A further insight into the adsorption mechanism of protein on hydroxyapatite by FTIR-ATR spectrometry Zhongyu Lin, Ren Hu, Jianzhang Zhou, Yiwen Ye, Zhaoxi Xu, Changjian Lin PII: DOI: Reference:
S1386-1425(16)30575-3 doi: 10.1016/j.saa.2016.09.050 SAA 14696
To appear in: Received date: Revised date: Accepted date:
4 March 2016 24 August 2016 26 September 2016
Please cite this article as: Zhongyu Lin, Ren Hu, Jianzhang Zhou, Yiwen Ye, Zhaoxi Xu, Changjian Lin, A further insight into the adsorption mechanism of protein on hydroxyapatite by FTIR-ATR spectrometry, (2016), doi: 10.1016/j.saa.2016.09.050
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ACCEPTED MANUSCRIPT A further insight into the adsorption mechanism of protein on hydroxyapatite by FTIR-ATR spectrometry
Changjian Lin a
Zhaoxi Xu b,
State Key Laboratory of Physical Chemistry of Solid Surfaces and Department of
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a
Yiwen Ye a,
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Ren Hu a, Jianzhang Zhou a,
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Zhongyu Lin a, *,
Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, PR China
Xiamen A ER TE System Engineering CO., LTD, Xiamen 361005, PR China
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b
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[email protected]
Abstract: The adsorption mechanism of bovine serum albumin (BSA) on hydroxyapatite (HA) for different time intervals has been studied by Fourier transform
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infrared (FTIR)-attenuated total internal reflectance (ATR) spectrometry in this paper.
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The difference spectra obtained in HA and BSA frequency regions demonstrate that
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the binding of P=O, from the phosphate (PO43-) of HA, to the hydrogen of methyl (–CH3), methene (–CH2) and amideⅡ (–CNH) in the protein appears to be much faster and stronger than that of the P–O group. In addition, Ca2+ must serve as a key
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role in the interaction of BSA with HA. The binding of Ca2+ to the oxygen of the peptide bond seems to induce a significant reconformation of polypeptide backbones from β-pleated sheet to α-helix and β-turn of helical circles. This alteration seems to have been accompanied by much hydrogen of polypeptides driven to bind PO43- and OH- of the HA actively and much –C=O and H–N– groups of the peptide bond freed from inter-chain hydrogen bonding to react on Ca2+ and combine strongly with the HA surface. This might be well expected to promote the HA biomineralization. Key words: BSA, HA, Protein adsorption, Biomineralization, FTIR-ATR, IR spectroscopy
1. Introduction Protein adsorption presents strong and weak bindings. The strong binding is 1
ACCEPTED MANUSCRIPT believed to produce a significant effect on biological processes and, the first step, on directing the response of biological systems to implanted materials. For better understanding of protein-surface reactions, the emphasis has been laid on determining
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the conformation of adsorbed proteins [1, 2] and on investigating the interactive
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process of proteins with biomaterials, especially with the active bioceramics – HA
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[3–6]. Still, the adsorption mechanism involving the true features of adsorbed proteins is in dispute and the additional data are needed to reason the molecular mechanisms active in the reaction of proteins on HA.
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We herein present, based on our previous study [6], a further on-the-spot investigation into the microcosmic process of BSA adsorbing onto the HA for
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different time intervals using FTIR-ATR technique. Water has a strong O–H absorption near 1643 cm-1 which overlaps to a certain extent the frequency range of
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the protein amide Ⅰ band, so the heavy water (D2O) has been used instead of the
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aqueous solution in this experiment. The FTIR-ATR provides a direct method for getting relevant information on the interaction of the protein with HA. Here, it should
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be noted that the thickness of the HA coating (near 20 – 40 nm thick) is much thinner than the depth of penetration of the IR evanescent wave (about 400 – 600 nm over the region of interest) and the reflected light can transmit further into the BSA solution.
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Thus, the recorded spectrum reflects a mixed absorption of adsorbed and non-adsorbed protein [7]. An IR spectral subtraction method [1, 7, 8] must be used to remove the spectral feature of non-adsorbed protein from the mixed absorption spectrum to obtain the information on really adsorbed protein. This method has provided reliable and reproducible results [9].
2. Materials and Methods 2.1 Main reagent BSA (0903, 99.0 %) was purchased from Shanghai Bio-Engineering Ltd. Inc. 2.2 Preparation of samples The preparation of germanium (Ge) ATR crystal covered with a HA coating was performed according to the reported method [3, 6]. The Ge crystal with dimensions of 2
ACCEPTED MANUSCRIPT 50 mm × 10 mm × 3 mm was immersed in a boiling saturated Ca(OH)2 solution for 40 min and then in a 20% H3PO4 solution adjusted to pH 2.0 – 2.4 at 90°C for 30 min, followed by 2 rinses with deionized water. Next, the HA coated on the two slopes (IR
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light pass through) of the crystal was carefully cleaned off by using silk moistened with thin hydrochloric acid, and then the bare slopes were cautiously rinsed twice
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with deionized water. After that this crystal was dried at room temperature prior to examination. The BSA solution was prepared, just before the IR determination, by dissolving the BSA powder in a 0.9% NaCl heavy water solution with its final
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concentration of 40 mg / ml. 2.3 FTIR-ATR spectrometry
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2.3.1 IR data collection
A Nicolet 740SX FTIR spectrophotometer (USA) with a MCT-B detector and
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ATR accessories was used for FTIR-ATR data collection. The IR spectrum of HA
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coated on Ge crystal was recorded just before that exposed to BSA solution. (The IR absorption of the HA coating on Ge crystal for 48 h was the same as that for 0 h just
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after the sample had been prepared). After 1 ml of the fresh BSA solution was injected into the cell with the HA-coated-Ge-crystal, IR data were collected every min from 0 to 10 min, thereafter for intervals of 15 min, 20 min, 30 min, 60 min, 5 h, 24 h and 30
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h. The spectra were recorded in the range 4000 – 625 cm-1 with a resolution of 4 cm-1, GAN 1 and 128 scans at room temperature less than 4℃. (This temperature could prevent the bacterial contamination during the interaction of BSA with HA for more than 48 h; the IR absorptions of the BSA dissolved in a NaCl heavy water solution for 24 and 48 h were each the same as that for 0 min. The IR absorptions of HA and BSA could each remain unchanged up to 48 h, so they most probably wouldn’t reveal any changes alone within the above-mentioned, much shorter time intervals). That the chemical properties of HA and BSA could each continue to be stable within the required time frames is the essential prerequisite for carrying out the on-the-spot investigation of the adsorption mechanism of BSA on HA by FTIR-ATR spectrometry. The time dependence experiment was performed in triplicate for quality control and statistical purposes. 3
ACCEPTED MANUSCRIPT 2.3.2 Spectra processing The recorded spectra were the mixed absorptions of adsorbed and non-adsorbed HA and BSA each, so the IR spectral subtraction method was used by modifying the
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subtraction factor (FCR) to remove the absorptions of non-adsorbed HA and BSA from the spectra of mixed absorptions respectively. For observation of the bound PO
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(P=O, P–O) absorptions, the subtraction of the blank HA spectrum was made from each of the spectra of HA reacted with BSA for 0 min, 30 min, 60 min, 5 h and 30 h in the HA frequency region 1170 – 900 cm-1 (Figure 3, curves 1–5); the subtraction
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factors were each adjusted to: 1) 0.8110 (0 min - HA), 2) 0.8653 (30 min - HA), 3) 0.8827 (60 min - HA), 4) 0.9233 (5 h - HA) and 5) 0.9802 (30 h - HA). For
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observation of the adsorbed BSA on HA, the subtraction of the spectrum of BSA reacted with HA for 0 min was made from each of those for 1 min, 30 min, 60 min, 5
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h and 24 h in the BSA absorption range 1750 – 1350 cm-1 (Figure 4, curves 1–5); the
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factors were: 1) 0.8610 (1 min - 0 min), 2) 0.8827 (30 min - 0 min), 3) 0.9900 (60min - 0 min), 4) 0.9950 (5 h - 0 min) and 5) 1.0000 (24 h - 0 min) respectively. And for
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better observation of the conformational changes of protein secondary structure after BSA reacted with HA for 30 h, the subtractions of the spectra of BSA reacted with HA for 0, 1, 2, 3, 5, 10 and 15 min were each made from that for 30 h in the range 1700 –
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1580 cm-1 (Figure 5, curves 1–7); the factors were: 1) 1.0000 (30 h - 0 min), 2) 0.9900 (30 h - 1 min), 3) 0.9802 (30 h - 2 min), 4) 0.9656 (30 h - 3 min), 5) 0.9466 (30 h - 5 min), 6) 0.9050 (30 h - 10 min) and 7) 0.8827 (30 h - 15 min) respectively. The result spectra were each smoothed by using the SMF command and choosing the parameter 13.
3. Results and discussions 3.1 Hydroxyapatite and that binding BSA 3.1.1 HA coated on Ge crystal The FTIR-ATR spectra of HA [Ca10(OH)2(PO4)6] coated on Ge crystal and that in contact with BSA for 0 min have been recorded respectively (Figure 1, curves 1 and 2). The main, characteristic absorptions of the PO43- group in the range 1100 – 4
ACCEPTED MANUSCRIPT 1000 cm-1 of curve 1 are roughly the same as that examined on the transmission [6]. This indicates that the HA really has been coated on the Ge crystal. Thus, there appear to be Ca2+, PO43- and OH- ions in the coating. The absorptions at 1098 and 1026 cm-1
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are ascribed to P=O and P–O stretching bands of the PO43- group respectively. The
1600
1400
962
1026
1094
962
1098
2
1200
1
1000
-1
Wavenumber (cm )
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1800
1208
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HA
1656
0 min
1547
1652
Absorbance
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obscuring of the amide Ⅰ absorption region by HA.
1026
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1656 cm-1 due to the O–H absorption of HA is a weak band, thereby avoiding the
Figure 1. FTIR-ATR spectra of 1) HA coated on Ge crystal and 2) that reacted with
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BSA for 0 min.
Figure 1, curve 2 shows HA reacted with BSA for 0 min; in fact, the absorption here occurred after 128 scans taking time of 64 second. Thus, it is an accumulated result of HA challenged with BSA from the first to the 64th second. There appear to be the absorptions of amide Ⅰ (1652 cm-1), amide Ⅱ (1547 cm-1), methene (1458 cm-1) and methyl (1446 cm-1) of the protein [10–12]; they are the mixed absorptions of adsorbed and non-adsorbed BSA on HA surface. Also mixed absorptions of bound and unbound PO (P=O and P–O) bands are each displayed at 1094 and 1026 cm-1. A mere red-shift of 4 cm-1 of the P=O band (from 1098 to 1094 cm-1) and no red-shift of the P–O (1026 cm-1) indicate only a small part of the PO groups participating in the bindings, and the red-shift regions of the bound PO bands each hidden completely from view by a respective bulk of unbound PO absorptions. The 1208 cm-1 band arises from the 5
ACCEPTED MANUSCRIPT O–D absorption of the heavy water. 3.1.2 Dissolution and re-crystallization of some HA ions The FTIR-ATR spectra of HA coating and that reacted with BSA for 5 min, 20
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min, 24 h and 30 h have been recorded respectively (Figure 2, curves 1–5). There is a
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distinct fall in the intensity of the P–O absorption at 1026 cm-1 within the first 5 min
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(curve 2) compared with the blank HA coating. It seems that the ion exchanges between BSA (NaCl/D2O) solution and HA coating occurred simultaneously with the adsorption of protein on the HA at the beginning. Na+ and Cl- ions of the solution
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might be incorporated/precipitated into the HA coating [13, 14] to liberate some HA ions (PO43-, Ca2+ and OH-) into the BSA solution, in which the protein could easily
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adsorb/incorporate the dissolved PO43-, Ca2+ and OH - ions, resulting in further release of these ions from the HA coating. Notably, the position of the PO43- dissolved
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in the solution here must have been beyond the penetration depth of the reflected light
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so that the intensities of PO absorptions decreased. The PO43- group contains three P–O and one P=O bonds (blank HA shows the intensity of the P–O absorption to be
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much stronger than that of the P=O band). Thus, the P–O band at 1026 cm-1 was reduced in intensity much more markedly than the P=O absorption near 1093 cm-1 when the PO43- group dissolved from the coating into the BSA solution within the
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initial 5 min.
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Absorbance
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1093
1026
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5
24 h
4
20 min
1
1170
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3 2
30 h
1080
5 min HA
990
900 -1
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Wavenumber (cm )
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Figure 2. FTIR-ATR spectra of 1) HA coating and that reacted with BSA for 2) 5 min,
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3) 20 min, 4) 24 h and 5) 30 h.
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Ong et al. [13] reported that the saline solution containing protein (albumin) liberated much more PO43- from Ca-P coating than the blank saline solution. Obviously, the BSA adsorption plays a key role in the dissolution of the HA coating [13, 15].
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Following the BSA binding to the HA coating surface, the adsorption of the protein for the dissolved HA ions from the solution occurred, with the result that those PO43-, OH- and Ca2+ ions quickly crystallized on the HA coating. Thus, the PO intensities got stronger (Figure 2, curves 3–5). The HA ions which were adsorbed onto and covered the protein surface, then, continued binding the BSA from the solution. The initial dissolution and re-crystallization of PO43-, OH-and Ca2+ [3, 16, 17] have caused the formation of the coating including the adsorbed BSA on HA from surfaceto subsurface-molecular layers [3], resulting in the interaction of BSA with HA in depth. 3.1.3 Binding of HA (PO43-) to BSA The IR spectra of the bound PO absorptions obtained by subtracting the blank HA spectrum from each of the spectra of HA reacted with BSA for intervals of 0 min, 7
ACCEPTED MANUSCRIPT 30 min, 60 min, 5 h and 30 h are shown in Figure 3, curves 1–5 respectively. Owing to removals of the respective unbound PO absorptions, the difference spectra (subtraction results) each exhibit the absorptions of really bound PO bands clearly.
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Two red-shifts of the P=O absorption from 1098 to 1090 and to 1064 cm-1 and a
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red-shift of the P–O from 1026 to 1017 cm-1 occurred after in contact with BSA for 0
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min (via 64 seconds’ scan). The obvious red-shifts of the PO bands within such a short time reflect that the interaction of HA with BSA is a chemical adsorption and a
1170
NU 962 962 962
60 min-HA
30 min-HA
962
1017
1012
1080
5 h-HA
962
1010
1010
1008
1064
1064 1064
1089
1064
1089
1089
1
30 h-HA
0 min-HA
990
900 -1
Wavenumber (cm )
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2
1064
3
1090
D
4
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Absorbance
5
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1089
rapid process as well.
Figure 3. FTIR-ATR difference spectra of HA reacted with BSA for different intervals:
1) 0 min - HA, 2) 30 min - HA, 3) 60 min - HA, 4) 5 h - HA and 5) 30 h - HA. The intensification of the bound P=O bands at 1089 and 1064 cm-1(curves 2–5) is more notable than that of the bound P–O at 1012 cm-1 (curve 2), 1010 cm-1 (curves 3 and 4) and 1008 cm-1 (curve 5). This indicates that the interaction of the P=O with protein seems to be considerably faster and stronger than that of the P–O group. The above-mentioned red-shifts must be due to the binding of PO bonds each to the hydrogen of –CH3, –CH2 and –CNH groups in the protein. Based on the PO bonding characteristic (the P–O is of σ-bond and the P=O has a π-bond besides the σ-bond; the π-bond is of higher chemical activity than the σ-bond), the P=O with the π-bond could 8
ACCEPTED MANUSCRIPT bind the hydrogen of –CH and –CNH more easily than the P–O group. This is in agreement with the findings of the IR experiment. The action of the P=O on protein presents both strong (1064 cm-1) and weak (1089 cm-1) bindings of the HA to BSA,
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the former appears more rapid than the latter. 3.2 Adsorption of BSA on HA
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The IR spectra of the adsorbed BSA on HA obtained by subtracting the spectrum of BSA reacted with HA for 0 min from each of those for 1 min, 30 min, 60 min, 5 h and 24 h are displayed in Figure 4, curves 1–5 respectively. Curve 1 presents the
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absorption at 1651 cm-1 due to the amide Ⅰ band (C=O absorption), suggesting that this band seems to contain the best part of the α-helix absorption centred at near 1653
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cm-1 [8, 18, 19] and a small portion of the β-pleated sheet centred at about 1632 cm-1 as well [19]. The latter here has been completely hidden from view by the α-helix
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absorption [1, 7–9, 18, 19]. Bands of 1458 and 1446 cm-1 each arise from the C–H
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deformation modes of –CH2 and –CH3 in polypeptides respectively. From 1 to 60 min in D2O solution (curves 1–3), bands at 1538–1578 cm-1 each have still been in the
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amide Ⅱ region [10, 12, 19] (the absorptions would have shifted to a much lower frequency near 1450 cm-1 if hydrogen-deuterium exchange had occurred), indicative of reduced H–D exchange; this is because of the amide hydrogen buried or joining in
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the strong hydrogen bonding [12, 19]. Noticeably, there appear to be blue-shifts of 1 – 2 cm-1 at each of 1578, 1568 and 1548 cm-1 of amide Ⅱ bands in Figure 4, curve 2 compared with the curve 1. The absorptions each result from a strongly-coupled vibration involving C–N stretching and N–H bending modes of the –C–N–H group [10, 11, 19]. The blue-shifts show that the bonds of C–N and N–H groups have strengthened. Similar blue-shifts of 4 – 6 cm-1 of amide Ⅱ bands (and only 1 cm-1 shift of the amide Ⅰ band) have been found in earlier reports [19, 20], which pointed out changes in much hydrogen bonding of protein, particularly in the β-pleated sheet regions due to the action of metal ions. It now seems clear that parts of the inter-chain hydrogen bonding of the β-pleated sheet changed in this system. Almost all –N–H and O=C– groups of the β-pleated sheet have joined in the inter-chain hydrogen bonding [21]. In this case, the strengthening 9
ACCEPTED MANUSCRIPT of both C–N and N–H bonds is reasonable to infer an increase in quantity of the unbound –N–H group. This suggests that a rupture of the inter-chain hydrogen bonds linking adjacent polypeptide backbones of the β-pleated sheet and the formation of
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helical circles of intra-chain hydrogen bonding between –N–H and O=C– groups of
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each 5 → 1 and/or 4 → 1 of peptide units must have occurred by the inductive effect
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of Ca2+ [21]. Thus, much of the –C=O group was freed from the inter-chain hydrogen bonding. Ca2+ could bind the carbonyl-oxygen quite easily and induce the change of the protein secondary structure to the α-helix version [22–27]. Furthermore, the
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regularity of the α-helical conformation can lead to the cooperativity in the course of helical folding. Once the first α-helical circle forms in the system, it will become
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easier and speedier that the following amino-acid residues keep joining in one after another [21]. In view of these reasons, the blue-shifts of amide Ⅱ bands (curve 2) are
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believed to reflect a reconformation of parts of peptide backbones from β-pleated
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sheet to α-helix in this system. Under the circumstances, the amide Ⅰ band should have been a blue-shift due to the unbound carbonyl on the increase; on the contrary, a
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red-shift from 1651 to 1644 cm-1 occurred in this absorption. Since the carbonyl-oxygen of the peptide bond could easily combine with Ca2+ [18, 28], perhaps the red-shift of the amide Ⅰ band had better be primarily associated with the
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coordination of Ca2+ with the unbound carbonyl-oxygen of the α-helix [1, 29]. The Ca/P ratio on the blank HA surface was 1.25 determined by XPS [3], showing the existence of a high Ca2+ content on the coating surface. Thus, more of the unbound carbonyl-oxygen could be bound by Ca2+. In this case, the frequency of the amide Ⅰ band appears to depend on two factors. One is the conformational change from β-pleated sheet to helical circles, in which case much of the carbonyl-oxygen freed from the hydrogen bonding could strengthen the C=O bond and result in a blue-shift of this band. The other is the likelihood of the C=O bond weakened by the complexation of Ca2+ with the oxygen. Of the two effects the latter must be stronger than the former, so the net result is a weakening of the C=O bond and a red-shift of this band. That the previous reports [30, 31] each showed a blue-shift of the amide Ⅰ band was in the case of lower metal-ion content, which had only a smaller influence 10
ACCEPTED MANUSCRIPT on the C=O bond while binding. Thus, the frequency shift of the amide Ⅰ band depended only on the conformation-change of protein. In the case of a high metal-ion content, the effect of that binding on the C=O bond must be taken into account when
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the molecular conformations of the adsorbed protein are defined. The band at 1644
1600
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1700
1500
1400
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Wavenumber (cm )
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1454 1446
1587 1572
1640
1575
24 h-0 min
5 5 h-0 min
4
1700
1600
1500
1400 -1
-1
Figure 4.
1666
30 min-0 min
1 min-0 min
1
1637
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60 min-0 min
MA 1458 1446
2
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1452 1443 1446
1573 1562 1538 1577 1567 1546
3
1578 1568 1548
1651
Absorbance
1644
1643
unbound and bound by Ca2+ on the HA surface.
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cm-1 (curve 2) most probably reflects a mixed absorption, in the α-helix, of the C=O
Wavenumber (cm )
FTIR-ATR difference spectra of BSA reacted with HA for different intervals:
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1) 1 min - 0 min, 2) 30 min - 0 min, 3) 60 min - 0 min, 4) 5 h - 0 min and 5) 24 h - 0 min.
Figure 4, curve 3 displays the IR spectrum of BSA reacted with HA for 60 min. A red-shift of the amide Ⅰ band occurred and especially of amide Ⅱ absorptions each compared with the curve 2, showing a further interaction of the BSA with HA. Figure 4, curve 4 presents the amide Ⅱ band centred on one band at 1575 cm-1 after in contact with HA for 5 h. The clear blue-shift of the amide Ⅱ band (compared with the curve 3) indicates that some hydrogen bonding of the β-pleated sheet continued being broken and shaped into the α-helical version (this is similar to the case of curve 2). The IR spectrum of BSA reacted with HA for 24 h (Figure 4, curve 5) shows a
11
ACCEPTED MANUSCRIPT new amide Ⅰ band at 1666 cm-1 most likely due to the absorption of β-turn [8, 19, 32], also a helical circle, characterized largely by the regular arrays of each 4 → 1 of intra-chain hydrogen bonding [21]; perhaps it also contains the contribution of a small
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part of the –C=O linked with Ca2+. The 1587 cm-1 absorption is assigned to the O)2ˉ stretching band of the carboxylate anion (–COO - ) from
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asymmetric C(
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side-chains and C-ends of peptide backbones [10, 12, 33–35]. This suggests that Ca2+ most probably attracted the –COO - group onto the HA surface via a strong electrostatic attraction and they both could be held together by the ionic bond [12, 19,
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33, 34]. The reaction of the carboxylate anion with Ca2+ is easier than that of the amide Ⅰ. Thus, it stands to reason that most of the –COO- group could be well
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coordinated with Ca2+ at the moment [22, 23, 35]. A blue-shift from 1587 to near 1605 cm-1 of the asymmetric stretching band of the complex –COO- should have occurred
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[33, 35]. As it was only in small quantity [34, 36], it therefore was covered over by
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the strong absorption of the amide Ⅰ [19]. The amide Ⅰ band in Figure 4 (curves 1–5) each could much probably involve the contribution of the complex –COO-. A
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red-shift of the amide Ⅰ band from 1643 cm-1 (1 h) to 1640 cm-1 (5 h) to 1637 cm-1 (24 h) and an intensification of the C=O absorption have both occurred with time prolonged; there seems to be more and more carbonyl-oxygen of the α-helix
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participating in the complexation with Ca2+. The intensities of CH3 (1443–1446 cm-1), CH2 (1452–1458 cm-1) and amide Ⅱ (1538–1578 cm-1) absorptions (curves 1–5) also increased with time, particularly of the CH2 absorption (curve 5, 1454 cm-1). It appears much more hydrogen of the protein adsorbing close against the HA surface. During the course, amide Ⅱ bands have continued in the normal range; a reduced rate of deuteration in the Ca2+-loaded state implies the formation of a more compact structure [37]. The findings make it clear that the BSA has a strong binding ability for HA and could combine strongly with the HA surface. For better revealing the adsorbed protein in its true colors, the changes of C=O absorptions for different intervals from 0, 1, 2, 3, 5, 10 and 15 min each to 30 h have been investigated. Removals of the spectra of BSA reacted with HA for 0, 1, 2, 3, 5, 10 and 15 min were each made from that for 30 h (Figure 5, curves 1–7). Three amide 12
ACCEPTED MANUSCRIPT Ⅰ bands are presented in Figure 5. Both frequencies of 1668 cm-1 (from much of the unbound C=O absorption in β-turn) and 1639 cm-1 (from the absorption of much bound C=O by Ca2+ in α-helix) remained unchanged, while the new band at 1649 cm-1
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has altered with the different intervals. It appears almost all the Ca2+ of the coating
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surface has joined in the coordination with both –COO- and –C=O groups and the
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reaction has achieved equilibrium at the moment, whereas the α-helix seems to continue being shaped. Chittur noted that in soluble proteins the α-helical band occurs at lower wave numbers, approximately 1650–1655 cm-1 [8]. With highly solvent,
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exposed helices in D2O and in the presence of Ca2+ for 24 h, the amide Ⅰ band (of
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CE P
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Absorbance
1668
D
1649 1639
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parvalbumin) can shift to a frequency as low as 1646–1647 cm-1 [37].
Figure 5.
30h-15min 30h-10min
7
30h-5min 30h-3min 30h-2min 30h-1min 30h-0min
6 5 4 3 2 1
1680
1640
1600 -1
Wavenumber (cm )
FTIR-ATR difference spectra of BSA reacted with HA for 30 h: 1) 30 h - 0 min, 2) 30 h - 1 min, 3) 30 h - 2 min, 4) 30 h - 3 min, 5) 30 h - 5 min, 6) 30 h - 10 min and 7) 30 h - 15 min.
The 1649 cm-1 absorption could therefore be well associated with much of the unbound C=O in α-helix. It presents a growing trend and a blue-shift of 1 – 4 cm-1 (curves 1–7), from 1645 cm-1 (curves 1–4) to 1646 cm-1 (curve 5), to 1648 cm-1 (curve 6) and to 1649 cm-1 (curve 7), mirroring the occurrence of the α-helix of polypeptide backbones clearly. This affords further evidence that the adsorbed BSA has a gain of α-helix (and a small amount of β-turn) and a loss of β-pleated sheet under the 13
ACCEPTED MANUSCRIPT inductive effect of Ca2+. 3.3 The interaction of protein with HA promoted by α-helix There are evidences that a marked reduction in the intensities of both amide Ⅰ
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band (1636 cm-1) and amide Ⅱ band (1545 cm-1) of the adsorbed immunoglobulin (IgG) on TiO2 film occurred after the phosphate buffer saline was added; besides, the
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former shifted from 1636 cm-1 to 1651 cm-1 [38]. It appears the amide Ⅰ band is largely due to the mixed absorption of a great part of the β-pleated sheet and a small portion of the α-helix component. Following the phosphate buffer saline added, P=O
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and P–O groups of that succeeded in replacing the most part of the weakly bound β-pleated sheet of the IgG; only the α-helix component of the amide Ⅰ band at 1651
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cm-1 remained on the TiO2 surface. The result suggests that the α-helix has a strong binding ability and the protein with the α-helical conformation could adsorb strongly
D
onto the biomaterial surface.
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Almost all of the peptide bond (–C=O and H–N– groups) of the β-pleated sheet participates in the hydrogen bonding; for α-helix and β-turn, only 20% and 25% of
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those groups each have shares in the hydrogen bonding. The conformational change from pleated sheet to helical circles is therefore believed to free much –C=O and H–N– groups of the peptide bond from the hydrogen bonding for easily bound as well
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as to alter the steric positions of side chains, peptide bond and hydrogen of polypeptides for more accessible by the target surfaces [39]. This could lead to a higher-affinity interaction of the protein with HA. Moreover, the α-helix periodicity (pitch) is similar to the 0.545 nm interatomic distance of the hexagonally disposed Ca2+ in the plane (001) of the HA lattice [40]. The highly significant, conformational feature of the adsorbed protein may favor the governing of its interaction with HA [22, 41, 42]. Apparently, the periodic spacing of the 0.54 nm pitch of the α-helix with the regularly repeated, unbound peptide bond and the carboxylate anionic side chains could be highly complementary to such lattice spacing of Ca2+ in HA. The reasons appear to explain the strong binding of the protein to HA surface very well. This intimate association of the protein with HA is important in mediating the fundamental reactions of cellular living tissue to the HA interface [43, 44] and in modulating the 14
ACCEPTED MANUSCRIPT cellular interactions, which play an important role in hard tissue regeneration [45–48].
4. Conclusions
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The findings of the FTIR-ATR spectrometry on BSA adsorbing onto HA reveal
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that the peptide protein of BSA seems to be activated by the inductive effect of Ca2+
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via the molecular re-conformation from β-pleated sheet to helical circles of peptide backbones and in turn reacts strongly on HA, resulting in a profound impact on the
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biomineralization of HA.
Acknowledgement
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The research was supported by the National Natural Science Foundation of China (Grant No. 51571169). The contributions of the research groups cited in this article
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and of the reviewers giving valuable advices for this manuscript are gratefully
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ACCEPTED MANUSCRIPT Figure Captions Figure 1. FTIR-ATR spectra of 1) HA coated on Ge crystal and 2) that reacted with BSA for 0 min.
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Figure 2. FTIR-ATR spectra of 1) HA coating and that reacted with BSA for 2) 5 min, 3) 20 min, 4) 24 h and 5) 30 h.
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Figure 3. FTIR-ATR difference spectra of HA reacted with BSA for different intervals: 1) 0 min - HA, 2) 30 min - HA, 3) 60 min - HA, 4) 5 h - HA and 5) 30 h HA.
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Figure 4. FTIR-ATR difference spectra of BSA reacted with HA for different intervals: 1) 1 min - 0 min, 2) 30 min - 0 min, 3) 60 min - 0 min, 4) 5 h - 0 min and 5)
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24 h - 0 min.
Figure 5. FTIR-ATR difference spectra of BSA reacted with HA for 30 h: 1) 30 h - 0
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min, 2) 30 h - 1 min, 3) 30 h - 2 min, 4) 30 h - 3 min, 5) 30 h - 5 min, 6) 30 h - 10 min
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and 7) 30 h - 15 min.
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30h-5min 30h-3min 30h-2min 30h-1min 30h-0min
6 5 4 3 2 1
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Absorbance
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Graphical Abstract
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Wavenumber (cm )
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Highlights
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The study of BSA adsorbing onto HA by FTIR-ATR spectrometry reveals that the peptide protein of BSA seems to be activated by the inductive effect of Ca2+ via the rearrangement of hydrogen bonding between –N–H and O=C– groups of the peptide backbone units from the conformation of β-pleated sheet to helical circles and in turn reacts strongly on HA, resulting in a profound impact on HA biomineralization.
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