aggregation kinetics

Biochemical Engineering Journal 63 (2012) 38–49

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Effect of (−)-epigallocatechin-3-gallate on human insulin fibrillation/aggregation kinetics Shi-Hui Wang, Xiao-Yan Dong, Yan Sun ∗ Department of Biochemical Engineering and Key Laboratory of Systems Bioengineering of the Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China

a r t i c l e

i n f o

Article history: Received 15 August 2011 Received in revised form 5 January 2012 Accepted 5 February 2012 Available online 14 February 2012 Keywords: Biophysical chemistry Protein Aggregation Kinetic parameters (−)-Epigallocatechin-3-gallate Inhibition

a b s t r a c t (−)-Epigallocatechin-3-gallate (EGCG), a food additive derived from green tea, has been reported to effectively inhibit the fibrillation of many amyloid proteins, but not insulin. So herein, the influences of EGCG on the fibrillation kinetics of human insulin at two conditions (pH 2.0, 60 ◦ C and pH 7.4, 37 ◦ C) were extensively studied. It was found that at pH 2.0 and 60 ◦ C the inhibitory effect increased with increasing EGCG concentration from 0.35 to 3.5 mmol/L but kept almost unchanged from 3.5 to 5 mmol/L EGCG. The addition of EGCG reduced the length and width of fibrils and kept part of insulin from fibrillation at this condition. At pH 7.4 and 37 ◦ C, however, EGCG altered the fibrillation pathway of insulin and redirected it into globular aggregates, and the inhibitory effect of EGCG on the aggregation reached maximum at about 0.1–0.2 mmol/L. In this case, part of insulin molecules were prevented from aggregation and existed as a mixture of monomer, dimer, tetramer, and hexamer in the solution. Circular dichroism spectroscopy indicated that EGCG slowed down the changes of the secondary structures of insulin in the aggregation. Finally, two physical models were proposed to explain the molecular interactions between insulin and EGCG at the two conditions. The research has clarified the kinetic mechanism of the inhibitory effect of EGCG on insulin fibrillation/aggregation. © 2012 Elsevier B.V. All rights reserved.

1. Introduction The misfolding/unfolding of amyloidogenic proteins under stressful conditions can lead to the formation of amyloid fibrils consisting of ␤-sheet rich structures, which is the hallmark of many diseases such as familial amyloid polyneuropathy, Alzheimer’s disease, Parkinson’s disease, and prion-associated encephalopathies [1–3]. Up to now, nearly 25 proteins and peptides that share no sequence homology have been identified as amyloidogenic proteins, like amyloid ␤-peptide (A␤), ␣-synuclein (␣S), insulin [4], islet amyloid polypeptide (IAPP) [5], transthyretin (TTR) and so on [6,7]. Though the fibrillation of these proteins is all based on a nucleation-dependent polymerization model, the aggregation mechanisms may vary due to different initial states of these proteins. For example, A␤ and ␣S are disordered and unfolded before aggregation [8], therefore the aggregation starts from a folding process. Insulin and TTR exist as natively folded oligomers, so they undergo dissociation and partially unfolding to form monomeric amyloidogenic intermediate before aggregation [3,9]. Besides, A␤ and ␣S are causative factors in diseases, but for

∗ Corresponding author. Tel.: +86 22 27404981; fax: +86 22 27406590. E-mail address: [email protected] (Y. Sun). 1369-703X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2012.02.002

insulin, the in vivo fibrillation is rare and does not seem to pose serious medical problems [10]. Fibrillar form of insulin was reported to be found at the site of frequent insulin injections [11]. In vitro, insulin is prone to fibrillation upon exposure to elevated temperatures, low pH, organic solvents, and agitation [9,12,13]. The amyloid fibril formed by insulin is characterized by ␤-sheet-rich structure [14,15] that binds to Congo Red [16] and Th T [17]. The kinetics of fibril formation can be described as a typical nucleation-dependent polymerization model involving two steps, nucleation and fibril elongation, characterized by lag time and fibrillation rate, respectively [18,19]. Therefore, prevention of insulin fibrillation can not only improve and optimize the therapeutic use of insulin but also shed some new light on the common aggregation mechanisms of similar amyloid proteins. (−)-Epigallocatechin-3-gallate (EGCG) (see Fig. 1 for the chemical structure) is a naturally occurring polyphenol derived from green tea and it is more stable at lower pH values [20]. It has been reported that EGCG binds to many proteins, such as ␣- and ␤-caseins, A␤, ␣S, and IAPP, by non-covalent and non-specific interactions and affects their functions [6,21–23]. Since EGCG contains three aromatic rings and eight hydroxyl groups (Fig. 1), it is inferred that the interactions between EGCG and proteins are mainly hydrogen bonding and hydrophobic interaction. Recently,

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Fig. 1. Chemical structure of EGCG.

this has been further verified by Hasni et al. [21] through FT-IR spectroscopy. With respect to insulin, the binding of EGCG has also been proved by isothermal titration calorimetry and dynamic light scattering experiments [24]. In addition, McGraw and Lindenbaum [25] reported that the interactions involved in the binding of phenols to insulin are mainly hydrogen bonding and hydrophobic interaction. Since EGCG is a polyphenol, it is thus considered that the interactions between EGCG and insulin are mainly mediated by hydrogen bonding and hydrophobic interaction. Though EGCG has been proved to prevent the fibrillation of many amyloid proteins, like A␤, ␣S [25], and IAPP [22], its behavior on the fibrillation of insulin is unclear. So we have herein studied the kinetics of insulin fibrillation/aggregation in the absence and presence of EGCG at different concentrations. The experiments were conducted either in 20% acetic acid (pH 2.0) at 60 ◦ C or phosphate buffered saline (pH 7.4) at 37 ◦ C (physiological condition), respectively [9]. The kinetics of fibril formation was determined by spectrophotometric measurement at 600 nm and the morphology was detected by transmission electron microscopy (TEM). The amounts of insulin in the supernatant were detected by size-exclusion chromatography (SEC) as a function of time. Furthermore, since conformational changes are commonly suggested to be the first stage in the fibrillation of amyloid proteins [26,27], in which partially destabilized and aggregation-prone structures appear [28], the effect of EGCG on the secondary structure of insulin was detected by circular dichroism (CD) spectroscopy. It is anticipated that the research could shed some new light on the molecular mechanisms for EGCG binding to insulin.

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Fig. 2. Effect of EGCG concentration on the fibrillation kinetics of insulin at pH 2.0 and 60 ◦ C. Insulin concentration was 2 mg/mL and EGCG concentrations are given in the figure. Experiments were carried out in 20% (v/v) acetic acid with 100 mmol/L NaCl (pH 2.0) at 60 ◦ C. Each experiment was repeated three to five times and the data were acquired as many as possible. All of the data were averaged and used for regression to Eq. (2), but only part of them were plotted in the figure to make them concise and clear.

[29,30]. Herein, the relationship between A600 and the dilution ratio for the insulin samples at the end of the kinetic experiments were measured to check whether A600 can be used as a direct measure of the mass of aggregated insulin. As a result, it is observed that the A600 values are in proportional to the reciprocal of dilution ratio in all trials (Fig. S1). Since the reciprocal of dilution ratio is proportional to the mass of aggregated insulin, the result confirms that A600 is linearly related to the mass of aggregated insulin in the absence and presence of EGCG at either pH 2.0, 60 ◦ C or pH 7.4, 37 ◦ C. Namely, A600 can be used to monitor insulin fibrillation/aggregation in this work. All solutions used for the fibril formation experiments were freshly prepared in glass vials prior to each experiment. Insulin solution was added to the same volume of EGCG solution to make a final concentration of 2 mg/mL insulin (0.34 mmol/L). The final concentrations of EGCG were in the range of 0–5.0 mmol/L. The sample was incubated either in 20% (v/v) acetic acid (pH 2.0) with 100 mmol/L NaCl at 60 ◦ C or in 10 mmol/L phosphate buffer (PB) containing 100 mmol/L NaCl at 37 ◦ C with an agitation of 150 rpm. At appropriate time intervals, the vial was first gently shaken to distribute the sample evenly, and then the sample was removed from the incubator and assayed by a Lambda 35 UV/VIS spectrophotometer (Perkin-Elmer, Shelton, CT, USA) at 600 nm to determine the A600 values. Each experiment was repeated three to five times and the data were acquired as many as possible. All of the data were averaged and used for regression to Eq. (2), but only part of them were plotted in Figs. 2 and 3 to make them concise and clear.

2. Materials and methods 2.1. Materials Zinc-free human insulin (99.9%) was a generous gift of Gan&Lee Pharmaceutical Ltd. (Beijing, China). EGCG was purchased from Sigma (St. Louis, MO). Phosphotungstic acid was purchased from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China). Other chemicals were all of the highest purity available from local sources. 2.2. Fibrillation kinetics The absorbance assay at 600 nm (A600) has been extensively used to determine the fibrillation kinetics of amyloid proteins

2.3. Analysis of kinetic parameters The fibrillation kinetics of insulin could be described as a sigmoidal time-dependent curve sequentially involving three stages: an initial lag phase, where no change in A600 intensity, a subsequent growth phase where A600 increases rapidly with time, and a final equilibrium phase, where A600 reaches a plateau indicating the end of fibril formation [31]. Therefore, the A600 intensities in this article were plotted as a function of incubation time and fitted by a sigmoidal curve described by Eq. (1) suggested by Nielsen et al. [31]. Y = yi + mi t +

yf + mf t 1 + e−[(t−t0 )/]

(1)

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S.-H. Wang et al. / Biochemical Engineering Journal 63 (2012) 38–49 Table 1 Effect of EGCG concentration on the kinetic parameters of insulin fibrillation at pH 2.0 and 60 ◦ C. EGCG (mmol/L)

T (h)

0 0.35 0.70 1.05 3.50 5.00

5.5 6.2 6.6 7.5 8.1 8.4

± ± ± ± ± ±

0.4 0.4 0.6 0.7 0.6 0.6

kapp (h−1 )

Ymax

± ± ± ± ± ±

1.10 0.98 0.93 0.74 0.67 0.65

2.8 2.4 1.8 1.6 1.3 1.1

0.3 0.3 0.2 0.2 0.2 0.1

yi ± ± ± ± ± ±

0.10 0.10 0.95 0.04 0.03 0.03

−0.01 0.01 −0.01 0 0 0

± ± ± ± ± ±

0.01 0 0.01 0 0 0

Insulin concentration was 2 mg/mL and EGCG concentrations are given in the table. Experiments were carried out in 20% (v/v) acetic acid with 100 mmol/L NaCl (pH 2.0) at 60 ◦ C.

2.6. Transmission electron microscopy Fig. 3. Effect of EGCG concentration on the fibrillation/aggregation kinetics of insulin at pH 7.4 and 37 ◦ C. Insulin concentration was 2 mg/mL and EGCG concentrations are given in the figure. Experiments were carried out in 10 mmol/L PB containing 100 mmol/L NaCl (pH 7.4) at 37 ◦ C with an agitation of 150 rpm. Each experiment was repeated three to five times and the data were acquired as many as possible. All of the data were averaged and used for regression to Eq. (2), but only part of them were plotted in the figure to make them concise and clear. The other lines are drawn to guide the eye.

where Y is the A600 intensity, t is time, t0 is the time to 50% of maximal A600, and yi , mi , yf , mf ,  are all coefficients. To keep the fitting lines horizontal, mi and mf are set as 0 [32]. Therefore, Eq. (1) is simplified to Eq. (2). Y = yi +

yf 1 + e−[(t−t0 )/]

(2)

Thus, the apparent rate constant for the growth of fibrils (kapp ), the lag time (T), and the A600 maximum (Ymax ) can be derived from Eq. (1) as follows. kapp =

1 

(3)

T = t0 − 2

(4)

Ymax = yf + yi

(5)

2.4. Size-exclusion chromatography Insulin sample was first centrifuged at 12,000 rpm for 10 min, and then the supernatant was filtered with a polyether sulfone membrane (nominal pore size, 0.45 ␮m). The insulin and EGCG left in the supernatant were separated by size-exclusion chromatography (SEC) with a Superdex 75 10/300 GL column equipped on the AKTA basic 100 system (GE Healthcare, Uppsala, Sweden). Before injection to the column, the supernatant at pH 2.0 was immediately diluted 10 times to pH 3 to avoid column corrosion. The column effluent was monitored at 280 nm. 2.5. Far-UV circular dichroism Far-UV CD spectra were collected using a Jasco 810 spectrophotometer (Jasco Inc., Tokyo, Japan) from 250 to 190 nm. Samples obtained at different time internals were injected into a 0.1-mm path length quartz cuvette. A background CD spectrum of buffer solution was subtracted from the sample spectra for baseline correction. Spectra were recorded using a resolution of 0.5 nm and a scanning speed of 100 nm/min, with a response time of 1 s and a bandwidth of 2 nm. Spectra presented were an average of three consecutive measurements. The spectra were analyzed by Jascow32, provided by the manufacturer. The percentages of ␣-helix, ␤-sheet, turn, and unordered coil of insulin were calculated by data fitting using SELCON3 (http://dichroweb.cryst.bbk.ac.uk/html/home.shtml).

Insulin samples were diluted to proper concentrations with deionized water and then ultrasounded for 20 min for fully dispersing. After that an aliquot of 10 ␮L sample was placed on grids covered by a carbon stabilized formvar film. After air drying, the grids were negatively stained with 0.5% (w/v) phosphotungstic acid in ethanol. The grids were then examined and photographed using a JEM 100CXII transmission electron microscopy (JEOL Inc., Tokyo, Japan). 2.7. Measurement of hydrodynamic radius The hydrodynamic radii of insulin at pH 2.0 and pH 7.4 were measured by dynamic light scattering (DLS) with Zetasizer Nano series (Malvern Instruments Ltd., Worcestershire, UK) at 25 ◦ C. The path length of the cuvette is 10 mm. Triplicate measurements were done and the averaged value of hydrodynamic radius was presented. 3. Results and discussion 3.1. Starting states of insulin at different pH values It is known that pH greatly influences the fibrillation kinetics of insulin by changing its oligomeric state [33]. Herein, the starting states of insulin were determined by DLS and SEC. The DLS results show that the hydrodynamic radius of insulin is 1.37 ± 0.08 nm at pH 2.0 and 2.69 ± 0.24 at pH 7.4. Since the radius of insulin monomer is 1.36 nm [34], it is speculated that insulin exists as monomer at pH 2.0 and dimer at pH 7.4. Further, the SEC results indicate that the elution volume of insulin is 15.5 mL at pH 2.0 and 12.2 mL at pH 7.4 (Fig. S2, black lines). By comparison with the elution volumes of different molecules on Superdex 75 10/300 GL column reported by the manufacturer (see the caption to Fig. S2 for the website), it is concluded that 15.5 mL elution volume stands for insulin monomer (molecular weight, 5807) and 12.2 mL elution volume stands for insulin dimer (molecular weight, 11,614). Thus, from the DLS and SEC data, it can be concluded that insulin exists as monomer at pH 2.0 and dimer at pH 7.4 before the kinetic experiments. 3.2. Fibrillation kinetics at pH 2.0 and 60 ◦ C The fibrillation kinetic curves in the absence and presence of EGCG at different concentrations are shown in Fig. 2 and the kinetic parameters derived from the fitting to Eq. (2) are listed in Table 1. It can be seen from the table that the lag time (T), the apparent rate constant (kapp ), and the A600 maximum (Ymax ) for insulin fibrillation alone are 5.5 h, 2.8 h−1 , and 1.1, respectively. The T and kapp are in good agreement with the literature values of 6.1 h and 2.6 h−1 , respectively [9]. By the addition of EGCG at 0.35–3.5 mmol/L, T

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increases and kapp as well as Ymax decrease with increasing EGCG concentration. However, further increase of EGCG concentration from 3.5 to 5 mmol/L has little effect on T, kapp , and Ymax . This phenomenon may be due to the fact that, at high EGCG concentrations, the excess EGCG cannot fully bind to insulin due to steric hindrance. More experiments at EGCG > 5 mmol/L could not be conducted due to the solubility limit of EGCG. Therefore, it can be concluded that the inhibitory effect increases with the increase of EGCG concentration from 0.35 to 3.5 mmol/L and become almost constant at 3.5–5 mmol/L. Besides EGCG, many other organic molecules were also reported to have inhibitory effect on the fibrillation of amyloid protein. On the one hand, Nayak et al. [29] reported that stabilizing osmolytes (sugars) slowed down the fibrillation of human insulin at pH 1.6 and 65 ◦ C; Arora et al. [35] suggested that some other osmolyte molecules such as ectoine, betaine, trehalose, and citrulline inhibited the fibrillation of bovine insulin at pH 2.0 and 50 ◦ C in varying degrees. Although the concentrations of insulin they used were the same as in our experiments, the concentrations of the osmolytes were 300 mmol/L, over 60 times higher than EGCG (0–5 mmol/L) used in this work. Moreover, Nayak et al. [29] also reported that sugars prolonged the lag time of insulin fibrillation up to 2 h, which is similar with the effect of EGCG, but they only decreased Ymax by less than 0.2, smaller than that of EGCG (0.37 at 5 mmol/L). Namely, the inhibitory effect of EGCG on the fibrillation of insulin is larger than those of the osmolytes. This can be explained by the preferential exclusion theory of stabilizing osmolytes from a peptide backbone [36], which results in the stabilizing effect on the native state of insulin, prolonging the lag time and finally delaying the fibrillation. As for EGCG, however, it slows down the fibril formation by preferentially binding to proteins [37]. As a result, the Ymax values are significantly reduced by EGCG, but were only little changed by sugars (see Fig. 2 in Ref. [29]). On the other hand, a group of polyphenols like catechins and flavonols were found to inhibit the fibrillation of carboxymethylated-␬casein and A␤ with a half maximal inhibitory concentration (IC50 ) of about 50 ␮mol/L or lower [38]. Recently, Thapa et al. [39] reported that biflavonoids are superior to monoflavonoids in inhibiting A␤ fibrillation with a decrease of Thioflavin T fluorescence intensity to about 50–60%. Moreover, several nonsteroidal anti-inflammatory drugs (NSAIDs) and structurally similar compounds, including flufenamic acid, diclofenac, flurbiprofen, and resveratrol, ortho-trifluoromethylphenyl anthranilic acid, and N(meta-trifluoromethylphenyl) phenoxazine 4,6-dicarboxylic acid, were found to inhibit the fibrillation of TTR [7]. The TTR fibrils were reduced to about 3–30%, depending on different NSAIDs. In summary, the order of the inhibitory effect is NSAIDs > polyphenols > osmolytes. One of the possible reasons might be that NSAIDs contain nitrogen, fluorine, and/or chlorine atoms, while polyphenols do not.

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Table 2 Effect of EGCG concentration on the kinetic parameters of insulin fibrillation/aggregation at pH 7.4 and 37 ◦ C. EGCG (mmol/L)

T (h)

kapp (h−1 )

Ymax

yi

0 0.001 0.005 0.01 0.05 0.10 0.20 0.35 0.70

50 ± 3 50 ± 3 51 ± 4 52 ± 5 53 ± 5 NAa NA 52 ± 4 52 ± 4

0.3 ± 0.1 0.3 ± 0.1 0.2 ± 0.0 0.2 ± 0.0 0.1 ± 0.0 NA NA 0.2 ± 0.0 0.2 ± 0.0

1.25 ± 0.05 1.08 ± 0.05 0.95 ± 0.02 0.54 ± 0.02 0.12 ± 0.01 NA NA 0.25 ± 0.01 0.36 ± 0.02

0.04 ± 0.01 0.03 ± 0.01 0.02 ± 0.01 0.01 ± 0 0 ± 0.01 NA NA −0.01 ± 0 −0.01 ± 0

Insulin concentration was 2 mg/mL and EGCG concentrations are given in the table. Experiments were carried out in 10 mmol/L PB containing 100 mmol/L NaCl (pH 7.4) at 37 ◦ C with an agitation at 150 rpm. a Data are not available in that the weak A600 intensity data cannot get a good fit to Eq. (2).

Fig. 4. Effect of EGCG on the far-UV circular dichroic spectra of insulin in the fibrillation at pH 2.0 and 60 ◦ C. Insulin concentration was 2 mg/mL and EGCG concentrations were 0 (a) and 1.05 mmol/L (b), respectively. The incubation times from solid lines [1] to [9] were 0, 3, 5, 7, 8, 9, 10, 11, and 13 h, respectively. Experiments were carried out in 20% (v/v) acetic acid with 100 mmol/L NaCl (pH 2.0) at 60 ◦ C.

indicating that the inhibitory effect is positively related to EGCG concentration in the range of 0–0.10 mmol/L EGCG. At EGCG concentrations of 0.10 and 0.20 mmol/L, the A600 is nearly 0, indicating that the fibrillation of insulin is almost completely suppressed by EGCG (Fig. 3). Since the A600 values are very small at the two EGCG concentrations, good fittings cannot be obtained from Eq. (2). By further increasing EGCG concentration from 0.20 to 0.70 mmol/L, it is found that Ymax turns to increase to some extent

3.3. Fibrillation/aggregation kinetics at pH 7.4 and 37 ◦ C The effect of EGCG concentration on the fibrillation/aggregation of insulin at pH 7.4 and 37 ◦ C was investigated to study the interactions of EGCG and insulin at physiological condition. The aggregation kinetic curves in the absence and presence of EGCG at different concentrations are shown in Fig. 3 and the kinetic parameters derived by fitting to Eq. (2) are given in Table 2. Table 2 lists the T and kapp values of insulin fibrillation without EGCG as 50 h and 0.3 h−1 , respectively, which are close to the literature values of 49.4 h and 0.2 h−1 , respectively [9]. It can be seen that the influences of EGCG on T and kapp are very small. However, Ymax rapidly decreases from about 1.25 to 0.02 with the increase of EGCG concentration from 0 to 0.10 mmol/L,

Fig. 5. Effect of EGCG on the far-UV circular dichroic spectra of insulin in the fibrillation/aggregation at pH 7.4 and 37 ◦ C. Insulin concentration was 2 mg/mL and EGCG concentrations were 0 (a), 0.01 (b), and 0.10 mmol/L (c), respectively. The incubation times from solid lines [1] to [9] were 0, 20, 45, 48, 51, 54, 57, 60, and 65 h, respectively. Experiments were carried out in 10 mmol/L PB with 100 mmol/L NaCl (pH 7.4) at 37 ◦ C with an agitation of 150 rpm.

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and the values become even larger than those at 0.05 mmol/L. This suggests that EGCG has dual effects, i.e. inhibition and acceleration, on the fibrillation of insulin. Similar results were also found with other agents. For instance, apoliprotein E4 (Apo E4) inhibits

the fibrillation of A␤ at low Apo E4/A␤ ratio and accelerates the fibrillation at high Apo E4/A␤ ratio [40–42]. In contrast, LVEALYL, a small peptide coming from the B-chain of insulin, was found to accelerate the fibrillation of insulin at low LVEALYL/insulin ratio,

Fig. 6. Effect of EGCG on the morphology of insulin in the fibrillation at pH 2.0 and 60 ◦ C. Insulin concentration was 2 mg/mL and EGCG concentrations of were 0 (a) and 1.05 mmol/L (b), respectively. Experiments were carried out in 20% (v/v) acetic acid with 100 mmol/L NaCl (pH 2.0) at 60 ◦ C.

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43

Fig. 6. (Continued)

but inhibit fibril formation at high LVEALYL/insulin ratio [15]. ␣1 Antichymotrypsin was found to accelerate the fibrillation of A␤ at ␣1 -antichymotrypsin/A␤ < 1:100, but inhibit the fibril formation at ␣1 -antichymotrypsin/A␤ = 1:10 [42,43]. To explore the mechanism behind the phenomenon, extensive experiments at higher EGCG concentrations were carried out. It was

found that EGCG > 1 mmol/L immediately induced insulin aggregation, which made the kinetic experiments impossible. Hence, it is concluded that at the physiological condition high-concentration EGCG can induce insulin aggregation, but the molecular mechanism is unclear. It will be a subject of further investigations. Anyway, the dual effect of EGCG on insulin aggregation leads to the

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presence of the optimum EGCG concentration (0.10–0.20 mmol/L) at which the inhibitory effect of insulin fibrillation reaches maximum.

3.4. Size-exclusion chromatography The aim of SEC experiments is to check whether all the insulin in the solution was eventually incorporated into fibrils. The results in Fig. S2a and S2c show that, in the absence of EGCG, the amount of insulin decreases with incubation time and finally all insulin incorporates into fibrils at both pH 2.0, 60 ◦ C and pH 7.4, 37 ◦ C. By the addition of EGCG, part of the insulin molecules are kept as monomer in the supernatant at the end of the fibrillation process at pH 2.0 and 60 ◦ C (Fig. S2b). Since pH 2.0 is too acidic to be stood by the column, samples were immediately diluted 10 times to pH 3 before injection. Hence, the results can only be used for estimation. At pH 7.4 and 37 ◦ C (see Fig. S2d and S2e), part of the insulin molecules

are also kept from aggregation by EGCG, but they exist as a mixture of monomer, dimer, tetramer, and hexamer. Since the forms of insulin are not simplex, its mass cannot be easily quantified either. As a result, it can only be qualitatively concluded from the comparisons of Fig. S2d, S2e and Fig. 3 that the better the inhibitory effect, the more insulin kept in the supernatant. Consequently, it can be concluded from Figs. 2 and 3 and S2 that the decreased Ymax stems from the fact that part of insulin molecules are not involved in the aggregation and kept in the supernatant by the addition of EGCG.

3.5. Far-UV circular dichroism Figs. 4a and 5a show the changes of secondary structure of insulin during the fibrillation without EGCG at the two solution conditions. It can be observed that insulin mainly maintains ␣-helical structure (double minima at 208 and 222 nm) at the

Fig. 7. Effect of EGCG on the morphology of insulin in the fibrillation/aggregation at pH 7.4 and 37 ◦ C. Insulin concentration was 2 mg/mL and EGCG concentrations were 0 (a), 0.01 (b), and 0.10 mmol/L (c), respectively. Experiments were carried out in 10 mmol/L PB containing 100 mmol/L NaCl (pH 7.4) at 37 ◦ C with an agitation of 150 rpm.

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Fig. 7. (Continued)

beginning of the incubation [44] and the structure almost does not change during the lag phase (solid lines [1]–[3]). In the fibril growth phase, however, the secondary structure of insulin gradually changes from ␣-helix to ␤-sheet (solid lines [4]–[7]), as characterized by the minimum at 216–218 nm. Finally, the CD spectra become unchanged, indicating that the secondary structure of insulin becomes constant at the end of fibrillation (solid lines [8] and [9]). These results are similar to those conducted at pH 2.5 and 57 ◦ C reported by Malisauskas et al. [17]. By the addition of EGCG (Figs. 4b, 5b and c), the changes of CD spectra are blocked in different degrees. To quantitatively analyze these changes, the CD data were fitted to SELCON3 to obtain the percentages of ␣-helix, ␤-sheet, turn, and unordered coil and the results are listed in Table S1. The data show that EGCG slows down the decrease of ␣-helix and the increase of ␤-sheet at both conditions.

In addition, by comparing the solid lines [1] in Figs. 4a and 5a, it can be found that the CD spectra of insulin at pH 2.0, 60 ◦ C and pH 7.4, 37 ◦ C are somewhat different, i.e., the spectrum at pH 7.4 and 37 ◦ C shows double minima at 208 and 222 nm, indicating a typical ␣-helical structure of insulin, while the minimum at 222 nm at pH 2.0, 60 ◦ C is not obvious. This phenomenon can be explained by the fact that though low pH does not cause any irreversible distortion of the native structure of insulin, low pH and high temperature result in chemical degradation of some amino acid residues such as Asn [9]. As a result, the secondary structures of insulin display a little difference at the two conditions. 3.6. Transmission electron microscopy The effect of EGCG on the morphology of insulin in the fibrillation/aggregation was investigated by TEM and the images at

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Fig. 7. (Continued)

two different conditions (pH 2.0, 60 ◦ C and pH 7.4, 37 ◦ C) are shown in Figs. 6 and 7, respectively. It can be observed from Fig. 6a that, there is no sizable object in solution at 0 h. After 3h incubation, insulin assembles into insoluble aggregates. After 5 h, protofibers start appearing with a length of about 50–100 nm. Fibrils occur at about 7 h and they only elongate from the two ends before 9 h, leading to fibrils of about 1 ␮m in length with an almost unvaried width. After 10 h, fibrils both grow from the two ends and assemble into bundles. At the end of the dynamic process (13 h, Fig. 6a), the network of fibrils with widths of about 100–200 nm is formed. Since the fibrils are very dense and extend across the images, exact lengths of them were not determined. By the addition of EGCG (1.05 mmol/L), as shown in Fig. 6b, the fibrils become shorter and thinner than those without EGCG at the same incubation time. At the end of the fibrillation (13 h, Fig. 6b), the fibrils formed are about 1–3 ␮m in length and 10–50 nm in width, which is obviously shorter and thinner than those without

EGCG. It can also be seen that the network of the fibrils is much thinner than that without EGCG. By comparing Figs. 6a and 7a, it can be found that the morphologies of insulin alone in the fibrillation are similar at pH 2.0, 60 ◦ C and pH 7.4, 37 ◦ C, except that the fibrils incubated at the physiological condition are thinner and shorter. However, by the addition of EGCG, the morphologies are essentially changed as compared to those without EGCG. As shown in Fig. 7b and c, globular aggregates appear instead of protofibers and fibrils. The final particle size is about 100–150 nm at 0.01 mmol/L of EGCG and 10–15 nm at 0.1 mmol/L of EGCG. Namely, it can be concluded that, at the physiological condition, EGCG not only prevents the fibrillation of insulin, but also alters the aggregation pathway and induces insulin into globular aggregates. Similar results with other small organic molecules and proteins have been reported in literature. For example, Kanapathipillai et al. [45] studied the effects of ectoine and hydroxyectoine on the

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fibrillation of A␤ in 10 mmol/L HCl with 5% (v/v) DMSO at 37 ◦ C and found that A␤ aggregated into insoluble particles in the presence of ectoine or hydroxyectoine; Nieva et al. [46] studied the effects of lipid-derived aldehydes on the fibrillation of antibody light chains at pH 7.4 and 37 ◦ C and reported that these aldehydes accelerated the aggregation of the light chain and induced them into amorphous aggregates; Ehrnhoefer et al. [37] studied the effects of EGCG on the fibrillation of A␤ and ␣S at pH 7.4 and 37 ◦ C and found that 0.1 and 1 mmol/L of EGCG induced the two amyloid proteins into amorphous aggregates. Meng et al. [22] reported that EGCG disaggregates IAPP fibrils into amorphous aggregates at pH 7.4 and 25 ◦ C. Those literature results, together with that in Fig. 7, indicate that many small organic molecules can alter the fibrillation pathway of amyloid proteins and redirect them into non-fibrillar aggregates. 3.7. Further discussion on the inhibition mechanisms The above results have shown that at pH 2.0 and 60 ◦ C the inhibitory effect of EGCG firstly increases with the increase of EGCG concentration and then reaches a plateau at about 3.5 mmol/L EGCG. EGCG both shortens and thins the fibrils and keeps part of insulin molecules from fibrillation. At pH 7.4 and 37 ◦ C, there is an optimum EGCG concentration (about 0.1–0.2 mmol/L) at which the inhibitory effect reaches the largest. In this case, EGCG changes the morphology of insulin aggregates from fibrils to globular ones and keeps dissolved insulin molecules as a mixture of monomer, dimer, tetramer, and hexamer. In addition, EGCG slows down the change of the secondary structure of insulin during the aggregation. Based on the results reported in this work and those in literature [9,37], two physical models are proposed to explain the different effects of EGCG on the fibrillation/aggregation of insulin at the two conditions. (1) At pH 2.0 and 60 ◦ C, insulin exists as monomer before fibrillation (Fig. 8a). In the absence of EGCG, it partially denatures, nucleates, and grows into fibrils (Fig. 8b–d). By the addition of EGCG, it binds to insulin, slows down the denaturation of insulin (Fig. 8e and f), and hinders the growth of fibrils by disturbing the interactions between insulin molecules, making the fibrils shorter and thinner (Fig. 8g). Besides, the binding of EGCG also inhibits part of insulin molecules from aggregation and keeps them in the solution (Fig. 8g), resulting in the decrease of Ymax . Insulin’s net charge is 5.4 at pH 2.0, so the relatively large electrostatic repulsion between insulin molecules prevents them from assembling into oligomers. As a result, the dissolved insulin molecules exist as monomer in the solution (Fig. 8g). (2) At pH 7.4 and 37 ◦ C, insulin mainly displays as dimer before aggregation (Fig. 9a). Without EGCG and under agitation, insulin first dissociates into monomer and then partially denatures, nucleates, and grows into fibrils (Fig. 9b–d). Since insulin’s net charge is −1.9 at pH 7.4, the electrostatic repulsion between them is relatively small. Therefore, when EGCG is added, it binds to part of the dimeric insulin and acts as bridges to assemble insulin molecules into tetramer and/or hexamer, which slows down the dissociation and denaturation of insulin. Since the molar concentration of EGCG is lower than that of dimeric insulin, EGCG can only bind to part of insulin (Fig. 9e and h). At the same time, the EGCG-free insulin molecules dissociate into monomer, partially denature, and nucleate (Fig. 9f and i), similar with that in Fig. 9c. Since native insulin is stable at physiological condition and does not self-associate [9,31], it is assumed that insulin oligomers can associate with denatured monomeric insulin and/or nuclei but cannot self-associate. Therefore, the insulin oligomers, denatured monomeric insulin, and nuclei associate with each other and form globular aggregates (Fig. 9g and j). At 0.01 mmol/L EGCG, the

Fig. 8. Schematic diagram of the aggregation kinetics of insulin in the absence and presence of EGCG at pH 2.0 and 60 ◦ C.

EGCG-free insulin molecules are more than those at 0.1 mmol/L EGCG, resulting in more nuclei and larger globular aggregates (Fig. 9g). At 0.1 mmol/L EGCG, more dimeric insulin is bound to EGCG and the nuclei are fewer, leading to relatively small aggregates (Fig. 9j). Anyway, since the native insulin oligomers do not interact with each other, part of them are kept in the solutions and lead to the decrease of Ymax at the two EGCG concentrations. The above mechanisms can also explain the CD results. Namely, at pH 2.0 and 60 ◦ C, insulin exists as monomer and can directly denature, so the effect of EGCG on the changes of insulin’s secondary structure is relatively small (Fig. 4 and Table S1). However, at pH 7.4 and 37 ◦ C, EGCG binds to insulin dimer and bridges them into oligomer, which hinders the denaturation and slows down the changes of CD spectra (Fig. 5 and Table S1). Especially at 0.1 mmol/L EGCG, most of insulin oligomers are left in the solution, leading to almost unchanged CD spectra (Fig. 5c).

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Fig. 9. Schematic diagram of the aggregation kinetics of insulin in the absence and presence of EGCG at pH 7.4 and 37 ◦ C.

4. Conclusions In this paper, the effects of EGCG on the fibrillation of insulin at two different conditions were investigated by TEM, size-exclusion chromatography, and CD spectroscopy. The results indicate that at pH 2.0 and 60 ◦ C the inhibitory effect increases with the increase of EGCG concentration at 0.35–3.5 mmol/L EGCG, and then keeps almost unchanged at 3.5–5 mmol/L EGCG. At this condition, the fibrils become shorter and thinner upon the addition of EGCG. However, at pH 7.4 and 37 ◦ C, there is an optimum EGCG concentration (about 0.1–0.2 mmol/L) at which the inhibitory effect reaches the largest. In this case, the morphology of insulin aggregation is essentially changed by EGCG from fibrils to globular aggregates. The addition of EGCG prevents part of insulin molecules from aggregation at both conditions, but they exist as monomer at pH 2.0, 60 ◦ C and a mixture of monomer, dimer, tetramer, and hexamer at pH 7.4 and 37 ◦ C. Moreover, the spectroscopic results suggested that EGCG slows down the change of the secondary structure of insulin during the fibrillation/aggregation. Based on

the results, two physical models were proposed to explain the molecular interactions between insulin and EGCG at the two conditions. The research has clarified the kinetic mechanism of the inhibitory effect of EGCG on insulin fibrillation/aggregation and would contribute to the development of more effective inhibitors for the fibrillation of similar amyloid proteins. Acknowledgements This work was supported by the Natural Science Foundation of China (20876111), National Basic Research Program of China (973 Program, No. 2009CB724705), and the Program of Introducing Talents of Discipline to Universities (No. B06006). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bej.2012.02.002.

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