Heparin Accelerates the Protein Aggregation via the Downhill Polymerization Mechanism: Multi-Spectroscopic Studies to Delineate the Implications on Proteinopathies.

Heparin is one of the members of the glycosaminoglycan (GAG) family, which has been associated with protein aggregation diseases including Alzheimer's disease, Parkinson's disease, and prion diseases. Here, we investigate heparin-induced aggregation of bovine serum albumin (BSA) using different spectroscopic techniques [absorption, 8-anilino-1-naphthalene sulfonic acid (ANS) and thioflavin T (ThT) fluorescence binding, and far- and near-UV circular dichroism]. Kinetic measurements revealed that heparin is involved in the significant enhancement of aggregation of BSA. The outcomes showed dearth of the lag phase and a considerable change in rate constant, which provides conclusive evidence, that is, heparin-induced BSA aggregation involves the pathway of the downhill polymerization mechanism. Heparin also causes enhancement of fluorescence intensity of BSA significantly. Moreover, heparin was observed to form amyloids and amorphous aggregates of BSA which were confirmed by ThT and ANS fluorescence, respectively. Circular dichroism measurements exhibit a considerable change in the secondary and tertiary structure of the protein due to heparin. In addition, binding studies of heparin with BSA to know the cause of aggregation, isothermal titration calorimetry measurements were exploited, from which heparin was observed to promote the aggregation of BSA by virtue of electrostatic interactions between positively charged amino acid residues of protein and negatively charged groups of GAG. The nature of binding of heparin with BSA is very much apparent with an appreciable heat of interaction and is largely exothermic in nature. Moreover, the Gibbs free energy change (ΔG) is negative, which indicates spontaneous nature of binding, and the enthalpy change (ΔH) and entropy change (ΔS) are also largely negative, which suggest that the interaction is driven by hydrogen bonding.

Introduction

Heparin [glycosaminoglycan (GAG) or heteropolysaccharides] is an extremely acidic sulfur-containing polysaccharide, composed of linear chains of repeating units of disaccharides comprising glucosamine and uronic acid.[1,2] It belongs to the family of GAGs or heteropolysaccharides and occurs in the liver, kidney, spleen, lungs including basophils and mast cells in the blood vessels, etc. It acts as the blood thinner that prevents blood from coagulation or blood clotting.[1,2] Structurally, heparin is formed from alternating units of N-sulfo D-glucosamine 6-sulfate and glucoronate 2-sulfate.[1]

Heparin has been suggested to accelerate the formation of amyloid fibrils of Aβ peptides which are the major agents involved in Alzheimer’s disease.[3] It has also been found to increase the aggregation of tau protein, which is one of the major aggregating proteins responsible for causing Alzheimer’s disease.[4] GAGs are consistently observed to be related with amyloid deposition in major amyloidosis diseases.[5] In vitro studies suggest that GAGs including heparin induce the phenomenon of amyloid formation in α-synuclein, which is the main aggregating protein in Parkinson’s disease.[5] Prion is a proteinaceous infectious isoform present ubiquitously throughout the mammalian body, especially in neurons, which can get converted into either misfolded protein or amyloids or amorphous aggregates through altering the conformation or shape.[6] The diseases associated with prions and affect the brain (encephalopathies) are called prion diseases or transmissible spongiform encephalopathies (TSEs).[7,8] Heparin has been related to misfolding and aggregation of prion protein.[9] Besides these instances, there is promotion of fibrillation rate in the various other proteins including transthyretin,[10] apomyoglobin,[11] gelsolin,[12] acyl carrier protein,[11] β-microglobulin,[4] and high-density lipoprotein-associated serum[13] due to heparin sulfate. Heparin has been suggested to increase the fibrillation of proteins and also stabilizes already present aggregates against proteolytic degradation,[14] and there are pieces of evidence which suggest that heparin is associated with both amyloid fibril formation and stabilization.[11] Some current studies suggest that when there is deposition of heparin in the serum, the level of amyloid A also elevates.[11] The loss of amyloid formation deposition is directly associated with inhibition of the biosynthesis of heparin sulfate.[11] Moreover, various studies have shown that the substantial level of polysaccharides including GAGs gets deposited in the tissues of the human body. Alzheimer’s disease, TSEs or prion diseases (including Creutzfeldt–Jakob disease, Gerstmann–Straussler syndrome, and scrapie), light-chain amyloidosis, and type II diabetes are some debilitating human complications which have been associated with the deposition of heparin.[15,16] However, both in vivo and in vitro, there are several reports that suggest that heparin inhibits protein aggregation. It was reported that there is inhibition of aggregation of insulin by heparin when aggregation occurs at low ionic strength and near its isoelectric point.[13,17] The different GAGs such as dermatan sulfate, chondroitin sulfate, hyaluronic acid, and chitin which are derived from heparin are referred to as heparinoids.[18] There are some pieces of evidence which suggest that heparin and heparinoids are involved in the suppression of cancer enhancement through serving the potent anti-cancer agent.[19] Heparin and these different types of GAGs are present in both extracellular and intracellular compartments of the cell. Heparin and associated different types of GAGs had been reported to accelerate the aggregation of various intracellularly existing peptide hormones. In the human body, any further normal physiological role associated with heparin molecules is still not known, and also, the exact mechanism behind heparin-induced protein aggregation is still not known. Thus, the investigation of aggregation behavior of bovine serum albumin (BSA) in the presence of heparin is an excellent topic to focus.

There are many suppositions which have been proposed to enlighten the phenomenon associated with heparin-induced aggregation, but the exact mechanism behind heparin-induced fibrillation is not fully deciphered and is also a topic to debate. Protein aggregation is the phenomenon of generic propensity among proteins, and its exact mechanism is still not deciphered. This phenomenon is involved in breaking the cellular quality-control mechanism and is associated with a number of human diseases such as Alzheimer’s disease, Parkinson’s disease, prion diseases (bovine spongiform encephalopathy and Creutzfeldt–Jakob disease), amyotrophic lateral sclerosis, Huntington’s disease, Down’s syndrome, cataract, and sickle cell disease.[20,21] These diseases are also referred to as proteopathies or proteinopathies, protein conformational disorders, or protein misfolding diseases.[22] According to some recent reports of 2019, the world’s suffering from Alzheimer’s disease is 50 million,[23] while that from Parkinson’s disease is 10 million.[24] The polypeptide folds into a functional, three-dimensional structure by a process called protein folding. The biological phenomenon in which protein fails to fold into native conformation and transform into an inactive and misfolded state is called protein misfolding. The misfolded protein conformation causes polymerization into aggregates which are either intracellular or extracellular, grow with time monotonically, and are functionally toxic or pathogenic in nature, causing a range of human pathological diseases. There are several factors such as temperature, pH, ionic strength, concentration of protein, denaturants, surfactants, and viscosity of solution which affect the phenomenon of protein aggregation.[25−28] The protein aggregation is also driven by the same forces which are responsible for stabilization of protein folding.[29] As a result, it is not possible to maintain the normal intermolecular interaction for the normal growth and development of the organism as there are instances wherein the abnormal interaction of proteins has resulted into the formation of misfolded or aggregated proteins, the cytotoxic effects of which are directly linked to pathologies of Alzheimer’s disease, Parkinson’s disease, etc.[30] The phenomenon of aggregation of biologically functional protein causes loss of functional activity, posing hindrances in various fields of research.[30] Thus, the major significance of these studies look at different structural changes in proteins that enroot to aggregation, the mechanism of action of different additives and different complications due to aggregates, in the fields of medicine, biotechnology, pharmaceuticals, industry, and food technology.[31]

Although BSA protein is not related to human diseases, still, it is an appropriate and excellent model to study in vitro protein aggregation. The structure of BSA is almost similar to that of human serum albumin (HSA). Under physiological conditions of pH and temperature, this protein results in amyloid-like fibrils. Moreover, it is an important transport protein in the circulatory system.[32] Inducing and preventing protein aggregation in vitro are important to study the different aspects of pathological complications caused by protein aggregation.[33] Therefore, the aim of this study was to investigate the aggregation behavior of BSA by inducing its aggregation at high temperature in the presence of heparin. The results from this study are likely to give an understanding of the mechanistic insights on the heparin-induced protein aggregation; for this, various in vitro approaches such as absorption,[34] fluorescence,[35] and circular dichroism (CD)[36] spectroscopies were exploited. In addition, to explain the nature of interaction of heparin with the protein and to understand the mechanism of action, binding studies were carried out using isothermal titration calorimetry (ITC).[37]

Results

Kinetic Measurements of BSA Aggregation in the Presence of Increasing Concentration of Heparin

The kinetics of BSA aggregation in the presence of heparin was checked by measuring the turbidity of the protein solution at 600 nm through time course measurements using a spectrophotometer. The kinetic experiment was done at a fixed 10 μM protein concentration solution in the presence of varying concentrations of heparin starting from 5 to 50 μM. Figure A shows the results of aggregation kinetics of BSA in the presence of increasing concentration of heparin at 60 °C. Figure A and Table depict that in the presence of heparin, the absorbance of BSA protein increases significantly from 0.0079 to 0.182 at 60 °C. Thus, it is evident that heparin increases the aggregation of BSA significantly. As the concentration of heparin is increased, the tendency for aggregation increases. The process of heparin-induced aggregation is depicted by a sigmoidal curve which is characterized by the negligible lag or nucleation phase, followed by the rapid growth phase and then ultimately the saturation phase. It should be noted that BSA did not show any aggregation in the presence of heparin at 25 °C (data not shown). The plot of maximum absorbance (Amax) versus concentration of heparin at 60 °C indicates that the heparin-induced aggregation is linearly dependent on the concentration shown in Figure B. The mechanism of aggregation was delineated using the log–lin plot of log absorbance versus time and the log–log plot of log absorbance versus log time shown in the Figure A,B. Using eq ,[21] the change in various kinetic parameters associated with the heparin aggregation of BSA was also calculated and is shown in Table .

Figure 1

(A) Aggregation kinetics of BSA (10 μM) in the presence of increasing concentration of heparin at 60 °C. (B) Effect of heparin concentration on the maximum absorbance (Amax) of heparin-induced BSA aggregation at 60 °C.

Table 1

Change in Kinetic Parameters Such as Maximum Absorbance (Amax), Initial Absorbance (yo), Rate Constant (b), Lag Time (tlag), and Reciprocal of Rate Constant (kapp min–1) Associated with Aggregation of BSA in the Presence of Varying Concentrations of Heparin at a Temperature of 60 °C

S. no.	[BSA + heparin], μM	a	y0	b (min–1)	t1/2 (min–1)	tlag (min–1)	kapp (min–1)
1	[10 + 0]	0.007 (±0.0005)	0.0023 (±0.0001)	6.69 (±0.68)	20.7 (±1.2)	7.00 (±0.41)	0.15 (±0.009)
2	[10 + 5]	0.032 (±0.004)	0.0022 (±0.0003)	3.66 (±0.09)	13.08 (±0.70)	5.75 (±0.004)	0.27 (±0.02)
3	[10 + 10]	0.062 (±0.001)	0.0015 (±0.0004)	1.94 (±0.004)	5.95 (±0.03)	2.16 (±0.03)	0.52 (±0.031)
4	[10 + 20]	0.068 (±0.0090)	0.0049 (±0.0007)	2.02 (±0.005)	6.2 (±0.04)	2.15 (±0.067)	0.50 (±0.04)
5	[10 + 30]	0.108 (±0.011)	0.0007 (±0.000013)	1.45 (±0.06)	4.85 (±0.013)	1.4 (±0.07)	0.68 (±0.06)
6	[10 + 40]	0.144 (±0.014)	0.0012 (±0.0009)	1.18 (±0.04)	3.73 (±0.20)	1.4 (±0.093)	0.84 (±0.057)
7	[10 + 50]	0.182 (±0.017)	0.0050 (±0.00002)	1.10 (±0.05)	3.75 (±0.021)	1.5 (±0.081)	0.90 (±0.063)

Figure 2

(A) Log–lin plot of absorbance vs time and (B) log–log of absorbance vs time for aggregation kinetics of BSA (10 μM) in the presence of increasing concentration of heparin at 60 °C.

Influence of Heparin on the Secondary and Tertiary Structure of BSA

Far-UV Circular Dichroism Measurements

To observe the structural changes in the secondary structure of BSA, the far-UV CD measurements were performed in which 5 μM BSA was first titrated with increasing concentrations of heparin, as shown in Figure A,B, at temperatures of 25 and 60 °C, respectively. The percentage change of the secondary structure of BSA in the presence of heparin was calculated through the secondary structural estimation application in JASCO’s Spectra Manager software to obtain the fraction of the helix, β-sheet, and random coil using Yang’s reference[38−40] in response to heparin at 25 and 60 °C, and the results are shown in Tables and 3, respectively.

Figure 3

(A) Far-UV CD spectra of BSA (5 μM) in the presence of increasing concentration of heparin at 25 °C. (B) Far-UV CD spectra of BSA (5 μM) in the presence of increasing concentration of heparin at 60 °C.

Table 2

Total Percentage Associated with the Change of the Secondary Structure of BSA in the Presence of Varying Concentrations of Heparin at 25 °C

S. no.	[BSA + heparin], μM	helix	β	random coil
1	5 + 0	59 (±3)	17.6 (±0.92)	18 (±1)
2	5 + 1	59 (±2)	17.2 (±0.89)	18 (±1)
3	5 + 5	59 (±3)	15.2 (±0.93)	20 (±1)
4	5 + 10	59 (±3)	12.1 (±0.94)	23 (±1)
5	5 + 20	60 (±3)	15 (±1)	20 (±1)
6	5 + 25	60 (±2)	15 (±1)	20 (±1)
7	5 + 30	60 (±3)	14 (±1)	20 (±1)
8	5 + 45	60 (±3)	14 (±1)	21 (±1)
9	5 + 50	61 (±4)	13.7 (±0.980)	20 (±2)

Table 3

Total Percentage Associated with the Change of the Secondary Structure of BSA in the Presence of Varying Concentrations of Heparin at 60 °C

S. no.	[BSA + heparin], μM	helix	random coil
1	5 + 0	57 (±4)	18 (±1)
2	5 + 10	64 (±6)	35 (±3)
3	5 + 20	58 (±5)	41 (±4)
4	5 + 30	61.6 (±5)	38 (±2)
5	5 + 50	55 (±4)	45 (±2)
6	5 + 60	58.1 (±2)	49.9 (±3)
7	5 + 80	60.6 (±5)	39.4 (±3)
8	5 + 90	59.0 (±4)	41.0 (±3)

Near-UV Circular Dichroism

To observe the structural changes in the tertiary structure of BSA in the presence of increasing concentration of heparin at 60 °C, the near-UV CD experiment was performed. Figure shows near-UV CD spectra of BSA in the presence of heparin at 60 °C. There is an inset in Figure , which shows the plot of the change in the mean residual ellipticity at 263 nm versus the concentration of heparin at 60 °C. It is seen in this figure that there is a considerable change in the CD signal, so the tertiary structure of protein was changed by heparin.

Figure 4

Near-UV CD spectra of BSA (20 μM) in the presence of increasing concentration of heparin at 60 °C. The inset in the figure shows the plot of the change in the mean residual ellipticity at 263 nm vs the concentration of heparin at 60 °C.

Fluorescence Measurements

The intrinsic fluorescence spectra of BSA in the presence of increasing concentration of heparin at 25 and 60 °C were recorded and are depicted in Figure A,B, respectively. The inset in Figure A shows a plot of maximum fluorescence of BSA at 347 nm, F347, versus concentration of heparin at 25 °C. The inset in Figure B shows a plot of maximum fluorescence of BSA at 346 nm, F346, versus concentration of heparin at 60 °C. F347 (25 °C) and F346 (60 °C) in the absence of heparin and in the presence of heparin were also calculated (see Table ). It is clear from Figure A,B that the fluorescence intensity of BSA is increased significantly upon addition of heparin, but the fluorescence intensity increased more at 60 °C upon addition of heparin.

Figure 5

(A) Fluorescence spectra of BSA (5 μM) in the presence of increasing concentration of heparin at 25 °C. The inset in (A) shows the plot of maximum fluorescence of BSA intensity at 347 nm vs concentration of heparin at 25 °C. (B) Fluorescence spectra of BSA (5 μM) in the presence of increasing concentration of heparin at 60 °C. The inset in (B) shows the plot of maximum fluorescence of BSA intensity at 346 nm vs concentration of heparin at 60 °C.

Table 4

Maximum Change in Structural Properties of BSA in the Absence and Presence of Heparin, 25 mM Phosphate Buffer, and pH 7.0

S. no.	structural change	protein without heparin	protein with heparin
1	F347 (at 25 °C)	97 (±5)	138 (±7)
2	F346 (at 60 °C)	236 (±8)	626 (±12)
3	ThT binding (Imax)	84 (±6)	120 (±8)
4	ANS binding (Imax)	234 (±9)	274 (±11)
5	[θ]208, deg cm2 dmol–1 (at 25 °C)	–20,786 (±7)	–23,071 (±210)
6	[θ]208, deg cm2 dmol–1 (at 60 °C)	–113,032 (±150)	–654 (±12)
7	[θ]263, deg cm2 dmol–1 (at 60 °C)	123 (±5)	–95 (±4)

Thioflavin T Binding Assay

Thioflavin T (ThT) fluorescence spectra in the presence of BSA–heparin are depicted in Figure A. The experiment of ThT binding was demonstrated at increasing concentration of heparin (0–10 μM); however, we showed only the final concentration effect of heparin (i.e., 10 μM) on the protein in Figure A. The excitation wavelength of the experiment was kept at 450 nm. The ratio of ThT with protein BSA was kept at 1:20 (5 μM BSA/100 μM ThT) throughout the ThT binding experiment in 25 mM phosphate buffer at pH 7.0. Figure A shows an overall significant increase of ThT fluorescence intensity in the presence of BSA–heparin.

Figure 6

(A) Fluorescence spectra of BSA (5 μM) in the presence of ThT and heparin. (B) Fluorescence spectra of BSA (5 μM) in the presence of increasing concentration of ANS and heparin.

8-Anilino-1-naphthalene Sulfonic Acid Fluorescence Assay

Figure B depicts the 8-anilino-1-naphthalene sulfonic acid (ANS) fluorescence spectra in the presence of BSA–heparin. The experiment of ANS binding was done at an excitation wavelength of 360 nm. The ratio of ANS to BSA was kept at 1:20 (5 μM BSA/100 μM ANS) throughout the ANS binding experiments in 25 mM phosphate buffer at pH 7.0. Figure B also displays the overall substantial increase in the ANS fluorescence intensity of BSA in the presence of BSA–heparin.

Isothermal Titration Calorimetry Measurements

To know the binding affinity, thermodynamic parameters, and type of interaction in this bimolecular binding between BSA and heparin, ITC measurements were taken. There are fewer reports in the literature regarding the study of energetics of interaction of inducers and inhibitors of aggregation of protein. Figure shows the titrated ligand (heparin) against the cell containing BSA. The upper-side panel of this figure shows the thermogram with raw data in power versus time, while the lower-side panel depicts the raw data in the power standardization to the amount of injections (kcal mol–1) versus its molar ratios with the addition of consecutive injections of the ligand to the protein. The quantity of heat released as a function of the mole ratio of the ligand to protein is depicted in the lower-side panel. The isotherms showing the profiles of heat change were fitted by the Origin software, which was provided by VP-ITC. The different parameters such as values of association constant (Ka), binding enthalpy (ΔH), and the equilibrium constant (Kd) associated with the ITC thermogram of BSA–heparin are given in Table . By using eq , the free-energy change (ΔG) was calculated. From the binding affinity equilibrium, the dissociation constant was also calculated (i.e., Kd = 1/Ka), as shown in Table .

Figure 7

Isothermal calorimetric titration of BSA protein (30 μM) in the presence of ligand heparin (900 μM), displaying calorimetric response as successive injection of ligand heparin added to the reaction cell (upper panel), and the resulting binding isotherms (lower panel) are shown for reverse titration at 25 °C.

Table 5

Thermodynamic Binding Parameters of BSA with Heparin Estimated from ITC Measurements at pH 7.0 and 25 °C

thermodynamic parameters (units)	Ka (M–1)	ΔH (cal mol–1)	ΔS (cal mol–1 deg–1)	ΔG (cal mol–1)
step 1	6.71 × 105 (±1.20 × 104)	–262.24 × 103 (±4.511 × 103)	–853	–8.046 × 103

Discussion

Generally, there are various factors by which protein aggregation can be affected including mutations in the cell, aging of cells, errors occurring during the process of transcription and translation, that is, protein synthesis, and various stress conditions in the cell such as temperature, pH, and oxidative stress triggered by free radicals in the cell.[41−43] Temperature is the one of the key factors on which protein stability is dependent.[44] Proteins exposed to higher temperatures result in their unfolding, and unfolded protein molecules interact with each other through their exposed hydrophobic residues, resulting into the formation protein aggregates.[44] Here, we investigated the effect of heparin on the protein aggregation. The results show that BSA does not aggregate at 60 °C in the absence of heparin. Heparin induces aggregation at 60 °C. The results obtained from the kinetics of thermal aggregation of BSA (at 60 °C) in the presence of heparin shown in Figure A depict that there is promotion of aggregation of BSA by heparin with an increase in absorbance and a decrease in lag phase or with no lag phase. Heparin-induced aggregation of BSA at 60 °C was observed to be linearly dependent on the concentration.

Usingeq , the change in various kinetic parameters such as rate constant (b), maximum absorbance (Amax), initial absorbance (yo), lag time (tlag), and reciprocal of rate constant (kapp min–1), associated with aggregation of BSA in the presence of heparin were calculated at 60 °C, as shown in Table . It is seen in the table that on increasing the concentration of heparin, there is an increase in aggregation of BSA as the maximum absorbance (Amax) increases from 0.0079 to 0.182, the rate constant (b) decreases from 6.69 to 1.10, the time at which the absorbance is half of its maximum (t1/2) decreases from 20.7 to 3.75 min–1, the lag time (tlag) also decreases from 7.00 to 1.5 min–1, and the reciprocal of rate constant (kapp min–1) increases from 0.15 to 0.90 min–1.

There are two important pathways which have been suggested to describe the mechanism of protein aggregation, that is, the nucleation-dependent polymerization mechanism and the downhill polymerization mechanism. However, the actual reason behind why some proteins follow the pathway of the nucleation-dependent polymerization mechanism, whereas some follow the pathway of the downhill polymerization mechanism, is still unexplored. To categorize the mechanism as nucleation-dependent polymerization or downhill polymerization, the log–lin plot of absorbance versus time and log–log of absorbance versus time can be used. This type of comparative mechanism was proposed by Librizzi and Rischel in 2005.[21]Figure A,B for the BSA–heparin system shows two phases of aggregation, that is, the growth or elongation phase and the stationary phase. By comparing the two plots, it is clearly observed from Figure A,B that the early growth phase curves of the log–log plot are straighter lines than the log–lin plot, which represents aggregation of BSA in the presence of heparin, indicating the downhill polymerization process. The pattern of heparin-induced aggregation of BSA can be characterized by the sigmoidal growth curve, which comprises negligible or no lag or nucleation phase and a well-defined growth phase, and finally stabilized by a saturation phase. Moreover, the non-existence of a well-established nucleus and a lag phase during the aggregation kinetics has also been found to be the characteristic property of amyloidogensis of other proteins such as acylphosphatase and β-2 microglobulin.[45] The other proteins such as serum albumins, human serum albumin(HSA), porcine serum albunin (PSA), sheep serum albumin (SSA), rabbit serum albumin (RSA),[46,47] zinc- and calcium-binding protein,[48] and head-induced protofibril by barstar are some examples in which aggregation has also been revealed without any lag phase.[49,50] The nucleation and growth of aggregation of BSA were affected by heparin. The kinetics of aggregation of BSA with a dearth of nucleation or lag phase in the presence of heparin indicates that the aggregation mechanism does not involve classic nucleation-dependent polymerization but involves the pathway of downhill polymerization.[51,52] In the downhill polymerization, the process starts without any addition of a high-energy multimeric nucleus or does not involve the rate-limiting step where each step is irreversible and is not dependent on the concentration of the monomer, and this is referred to as classic coagulation. This type of mechanism is independent of the initial size of the molecule and can also be described by a relative change in one quantity that results in a proportional relative change in another through power law growth.[49,53−55] In the presence of heparin, the charge on the surface of the BSA molecule may be minimized, which results in an increase in the rate of association of protein molecules, thus promoting the protein aggregation, which is also the cause of the absence of the lag phase. The abolishment of the lag phase can also be due to the accumulation of an adequate number of preformed fibrils or seeds.[56] Thus, we hypothesize that heparin acts as the template which is involved in association of monomers or oligomer proteins, thus abolishing the lag phase of BSA aggregation. Moreover, the nucleation phase is characterized by an unfavorable step in the form of a bottleneck (i.e., delay in the process of aggregation), and this step is regulated by the concentration and size of aggregated species.[57]

The effect of heparin on the secondary structure of BSA was studied by using far-UV CD measurements. This measurement ranges from 250 to 200 nm and relates the absorption of the peptide bond which has an asymmetric conformation. Therefore, molecules having an asymmetric conformation exhibit the phenomenon of CD. Importantly, far-UV CD gives knowledge regarding the secondary structure of protein which comprises the α-helix and β-sheet, turn, and random coil.[58]Figure A,B depicts the far-UV CD measurements to determine the effect of heparin on the secondary structure of BSA at temperatures of 25 and 60 °C, respectively. The total percentage associated with the change of the secondary structure of BSA in the presence of varying concentrations of heparin at 25 and 60 °C were also calculated and is shown in Tables and 3. From Figure A,B and Tables and 3, it is clear that a significant change in the secondary structure of the protein in the presence of increasing concentration of heparin was observed. At 25 °C, the ellipticity of protein at 208 nm changes approximately from −20,786 to −23,071 in the presence of heparin, as shown in Figure A and Tables and 4. On the other hand, a complete shift in the secondary structure of the protein at 60 °C in the presence of increasing concentration of heparin was observed, as shown in Figure B and Tables and 4. The ellipticity of protein at 208 nm changes approximately from −113,033 to 654 in the presence of increasing concentration of heparin at the 60 °C temperature, as shown in Tables and 4. Far-UV CD measurements of BSA at 60 °C in the presence of heparin show that there is the loss of the peak at 220 nm accompanied by shifting spectral of protein and the formation of a new peak at 230 nm at 60 °C. The peak at 230 nm is suggestive of aggregation or disordered proteins. This disordered conformation is unstructured to partially structured, dominated by random coils and pre-molten globules,[59] and similar to intrinsically disordered proteins (IDPs) which are without a regular secondary structure or have an absence of a three-dimensional structure and is detected by CD as “random coil”, “unordered”, or “disordered”. This study may be extrapolated to some IDPs or provide some clues to diseases caused by protein misfolding.[60−64] The disordered secondary structure mainly with the α-helix formation from the random coil is reported by other surfactants such as sodium dodecyl sulfate titration of acid-induced denatured cytochrome c as revealed by far-UV CD measurements.[65,66] Moreover, studies had also described that this peak at 230 nm in the far-UV CD measurement of protein indicates that tryptophan is involved in the cation−π interactions with other residues of proteins.[67,68] Also, aggregation studies on the monoclonal antibody using the CD spectroscopic approach showed the peak around 230 nm. The study concluded that this shift has been due to perturbation and rearrangement of the secondary structure of the protein, which confirms that it is due to aggregation.[69] On the other hand, near-UV CD spectra at 60 °C in the presence of heparin at 60 °C depict that there are considerable changes in the CD signal. The ellipticity of protein at 263 nm changes from −123 to −95 in the presence of heparin. Thus, it can be concluded that the secondary structure and tertiary structure of protein get altered significantly in the presence of heparin.

The intrinsic fluorescence measurements of BSA due to the presence of aromatic amino acids (predominantly tryptophan) showed an increase in the fluorescence emission in the presence of heparin, which depicts the significant change in the protein at 25 °C (see Figure A). These changes in values of fluorescence intensity are from ∼97 to ∼138 at 25 °C upon addition of heparin, which are given in Table . Moreover, it was observed that the figure shows an isosbestic point where protein in the absence and presence of heparin shows similar characteristics of the structure throughout the bimolecular reaction. On the other hand, intrinsic fluorescence measurements of BSA in the presence of increasing [heparin] showed a massive increase in fluorescence emission change at 60 °C (see Figure B). The value of fluorescence intensity changes from ∼236 to ∼627 at 60 °C due to heparin, as given in Table .

Protein folding intermediates, surface hydrophobicity, and aggregation or fibrillation of proteins can be investigated through fluorescent dyes such as ANS and ThT.[70] ANS is a fluorescent probe used for investigation of changes in conformation, molten-globule formation, folding and unfolding of proteins, and amorphous aggregate formation.[11,49] It does not bind with the native (which usually does not have exposed hydrophobic patches) and the denatured state (which is highly mobile) but recognizes the exposed hydrophobic sites of proteins in the aggregates.[71] The results obtained from the ThT binding assay can be used to confirm the existence of amyloid aggregates (ordered aggregates dominated by the β-sheet structure) of BSA in the presence of heparin. When the ThT dye comes in the contact with BSA in the presence of heparin, there is a significant increase in ThT fluorescence intensity. The ThT fluorescence intensity of BSA increases from ∼84 to ∼120 in the presence of heparin (see Figure A and Table ). Thus, it is clear from this assay that there is also the formation of amyloids of BSA in the presence heparin. Amyloids are insoluble, heterogeneous, and resistant to degradation and structurally dominated by β-sheets and are readily found in the Alzheimer’s disease and prion diseases.[3,72] Further, the results from ANS measurement assays showed that when ANS interacts with BSA, there is a marked increase in ANS fluorescence intensity from ∼234 to ∼275 (see Figure B and Table ). This significant enhancement in the ANS fluorescence intensity indicates the existence of some specific binding sites located on BSA for ANS. It has been reported that at pH 7.0, BSA possesses five hydrophobic sites for binding the ANS dye.[49,73] ANS binding to BSA in the presence of heparin also showed a major boost in the ANS fluorescence intensity. This infers us that the increase in the ANS fluorescence intensity indicates the formation of amorphous aggregates.[49,73] Nitani et al. observed the enhancement of amorphous aggregates in the hen egg white lysozyme, while a slight enhancement of amyloid aggregates was also seen in the protein in the presence of heparin.[74] This study was comparable to the above literature studies as the results show the formation of amorphous aggregates and amyloid aggregates in the presence of heparin. External stress factors always turn the native functional proteins into an unfolded state where hydrophobic regions are exposed, which changes the thermodynamically stable states. This subsequently causes the misfolding-coupled irreversible protein aggregation. Although ITC may not provide direct information about protein aggregation, surely, it may provide some crucial information about the interaction of the protein with heparin that may give some clues toward the mechanism of aggregation. The information obtained from ITC about the interaction may serve the useful approach for establishing the treatment and averting the different diseases associated with protein aggregation.[75,76]Figure B illustrates a typical ITC thermogram obtained from titration of BSA with heparin. The nature of binding of heparin with BSA is largely exothermic in nature with negative heat pulses, as shown in the upper panel. The ITC thermogram data provide best fitting with the one-site binding model. Furthermore, from the data obtained from the ITC measurement, it was evaluated that each heparin molecule interacts with 2.5 molecules of BSA protein, equivalent to 20–25 monosaccharide units of heparin.[77] The propensity to prompt any conformational change in the protein molecule has been proposed to be reliant on the size of the heparin molecule. It has also been observed that there should be at least 10–14 heparin monosaccharide units for each heparin molecule which makes it necessary to assist as a template that leads to aggregation.[77]

Heat is an important factor to study quantitative and qualitative thermodynamic behavior during the comprehensive study of protein aggregation.[76] The type and formation of aggregates can be determined through observing the heat reaction properties such as sign, magnitude, pattern, and shape on the thermogram. Such heat reaction properties are associated with the aggregation kinetics of proteins and are also useful in the development of molecules which act as inhibitors of protein aggregation. Heat is an important factor to study quantitative and qualitative thermodynamic behavior during the comprehensive study of protein aggregation.[76] In our case, the binding affinity between heparin and BSA protein is very much apparent with an appreciable heat of interaction or a specific heat pattern. The enthalpy change is largely negative with an overall exothermicity of ∼ ΔH = −262.24 × 103 cal mol–1, and the change in Gibbs free energy (ΔG) is also negative, which indicates that the reaction is spontaneous in nature and ΔH and ΔS are largely negative, representing that binding is of heparin with BSA and is driven by hydrogen bonding interactions,[78] as shown in Table . This exothermic nature can be directly associated with aggregation of BSA induced by heparin as there are already reports which suggest that aggregation of protein is a exothermic in nature.[79] It has been observed that exothermic heat is associated with the growth of amyloid fibrils in the seed-dependent aggregation process in β2-microglobulin.[76] There are other instances including heparin–apomyoglobin, which are most likely with electrostatic interactions and with exothermic heat of interaction. It has also been proposed that exothermic binding interactions are expected as a result of various interactions of positively charged amino acid residues of protein with the negatively charged sulfate and carboxyl groups of heparin.[80] It is already reported that BSA protein comprises three domains with different charge densities that affect the adsorption on its surface. There is an existence of negative charge on the entire BSA at the neutral pH; still, one domain holds negatively charged amino acids such as glutamic acid or aspartic acid (approximately constitutes 18%) and the other domain holds positively charged amino acids such as lysine or histidine which are (approximately constitutes 14%) with 4.8 pI. Thus, BSA is negatively charged at pH 7.2 or positively charged below pH 4.7.[81−83] It has been observed that heparin is the only biological molecule that possesses the maximum negative charge density of any other identified molecule in the living system.[84] Therefore, the positively charged side chains of BSA and negatively charged groups of GAG, that is, heparin, bind through electrostatic interactions, resulting into the formation of the protein–GAG complex.[60,85−88] Such a type of complex, especially the tau–GAG complex, is also supposed to be the major promoter involved in the formation of proteinopathies including Alzheimer’s disease and tauopathies. There are many sulfate moieties present in a regularly spaced pattern on the heparin molecule with which the protein molecule may interact to form a heparin–protein complex. These interactions within the protein molecules can result in shielding of charge–charge repulsion moderately, causing the promotion of a local concentration of proteins to induce nucleation, hence triggering the aggregation, and this type of aggregation is referred to as facilitated aggregation. This facilitated aggregation has been observed to change the orientation of the protein molecule which is referred as per se aggregation. Reports suggest that both these processes accelerate the oligomerization and fibril formation of proteins.[4] It has been suggested that it is due to the high content of sulfate groups in heparin which are negatively charged, protein aggregation is promoted.[11] Thus, the result obtained from the ITC measurements validates the results observed from aggregation kinetic studies using spectrophotometric measurements.

Conclusions

Kinetic measurements revealed that heparin accelerates aggregation of BSA significantly. There is dearth of the lag phase and a considerable change in rate constant, which provides the conclusive evidence that the BSA aggregation mechanism in the presence of heparin involves the pathway of downhill polymerization. Heparin showed a considerable change in the secondary and tertiary structure of the protein. There is a considerable enhancement of fluorescence intensity of BSA due to heparin. The results from the ITC measurement showed that electrostatic interactions (between positively charged amino acid residues of protein and negatively charged groups of GAG) and protein–GAG complexes were the key factors leading to aggregation. The nature of binding of heparin with BSA was largely exothermic, spontaneous, and driven by hydrogen bonding. Thus, our in vitro novel study provides exclusive evidence regarding the inherent effects of heparin on the aggregation behavior of BSA; hence, there is a chance that the serum albumin in the human blood may get aggregated on interaction with heparin under in vivo conditions too. Thus, our findings can also provide a platform to understand the impact of heparin on the aggregation of proteins, which can help us to develop a heparin-based drug against protein aggregation diseases including Alzheimer’s disease, Parkinson’s disease, and prion diseases.

Materials and Methods

Materials

Chemicals such as lyophilized BSA (UniProtKB-A0A140T897_BOVIN: A0A140T897), heparin, ANS, and ThT were purchased from Sigma-Aldrich. Disodium hydrogen phosphate (Na2HPO4) and sodium dihydrogen phosphate (NaH2PO4) were purchased from Merck (India). These chemicals were used without further purification, and all the additional chemicals used were of analytical grade. All the experiments were performed in the phosphate buffer solution (25 mM) at pH 7.0, which was prepared by dissolving an appropriate amount of disodium hydrogen phosphate and sodium dihydrogen phosphate in Mill-Q water from the Millipore system. A stock solution of 15 mg/mL of BSA protein was prepared in the phosphate buffer, and its concentration was determined by taking the absorbance spectra using a Jasco V-660 UV–vis spectrophotometer at 278 nm using a molar extinction coefficient of 44,000 M–1 cm–1. The stock solution of 1 mg/mL of heparin was also prepared in the phosphate buffer at pH 7.0. A Toshcon digital pH meter CL-54 was used to determine the pH of all the above stock solutions.

Methods

Kinetic Measurements

The measurement of the kinetics of BSA was carried out using the Jasco V-660 UV–vis spectrophotometer (JASCO Corporation 2967-5, Ishikawa-machi, Hachioji-shi, Tokyo, Japan). The temperature of the spectrophotometer was regulated using a programmable Peltier-type temperature controller (ETCS61). The kinetics of BSA was obtained by the measuring the turbidity of the protein solution at 600 nm in the presence of heparin from time course measurements using the spectrophotometer. The concentration of heparin was kept at 1 mg/mL in 25 mM phosphate buffer, pH 7.0. Experiments of aggregation kinetics of BSA in the presence of heparin were performed at two different temperatures (25 and 60 °C). Usually, the turbidity assay of protein solutions is performed at wavelengths higher than 400 nm in order to avoid interference from the optical phenomena such as chromophoric absorption by the residues. It has been found that protein aggregates have a high optical density as well as high turbidity. The obtained data from measurements of kinetic aggregation were fitted with a four-parameter sigmoidal curve represented by the following equation[21]where y is the absorbance at any time t, yo is initial absorbance value, a is the maximum absorbance, t1/2 is the time at which the absorbance is half of its maximum, b is 1/kapp (reciprocal apparent rate constant), the apparent rate constant kapp is 1/b, and the lag time tlag = t1/2 – 2b.

Structural Measurements

Fluorescence Measurements

A Jasco FP-6200 spectrofluorometer (JASCO Corporation 2967-5, Ishikawa-cho, Hachioji, Tokyo 192-8537, Japan) was used for the fluorescence measurements in the 1 cm quartz cell at 25 °C, with both emission and excitation slit bandwidths of 10 nm, a data pitch 1 nm, and a scanning speed of 125 nm min–1. The temperature was regulated using an externally placed thermostated water bath. Fluorescence spectra were recorded by excitation at a wavelength of 280 nm, and the wavelength range for the emission spectra was 300–400 nm. Each sample solution prepared for experiments of structural measurements including far-UV CD, near-UV CD, and fluorescence including ANS and ThT fluorescence binding was carefully mixed and incubated overnight at a 25 °C temperature.

ThT Binding Assay

The formation of BSA amyloids with the benzothiazole salt (ThT) in the presence of different concentrations of heparin was monitored through fluorescence emission measurements using the Jasco FP-6200 spectrofluorometer in the 1 cm quartz cell at 25 °C, with both emission and excitation slit widths fixed at 10 nm, a data pitch of 1 nm, and a scanning speed of 125 nm min–1. Experiments of ThT binding were done at the excitation wavelength of 450 nm. The molar ratio of ThT with protein BSA was kept at 20:1 (100 μM ThT/5 μM BSA) for all the ThT binding experiments in 25 mM phosphate buffer at pH 7.0.

ANS Fluorescence Assay

The formation of BSA aggregates with heparin was monitored through the ANS fluorescence emission measurements using the Jasco FP-6200 spectrofluorometer at 25 °C using a 1 cm cuvette, with both emission and excitation slit widths fixed at 10 nm, a data pitch of 1 nm, and a scanning speed of 125 nm min–1.[89] Experiments of ANS binding were done at the excitation wavelength of 360 nm. The molar ratio of ANS to protein BSA was kept as 20:1 (100 μM ANS/5 μM BSA) for all the ANS binding experiments in 25 mM phosphate buffer at pH 7.0.[40,44,90]

Circular Dichroism Measurements

CD measurements were recorded on a Jasco J-1500 CD Spectropolarimeter (JASCO International Co., Ltd., Tokyo, Japan) connected to a circulatory water bath (MCB100).[91,92] Far-UV CD spectra were recorded at a protein concentration of 5 μM in a 0.1 cm path length cuvette, and near-UV CD spectra were recorded at a protein concentration of 20 μM in the 1.0 cm path length cuvette.[74,93,94]d-10-Camphorsulphonic acid was routinely used to calibrate the machine. Spectra recorded here are the averages of five scans to get an accurate signal. The raw CD signal in millidegrees at the wavelength λ was corrected for the background CD signal and changed into molar ellipticity [θ]λ by applying the following equation[37]where θλ is the molar ellipticity in millidegrees at the wavelength λ, M0 is the mean residue weight of protein, c is the concentration of the protein in grams per cubic centimeter, and l is the cell path length in centimeters.

Isothermal Titration Calorimetry Measurements

ITC is a technique used for the determination of binding affinity and thermodynamic parameters of bimolecular interactions in the solution by measuring the heat released or absorbed. A VP-ITC calorimeter (MicroCal, 22 Industrial Drive East, Northampton, MA 01060, United States) instrument was used for ITC measurements at 25 °C, in which the calorimeter cell was injected with a fixed concentration of 30 μM BSA protein in 25 mM phosphate buffer (pH 7.0). The ligand with a concentration of 900 μM heparin was titrated against the cell containing 30 μM BSA solution. The ligand solution was loaded with 10 μL aliquots in each step in 260 s through the syringe, and each ligand was loaded into the phosphate buffer as a control. The normalization of data was done with the results of titration of the respective ligands, and MicroCal Origin ITC software was used for fitting the data to produce the profile of heat change. From the measured heat changes, the stoichiometry (N), binding enthalpy (ΔH), and association constant (Ka) were calculated on binding of heparin with BSA. The standard Gibbs-free-energy changes (ΔG) were also calculated from the measured heat changes using the following equation[37]where ΔH and ΔS are the enthalpy and entropy changes, R is the gas constant, and T is the absolute temperature.