Prevention and Disintegration of Human Serum Albumin Fibrils under

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Prevention and Disintegration of Human Serum Albumin Fibrils Under Physiological Conditions: Biophysical Aspects Achal Mukhija, and Nand Kishore J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b07140 • Publication Date (Web): 05 Oct 2018 Downloaded from http://pubs.acs.org on October 7, 2018

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The Journal of Physical Chemistry

Prevention and Disintegration of Human Serum Albumin Fibrils under Physiological Conditions: Biophysical Aspects AchalMukhija, Nand Kishore* Department of Chemistry, Indian Institute of Technology Bombay, Powai, Mumbai – 400 076, India. *Corresponding author. Email: [email protected] ABSTRACT

An anomaly in the protein folding process can lead to aggregation or fibrillation of proteins which has been related to neurodegenerative and peripheral diseases. Therefore, it is important to understand the mechanism of prevention of aggregation/fibrillation and to design suitable inhibitors for this process. Literature information suggests that most of the work on these systems has been done on heat induced fibrils (57oC to 65oC). As a step ahead, in the present study, efforts have been made to understand the inhibition process under physiological conditions (37oC, pH 7.4) which is more relevant to the fibrils formed under natural cellular environment. The qualitative and quantitative aspects of the interactions of the surfactant sodium dodecyl sulphate and anti-inflammatory drug diclofenac sodium (DCF) with human serum albumin at different stages of the fibrillation process have been studied employing a combination of spectroscopic, calorimetric and microscopic techniques. Fibril formation understudied conditions was confirmed by TEM images and Thioflavin T binding assay along with DLS measurements. Energetics from isothermal titration calorimetry provided insights into the nature of interactions and mechanism of inhibition. We found inhibition efficiency of the additives in the order, micellar SDS > 45 mM DCF > monomeric SDS > 5 mM DCF. The energetics of interaction, correlated with the molecular structure of inhibitors provide guidelines for effective synthesis and design of inhibitors. ITC results have imparted important relationship between inhibition efficiency and exothermicity of interactions and have demonstrated the significance of polar interactions in fibril prevention by these inhibitors. Interestingly it was found that the micellar SDS not only inhibits the process but also effectively disintegrates the formed fibrils.

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INTRODUCTION Amyloid fibrillation leads to at least 27 diseases including Alzheimer’s, Parkinson’s, Huntington’s and diabetes due to aberrations in the folding process of proteins and deposition of fibrillar structure in cells or tissues.1-3 This occurs due to misfolding of the protein which results in their inability to perform their requisite functions or they escape the protective mechanisms of the cell and form intractable fibrils and aggregates within the extracellular and intracellular environments of the cells.4 The protein aggregation process may lead to the formation of either highly ordered amyloid fibrils or amorphous aggregates depending upon the surrounding conditions and amino acid sequence of the protein.5,6 These two pathways of formation of fibrils or aggregates are not mutually dependent on each other, but one pathway can prevail under specific conditions. In recent reports, amyloid formation is shown as a generic feature of all peptide sequences and attempts have been made to make functional use of these amyloid fibrils e.g. use of amyloid fibrils as a depot for controlled continued release of peptide or peptide based drugs.7 Amyloid fibrils have cross beta sheets which give rise to characteristic x-ray diffraction pattern and shows specific binding to thioflavin T and congo red dye which can be used to distinguish the fibril formation process from non amyloid deposits. Proteins have been classified into two groups – globular protein and natively unfolded protein, based on their native conformation. They show two different kinds of transition forms during the fibrillation process. The first group which includes lysozyme, serum albumins and 2-microglobulin forms partially unfolded unstable transition form, whereas another group which includes amyloid β-protein and α-synuclein undergoes partial folding.8 Above facts give the impression on the need to understand the process and mechanism of fibril formation and its inhibition. Folding process of protein can be interrupted by many factors such as temperature, pH or the presence of some reducing agents or other molecules9,10 and can lead to fibril formation in cellular environment thus affecting the cellular functioning of protein which makes inhibition or disintegration studies more important. Many reports have been reported on these inhibitions using different molecules such as osmolytes, drugs, nanoparticles, polyphenols, and micelles.11-15 The most reported studies in this regard are based on the inhibition of heat induced fibrils and which can be different from those formed under natural cellular conditions. In the present study, we report inhibition of human serum albumin by anionic surfactant sodium dodecyl sulfate and antiinflammatory drug diclofenac sodium under physiological conditions at 37 ºC and pH 7.4. Micelles can mimic the membrane structure 16–22 and SDS having similar properties with the lipid membrane, is the most widely used surfactants to mimic such an environment.22 SDS is an 2 ACS Paragon Plus Environment

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organo sulfate consisting of a 12-carbon tail attached to a sulfate group. It is a compound with amphiphilic properties

due

to

the

presence

of hydrocarbon tail

and polar head

group which makes it useful as a detergent. SDS is a common component of many domestic cleaning, personal hygiene, cosmetic, pharmaceutical, and food products, as well as of industrial and commercial cleaning and product formulations.23-25 Diclofenac sodium is an anti inflammatory drug and has been widely used in the treatment of arthritis.26 It has also been reported as potent inhibitor of human transthyretin amyloid fibrils.27 Human Serum Albumin is a globular protein of 583 amino acids with 3 domains consisting of 2 sub domains in each. It has 60 % α-helix character and 35 SH groups out of which 34 participate in the formation of 17 disulfide bonds leaving one free SH group. The available structural information on HSA present in the literature makes it important for studying the amyloid fibrillation. Further HSA has been extensively used as a model protein to understand deep insights of fibrillation process, as it easily forms fibrils under the influence of external factors such as pH, ionic strength, temperature, and in the presence of different solvents 28-30 and its inhibition in presence of many molecules like polyphenols, nanoparticle, even in presence of metal ions or metal complexes.14,15,31,32 In the present study HSA fibrillation and inhibition process has been studied under physiological conditions pH 7.4 and 37 ºC. For this HSA has been treated with DTT, which convert its α-helical structure to β-sheet by reducing disulfide bonds and upon incubation these β-sheets stack on each other to form fibrils. DTT is a well known reducing agent to reduce disulfide bonds of protein by interacting with s-s bonds and converting it to SH group.33,34 For the inhibition of this fibrillation process, SDS and DCF at different concentrations have been used to study the mechanism of inhibition with an ultimate goal to develop suitable / efficient inhibitors of the process by knowing the structure-property-energetics relationships.

EXPERIMENTAL Materials and methods

Sodium dodecyl sulphate (SDS, C 12 H 25 NaO 4 S), diclofenac sodium (DCF, C 14 H 10 Cl 2 NNaO 2 ), DL-dithiothreitol (DTT, C 14 H 10 O 2 S 2 ), thioflavin T (ThT, C 17 H 19 ClN 2 S), 8-anilino-1naphthalenesulfonic acid (ANS, C 16 H 13 NO 3 S) and morpholinopropane sulfonic acid (MOPS, 3 ACS Paragon Plus Environment


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C 7 H 15 NO 4 S) of best available purity were procured from Sigma-Aldrich Chemicals Pvt. Ltd. Figure 1 represents the molecular structure of these compounds. Human serum albumin (HSA,

66.5 kDa) was extensively dialysed overnight at 4 ºC against MOPS buffer (pH 7.4) with

minimum of 4 changes. The dialysate buffer was used to prepare all other solutions.

Figure 1.Molecular structures of (A)sodium dodecyl sulphate, (B) dithiothreitol, and(C) diclofenac sodium.

UV-visible spectroscopy For quantitative analysis, it is important to know the exact concentration of differently used substances including protein and dyes which are partially soluble. A JASCO V-550 UV-visible spectrometer was used to determine the concentrations of protein, ThT, and ANS. All solutions were prepared in 20 mM MOPS buffer at pH 7.4. The concentration of the dialysed HSA was 35 determined at 280nm (𝐴𝐴1% and diluted to a final concentration of 60µM. For ThT, 1𝑐𝑐𝑐𝑐 = 5.3)

and ANS, the concentrations were determined by using molar extinction coefficients 𝜀𝜀 = 26,620M-1 cm-1 (at 412 nm)36 and 5000 M-1 cm-1(at 350 nm),37 respectively.

HSA fibril formation and inhibition 4 ACS Paragon Plus Environment

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To study the fibrillation process of HSA under physiological conditions (pH 7.4 and 37oC) DLdithiothreitol (DTT) was used as a reducing agent. For fibril formation, 60 µM HSA solution was incubated at 37 ºC for 1500 min in presence of 10 mM DTT and this was confirmed by ThT and ANS fluorescence, DLS, CD spectroscopy and TEM images along with the calorimetry studies. For inhibition of fibrillation, small molecules like anionic surfactant SDS and anti-inflammatory drug DCF were used at different concentrations. SDS was used in both the monomeric and micellar forms at concentrations of 2 mM and 16 mM, respectively. For inhibition studies with DCF, experiments were performed in 5 mM and 45 mM concentration. Inhibition of the fibrillation process was studied by incubating DTT treated HSA in presence of above mentioned concentrations of inhibitors under similar conditions. Fluorescence Studies

ThT Assay Thioflavin T (ThT) is a well - known dye which binds specifically with fibrils,38 therefore, to confirm the fibril formation and extent of fibrillation, ThT fluorescence was used. For fluorescence studies, a Cary Eclipse fluorescence spectrophotometer from Varian, USA with Cary temperature controller water bath was used at 37ºC. ThT was excited at 450 nm and its emission was recorded from 460 nm to 600 nm with both excitation and emission slit widths set at 5 nm. The excitation wavelength was kept at450 nm instead of 412 nm (λ max of ThT absorbance) due to red shift in λ max of ThT upon binding to fibrils. The final concentration of ThT used in the fluorescence study was 50 µM. As the presence of ThT can affect the fibrillation process, the endeavour has been made to study this fibrillation process by incubating protein in presence and absence of ThT molecules. For inhibition experiments, ThT fluorescence was recorded in the presence of inhibitors (2mM and 16 mM for pre and post micellar concentrations for SDS and 5 and 45 mM for DCF). For the control experiments, only protein and only ThT fluorescence were recorded under similar conditions and subtracted to get the final spectra.

ANS Assay



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8-Anilino-1-naphthalenesulfonic acid (ANS) is a fluorescent dye which binds to hydrophobic groups and demonstrates the exposure of these groups on the surface of proteins.39 For this, ANS emission was also recorded by Cary Eclipse fluorescence spectrophotometer from varian, USA at 37 ºC. The ANS emission fluorescence spectra were recorded from 360 nm to 600 nm by exciting it at 350 nm with emission and excitation slit fixed at 5 nm. Final concentrations of protein and ANS in these experiments were 60 µM and 50 µM, respectively. The ANS emission was also recorded in the presence of inhibitors to understand the changes in surface hydrophobicity. The ANS emission was also recorded in the absence of the protein as control experiment. Circular Dichroism spectroscopy The CD experiments were done to study the changes in the secondary and tertiary structures of the protein in the presence of additives and also to illustrate the mechanism of fibrillation/aggregation by these molecules. A Jasco-810 spectropolarimeter was used to carry out these experiments. The far UV-CD spectra were recorded to monitor the secondary structure of the protein from 200 nm to 260 nm in 0.2 cm path length cuvette and near UV CD spectra were recorded to monitor tertiary structure of protein from 260 nm to 360 nm in 1 cm path length cuvette at a scan rate of 100 nm min-1 and continuous nitrogen purging. The concentrations of the protein used were 5 µM and 15 µM for far and near UV-CD spectra, respectively. The molar ellipticity values were calculated using following equation: 𝜃𝜃 = 100 × (𝜃𝜃/(𝐶𝐶 × 𝑙𝑙))

Where C is the concentration of protein, l is the path length and 𝜃𝜃 is the ellipticity.

Dynamic Light Scattering

The fibrillation process is associated with the changes in the structure of the protein which can be monitored by studying the changes in the size of the protein as the fibrillation process proceeds. A Malvern Zetasizer Nano ZS Zen 1600 was used to perform the Dynamic Light Scattering (DLS) measurements. In a typical experiment, laser beam is passed through the protein sample and the scattered light is detected at 90º, and the hydrodynamic diameter is measured. The DLS experiments were done with 4 mg ml-1 HSA in the presence and absence of different additives at different incubation time.

Transmission Electron Microscopy 6 ACS Paragon Plus Environment

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To get the real-time images of the fibrillation process and to see the morphology of the fibrils, a JEM 2100F JEOL field emission microscope working at an accelerating voltage of 200 keV was used. For this, a sample of 4 mg ml-1 protein in the presence or absence of the inhibitor at different stages ofthe fibrillation was loaded on a formvar-coated 300 mesh copper grid. To increase the contrast between sample and background, negative staining was done using 2% phosphotungstic acid at pH 7.4. The staining solution was centrifuged and filtered through a 0.22 µm filter.

Calorimetric Studies Isothermal titration calorimetric experiments were done to understand the nature of interactions and further to illustrate the mechanism of inhibition process. A Nano ITC from TA Instruments, New Castle, DE, USA was used to carry out the ITC experiments for a better understanding of the interaction of inhibitors with protein at different stages of fibrillation process and to obtain associated thermodynamic signatures. A total of 25 injections, each of 10 µL from the computer controlled syringe containing 60 µM protein at different stages of fibrillation were injected into the cell containing different concentrations of inhibitors (2 mM and 16 mM SDS for pre and post micellar SDS conditions respectively and 5 mM or 45 mM DCF). Thus a total of 250 µL of the protein was injected in 25 injections with an interval of 5 minutes between successive injections. To ensure proper mixing of the injected solution into the cell solution, stirring was done at 250 rpm. The instrument was equilibrated for a constant time before the start of injections to acquire the same stage of fibrillation process in each case. To obtain the heat of interaction between the inhibitors and the protein at different stages of the fibrillation process, the corresponding dilution experiments were also performed. The heats of dilution of the protein into the buffer and buffer into the inhibitor solution were subtracted from the main experiments. The Nano Analyzer software provided along with the instrument was used to analyze the titration profiles and integrated heats were obtained. All the experiments were repeated twice with freshly prepared solutions to check the reproducibility of results.

RESULTS AND DISCUSSION ThT binding



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The explicit property of ThT to bind with amyloid fibrils was used to confirm the fibril formation and also to estimate the extent of fibril formed. ThT has been used as a standard probe to detect amyloid fibrils as it binds to the channels running parallel to the long axis of the fibrils.40,41 ThT binds to a cavity of minimum diameter of 8-9 Å present in the amyloid fibrils, to give rise to its characteristic emission fluorescence peak around 485 nm upon excitation at 450 nm.41,42 The kinetics of binding of ThT to formed amyloid fibrils is sufficiently fast that it completes within a time period of 30 s.43 The C-C torsion angle between benzothiazole and benzamine in non-bound ThT is around 35º which changes to planar or near to planar structure upon binding to fibrils due to change in conformation of ThT.44-46 ThT mainly binds to fibrils in its monomeric or dimeric molecular forms.40,44 The red shift from 412 to 450 nm in the absorbance of ThT was observed upon binding to fibrils, due to the molecular rotor behavior of monomer or the excimer formation in the cavities of fibrils 40 thus the samples were excited at 450 nm wavelength instead of 412 nm.Figure 2 (A) shows the ThT kinetics of 60 µM HSA with 50 µM ThT incubated at 37ºC in presence and absence of 10 mM DTT over a period of 0 to 1500 min. The figure illustrates that in the presence of DTT, the emission of ThT increases drastically with time and shows the absence of lag phase in the fibrillation process which is consistent with previous reports on fibrillation of serum albumins47 and saturates after about 1000 min. This increment in the intensity of ThT emission with time shows increase in fibrillar concentration as the intensity of ThT emission fluorescence is proportional to the concentration of fibrils in the specific conditions for a given protein. 41,47 As it is also clear from the figure that the native protein in the absence of DTT does not show any change in the ThT emission and thus the absence of fibril formation. The presence of ThT can also affect the fibril formation so the experiment has also been done by incubating the sample in similar conditions but without ThT and aliquots were taken out at a different time and then ThT was added to record the ThT emission. Figure 2(B) represents the

ThT emission spectra of HSA incubated in the presence of DTT at different incubation time when aliquots of ThT were added after the incubation and Figure 2(C) shows change in

intensity of ThT fluorescence emission as a function of time. Figures 2(A) and (C) exhibited

similar changes and denote that the presence of ThT does not show any effect on the fibrillation process.


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Figure 2. Kinetics of 60 µM HSA monitored through ThT fluorescence emission in presence and absence of 10 mM DTT (A) when HSA was incubated with ThT and (C) ThT was added after incubation at 370 C. 2 (B) ThT fluorescence emission spectra at different incubation time.



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For inhibition of fibrillation process, experiments were done by incubating the protein sample in the presence of inhibitors. Figure 3 (A) shows the ThT emission kinetics in the absence and

presence of 2 mM and 16 mM SDS. It is clear from the figure that the intensity of ThT fluorescence decreases significantly in the presence of 2 mM SDS and it becomes almost zero when the concentration of SDS is 16 mM in solution where SDS is in its micellar form. In the presence of both pre and post micellar SDS, the decrement in ThT fluorescence was drastic. The decrease in fibril formation was 35% and 95% respectively in the presence of pre and post micellar concentrations of SDS after 1500 min of incubation. The control experiment was also done to see the effect of pre and post micellar concentrations of SDS on HSA fibril formation in the absence of DTT and it was found that the ThT fluorescence emission remains constant during the experiment thus suggesting absence of any fibril formation without DTT. Figure

3(B) represents the ThT fluorescence emission kinetics of HSA in the absence and presence of 5 mM and 45 mM DCF. The figure illustrates that the changes in ThT fluorescence emission intensity are more prominent in the presence of 45 mM DCF. The intensity of ThT decreases significantly and shows the inhibition of the fibril formation process by 29 % and 70 % respectively, in the presence of 5 mM and 45 mM DCF after 1500 min of incubation. This suggests that the presence of SDS and DCF decreases the extent of fibrillation establishing that SDS and DCF are effective inhibitors of the fibrillation process.


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Figure 3. Kinetics of DTT treated HSA monitored through ThT fluorescence emission in absence and presence of 2 mM, 16 mM SDS (A), 5 mM, 45 mM DCF (B) and histogram for the percentage of fibril present in presence of different inhibitors(C).



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ANS Assay To study the formation of amorphous aggregates or changes in the conformation of HSA by the presence of different molecules and incubation time in the given conditions, ANS fluorescence was recorded by exciting it at 350 nm. ANS interacts with protein by both ionic and hydrophobic interactions, its negatively charged sulfonate group interacts with polar groups of protein whereas its hydrophobic aromatic groups interact with the hydrophobic groups of the protein48 and both of these interactions are responsible for its characteristic fluorescence emission. ANS specifically binds to the hydrophobic groups of amorphous aggregates or molten globule state or partially folded states of the proteins.39 However it has been reported that in some cases it also binds to the native state of the protein and shows its characteristic fluorescence.49 Figure 4 (A) represents the ANS kinetics of HSA in the presence and absence of DTT which shows that the intensity of ANS in absence of DTT remains constant over the incubation time of 1500 min and denotes the absence of amorphous aggregates by incubating it under physiological conditions. In the presence of DTT, fluorescence intensity decreases with time which indicates change in the structure of HSA upon interaction with DTT and supports the ThT kinetics observation of fibril formation. The decrease in intensity could be due to the conversion of native HSA structure into fibrillar structures upon incubation. As hydrophobic groups of HSA get buried inside to form fibrils and thus no longer available for ANS binding. Therefore, ANS fluorescence decreases with time. Figures 4 (B) and (C)

represent the ANS kinetics of DTT treated HSA in the presence and absence of SDS (2 mM and 16 mM) and DCF (5 mM and 45 mM), respectively. The figure illustrates insignificant

change in the intensity of ANS with incubation time. Experiments were also performed with higher molar ratio of protein to ANS (1:2.5, 1:7.5 and 1:15) (Supporting information) and

even at higher ANS concentration, no significant change was observed in the ANS fluorescence emission in the presence of post micellar SDS or 45 mM DCF with incubation time which indicates absence of amorphous aggregates or change in the conformation by incubating HSA in the presence of these inhibitors.


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Figure 4. Kinetics of HSA monitored through ANS emission (A) in presence and absence of 10 mM DTT, DTT treated HSA in absence and presence of 2 mM, 16 mM SDS (B) and 5mM, 45 mM DCF (C) Circular Dichroism Spectroscopy Change in conformation of the protein along the fibrillation process can be helpful to understand the mechanism of fibrillation. The CD spectroscopy is one of the best tools to study the conformational changes in proteins. Far and near UV-CD spectra were recorded for HSA in the presence and absence of DTT and SDS to study the changes in the secondary and tertiary structure of the protein upon interaction with these molecules. Figure 5 (A), and (B), represent the far and near UV-CD spectra of HSA in the presence and absence of 10 mM DTT at pH 7.4. Figure 5 (A) shows significant change in the peak position in far UV-CD spectra from 208 and 222 nm which are characteristic peaks of α-helix to 218 nm which corresponds to the characteristic peak of β-sheet whereas the change in the tertiary structure is not appreciable [Figure 5 (B)]. This suggests that DTT induces conformational transition in the secondary structure of HSA from α-helix to β-sheet, which leads to formation of mature amyloid fibrils by stacking upon each other. These results are in agreement with the previously reported heat induced fibrillation process which also suggests the formation of β-sheets during the fibrillation process.10 The CD spectra of HSA was also recorded at different incubation time in presence and absence of 10 mM DTT to understand the changes in conformation of HSA with incubation time (see Supporting information). Insignificant change in the spectra of HSA is observed in the absence of DTT, whereas in the presence of DTT the peak position of β-sheet was maintained with reduction in ellipticity upon incubation, suggesting formation of fibrils. SDS is a well known denaturant for proteins and has been used in SDS-PAGE to denature the protein.50 To confirm that the concentrations of SDS used in this study donot denature the protein, the CD spectrum of HSA was also recorded under these conditions (Supporting 13 ACS Paragon Plus Environment


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information). As the figure illustrates, although the presence of SDS decreases the molar ellipticity leading to partial reduction in the secondary structure, but the characteristic peaks of α-helix are preserved and suggest that the concentrations of SDS used in this work do not lead to denaturation of the protein. CD spectra of HSA in presence of DCF could not be reported due to the high HT voltage (HT vol> 800) issue which leads to scattering of data.

Figure 5.(A) Far and (B) near UV-CD spectra of HSA in absence and presence of 10 mM DTT. DynamicLight Scattering Along with the conformation of the protein, the size also changes as the fibrillation proceeds which can be monitored by dynamic light scattering measurements. Hydrodynamic diameter of HSA in the presence and absence of additives at different stages of the fibrillation process is tabulated in Table 1. The number average size of HSA increases upon incubating it for 1500 min in the presence of DTT. The size of the native HSA in the absence of DTT was found to be (5.1±0.2) nm and in the presence of DTT it was (3.6±0.4) nm which constantly increased with incubation time. After duration of 75 min, it increased to (11.0±0.7) nm which can be due to the presence of partially unfolded conformation of protein in the elongation stage. It further increased to (25.5±2.4) nm with the formation of mature fibrils after about 1500 min. This constant increase in the size of the protein with incubation time provides additional support to ThT binding results and confirms fibril formation. The DLS measurements were also done for the inhibition experiments. For this the size of HSA was measured in the presence of inhibitors along the progress of fibrillation process. The size of HSA increased in the presence of 14 ACS Paragon Plus Environment

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monomeric SDS as well but the extent of increase was significantly low from (4.7±0.3) nm to (7.6±1.2) nm suggesting the formation of fibrils at low rate with less extent of fibrillation. Insignificant change in the size of HSA was observed when the protein was incubated with post micellar of SDS. This illustrates the absence of fibril formation in this case and it is in accordance with ThT kinetics which exhibited similar changes in the presence of micellar SDS. Further, the DLS experiments were performed in the presence of 45 mM DCF and change in size of the protein was found to be negligible (from 4.7±0.4 to 4.5±0.6) which provides support to ThT binding results with no change in the ThT intensity with incubation time in presence of the drug (Plots are presented in Supporting Information). Table 1. Size distribution of HSA protein in presence and absence of 10 mM DTT and inhibitors at different stages of fibrillation process

Sample (stages of fibrillation process)

Size / nm (Number Avg.)

HAS

5.1±0.2

HSA + DTT (Nucleation stage)

3.6±0.4

HSA + DTT (Elongation stage)

11.0±0.7

HSA + DTT (Maturation stage)

25.5±2.4

HSA + pre micellar SDS

5.2±0.2

HSA + DTT + pre micellar SDS (Nucleation stage)

4.7±0.3

HSA + DTT + pre micellar SDS (Elongation stage)

5.9±0.5

HSA + DTT + pre micellar SDS (Maturation stage)

7.6±1.2

HSA + post micellar SDS

4.4±0.4

HSA + DTT + post micellar SDS (Nucleation stage)

4.3±0.5

HSA + DTT + post micellar SDS (Elongation stage)

4.3±0.3

HSA + DTT + post micellar SDS (Maturation stage)

4.4±0.9

HSA + 45 mM DCF

5.0±0.3

HSA + 45 mM DCF (Nucleation stage)

4.7±0.4

HSA + 45 mM DCF (Elongation stage)

4.5±0.7

HSA + 45 mM DCF (Maturation stage)

4.5±0.6



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Transmission Electron Microscopy Morphology of the fibrils can be seen by taking the real time images of the protein along the progress of fibrillation process. For this, 4 mg ml-1 HSA was incubated with 10 mM DTT. After incubation, the sample was loaded on a carbon copper mesh and then stained with PTA (phosphotungstic acid). The stained sample loaded grid was dried in desiccators for 3 days before taking imaging. FEG-TEM images of HSA are shown in Figure 6 in the presence of

DTT incubated at 37ºC for (A) 0 min and (B) 1500 min. The TEM images of protein incubated in presence of DTT for 0 min shows the presence of spherical particles whereas TEM image of the protein incubated for 1500 min shows the presence of fibrillar structure which confirms the fibril formation upon incubating HSA in presence of DTT for 1500 min at 37 ºC. The TEM images were also taken in presence of inhibitors. Figure 6 (C) and (D) represents the TEM

images of HSA incubated for 1500 min in presence of 16 mM SDS and 45 mM DCF, respectively which shows the absence of fibrils under these conditions.


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Figure 6.TEM images of 0.06 mM HSA incubated in presence of 10 mM DTT at 37 ºC for (A) 0 min, (B) 1500 min, and in presence of (C) 16 mM SDS, (D) 45 mM DCF.

Isothermal titration calorimetry of interaction of inhibitors with HSA at different stages offibrillation process A knowledge of mode of interaction of the protein at different stages of fibrillation with inhibitors can be helpful in understanding the mechanism of inhibition and further to develop more efficient inhibitors by establishing structure – property – energetics relationships. With this objective, the interaction studies were done by using isothermal titration calorimetry. The interaction studies of the inhibitors were done with the native protein, with the protein at elongation stage (obtained after 75 min of incubation with DTT), and with the protein at 17 ACS Paragon Plus Environment


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maturation stage of fibrillation process (obtained after incubation of 1500 min). The concentrations of inhibitors used were 2 mM and 16 mM SDS corresponding to pre and post micellar concentrations of SDS as both the stages possess different conformations and can interact differently with the protein. In the case of DCF, experiments were performed at 5 mM and 45 mM (maximum solubility) concentrations of the drug. ITC profiles for dilution of protein at different stages of fibrillation process are presented in Supporting Information. These dilutions were found to be exothermic and exothermicity increases as the fibrillation

proceeds. The values of enthalpy of dilution of native HSA, HSA at elongation stage and HSA 0 0 at maturation stage of fibrillation process are [∆𝐻𝐻𝑚𝑚 = -(2.6±0.9) kJ mol-1], [∆𝐻𝐻𝑚𝑚 = -(242.6±4.3)

0 = -(170.2±9.5) kJ mol-1] respectively. Dilution heats of protein at kJ mol-1] and [∆𝐻𝐻𝑚𝑚

elongation stage were found to be more than the dilution of native HSA or mature fibrils. As the protein at elongation stage is in partially folded state which provides more surface area / interacting groups for dilution, than native state or mature fibrils, thus the dilution is more 0 exothermic. The standard molar enthalpies (∆𝐻𝐻𝑚𝑚 ) of the interaction of protein at different

stages of fibrillation process with different inhibitors are tabulated in Table 2. These values correspond to the intercept obtained by fitting the data to appropriate polynomial or linear fit equations. These enthalpy values provide knowledge about the nature of interaction and help in proposing the mechanism. As seen from the Table 2 the exothermicity of interaction of the

native protein with inhibitors is maximum for micellar SDS followed by 45 mM DCF, followed by monomeric SDS, and minimum for 5 mM DCF. The exothermicity order is proportional to the efficiency order of these molecules to inhibit the fibrillation process of HSA under studied conditions as seen from the ThT binding assay. This provides a strong correlation between the efficiency of inhibitor to inhibit fibrillation process with exothermicity of interaction with native protein. Higher the exothermicity of interaction between inhibitor molecule and native protein, more its efficiency towards inhibition of fibrillation process.

Figure 7{(A) and (B)} represents the ITC profiles for the interaction of HSA at different stages of fibrillation process with pre and post micellar SDS, respectively. The interaction enthalpies for the interaction of monomeric SDS with the native protein, protein at elongation stage and 0 0 maturation stage of fibrillation process are [∆𝐻𝐻𝑚𝑚 =-(130.6±5.2) kJ mol-1], [∆𝐻𝐻𝑚𝑚 =-

0 (419.2±22.4) kJ mol-1], and [∆𝐻𝐻𝑚𝑚 = -(380.0±25.3) kJ mol-1] respectively. These interaction

enthalpies are found to be exothermic and suggest predominance of polar interactions between

protein and monomers. Exothermic heat of interaction implying dominance of polar 18 ACS Paragon Plus Environment

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interactions between the inhibitor molecules (osmolyte) and those of insulin, bovine serum albumin, and lysozyme, have been reported earlier11,12,45 to play significant roles in the prevention of fibrillation of the proteins. Polar interaction between inhibitor molecules and protein disrupts / influences the hydrogen bonds between beta sheets of fibrils thus inhibit the fibrillation process. As can be seen from the Table 2, the interactions of SDS monomers with 0 native HSA [∆𝐻𝐻𝑚𝑚 =-(130.6±5.2) kJ mol-1] are less exothermic than that with the protein at

0 elongation stage [∆𝐻𝐻𝑚𝑚 = - (419.2±22.4) kJ mol-1], which illustrates increased polar interactions

between the two. The reason of higher enthalpy of interaction of protein at elongation stage with monomeric SDS could be the presence of partially unfolded conformation of the protein in the elongation stage, which allows negatively charged monomers to interact the with a

positively charged alkyl group of basic amino acids of the protein. The value of interaction 0 0 enthalpy decreases from [∆𝐻𝐻𝑚𝑚 =-(415.2±20.5) kJ mol-1] to [∆𝐻𝐻𝑚𝑚 = -(153.3±8.3) kJ mol-1] as

the titration proceeds which indicates unavailability of the monomers to interact with the incoming protein. The interactions of monomeric SDS with the protein at maturation stage are 0 less exothermic [∆𝐻𝐻𝑚𝑚 =-(380.0±25.3) kJ mol-1] than with protein at elongation stage and

indicates the unapproachability of the monomers to the interacting groups of protein, as at this

stage the protein is in mature fibrillar structure[ensemble of β –sheets]. These interactions decrease drastically along the titration and the value of enthalpy becomes less exothermic.



Page 20 of 31

Figure 7. Enthalpies for the interaction of 0.06 mM protein at different stages of fibrillation process with (A) monomeric SDS (2 mM) and (B) micellar SDS (16 mM) at 37 ºC.


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Interactions of the micellar SDS with 0.06 mM protein at different stages are shown in Figure 0 = 7 (B). The interactions of micellar SDS with native HSA are more exothermic [∆𝐻𝐻𝑚𝑚

(371.2±7.5) kJ·mol-1] indicating presence of stronger polar interactions of the protein with SDS micelles than with the monomeric SDS. As monomeric SDS can interact with protein by hydrophobic as well as hydrophilic interactions whereas in micellar SDS hydrophobic groups are no longer available for interaction, they are buried inside the core of the micelles, thus only hydrophilic head part is available for interaction and shows higher exothermic enthalpy of interaction employing higher polar interactions. The interactions for the protein at elongation stage of fibrillation process with micellar SDS are also exothermic, with larger value of interaction enthalpy, indicating stronger interactions between SDS micelles with partially folded protein. This helps in prevention of the fibril formation thus shows the lesser intensity for the ThT binding assay in the presence of these SDS micelles. The interaction of mature fibrils with SDS micelles shows the highest value of exothermic enthalpy and the trend is opposite to that observed for the interaction with monomeric SDS as the interactions of the monomeric SDS with mature fibrils are less exothermic than with the protein at elongation 0 = -(1275.3±31.9) kJ mol-1] for the stage of fibrillation process. A high enthalpy value[∆𝐻𝐻𝑚𝑚

interaction of micellar SDS with protein at maturation stage of fibrillation process suggests

even stronger polar interactions than the protein at elongation stage which is possible if the fibrils are disintegrating in the presence of these micelles. Since the protein in the fibrillar form will not be able to interact with this high exothermic heat with negatively charged SDS 0 micelles. It may be noted that the values of ∆𝐻𝐻𝑚𝑚 are high as these are expressed per mole of

the protein having a molecular weight of 66.5 kD. The disintegration of protein makes its interacting groups approachable to the SDS micelles and leads to higher exothermic interactions. Although the ThT assay confirms the inhibition of fibrillation in the presence of SDS micelles, the ThT binding studies were also done to confirm the disintegration of the formed fibrils by the addition of SDS micelles and to justify this high exothermic enthalpy. Figure 8 represents the ThT binding for the fibrils formed by the process described in “section 3.3”in presence and absence of SDS micelles incubated for 0 or 75 min. As the figure illustrates, ThT shows sufficient emission intensity at 485 nm by exciting it at 450 nm in the absence of SDS micelles but as soon as the SDS micelles were introduced in the system, the ThT emission intensity decreased significantly which confirms disintegration of the fibrils by SDS micelles. This supports ITC observations of high exothermic heats of interaction between the protein at maturation stage of fibrillation process and SDS micelles.



Page 22 of 31

Figure 8.ThT fluorescence emission for the binding of ThT to formed fibrils in presence and absence of micellar SDS incubated for 0 or 75 min.

0 Table 2.Limiting standard molar enthalpies (∆𝐻𝐻𝑚𝑚 / kJ mol-1) of interaction of HSA at

different stages of fibrillation process with inhibitors at 37 ºC

ITC cell content

Native HSA

Elongation stage

Maturation stage

Buffer

-(2.6±0.9)

-(242.6±4.3)

-(170.2±9.5)

Pre micellar SDS (2 mM)

-(130.6±5.2)

-(419.2±22.4)

-(380.0±25.3)

Post micellar SDS (16 mM)

-(371.2±7.5)

-(902.4±10.0)

-(1275.7±31.9)

5 mM DCF

-(93.2±1.0)

-(274.7±4.8)

-(200.7±3.7)

45 mM DCF

-(221.9±4.0)

-(393.5±22.5)

-(425.8±24.3)


Page 23 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 9 {(A) and (B)} represents the ITC profile for the interaction of 0.06 mM native HSA

or HSA at different stages of fibrillation process with 5 mM and 45 mM DCF at 37 ºC. The interactions of native HSA with 5 mM DCF are exothermic with the heat of interaction starting 0 0 from [∆𝐻𝐻𝑚𝑚 =-(95.2±15.8) kJmol-1] which decreases along the titration and becomes [∆𝐻𝐻𝑚𝑚 =-

(49.1±13.4) kJ mol-1] at the end of 25 injections. This exothermic enthalpy indicates that the DCF molecules bind to native HSA by polar interactions. These polar interactions affect the fibrillation process as also seen from the low ThT kinetics and lesser binding of the protein to ThT in the presence of 5 mM DCF as compared to the absence of any inhibitors (Figure 3(b)).

The interaction heats become more exothermic for the interaction of the protein at elongation stage of the fibrillation process with 5 mM DCF, suggesting stronger polar interactions between DCF and partially unfolded protein present at the elongation stage. The heats for the interaction of 5 mM DCF with HSA at maturation stage becomes less exothermic as compared to the interactions with the protein at elongation stage.This is due to presence of mature fibrils where interacting groups of the protein are unapproachable to DCF molecules for interaction. The interaction of native HSA with 45 mM DCF is even more exothermic which makes it more efficient inhibitor than 5 mM DCF. This is supported by ThT fluorescence results as in the presence of 45 mM DCF, theThT binding assay of protein decreases by 70 %. This 70 % reduction in the fibrillation process is accompanied by an increase in exothermicity of 0 0 interaction from [∆𝐻𝐻𝑚𝑚 =-(93.2±1.0) kJ mol-1] to [∆𝐻𝐻𝑚𝑚 = -(221.9±4.0)kJ.mol-1]. This

exothermic heat suggests interaction between basic amino acids of HSA with the negatively charged DCF molecules (pI – 4.2).51 Although over all charge on protein at pH 7.4 is negative,

still the side chains of basic amino acids are positively charged (pk a > 8) and can interact with negatively charged DCF molecules. The heat becomes more exothermic for the interaction of protein at elongation stage of fibrillation process with 45 mM DCF. The trend of interaction heat is same as that observed with 5 mM DCF but the values are greater for 45 mM DCF due to stronger polar interactions with partially unfolded protein. The fraction of partially unfolded protein is more in the case of 45 mM DCF than 5 mM DCF due to lesser fibril formation and thus interacts strongly with 45 mM DCF. 45 mM DCF interacts with mature fibrils with similar exothermic heat as with the protein at elongation stage of fibrillation process. Similar exothermic heat suggests similar interactions between 45 mM DCF and HSA at elongation stage and maturation stage of the fibrillation process, and supports the ThT binding assay which shows constant ThT fluorescence intensity in presence of 45 mM DCF along the incubation time. To further confirm this, the formed fibrils were also incubated in presence of 45 mM



Page 24 of 31

DCF, and the ThT emission fluorescence spectra of formed fibrils at different time of incubation are presented in Supporting Information. As clearly seen from the figure, the intensity of ThT emission fluorescence remains constant overtime, suggesting presence of similar structure of the protein at elongation and maturation stage of the fibrillation process. This supports similar exothermic heats of interaction of 45 mM DCF with HSA at elongation and maturation stages of the fibrillation process. Both SDS and DCF have anionic terminal functional groups (fig 1) and exhibit exothermic interactions with the native as well as with fibrillar form. Further SDS is known to form micelles at 8.2 mM concentration in aqueous solution52 and DCF also forms micelle-like aggregates in water above a concentration of 35 mM.53 As observed micelles of both SDS and DCF are more effective in the inhibition of fibrillation of HSA compared to their monomeric forms. A comparison of molecular structure of SDS and DCF suggests that well formed micelles of SDS are better inhibitor than those formed by DCF molecules in as established by ThT fluorescence measurements.


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Figure 9. Enthalpies for the interaction of 0.06 mM protein at different stages of fibrillation process with (A) 5 mM and (B) 45 mM DCF at 37 ºC.



Page 26 of 31

CONCLUSION AND FUTURE PERSPECTIVES The Amyloid fibrillation process of HSA has been studied under physiological conditions (37 ºC, pH 7.4) by incubating HSA in the presence of DTT. The ThT binding assay along with ANS binding, DLS measurements, and TEM images confirm the fibril formation in the presence of DTT. The ThT binding assay confirms formation of mature fibrils in about 1000 minutes under these conditions. The decrease in ANS fluorescence emission and increase in hydrodynamic diameter of HSA in presence of DTT further confirms fibril formation. The TEM images allowed visualizing the morphology of fibrils. It was observed that the presence of 2 mM SDS, 16 mM SDS, 5 mM DCF and 45 mM DCF inhibit the process of fibrillation. SDS in the micellar form inhibits fibrillation more efficiently than the surfactant monomers by interacting strongly with the protein. The ITC experiments show that native protein interacts exothermically with these inhibitors. The trend of exothermicity follows the order of these interactions with micellar SDS > 45 mM DCF > monomeric SDS > 5 mM DCF, which is similar to the trend of their inhibition efficiency of fibrillation process as observed by ThT kinetics. The ITC results for the interaction of 45 mM DCF with HSA at elongation and maturation stage of fibrillation process shows the presence of similar structure of protein along the incubation time. Constant ThT emission fluorescence intensity in presence of 45 mM DCF further confirms these observations. The ITC results for the interaction of micellar SDS with HSA at maturation stage of the fibrillation process show the disintegration of the formed fibrils, and ThT binding to the formed fibrils in presence and absence of micellar SDS supports these observations. The presence of SDS micelles disintegrates the formed fibrils as soon as it is introduced to the fibrils via polar interaction between negatively charged sulfate groups with basic amino acids of the protein backbone.

ACKNOWLEDGEMENTS The authors are thankful to Indian Institute of Technology, Bombay and University Grant Commission for providing financial support. The authors also acknowledge the Sophisticated Analytical Instrument Facility (SAIF) at Indian Institute of Technology Bombay, Mumbai for providing Transmission Electron Microscopic facilities.


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 SUPPORTING INFORMATION Kinetics of HSA monitored through ANS emission in the presence of 16 mM SDS and 45 mM DCF with different protein to ANS molar ratio; CD spectra of HSA in absence and presence of different concentrations of SDS; Far and near UV-CD spectra of HSA at different incubation time (0 min, 45 min, 1500 min) in absence and presence of 10 mM DTT; size distribution of HSA in presence and absence molecules at different stages of fibrillation; enthalpies for the dilution of protein at different stages of fibrillation process; ThT fluorescence emission for the binding of ThT to formed fibrils in presence and absence of 45 mM.

REFERENCES 1. AguzziA.; O’ConnorT., Protein aggregation diseases: pathogenicity and therapeutic perspectives, Nat. Rev. Drug Discov., 2010, 9, 237–248. 2. PetterssonT.; KonttinenYT., Amyloidosis-recent developments, Semin Arthritis Rheum. 2010,39 (5), 356-68. 3. Lemkul, J. A.; Bevan, D. R. Assessing the stability of Alzheimer’s amyloid protofibrils using molecular dynamics. J. Phys. Chem. B, 2010, 114, 1652−1660. 4. DobsonC.M.; SaliA.; KaplusM., Protein folding: a perspective from theory and experiment, Angew. Chem. Int. Ed. Engl., 1998,37, 868-893. 5. ChitiF.; StefaniM.;TaddeiN.;RamponiG.; C.M. Dobson, Rationalization of theeffects of mutations on peptide andprotein aggregation rates, Nature, 2003, 424, 805–808. 6. ChitiF.; DobsonC.M., Protein misfolding, functional amyloid, and humandisease, Annu. Rev. Biochem.2006,75, 333–366. 7. Maji S.; Schubert D.;Rivier C.; Lee S.;Rivier J.;Riek R., Amyloid as a Depot for the Formulation of Long-Acting Drugs, PLoSBiol,2008,6, 240-252. 8. FezouiY.;TeplowD.B.,Kinetic Studies of Amyloid β-Protein Fibril Assembly, J. Biol. Chem.2002,277,36948–36954. 9. WangW.;NemaS.;Teagarden D, Protein aggregation pathways and influencing factors,Int J Pharm, 2010,390, 89-99. 10. WangY.;TruexN. L.;VoN.D.P.;NowickJ. S., Effects of charge and hydrophobicity on the oligomerization of peptides derived from IAPP.Bioorg Med Chem.2018,26,11511156. 11. ChoudharyS.; KishoreN.;HosurR. V., Inhibition of insulin fibrillation by osmolytes: Mechanistic Insights. Sci Rep.,2015,5, 17599. 27 ACS Paragon Plus Environment


Page 28 of 31

12. Sen S.;Gupta S. D.;Gupta S. D.; Does Surface Chirality of Gold Nanoparticles Affect Fibrillation of HSA? J. Phys. Chem. C,2017, 121, 18935-18946. 13. DizajiN. M.;beigiH. M.;AliakbariF.;MarvianA. T.;ShojaosadatiS. A.;MorshediD., Inhibition of lysozyme fibrillation by human serum albuminnanoparticles: Possible mechanism Negar, Int. J. Biol. Macromol., 2016,93, 1328–1336. 14. PoratY.; AbramowitzA.; GazitE..Inhibition

of

amyloid

fibril

formation

by

polyphenols: structural similarity and aromatic interactions as a common inhibition mechanism, ChemBiol Drug Des. 2006,67, 27-37. 15. PandeyN. K.;GhoshS.;DasguptaS., Effect of surfactants on preformed fibrils of human serum albumin, Int. J. Biol. Macromol.2013,59, 39– 45. 16. GullN.;SenP.; KhanR.H., DinK., Spectroscopic studies on the comparative interaction of cationic single-chain and gemini surfactants with human serum albumin, J Biochem2009,145 (1), 67–77. 17. GeY.S.; TaiS.X.;XuZ.Q.; LaiL.;TianF.F.; LiD.W.; JiangF.L.; LiuY.;GaoZ.N., Synthesis of Three Novel Anionic Gemini Surfactants and Comparative Studies of Their

Assemble

Behavior

in

the

Presence

of

Bovine

Serum

Albumin,Langmuir,2012,28, 5913–5920. 18. FendlerJ.H., Membrane Mimetic Chemistry, Wiley, New York, 1982. 19. KunitakeT., in: MittalK.L., BothorelP. (Eds.), Surfactants in Solution, Plenum Press, New York, 1986. 20. VieiraO.V.; HartmannD.O.; CardosoC.M.;OberdoerferD.;BaptistaM.; SantosM.A.; AlmeidaL.;Ramalho-SantosJ.;VazW.L., Surfactants as microbicides and contraceptive agents: a systematic in vitro study,PLoS ONE, 2008,3, e2913. 21. HenryG.D.; SykesB.D., Methods to study membrane protein structure in solution, Methods Enzymol, 239, 1994, 515–535. 22. RiversR.C.;KumitaJ.R.;TartagliaG.G.;DedmonM.M.;PawarA.;VendruscoloM.; DobsonC.M.; ChristodoulouJ.,Molecular determinants of the aggregation behavior of alpha- and beta-synuclein. Protein Sci.2008,17, 887–898. 23. 7 Final report on the safety assessment of sodium lauryl sulfate and ammonium lauryl sulfate. Int J Toxicol., 1983,2, 127–281. 24. Annual review of cosmetic ingredient safety assessment – 2002/2003.Int J Toxicol.2005,24,1–102. 25. Myers, D, Surfactant science and technology. Wiley-International science: Hoboken, N.J, 2006 28 ACS Paragon Plus Environment

Page 29 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


26. AbramsG.J.; SolomonL.; MeyersO.L., A long-term study of diclophenac sodium in the treatment of rheumatoid arthritis and osteo-arthrosis, SAfr Med Jl, 1978, 53, 442-445. 27. Klabunde T.;Petrassi H. M.;Oza V. B.; Raman P.;Kelly J. W.;Sacchettini J. C., Rational design of potent human transthyretin amyloid disease inhibitors, natstructbiol, 7, 2000, 312-321. 28. TaboadaP.; BarbosaS.; CastroE.;MosqueraV., Amyloid fibril formation and other aggregate species formed by human serum albumin association, JPhysChem B, 2006,110, 20733–20736. 29. JuarezJ.;TaboadaP.;

Goy-LopezS.;CambonA.;MadecM.B.;YeatesS.G.;MosqueraV.,

Additional supra-self-assembly of human serum albumin under amyloid-like-forming solution conditions, J PhysChemB,2009,113, 12391–12399. 30. JuarezJ.;

M.Alatorre-Meda;CambonA.;TopeteA.;

BarbosaS.;TaboadaP.;

V.Mosquera,Hydration effects on the fibrillation process of a globular protein: the case of human serum albumin,Soft Matter, 2012,8, 3608–3619. 31. PandeyN.K.;GhoshS.;DasguptaS., Fibrillation in human serum albumin is enhanced in the presence of copper(II),J PhysChem B,2010,114 (31), 10228–10233. 32. Stirpe A.;PantusaM.;RizzutiB.;SportelliL.;BartucciR.;GuzziR.,Early stage aggregation of human serum albumin in the presence of metal ions, Int J BiolMacromol,2011,49, 337–342. 33. Braakman I.;Helenius J.;Helenius A., Manipulating disulfide bond formation and protein folding in the endoplasmic reticulum. EMBO J, 1992, 11, 1717–1722. 34. Konigsberg W., Reduction of disulfide bonds in proteins with dithiothreitol, Methods enzymol, 1972, 25, 185-188. 35. Clark P.;Rachinsky M. R.; Foster J. F., Moving boundary electrophoresis behaviour and acid isomerization of human mercaptalbumin, J. Biol. Chem, 1962, 237, 25092513. 36. KachooeiE.;MoosaviMovahediA.A.;KhodagholiF.;RamshiniH.;ShaerzadehF.;SheibaniN.,Oligomeric forms of insulin amyloid aggregation disrupt outgrowth and complexity of neuron-Like PC12 cells. PLoS One. 2012,7, e41344. 37. StryerL., The interaction of a naphthalene dye with apomyoglobin and apohemoglobin. A fluorescent probe of non-polar binding sites, J Mol Biol., 1965, 13,482-495.



Page 30 of 31

38. KhuranaR.; ColemanC.;Ionescu-ZanettiC.; CarterS. A.; KrishnaV.; GroverR.; Roy, R; Singh, S., Mechanism of thioflavinT binding to amyloid fibrils. J Struct Biol., 2005, 151,229-238. 39. CardamoneM.;Puri N. K.,Spectrofluorimetric assessment of the surface hydrophobicity of proteins.Biochem. J.,1992,282,589–593. 40. Krebs M. R. H.; Bromley E. H. C.; Donald A. M., The binding of thioflavin-T to amyloid fibrils: localisation and implications, J StructBiol,2005,149,30–37. 41. GroenningM.;NorrmanM.;FlinkJ. M.; Van deWeert,M.;BukrinskyJ. T.; Schluckebier, G.;FrokjearS., Binding mode of Thioflavin T in insulin amyloid fibrils, J StructBiol,2007,159, 483–497. 42. Wall, J; Murphy C.L.; Solomon, A., In vitro immunoglobulin light chain fibrillogenesis,Methods Enzymol, 1999,309,204–217. 43. LeVine, H.,3rd, Stopped-flow kinetics reveal multiple stages of thioflavin T binding to alzheimer beta (1-40) amyloid fibrils, Arch BiochemBiophys, 1997,342, 306–316. 44. Groenning,M.; Olsen,L.; van de Weert,M.;Flink,J. M.;Forkjear,S.; Jorgensen,F.,Study on the binding of thioflavin T to β-sheet-rich and non-β-sheet cavities. J StructBiol, 2007,158, 358–369. 45. Stsiapura,V.

I.;Maskevich,A.

A.;Kuzmitskyl,V.

A.;Turoverov,K.;Kuznetsova,I.,

Computational study of thioflavin T torsional relaxation in the excited state.J PhysChem A, 2007,111, 4829–4835. 46. Voropai,E.;Samtsov,M.;Kaplevskii,K.;Maskevich,A.;Stepuro,V.;Povarova,O.;Kuznet sova,I. M.;Turoverov,K.; Fink,A.;Uverskiid,V.,Spectral Properties of Thioflavin T and Its Complexes with Amyloid Fibrils, J Appl, Spectrosc, 2003,70(6),868–874. 47. Dasgupta,M.; Kishore,N.,Selective inhibition of aggregation/fibrillation of bovine serum albumin by osmolytes:Mechanistic and energetics insights, PLoS One. 2017, 12(2),e0172208. 48. Royer,C.A.,

Probing

protein

folding

and

conformational

transitions

with

fluorescence.Chem. Rev. 2006,106, 1769–1784. 49. Daniel, E.; Weber, G.,Cooperative effects in binding by bovine serum albumin. I. The binding

of

1-anilino-8-naphthalenesulfonate.

Fluorimetric

titrations,

Biochemistry, 1966,5, 1893–1900. 50. Nowakowski, A. B.;Wobig,W.; Petering,D., Native SDS-PAGE: high resolution electrophoretic separation of proteins with retention of native properties including boundmetal ions.Metallonics, 2014, 6(5),1068–1078. 30 ACS Paragon Plus Environment

Page 31 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


51. Ruiz,R.;Roses,M.;Rafols,C.; Bosch,E., Critical validation of a new simpler approach to estimate aqueous pK a of drugs sparingly soluble in water, Anal ChimActa, 2005, 550, 210–221. 52. Marcolongo, J.; Mirenda M., Thermodynamics of sodium dodecyl sulphate (SDS) micellization

:

An

undergraduate

laboratory

experiment.

J.

Chem.

Educ., 2011, 88, 629-633. 53. Kozlowska, M.; Rodziewicz, P.; Utesch, T.; Mroginski M. A.; Kaczmarek-Kedziera, A. Solvation of diclofenac in water from atomistic molecular dynamics simulations – interplay between solute-solute and solute-solvent interactions. Phys. Chem. Chem. Phys., 2018, 20, 8629−8639.

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