang Xiao-Jun,£
†
Department of Chemistry, Key Laboratory of Advanced Textile Materials and
Manufacturing Technology of Education Ministry, Zhejiang Sci-Tech University, Hangzhou 310018, China ‡
Biosciences Group, Transportation Research Institute, University of Michigan, Ann
Arbor 48109, Michigan, USA §State
Key Laboratory of Bioelectronics, School of Biological Science and Medical
Engineering, Southeast University, Nanjing 210096, China £
Key Laboratory of Macromolecular Synthesis and Functionalization (Ministry of
Education), Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China
*Corresponding Author E-mail : [email protected]
Graphical abstract
1
Hightlight
A novel method of oriented immobilization was presented: affinity Langmuir-Blodgett (LB) technique.
The specific activity of HRP immobilized by affinity LB (182.1 ± 14 U/mg) was higher than that by adsorption (40.5 ± 5 U/mg).
This method is easy to handle, and could be universally applied to the oriented immobilization of various types of enzymes and antibodies.
Abstract: A novel method of oriented immobilization was presented: affinity Langmuir-Blodgett (LB) technique. Firstly, a long carbon chain was bond to a ligand of Horseradish Peroxidase (HRP). The ligand derivative appears surface activity with the hydrophobic carbon chain oriented to air and the hydrophilic ligand faced to water. Then, this derivative was put onto the water/air surface to assemble a LB film and formed the affinity interaction with the active site of HRP. After that, the affinity LB film with the enzyme was transferred onto the support to obtain the oriented immobilized HRP. The specific activity of HRP immobilized by affinity LB (182.1 ± 2
14 U/mg) was higher than that by adsorption (40.5 ± 5 U/mg). HRP immobilized by affinity LB could maintain a more native conformation, compared to that by adsorption. This method could be effectively used to immobilize protein with orientation and show widely promising applications in many fields including biosensor and bioreactor.
1 Introduction The interest has grown greatly in controlling the orientation of protein, such as enzyme and antibody, immobilized on solid surface to optimize the interactions with the solid surface, as well as with the corresponding target molecules in solution.1-4 Some successful procedures have been used for oriented immobilization of protein, based on protein modification to introduce a specific affinity motif that subsequently binds to a functionalized surface.5 This method needs a elaborate knowledge of the modification position introduced in the protein and that of the distribution of the binding motifs on the functionalized surface. Concanavalin A (Con A) can conjugate via strong biospecific affinity with the sugar residues of protein, so it has been extensively investigated for the oriented immobilization of protein.6-8 However, due to its relatively high cost, the application is limited. Langmuir-Blodgett (LB) technology can effectively construct a single molecular layer on the molecular level.9-12 It has also been employed for oriented immobilization of enzymes13. Since the hydrophobic regions of bovine testicular hyaluronidase are located in the opposite side of its active center, the hydrophobic moiety can interact with the LB film, resulting in a preferential orientation of the enzyme, exposing its active center to solution.14 But this method applies to only a few enzymes. To overcome these drawbacks, we presented a novel method: affinity Langmuir-Blodgett (as Figure 1). Firstly, a long carbon chain was bond to a ligand of enzyme, which has a similar molecular structure with the enzyme substrate, forms affinity interaction with the active site of the enzyme and shows strong water solubility. The ligand derivative presents surface activity, which can assemble on water/air surface, with the long carbon chain oriented to air and the ligand faced to water. Then, this derivative was put onto water surface to assemble a LB film and formed affinity interaction with the active site of the enzyme. After that, the affinity
3
LB film with the enzyme was transferred onto the support to obtain the oriented immobilized enzyme, with the active site faced to air. At last, the immobilized enzyme was submerged into a substrate solution, and the substrate could readily replace the ligand derivative. For this method, it is a key point to choose a suitable ligand. In pharmacology and biochemistry, a ligand is usually a small molecule, which forms a complex with a biomolecule to serve a biological purpose.15-20 Ligands include substrates, inhibitors, activators and neurotransmitters. Horseradish Peroxidase (HRP) is an important enzyme in biochemistry in virtue of its ability to amplify a weak signal.21-25 In our work, this enzyme was employed as model enzyme to immobilize by affinity LB. Normally, a substrate oxidized by HRP using H2O2 as the oxidizing agent, and produces a characteristic variation that is measurable by spectrophotometric techniques.26 In fact, hydrogen peroxide is the real substrate, the substrate is electron donor. The affinity Langmuir-Blodgett technique could be effectively to realize the oriented immobilization of enzyme and might be applied to other biomolecules, such as antibody. This technique shows widely promising applications in many fields including biosensor and bioreactor.
2 Materials and methods 2.1 Materials. Horseradish Peroxidase (HRP) and Bovine serum albumin (BSA) were purchased from Sigma Company. All the other chemicals were of analytical grade and used without further purification. Pure water (purified with a Milli-Q purification system (Millipore Corp., USA) to a resistivity of 18 MΩ·cm) was used in this experiment. 2.2 Experiments methods 2.2.1 Synthesis of stearyl DOPA ester Firstly, to synthesize stearyl DOPA (3,4-dihydroxyphenylalanine) ester, L-DOPA (5.5 mmol) and octadecanol (13.9 mmol) was solved in a 122 oC oil bath and then added methanesulfonic acid (13.9 mmol), according to Penney’s method.27 2.2.2 Oriented immobilization of HRP To fabricate affinity Langmuir-Blodgett (LB) film, stearyl DOPA ester was mixed
4
with 1-Hexadecanol in molar ratio 1:8, then the mixture was solved (1mM) in chloroform and spread on a phosphate buffer (25 mM, pH 7.4, prepared by potassium dihydrogen phosphate and disodium hydrogen phosphate) in a Langmuir-Blodgett trough (Microthough X, Kibron Instruments, Finland). The surface pressure was evaluated with a Wilhelmy balance at the air/water interface. 50 μl HRP solution (1 mg/ml, phosphate buffer, 25 mM, pH 7.4) was inserted onto the subphase interface, after stearyl DOPA ester solution was spread onto the subphase interface for 10 minute. The pressure-area (π-A) isotherm was measured at a barrier speed of 15 cm2/minute, when the interaction equilibrium between the monolayer and HRP was established (1 h). By emerging polystyrene, which was previously immersed in the solution, perpendicularly to the interface at the dipping speed of 3 mm minute-1, the monolayer was transfered onto polystyrene at the surface pressure of 30 mN m-1. Finally, the support was immersed into substrate solution (10 mL 20 mM o-phenylenediamine and 0.2 mL 30% wt hydrogen peroxide) to obtain the oriented-immobilized enzyme. HRP was also immobilized on the same support by adsorption. The polystyrene support (20 × 20 mm) was submerged into 10 mL HRP solution (0.15 mg/mL, phosphate buffer, 25 mM, pH 7.4) and shaken gently for 1 hour. Then, the support was taken out, and thoroughly rinsed with the phosphate buffer (25 mM, pH 7.4). The sum frequency generation (SFG) vibrational spectra were recorded with a custom-designed EKSPLA SFG spectrometer that has been reported in previous papers.28-30 The incident angles of the IR beam and the visible beam were 53° and 60°, respectively. Both the IR and visible beams were focused on the sample surface with a pulse width of 30 ps and a repetition rate of 50 Hz with diameters of 0.5 mm. The SFG spectra as a function of the input infrared frequency were normalized by the input visible and infrared lasers beams. The SFG spectra with an ssp (an s-polarized sum frequency output, an s-polarized visible input, and a p-polarized infrared input) polarization combination were obtained at the air/solution interface. During the SFG experiments, the surface pressure was maintained at 30 mN/m. 2.2.3 Characterization of immobilized HRP 5
The amount of immobilized protein was measured with fluorescence spectroscopy (Perkin Elmer, PE, LS 45), by using HRP labelled with fluorescein isothiocyanate (FITC).31 The corresponding fluorescence emission intensity (515 nm) of different concentration of HRP-FITC was measured for the construction of calibration curve. The labelling of the enzyme was transferred to carrier and eluted by surfactant solution (0.5 % SDS /5 mM NaOH). The quantity of immobilized HRP was characterized by the difference between initial concentration of HRP-FITC and amount of surfactant solution. The molecular structure of immobilized protein was analyzed with Fourier transform infrared spectroscopy (FT-IR, Nicolet Avatar370). To determine the enzyme activity, enzyme was inserted in a media containing 10 mL 20 mM o-phenylenediamine and 0.4 mL 30wt % hydrogen peroxide at 30 oC. The absorbance was monitored after 15 min at 450 nm.32 XPS experiments were performed on a PHI-5000C ESCA system (PerkinElmer) with Al Kα radiation (hν = 1486.6 eV).
3 Results and discussion 3.1 Oriented immobilization of HPR
L-DOPA (L-3,4-dihydroxyphenylalanine) is produced from L-tyrosine by tyrosine hydroxylase. L-DOPA is a derivative of phenol, which is a classic substrate of Peroxidase. This molecule could occupy the active site of HRP with affinity interaction.33-35 And the oxidation of L-DOPA by HRP is very slow at the absence of hydrogen peroxide as the oxidizing agent. Meanwhile, L-DOPA contain carboxyl group, which can easily react with other molecule to bind a long carbon chain. Figure 2 shows the effect of the enzyme concentration on the production of 2,3-diaminophenazine (DAP). It could be found that there is an obviously decrease of the production of DAP in the presence of L-DOPA (Figure 2b), which could be attributed to the competitive binding of L-DOPA to the active site of HRP. A similar result could be found for stearyl DOPA ester (Figure 2c), which indicate stearyl DOPA ester could also occupy the active site of HPR with affinity interaction. Thus, 6
L-DOPA was chosen as the ligand of HRP in this work. Surface pressure-area isotherms of stearyl DOPA ester/1-Hexadecanol, stearyl DOPA ester/1-Hexadecanol/HRP and 1-Hexadecanol is shown in Figure 3. It could be found that for all of these isotherms, the monolayers adpoted several distinct phases as the area per lipid molecule decreased and the lateral pressure increased: the gas, liquid-expanded, liquid-condensed and solid phases. At high pressure, the monolayer surfaces were rippled; upon further compression, the monolayers underwent collapses.36 The Surface pressure-area isotherms are the significant indicators of monolayer properties. To keep constant surface pressure is necessary to fabricate uniform LB films during deposition. 37,38 Figure 4 shows the SFG spectra of the Langmuir film at the air/water interface. For the Langmuir film of stearyl DOPA ester/1-Hexadecanol after inserting HRP solution, in the spectral region from 1600 to 1760 cm−1, the vibrational bands at 1655 and 1735 cm−1 were found and could be attributed to amide I and to carboxylic ester vibrations, respectively.28,29 The amide I band originates from R-C=O carbonyl vibrations of molecular groups in protein backbone.39 The carboxylic ester vibration comes from stearyl DOPA ester. While for Langmuir film of stearyl DOPA ester/1-Hexadecanol, there was only the vibrational bands at 1735 cm−1 which comes from stearyl DOPA ester. For the Langmuir film of 1-Hexadecanol after inserting HRP solution, the vibrational band at 1655 cm−1 was observed and could be attributed to amide I, which comes from HRP backbone. For the Langmuir film of stearyl DOPA ester/1-Hexadecanol, there was not any vibrational band. After inserting protein, the vibrational band at 1655 cm−1 which comes from the amide I of HRP for the Langmuir film of stearyl DOPA ester/1-Hexadecanol was stronger than that of 1-Hexadecanol, indicating the amount of protein under the Langmuir film of stearyl DOPA ester/1-Hexadecanol was larger than that under the Langmuir film of 1-Hexadecanol . This could be ascribed to the formation of the affinity interaction between stearyl DOPA ester and the active site of HRP. 3.2 Activity and Conformation of HRP 7
After enzyme immobilization, there always is a decrease of enzyme activity . This is owing to the limitations imposed by slow mass transfer of substrate or product, the presence of random immobilization and the modification in the enzyme three-dimension structure.40-44 As shown in Table 1, it could be found that the specific activity of HRP by affinity LB (182.1 ± 14 U/mg) was higher than that by adsorption (40.5 ± 5 U/mg). These results could be due to the fact that the active site of HRP immobilized by affinity LB oriented to the substrate solution, which was favorable to maintain enzyme activity, while the active site of HRP immobilized by adsorption was random and easily formed interaction with the support to decrease its activity. The amount of immobilized protein by affinity LB (517 ± 26 ng/cm2) was lower than that by adsorption (602 ± 32 ng/cm2). The reason for this might be the multilayer adsorption by adsorption. Additionally, for the affinity LB method, there existed adsorbed protein on the support surface, which adsorbed when the support was submerged into the solution, and the amount of the adsorbed protein was 46 ± 8 ng/cm2. The kinetic parameters (Km and Vmax) of the free and immobilized HRP are listed in Table 1. Compared to the free HRP, there were noticeable increases in Km value for the immobilized HRPs. Km depends on diffusional effects and indicates the enzyme characteristics.45 The increase in Km values is owing to the modification of enzyme conformation and the increased diffusion limitation. The Km value for the immobilized HRP by adsorption was higher than that by affinity LB. Thus, it was easier for the immobilized HRP by affinity LB to form the substrate-enzyme complex, compared to that by adsorption. Vmax is defined as the highest possible reaction velocity when the enzyme is saturated with its substrate. In Table 1, it could be found that after immobilization Vmax values demonstrated a decrease. The Vmax value of the immobilized HRP by adsorption was lower than that by affinity LB, which corresponded to the specific activity of the immobilized HRPs, and this result could be explained by the same reason. Figure 5 shows the XPS spectra of the PS supports. In the case of the PS supports
8
after the enzyme immobilization (Figure 5a, b), there were three peaks at 536, 404 and 289 eV, corresponding to O1S, N1S and C1S. The increased intensities of O1S and N1S peak and the decreased intensity of C1S peak were found upon the introduction of protein on the support surfaces. For the support after the enzyme immobilization by affinity LB, the intensities of O1S and N1S peak were relatively small, which indicated that there was a smaller amount of protein immobilized on the support surface and was in accord with the amount of immobilized protein measured by fluorescence spectroscopy. ATR-Infrared spectroscopy is an effective method to investigate the conformation of adsorbed protein. The amide I band at 1700-1600 cm-1 is mainly owing to C=O stretching vibrations, which is sensitive to the modifications in protein secondary structure. Figure 6 shows the results of curve fitting for the IR spectra of the free and immobilized HRPs. In Figure 6A five bands of the free HRP are identified at 1620, 1638, 1657, 1674 and 1690 cm-1, which are attributed to random coil structures, β-sheet structures, α-helical structures, and other structures.46,47 After HRP adsorption, the bands at 1657 cm-1 owing to the α-helix became weaker, while other bands owing to the β-sheet (1638 cm-1) and random coil (1620 cm-1) became much stronger (Figure 6B). These results indicated that the α-helicals of HRP were lost due to the interaction between HRP and the support surfaces, with the increases of the β-sheet and random coil structures. It could also be found that the decrease of the α-helical structures and the increase of the β-sheets and random coil structures of the immobilized HRP by affinity LB were smaller than that by adsorption. This phenomenon illustrated that HRP immobilized by affinity LB could maintain a more native conformation, compared to that by adsorption.
4 Conclusion Bio-affinity interaction is a highly specific interaction between biological substances, such as that between enzyme and substrate, or receptor and ligand. Based on this interaction, some motheds of separating biochemical mixtures have been developed and applied, such as affinity chromatography. 9
In our study, to control the orientation of immobilized enzyme, a novel method was presented: affinity Langmuir-Blodgett (LB). A ligand derivative of the enzyme was synthesized by bind a long carbon chain to a ligand. The ligand derivative can forms affinity interaction with the active site of the enzyme and appears surface activity to assemble on the water/air surface. The specific activity of HRP immobilized by affinity LB (182.1 ± 14 U/mg) was higher than that by adsorption (40.5 ± 5 U/mg). HRP immobilized by affinity LB could maintain a more native conformation, compared to that by adsorption. These results demonstrated that the novel affinity LB was an effective method to control the orientation of the immobilized enzyme and obtain higher enzyme activity. This method could be effectively to realize the oriented immobilization of enzyme and might be applied to other biomolecules, such as antibody
AUTHOR INFORMATION Corresponding Author *E-mail : [email protected] Author Contributions The manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest
Acknowledgements. The work was funded by the National Natural Science Foundation of China (NSFC, Nos. 51473148, 51003097, 50703034, 51173169 and 31200746), the Natural Science Foundation of Zhejiang Province (No. Y407275). 10
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Figure 1. The schematic diagram of the oriented immobilization of Horseradish Peroxidase (HRP) by affinity LB method. (A, to form a monolayer of surfactant on the water/air surface; B, to form the affinity interaction between the ligand derivative and the active site of the enzyme; C, to deposite the affinity LB film with the enzyme onto the support; D, to obtain the oriented immobilized enzyme; E, to replace the ligand derivative with the substrate of the enzyme)
Figure 2. Effect of the enzyme concentration on the production of DAP, without ligand (a), in the presence of L-DOPA (b) and stearyl DOPA ester (c).
16
Figure 3. Surface pressure-area isotherms of stearyl DOPA ester/1-Hexadecanol (a), stearyl DOPA ester/1-Hexadecanol/HRP (b) and 1-Hexadecanol (c).
Figure 4. SFG spectra of the Langmuir film at the air/water interface. (a) stearyl DOPA ester/1-Hexadecanol after inserting HRP solution; (b), stearyl DOPA ester/1-Hexadecanol; (c), 1-Hexadecanol after inserting HRP solution ; (d) 1-Hexadecanol.
Figure 5. the XPS survey scan spectra of the PS supports (a, after the enzyme immobilization by 17
affinity LB; b, after the enzyme immobilization by adsorption; c, nascent PS support).
Figure 6. FT-IR spectra of HRPs in the amide I region with the respective best fitted individual component bands for the free (A) and immobilized HRPs by adsorption (B) and by affinity-LB (C), respectively. Solid and dotted lines indicate the experimental data and individual Gaussian components, respectively.
18
Table 1. Activity and kinetic parameters of the free and immobilized HRPs Sample
Free
Adsorption
Affinity LB
Specific activity (U/mg)
2250.0 ± 55
40.5 ± 5
182.1 ± 14
Absorbed amount (ng/cm2） -
602 ± 32
517 ± 26
Vmax (U/mg)
2389.0
44.3
192.7
Km (mM)
1.15
2.63
1.48
19
S0169-4332(16)32699-X http://dx.doi.org/doi:10.1016/j.apsusc.2016.11.237 APSUSC 34542
To appear in:
APSUSC
Received date: Revised date: Accepted date:
25-8-2016 30-10-2016 30-11-2016
Please cite this article as: Ye Peng, Hu Ling-Ling, Du Yu-Zhi, Xu Yong-Juan, Ni HuaGang, Chen Cong, Lu Xiao-Lin, Huang Xiao-Jun, Hooking Horseradish Peroxidase by using the affinity Langmuir-Blodgett technique for an oriented immobilization, Applied Surface Science http://dx.doi.org/10.1016/j.apsusc.2016.11.237 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Hooking Horseradish Peroxidase by using the affinity Langmuir-Blodgett technique for an oriented immobilization
Ye Peng,*,† Hu Ling-Ling,† Du Yu-Zhi,† Xu Yong-Juan,† Ni Hua-Gang,† Chen Cong,‡ Lu Xiao-Lin, § and Huang Xiao-Jun,£
†
Department of Chemistry, Key Laboratory of Advanced Textile Materials and
Manufacturing Technology of Education Ministry, Zhejiang Sci-Tech University, Hangzhou 310018, China ‡
Biosciences Group, Transportation Research Institute, University of Michigan, Ann
Arbor 48109, Michigan, USA §State
Key Laboratory of Bioelectronics, School of Biological Science and Medical
Engineering, Southeast University, Nanjing 210096, China £
Key Laboratory of Macromolecular Synthesis and Functionalization (Ministry of
Education), Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China
*Corresponding Author E-mail : [email protected]
Graphical abstract
1
Hightlight
A novel method of oriented immobilization was presented: affinity Langmuir-Blodgett (LB) technique.
The specific activity of HRP immobilized by affinity LB (182.1 ± 14 U/mg) was higher than that by adsorption (40.5 ± 5 U/mg).
This method is easy to handle, and could be universally applied to the oriented immobilization of various types of enzymes and antibodies.
Abstract: A novel method of oriented immobilization was presented: affinity Langmuir-Blodgett (LB) technique. Firstly, a long carbon chain was bond to a ligand of Horseradish Peroxidase (HRP). The ligand derivative appears surface activity with the hydrophobic carbon chain oriented to air and the hydrophilic ligand faced to water. Then, this derivative was put onto the water/air surface to assemble a LB film and formed the affinity interaction with the active site of HRP. After that, the affinity LB film with the enzyme was transferred onto the support to obtain the oriented immobilized HRP. The specific activity of HRP immobilized by affinity LB (182.1 ± 2
14 U/mg) was higher than that by adsorption (40.5 ± 5 U/mg). HRP immobilized by affinity LB could maintain a more native conformation, compared to that by adsorption. This method could be effectively used to immobilize protein with orientation and show widely promising applications in many fields including biosensor and bioreactor.
1 Introduction The interest has grown greatly in controlling the orientation of protein, such as enzyme and antibody, immobilized on solid surface to optimize the interactions with the solid surface, as well as with the corresponding target molecules in solution.1-4 Some successful procedures have been used for oriented immobilization of protein, based on protein modification to introduce a specific affinity motif that subsequently binds to a functionalized surface.5 This method needs a elaborate knowledge of the modification position introduced in the protein and that of the distribution of the binding motifs on the functionalized surface. Concanavalin A (Con A) can conjugate via strong biospecific affinity with the sugar residues of protein, so it has been extensively investigated for the oriented immobilization of protein.6-8 However, due to its relatively high cost, the application is limited. Langmuir-Blodgett (LB) technology can effectively construct a single molecular layer on the molecular level.9-12 It has also been employed for oriented immobilization of enzymes13. Since the hydrophobic regions of bovine testicular hyaluronidase are located in the opposite side of its active center, the hydrophobic moiety can interact with the LB film, resulting in a preferential orientation of the enzyme, exposing its active center to solution.14 But this method applies to only a few enzymes. To overcome these drawbacks, we presented a novel method: affinity Langmuir-Blodgett (as Figure 1). Firstly, a long carbon chain was bond to a ligand of enzyme, which has a similar molecular structure with the enzyme substrate, forms affinity interaction with the active site of the enzyme and shows strong water solubility. The ligand derivative presents surface activity, which can assemble on water/air surface, with the long carbon chain oriented to air and the ligand faced to water. Then, this derivative was put onto water surface to assemble a LB film and formed affinity interaction with the active site of the enzyme. After that, the affinity
3
LB film with the enzyme was transferred onto the support to obtain the oriented immobilized enzyme, with the active site faced to air. At last, the immobilized enzyme was submerged into a substrate solution, and the substrate could readily replace the ligand derivative. For this method, it is a key point to choose a suitable ligand. In pharmacology and biochemistry, a ligand is usually a small molecule, which forms a complex with a biomolecule to serve a biological purpose.15-20 Ligands include substrates, inhibitors, activators and neurotransmitters. Horseradish Peroxidase (HRP) is an important enzyme in biochemistry in virtue of its ability to amplify a weak signal.21-25 In our work, this enzyme was employed as model enzyme to immobilize by affinity LB. Normally, a substrate oxidized by HRP using H2O2 as the oxidizing agent, and produces a characteristic variation that is measurable by spectrophotometric techniques.26 In fact, hydrogen peroxide is the real substrate, the substrate is electron donor. The affinity Langmuir-Blodgett technique could be effectively to realize the oriented immobilization of enzyme and might be applied to other biomolecules, such as antibody. This technique shows widely promising applications in many fields including biosensor and bioreactor.
2 Materials and methods 2.1 Materials. Horseradish Peroxidase (HRP) and Bovine serum albumin (BSA) were purchased from Sigma Company. All the other chemicals were of analytical grade and used without further purification. Pure water (purified with a Milli-Q purification system (Millipore Corp., USA) to a resistivity of 18 MΩ·cm) was used in this experiment. 2.2 Experiments methods 2.2.1 Synthesis of stearyl DOPA ester Firstly, to synthesize stearyl DOPA (3,4-dihydroxyphenylalanine) ester, L-DOPA (5.5 mmol) and octadecanol (13.9 mmol) was solved in a 122 oC oil bath and then added methanesulfonic acid (13.9 mmol), according to Penney’s method.27 2.2.2 Oriented immobilization of HRP To fabricate affinity Langmuir-Blodgett (LB) film, stearyl DOPA ester was mixed
4
with 1-Hexadecanol in molar ratio 1:8, then the mixture was solved (1mM) in chloroform and spread on a phosphate buffer (25 mM, pH 7.4, prepared by potassium dihydrogen phosphate and disodium hydrogen phosphate) in a Langmuir-Blodgett trough (Microthough X, Kibron Instruments, Finland). The surface pressure was evaluated with a Wilhelmy balance at the air/water interface. 50 μl HRP solution (1 mg/ml, phosphate buffer, 25 mM, pH 7.4) was inserted onto the subphase interface, after stearyl DOPA ester solution was spread onto the subphase interface for 10 minute. The pressure-area (π-A) isotherm was measured at a barrier speed of 15 cm2/minute, when the interaction equilibrium between the monolayer and HRP was established (1 h). By emerging polystyrene, which was previously immersed in the solution, perpendicularly to the interface at the dipping speed of 3 mm minute-1, the monolayer was transfered onto polystyrene at the surface pressure of 30 mN m-1. Finally, the support was immersed into substrate solution (10 mL 20 mM o-phenylenediamine and 0.2 mL 30% wt hydrogen peroxide) to obtain the oriented-immobilized enzyme. HRP was also immobilized on the same support by adsorption. The polystyrene support (20 × 20 mm) was submerged into 10 mL HRP solution (0.15 mg/mL, phosphate buffer, 25 mM, pH 7.4) and shaken gently for 1 hour. Then, the support was taken out, and thoroughly rinsed with the phosphate buffer (25 mM, pH 7.4). The sum frequency generation (SFG) vibrational spectra were recorded with a custom-designed EKSPLA SFG spectrometer that has been reported in previous papers.28-30 The incident angles of the IR beam and the visible beam were 53° and 60°, respectively. Both the IR and visible beams were focused on the sample surface with a pulse width of 30 ps and a repetition rate of 50 Hz with diameters of 0.5 mm. The SFG spectra as a function of the input infrared frequency were normalized by the input visible and infrared lasers beams. The SFG spectra with an ssp (an s-polarized sum frequency output, an s-polarized visible input, and a p-polarized infrared input) polarization combination were obtained at the air/solution interface. During the SFG experiments, the surface pressure was maintained at 30 mN/m. 2.2.3 Characterization of immobilized HRP 5
The amount of immobilized protein was measured with fluorescence spectroscopy (Perkin Elmer, PE, LS 45), by using HRP labelled with fluorescein isothiocyanate (FITC).31 The corresponding fluorescence emission intensity (515 nm) of different concentration of HRP-FITC was measured for the construction of calibration curve. The labelling of the enzyme was transferred to carrier and eluted by surfactant solution (0.5 % SDS /5 mM NaOH). The quantity of immobilized HRP was characterized by the difference between initial concentration of HRP-FITC and amount of surfactant solution. The molecular structure of immobilized protein was analyzed with Fourier transform infrared spectroscopy (FT-IR, Nicolet Avatar370). To determine the enzyme activity, enzyme was inserted in a media containing 10 mL 20 mM o-phenylenediamine and 0.4 mL 30wt % hydrogen peroxide at 30 oC. The absorbance was monitored after 15 min at 450 nm.32 XPS experiments were performed on a PHI-5000C ESCA system (PerkinElmer) with Al Kα radiation (hν = 1486.6 eV).
3 Results and discussion 3.1 Oriented immobilization of HPR
L-DOPA (L-3,4-dihydroxyphenylalanine) is produced from L-tyrosine by tyrosine hydroxylase. L-DOPA is a derivative of phenol, which is a classic substrate of Peroxidase. This molecule could occupy the active site of HRP with affinity interaction.33-35 And the oxidation of L-DOPA by HRP is very slow at the absence of hydrogen peroxide as the oxidizing agent. Meanwhile, L-DOPA contain carboxyl group, which can easily react with other molecule to bind a long carbon chain. Figure 2 shows the effect of the enzyme concentration on the production of 2,3-diaminophenazine (DAP). It could be found that there is an obviously decrease of the production of DAP in the presence of L-DOPA (Figure 2b), which could be attributed to the competitive binding of L-DOPA to the active site of HRP. A similar result could be found for stearyl DOPA ester (Figure 2c), which indicate stearyl DOPA ester could also occupy the active site of HPR with affinity interaction. Thus, 6
L-DOPA was chosen as the ligand of HRP in this work. Surface pressure-area isotherms of stearyl DOPA ester/1-Hexadecanol, stearyl DOPA ester/1-Hexadecanol/HRP and 1-Hexadecanol is shown in Figure 3. It could be found that for all of these isotherms, the monolayers adpoted several distinct phases as the area per lipid molecule decreased and the lateral pressure increased: the gas, liquid-expanded, liquid-condensed and solid phases. At high pressure, the monolayer surfaces were rippled; upon further compression, the monolayers underwent collapses.36 The Surface pressure-area isotherms are the significant indicators of monolayer properties. To keep constant surface pressure is necessary to fabricate uniform LB films during deposition. 37,38 Figure 4 shows the SFG spectra of the Langmuir film at the air/water interface. For the Langmuir film of stearyl DOPA ester/1-Hexadecanol after inserting HRP solution, in the spectral region from 1600 to 1760 cm−1, the vibrational bands at 1655 and 1735 cm−1 were found and could be attributed to amide I and to carboxylic ester vibrations, respectively.28,29 The amide I band originates from R-C=O carbonyl vibrations of molecular groups in protein backbone.39 The carboxylic ester vibration comes from stearyl DOPA ester. While for Langmuir film of stearyl DOPA ester/1-Hexadecanol, there was only the vibrational bands at 1735 cm−1 which comes from stearyl DOPA ester. For the Langmuir film of 1-Hexadecanol after inserting HRP solution, the vibrational band at 1655 cm−1 was observed and could be attributed to amide I, which comes from HRP backbone. For the Langmuir film of stearyl DOPA ester/1-Hexadecanol, there was not any vibrational band. After inserting protein, the vibrational band at 1655 cm−1 which comes from the amide I of HRP for the Langmuir film of stearyl DOPA ester/1-Hexadecanol was stronger than that of 1-Hexadecanol, indicating the amount of protein under the Langmuir film of stearyl DOPA ester/1-Hexadecanol was larger than that under the Langmuir film of 1-Hexadecanol . This could be ascribed to the formation of the affinity interaction between stearyl DOPA ester and the active site of HRP. 3.2 Activity and Conformation of HRP 7
After enzyme immobilization, there always is a decrease of enzyme activity . This is owing to the limitations imposed by slow mass transfer of substrate or product, the presence of random immobilization and the modification in the enzyme three-dimension structure.40-44 As shown in Table 1, it could be found that the specific activity of HRP by affinity LB (182.1 ± 14 U/mg) was higher than that by adsorption (40.5 ± 5 U/mg). These results could be due to the fact that the active site of HRP immobilized by affinity LB oriented to the substrate solution, which was favorable to maintain enzyme activity, while the active site of HRP immobilized by adsorption was random and easily formed interaction with the support to decrease its activity. The amount of immobilized protein by affinity LB (517 ± 26 ng/cm2) was lower than that by adsorption (602 ± 32 ng/cm2). The reason for this might be the multilayer adsorption by adsorption. Additionally, for the affinity LB method, there existed adsorbed protein on the support surface, which adsorbed when the support was submerged into the solution, and the amount of the adsorbed protein was 46 ± 8 ng/cm2. The kinetic parameters (Km and Vmax) of the free and immobilized HRP are listed in Table 1. Compared to the free HRP, there were noticeable increases in Km value for the immobilized HRPs. Km depends on diffusional effects and indicates the enzyme characteristics.45 The increase in Km values is owing to the modification of enzyme conformation and the increased diffusion limitation. The Km value for the immobilized HRP by adsorption was higher than that by affinity LB. Thus, it was easier for the immobilized HRP by affinity LB to form the substrate-enzyme complex, compared to that by adsorption. Vmax is defined as the highest possible reaction velocity when the enzyme is saturated with its substrate. In Table 1, it could be found that after immobilization Vmax values demonstrated a decrease. The Vmax value of the immobilized HRP by adsorption was lower than that by affinity LB, which corresponded to the specific activity of the immobilized HRPs, and this result could be explained by the same reason. Figure 5 shows the XPS spectra of the PS supports. In the case of the PS supports
8
after the enzyme immobilization (Figure 5a, b), there were three peaks at 536, 404 and 289 eV, corresponding to O1S, N1S and C1S. The increased intensities of O1S and N1S peak and the decreased intensity of C1S peak were found upon the introduction of protein on the support surfaces. For the support after the enzyme immobilization by affinity LB, the intensities of O1S and N1S peak were relatively small, which indicated that there was a smaller amount of protein immobilized on the support surface and was in accord with the amount of immobilized protein measured by fluorescence spectroscopy. ATR-Infrared spectroscopy is an effective method to investigate the conformation of adsorbed protein. The amide I band at 1700-1600 cm-1 is mainly owing to C=O stretching vibrations, which is sensitive to the modifications in protein secondary structure. Figure 6 shows the results of curve fitting for the IR spectra of the free and immobilized HRPs. In Figure 6A five bands of the free HRP are identified at 1620, 1638, 1657, 1674 and 1690 cm-1, which are attributed to random coil structures, β-sheet structures, α-helical structures, and other structures.46,47 After HRP adsorption, the bands at 1657 cm-1 owing to the α-helix became weaker, while other bands owing to the β-sheet (1638 cm-1) and random coil (1620 cm-1) became much stronger (Figure 6B). These results indicated that the α-helicals of HRP were lost due to the interaction between HRP and the support surfaces, with the increases of the β-sheet and random coil structures. It could also be found that the decrease of the α-helical structures and the increase of the β-sheets and random coil structures of the immobilized HRP by affinity LB were smaller than that by adsorption. This phenomenon illustrated that HRP immobilized by affinity LB could maintain a more native conformation, compared to that by adsorption.
4 Conclusion Bio-affinity interaction is a highly specific interaction between biological substances, such as that between enzyme and substrate, or receptor and ligand. Based on this interaction, some motheds of separating biochemical mixtures have been developed and applied, such as affinity chromatography. 9
In our study, to control the orientation of immobilized enzyme, a novel method was presented: affinity Langmuir-Blodgett (LB). A ligand derivative of the enzyme was synthesized by bind a long carbon chain to a ligand. The ligand derivative can forms affinity interaction with the active site of the enzyme and appears surface activity to assemble on the water/air surface. The specific activity of HRP immobilized by affinity LB (182.1 ± 14 U/mg) was higher than that by adsorption (40.5 ± 5 U/mg). HRP immobilized by affinity LB could maintain a more native conformation, compared to that by adsorption. These results demonstrated that the novel affinity LB was an effective method to control the orientation of the immobilized enzyme and obtain higher enzyme activity. This method could be effectively to realize the oriented immobilization of enzyme and might be applied to other biomolecules, such as antibody
AUTHOR INFORMATION Corresponding Author *E-mail : [email protected] Author Contributions The manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest
Acknowledgements. The work was funded by the National Natural Science Foundation of China (NSFC, Nos. 51473148, 51003097, 50703034, 51173169 and 31200746), the Natural Science Foundation of Zhejiang Province (No. Y407275). 10
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Figure 1. The schematic diagram of the oriented immobilization of Horseradish Peroxidase (HRP) by affinity LB method. (A, to form a monolayer of surfactant on the water/air surface; B, to form the affinity interaction between the ligand derivative and the active site of the enzyme; C, to deposite the affinity LB film with the enzyme onto the support; D, to obtain the oriented immobilized enzyme; E, to replace the ligand derivative with the substrate of the enzyme)
Figure 2. Effect of the enzyme concentration on the production of DAP, without ligand (a), in the presence of L-DOPA (b) and stearyl DOPA ester (c).
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Figure 3. Surface pressure-area isotherms of stearyl DOPA ester/1-Hexadecanol (a), stearyl DOPA ester/1-Hexadecanol/HRP (b) and 1-Hexadecanol (c).
Figure 4. SFG spectra of the Langmuir film at the air/water interface. (a) stearyl DOPA ester/1-Hexadecanol after inserting HRP solution; (b), stearyl DOPA ester/1-Hexadecanol; (c), 1-Hexadecanol after inserting HRP solution ; (d) 1-Hexadecanol.
Figure 5. the XPS survey scan spectra of the PS supports (a, after the enzyme immobilization by 17
affinity LB; b, after the enzyme immobilization by adsorption; c, nascent PS support).
Figure 6. FT-IR spectra of HRPs in the amide I region with the respective best fitted individual component bands for the free (A) and immobilized HRPs by adsorption (B) and by affinity-LB (C), respectively. Solid and dotted lines indicate the experimental data and individual Gaussian components, respectively.
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Table 1. Activity and kinetic parameters of the free and immobilized HRPs Sample
Free
Adsorption
Affinity LB
Specific activity (U/mg)
2250.0 ± 55
40.5 ± 5
182.1 ± 14
Absorbed amount (ng/cm2） -
602 ± 32
517 ± 26
Vmax (U/mg)
2389.0
44.3
192.7
Km (mM)
1.15
2.63
1.48
19