# Spectroscopic Investigations of Pentobarbital Interaction with Transthyretin

Authors: Saqer M. Darwish; Jafar Ghithan; Musa M. Abuteir; Mariam Faroun; Mahmoud M. Abu-hadid
Journal: Journal of Spectroscopy
 (2013)

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## Abstract

Transthyretin (TTR) aggregation has been characterized to be responsible for several amyloid diseases. Fourier transform infrared (FTIR) spectroscopy, fluorescence, and atomic force microscopy (AFM) are used to investigate secondary structure changes in transthyretin, induced upon thermal denaturation and interaction with pentobarbital. Spectral analysis revealed a strong static quenching of the intrinsic fluorescence of TTR by pentobarbital with a binding constant (K) estimated at2.092×103M-1. Fourier self-deconvolution (FSD) technique is used to evaluates intensity changes in the spectra of the component bands in the amide I and amide II regions due to the changes in pentobarbital concentration in the protein complex. The increases of the relative intensities of the peaks at 1614 cm−1 and 1507 cm−1 are due to the increase of pentobarbital concentrations which is linked to the formation of oligomers in the protein.


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## Body

## 1. Introduction

Transthyretin (TTR) is a plasma protein composed of 127-residue subunits mainly composed ofβ-sheet structures [1]. It is present in both human plasma and cerebrospinal fluid (CSF) with concentrations of (0.1–0.4 mg/mL) in human plasma and (0.017 mg/mL) in CSF [2]. X-ray crystal structure studies have shown that human TTR have a molecular weight of 55 kDa in a tetramer form with four identical subunits [3].TTR is synthesized by the liver and released in the plasma, while the TTR in CSF is mostly produced by the choroid plexus [4–6]. It is considered to be the primary transporter of thyroid hormones in the form of thyroxine in the CSF and it carries retinol via interaction with the retinol-binding protein (RBP) [7].Other additional function of TTR has been detected in the development of the central nervous system due to the high concentration during the prenatal and postnatal life [8]. Several research groups have shown cerebral TTR expression to rise during the course of experimental Alzheimer disease (AD) in mice and in response to the intake of some drug or mixtures of compounds such as gingko extracts or dietary fatty acids [9–11].The process of transthyretin amyloidogenesis or amyloid fibril formation seems to be associated with some amyloid diseases. It is not understood precisely how TTR forms amyloids, but several biophysical studies on wild-type (WT) TTR reveals that tetramer dissociation is rate limiting for amyloidogenesis [12–14]. All amyloid diseases are characterized by misfolded proteins that undergo aggregation causing a deposition of insoluble amyloid fibrils either systemically or in specific organs as the brain [15–18]. It is believed that such disorders can lead to several diseases such as in familial amyloid polyneuropathy, Alzheimer, Parkinson’s, Huntington’s, and type II diabetes [15, 19–23]. Familial amyloid polyneurophy and senile systematic amyloidosis are two diseases associated with formation of TTR fibrils due to the lack of stability in parallel β-sheets. The precise mechanisms of TTR amyloid fibril formations at the molecular level are not known, Quintas et al. suggested that amyloid fibril formation might be triggered by tetramer dissociation to a compact nonnative monomer with low-conformational stability, which results in partially unfolded monomeric species with a high tendency for ordered aggregation into amyloid fibrils [24].Although understanding the mechanisms of protein aggregation is remained to be a very crucial issue, current research supports the notion that small toxic oligomers are formed during the aggregation results in cellular dysfunction and death [20, 21].Recent studies suggest that neurodegenerative disorders, such as Alzheimer’s and Parkinson’s diseases, might be accelerated by the intake of anesthetic drugs [25–28]. It is been suggested that some small ligands can bind strongly to wild-type TTR forming a stable folded state leading to an amoloid formation [29, 30]. The commonly used inhalation anesthetic isoflurane has been reported to induce apoptosis, which, in turn, increases aggregation of amyloid β-protein in cultured cells [28].Infrared (IR) spectroscopy is one of the oldest and well-established experimental techniques for the analysis of secondary structure of polypeptides and proteins [31–34]. FTIR spectroscopy revealed that certain vibration-frequency bands can be used to follow the aggregation and to explore environmental properties and temperature effects on the structure of proteins [24]. The presence of β-sheets in the core of amyloid fibrils focused the investigations to β-sheet containing proteins such as transthyretin to find if such proteins are predisposed to form such fibrils. However, any differences in the molecular structure of the protein can be detected from the changes in the amide I region of the infrared spectrum by measuring changes in the intensities of the absorption bands of β-sheets [34].In this work we investigated possible changes in the intensity of the component bands of the secondary structure by changing temperatures and concentrations of pentobarbital in the protein complexes. The binding of pentobarbital to TTR was investigated by means of Fluorescence and FTIR spectroscopy. In addition we used atomic force microscopy (AFM) to monitor physical changes of the molecular structure of the complex-protein [35, 36].


## 2. Materials and Methods

Pentobarbital in salt form with chemical structure shown in Figure1, and transthyretin in Lyophilized powder were purchased from Sigma Aldrich chemical company and used without further purification.Figure 1
Chemical structure of pentobarbital.


### 2.1. Preparation of Stock Solutions

TTR was dissolved in 99.9% D2O at a concentration of 5 mg/mL in 10 mM phosphate buffer saline at physiological pH 7.4. Pentobarbital with molecular weight of (226.3 g/moL) was dissolved in 99.9% D2O and 10 mM phosphate buffer saline to attain the desired drug concentration of 15.36 mM.In the final step drug solution was added to an equal volume of the protein solution to attain the desired pentobarbital concentrations of 7.68, 3.84, 1.92, and 0.96 mM with a final protein concentration of 2.5 mg/mL.


### 2.2. Fluorescence Spectroscopy

The fluorescence measurements were performed by a NanoDrop ND-3300 Fluorospectrometer at 25°C. The excitation source comes from one of three solid-state light emitting diodes (LED’s). The excitation source options include UV LED with maximum excitation 365 nm, Blue LED with excitation 470 nm, and white LED from 500 to 650 nm excitation. A 2048-element CCD array detector covering 400–750 nm is connected by an optical fiber to the optical measurement surface. The excitation is done on the wavelength of 360 nm and the maximum emission wavelength is at 423 nm.


### 2.3. FTIR Spectroscopic Measurements

FTIR measurements were obtained on a Bruker IFS 66/S spectrophotometer equipped with liquid nitrogen cooled MCT detector and a KBr beam splitter. The spectrometer was continuously purged with dry air during the measurements. Samples are prepared after 2 h of incubation of TTR with pentobarbital solution at room temperature, 50μL of the sample were placed on a certain area on a silicon window plate and left to dry at room temperature inside an incubator. The dehydrated films are made with desired concentrations of pentobarbital while keeping the same protein content.The absorption spectrum were recorded in mid-infrared range (4000–400 cm−1), as an average of 60 scans to increase the signal to noise ratio, and the spectral resolution was at 4 cm−1. The aperture used in this study was 8 mm, since we found that this aperture gives the best signal to noise ratio. Baseline correction and normalization were performed for all the spectra by OPUS software.For the temperature dependence studies, protein-pentobarbital complexes on silicon windows were placed into an infrared cell window. The temperature in the cell was controlled by an external water path and was increased gradually between 20°C and 80°C at 3°C per 10 minute scan rate.Peak positions were determined using the second derivative by OPUS software. Calculations of the areas under the different curves were performed using FSD techniques by OPUS software.


### 2.4. AFM Measurements

The prepared stock solutions of pentobarbital-TTR complex are incubated for 2 h at room temperature. A 10μL of the solution is placed on certain area of a freshly cleaved mica substrate and left to dry at room temperature. TTR free and TTR-pentobarbital solutions were characterized by AFM equipped with 4 × 4 μm2 piezoelectric scanner. Analysis were carried out using tapping mode at room temperature using AFM cantilevers with a spring constant 2 N/m (Micro cantilever, OLYMPUS). All scanned images were analyzed using WSxM 5.0 Develop 3.2 software.


## 2.1. Preparation of Stock Solutions

TTR was dissolved in 99.9% D2O at a concentration of 5 mg/mL in 10 mM phosphate buffer saline at physiological pH 7.4. Pentobarbital with molecular weight of (226.3 g/moL) was dissolved in 99.9% D2O and 10 mM phosphate buffer saline to attain the desired drug concentration of 15.36 mM.In the final step drug solution was added to an equal volume of the protein solution to attain the desired pentobarbital concentrations of 7.68, 3.84, 1.92, and 0.96 mM with a final protein concentration of 2.5 mg/mL.


## 2.2. Fluorescence Spectroscopy

The fluorescence measurements were performed by a NanoDrop ND-3300 Fluorospectrometer at 25°C. The excitation source comes from one of three solid-state light emitting diodes (LED’s). The excitation source options include UV LED with maximum excitation 365 nm, Blue LED with excitation 470 nm, and white LED from 500 to 650 nm excitation. A 2048-element CCD array detector covering 400–750 nm is connected by an optical fiber to the optical measurement surface. The excitation is done on the wavelength of 360 nm and the maximum emission wavelength is at 423 nm.


## 2.3. FTIR Spectroscopic Measurements

FTIR measurements were obtained on a Bruker IFS 66/S spectrophotometer equipped with liquid nitrogen cooled MCT detector and a KBr beam splitter. The spectrometer was continuously purged with dry air during the measurements. Samples are prepared after 2 h of incubation of TTR with pentobarbital solution at room temperature, 50μL of the sample were placed on a certain area on a silicon window plate and left to dry at room temperature inside an incubator. The dehydrated films are made with desired concentrations of pentobarbital while keeping the same protein content.The absorption spectrum were recorded in mid-infrared range (4000–400 cm−1), as an average of 60 scans to increase the signal to noise ratio, and the spectral resolution was at 4 cm−1. The aperture used in this study was 8 mm, since we found that this aperture gives the best signal to noise ratio. Baseline correction and normalization were performed for all the spectra by OPUS software.For the temperature dependence studies, protein-pentobarbital complexes on silicon windows were placed into an infrared cell window. The temperature in the cell was controlled by an external water path and was increased gradually between 20°C and 80°C at 3°C per 10 minute scan rate.Peak positions were determined using the second derivative by OPUS software. Calculations of the areas under the different curves were performed using FSD techniques by OPUS software.


## 2.4. AFM Measurements

The prepared stock solutions of pentobarbital-TTR complex are incubated for 2 h at room temperature. A 10μL of the solution is placed on certain area of a freshly cleaved mica substrate and left to dry at room temperature. TTR free and TTR-pentobarbital solutions were characterized by AFM equipped with 4 × 4 μm2 piezoelectric scanner. Analysis were carried out using tapping mode at room temperature using AFM cantilevers with a spring constant 2 N/m (Micro cantilever, OLYMPUS). All scanned images were analyzed using WSxM 5.0 Develop 3.2 software.


## 3. Results

The intrinsic fluorescence of many proteins is mainly contributed by tryptophan alone, because phenylalanine has very low quantum yield and the fluorescence of tyrosine is almost totally quenched if it is ionized or near an amino group, a carboxyl group, or a tryptophan residue [35]. The fluorescence spectra of TTR at various concentrations of pentobarbital (0.03, 0.06, 0.24, 0.48, 0.96, and 1.92) × 10−3 moL L−1 are shown in Figure 2. The fluorescence intensity of TTR decreased regularly with the increase of pentobarbital concentration, while the peak position shows little or no change at all.Figure 2
Fluorescence emission spectra of TTR pentobarbital complexes at different pentobarbital concentrations.The dynamic quenching process can be described by the Stern-Volmer equation [37]:
(1)F0F=1+Kqτ0(L)=1+Ksv(L),
where F and F0 are the fluorescence intensity with and without quencher, Kq is the quenching rate constant of the biomolecule, Ksv is the Stern-Volmer quenching constant, τ0 is the average lifetime of the biomolecule without quencher, and (L) is the concentration of pentobarbital. As can be seen from Figure 3, the Stern-Volmer plot is linear and the slope is equal to Ksv (5.66 × 102 L moL−1). Fluorescence quenching can be induced by different mechanisms, which were usually classified into dynamic quenching and static quenching. Dynamic quenching arises from collisional encounters between the fluorophore and quencher and static quenching results from the formation of a ground state complex between the fluorophore and the quencher [38]. The quenching rate constant Kq, is calculated using the fluorescence life time of 10 ns [39] at (Kq = 5.66 × 1010 L moL−1 s−1). This value is larger than the maximum dynamic quenching constant for various quenchers with biopolymer (2 × 1010 L moL−1 s−1) [40], which confirms that a static quenching is dominant in the formed complexes [41].Figure 3
The Stern-Volmer plot for pentobarbital-TTR complexes.When the static quenching equation is used [38]
(2)1F0-F=1F0K(L)+1F0,
where K is the binding constant of pentobarbital with TTR. The value of K has been determined from the slope and the intercept in Figure 4 and is calculated at 2.092 × 103 M−1 which supports the effective role of static quenching.Figure 4
The plot of1/(F0-F) versus (1/[L]) for pentobarbital-TTR complexes.The FTIR absorption spectra in the amide I and amide II regions are taken for different concentrations of TTR-pentobarbital samples. The analysis of the secondary structure of TTR in the amide I which occurs between 1600 cm−1 and 1700 cm−1 yields several components. The peaks of these components correspond to the C=O stretching vibrations of the amide group, coupled to the C–N stretching and C–C–N deformation mode [32]. The peaks in the amide II (1600–1480) cm−1 region are due to the coupling of the N–H in-plane bending and C–N stretching modes [42].The component bands of the amide I and amide II regions were determined using FSD and second derivative resolution with curve fitting procedures. This procedure has provided a basis for the quantitative estimation of protein secondary structure [32–34, 43].In this work a quantitative analysis of the protein secondary structure for TTR and pentobarbital-TTR complex in dehydrated films is determined from the shape of amide I and amide II bands. Figure5 shows the spectra and its second derivative for the amide I and the amide II regions. The second derivative resolution enhancement and the curve-fitted graphs for the amide I and amide II regions are shown in Figure 6. The curve fitted graphs show the secondary structure determinations of the TTR free and pentobarbital-TTR complexes. The exact frequency of the vibrations depend on the nature of the hydrogen bonding involving the amide group, and by the particular secondary structure adopted by the protein. In Table 1 we have listed the peak positions of the separate components of the amide I and amide II regions using both second derivative and FSD procedures.Table 1
Band assignment in the absorption spectra of TTR with different pentobarbital concentrations for amide I and amide II regions.
Bands
TTR free
TTR + pento.  0.12 mM
TTR + pento. 0.48 mM
TTR + pento. 0.96 mM
Amide I1600–1700 cm−1
1611
1614
1615
1614
1631
1631
1631
1632
1648
1644
1645
1642
1660
1658
1658
1658
1677
1674
1674
1673
1694
1692
1692
1691
Amide II1490–1590 cm−1
1499
1499
1501
1501
1507
1519
1517
1517
1536
1538
1540
1540
1561
1561
1560
1560
1582
1581
1579
1578(a) Absorbance spectra of TTR-pentobarbital complexes. (b) Second derivative of TTR-pentobarbital complexes spectra. (A, B, C, and D) correspond to pentobarbital concentrations of (0.0, 0.12, 0.48, and 0.96 mM), respectively.
(a)
(b)Figure 6
Curve-fitted graphs for (a) free TTR in amide I region, (b) TTR-pentobarbital complex with 0.96 mM pentobarbital concentrations in amide I region, (c) free TTR in amide II region, and (d) TTR-pentobarbital complexes with 0.96 mM pentobarbital concentration in amide II region.The quantitative estimation of protein secondary structure is based on the assumption that the total absorption due to C=O vibration can be represented by the sum of the individual absorptions due to the vibrations of the separate elements of the band [33].The percentages of each secondary structure of the pentobarbital TTR complexes were calculated from the integrated areas of the component bands in amide I and amide II respectively. Table2 shows the relative intensity of each secondary structure component of TTR before and after the interaction with pentobarbital at different concentrations. The percentage values of the relative intensities of the components of amide I for free TTR are in agreement with the results of other recent spectroscopic studies [32, 34]. The peaks of the amide II showed similar proportionalities in their percentage values to that of amide I.Table 2
Percentages  of secondary structure components in the amide I and amide II regions  of TTR  and its pentobarbital complexes.
Bands
TTR  free
TTR + pento  0.12 mM
TTR + pento  0.48 mM
TTR + pento 0.96 mM
Amide I
Parallelβ-sheet (1621–1640 cm−1)
51.6
31.3
16.9
14.7
Random Coil (1640–1652 cm−1)
1.7
3.4
4.2
4.0
α-helix (1652–1666 cm−1)
6.3
8.3
7.8
8.9
β-turns (1666–1685 cm−1)
21.3
33.4
36.6
33.9
Antiparallelβ-sheets (1600–1621 cm−1) (1685–1700 cm−1)
8.910.1
13.410.3
16.018.5
20.218.3
Amide II
Parallelβ-sheet (1490–1507 cm−1)
45.0
27.6
6.4
5.2
Antiparallelβ-sheet (1507–1528 cm−1)
0.14
12.1
35.2
32.6
α-helix (1528–1550 cm−1)
23.2
18.9
17.4
18.9
β-turns (1550–1570 cm−1)
25.3
25.5
21.6
20.17
Antiparallelβ-sheets (1570–1590 cm−1)
6.3
15.9
19.3
23.2The relative intensity of theβ-sheet band with its peak at (1631 cm−1) shows a remarkable decrease with the increase in pentobarbital concentrations, shown in Figure 7(a). A similar observation can be made for the β-sheet band at (1499 cm−1) in the amide II region as shown in Figure 7(b). On the other hand an increase in the relative intensities of the bands at (1694 cm−1, 1611 cm−1, 1582 cm−1, and 1507 cm−1) are noted with the increase of pentobarbital concentrations in the protein complexes as shown in the same figures. The changes in the relative intensities can also be observed directly from the curve fitted graphs in Figure 6.Relative intensity variation for different concentrations of TTR-pentobarbital complexes in (a) amide I region and (b) amide II region.
(a)
(b)The variation of intensities with temperature change for the parallel and antiparallelβ-sheets in TTR free and pentobarbital-TTR complexes are shown in Figure 8(a) for the amide II region and Figure 8(b) for the amide I region. The relative intensity increases as temperature increases for the antiparallel β-sheets while it remarkably decreases for the parallel β-sheets. The reduction of parallel β-sheets intensity percentage in favour of the increase of the antiparallel β-sheets structures are believed to be due to the unfolding of the protein in the presence of pentobarbital as a result of the formation of H-bonding between TTR and pentobarbital [44].Relative intensity versus temperature change pentobarbital 1.92 mM complexes (a) the bands at 1630 cm−1 and 1611 cm−1 (b) the band at 1507 cm−1 and 1499 cm−1.
(a)
(b)AFM techniques are used as additional method to investigate the effect of pentobarbital on TTR by taking images for TTR solution and then for TTR-pentobarbital complex as shown in Figure9. These figures confirm the formation of oligomers seen as spherical shaped objects (30–50 nm in diameter) which support previously established characterization of oligomers [36, 43, 45].AFM Images of (a) TTR free and (b) TTR + Pentobarbital 7.68 mM concentration.
(a)
(b)


## 4. Discussion

The difference in behavior for the two types ofβ-sheets is mainly due to the different amino acids and their preferred secondary structural arrangements. It has been reported that the two forms of β-sheets have different thermodynamic propensity scales [46]. The gradual decrease in intensity for the bands of parallel β-sheets (1631 cm−1 and 1499 cm−1) with increasing temperatures are believed to be mainly due to the unfolding of the protein as a result of the formation of H-bonding between TTR and pentobarbital. The newly formed H-bonding causes the C–N bond to assume a partial double bond character due to electrons flow from the C=O to the C–N bond which decreases the intensity of the molecular vibrations [47]. On the other hand the increase of intensity for antiparallel β sheets as shown in Figure 8 implies more stability and less conversions of the C=O bond as a result of the interaction with pentobarbital. The parallel arrangement is less stable because the geometry of the individual amino acid molecules forces the hydrogen bonds to occur at an angle, making them longer and thus weaker. Contrarily, in the antiparallel arrangement the hydrogen bonds are aligned directly opposite to each other and it allows the interstrand hydrogen bonds between carbonyls and amines to be planar making stronger and more stable bonds [48].According to Giorgia Zandomeneghi there is structural difference betweenβ-sheets in native proteins and amyloid fibrils [34]. In their work, parallel β-sheet reported at 1630 cm−1 while amyloid fibrils are reported at 1615 cm−1. The analysis of the absorption spectra of pentobarbital-TTR complex has shown a decrease in the intensity of the parallel β-sheet band at 1631 cm−1 accompanied by a simultaneous increase in the intensities of the bands at 1611 cm−1 and 1694 cm−1 Figure 6, which is typical for antiparallel β-sheet formation. This noticeable increase in intensity of the 1611 cm−1 peak can be related to the formation of fibrils or oligmers as a result of the TTR interaction with pentobarbital. A similar effect has been observed in the amide II region where an increase in the relative intensity of the peak at 1507 cm−1 is accompanied with a decrease in the relative intensity of the peak at 1499 cm−1. The increase of pentobarbital concentration in the protein complex has shown a larger increase in the relative intensities of the peaks at 1611 cm−1, 1694 cm−1, and 1507 cm−1 while the intensities of the two peaks at 1630 and 1499 cm−1 have decreased as shown in Figure 7.The slight shifts in the peak positions of (1611 cm−1 and 1507 cm−1) are mainly due to the interaction of pentobarbital with TTR turning more β-sheets into oligomer structure which causes permanent unfolding of the sheets producing insoluble structure [49]. The nature of this interaction can be explained in terms of the electrostatic interactions among surface charges on the protein [50, 51]. The charge-charge interaction contributes to polarization of the structure in between these surfaces creating large stable structures in the form of oligomers as suggested by Ferández [52].In our case we suggest that oligomer structure can be formed by assuming that pentobarbital molecules play the role of polarized dielectric material sandwiched between the unfoldedβ-sheets creating a capacitor-like structure, which raises the internal energy of the arrangement leading to a more stable complex. The developed rigid structure is expected to stop the β-sheets from folding due to the additional constrained on its surface leading to an insoluble oligomer. The size of the formed structure are determined by AFM measurements to be in the range of (30–50 nm in diameter) which indicates to some extent that the structure is more likely in the form of oligomers as supported by other previous works [53–56]. Other studies have proposed that some ligands can work as a kinetic stabilizer by binding to the hormone binding sites in TTR which increase the free-energy barrier for native tetramer dissociation [57]. All of these research results demonstrate the feasibility of using small molecules to stabilize the native fold of a potentially amyloidogenic human protein, thus preventing the conformational changes, which appear to be the common link in several human amyloid diseases [58].In conclusion, the binding of pentobarbital to TTR has been investigated by fluorescence and FTIR spectroscopy. The value of the binding constant has been determined, the results indicate that the intrinsic fluorescence of TTR was quenched by pentobarbital through static quenching mechanism. We have demonstrated by using AFM and FTIR spectroscopy that pentobarbital interaction with TTR can trigger the conversion ofβ-sheets into insoluble oligimor structure. In addition we proposed that TTR amyloidogenesis can easily be the result of polymerization mechanism due to protein interaction with small anesthetics molecules leading to oligomerization. Current research indicates that oligomer formation leads to memory impairment causing Alzheimer and other brain diseases [59–61]. Further investigations are still needed to explain the role of anesthetics in initiating oligomer formation in proteins, and more clinical studies are needed to test these expectations.



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