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Thioflavin T and Birefringence Assays to Determine the Conversion of Proteins into Fibrils Suzanne G. Bolder,†,‡ Leonard M. C. Sagis,‡ Paul Venema,‡ and Erik van der Linden*,‡ DMV International b.V., P.O. Box 13, 5460 BA Veghel, The Netherlands, and Food Physics Group, Department of Agrotechnology and Food Sciences, Wageningen UniVersity, P.O. Box 8129, 6700 EV Wageningen, The Netherlands ReceiVed October 17, 2006. In Final Form: January 18, 2007 The conversion of protein monomers into fibrils can be determined using the centrifugal filtration method. The results of this method were used to calibrate steady-shear birefringence and Thioflavin T fluorescence measurements. For both measurements, a linear correlation with the fibril concentration was extracted, resulting in two fast assays to determine the fibril concentration quantitatively. From birefringence measurements and the conversion determined using the centrifugal filtration method, we were able to calculate more precise values for the birefringence per unit length of the fibrils (M) and the flexibility of the fibrils (β).
Introduction Assembly of proteins into fibrils is a subject of research in a wide range of scientific areas (e.g., biomedical research, biotechnological research, and materials science).1-10 The structure of fibril assemblies is extensively studied as well as the mechanism and kinetics of fibril formation (e.g., refs 11-14). Common methods to identify the presence of fibrils are Thioflavin T (ThT) fluorescence, Congo red binding, transmission electron microscopy, and atomic force microscopy.11,13-26 These methods, * To whom correspondence
[email protected]. † DMV International b.v. ‡ Food Physics Group.
should
be
addressed.
E-mail:
(1) Bucciantini, M.; Giannoni, E.; Chiti, F.; Baroni, F.; Formigli, L.; Zurdo, J.; Taddei, N.; Ramponi, G.; Dobson, C. M.; Stefani, M. Nature 2002, 416, 507-511. (2) Dobson, C. M. Semin. Cell DeV. Biol. 2004, 15, 3-16. (3) Lowe, C. R. Curr. Opin. Struct. Biol. 2000, 10, 428-434. (4) Merkle, R. C. Trends Biotechnol. 1999, 17, 271-274. (5) Reches, M.; Gazit, E. Science 2003, 300, 625-627. (6) Uversky, V. N.; Fink, A. L. Biochim. Biophys. Acta 2004, 1698, 131-153. (7) Viney, C. Curr. Opin. Solid State Mater. Sci. 2004, 8, 95-101. (8) Waterhouse, S. H.; Gaerrard, J. A. Aust. J. Chem. 2004, 57, 519-523. (9) Zhang, S. Nat. Biotechnol. 2003, 21, 1171-1178. (10) Zhang, S.; Marini, D. M.; Hwang, W.; Santoso, S. Curr. Opin. Chem. Biol. 2002, 6, 865-871. (11) Gosal, W. S.; Clark, A. H.; Ross-Murphy, S. B. Biomacromolecules 2004, 5, 2408-2419. (12) Kelly, J. W. Nat. Struct. Biol. 2000, 7, 824-826. (13) Naiki, H.; Gejyo, F. In Methods Enzymology: Amyloids, Prions and other Protein Aggregates; Wetzel, R., Ed.; Academic Press: London, 1999; Vol. 309, pp 305-318. (14) Nielsen, L.; Khurana, R.; Coats, A.; Frokjaer, S.; Brange, J.; Vyas, S.; Uversky, V. N.; Fink, A. L. Biochemistry 2001, 40, 6036-6046. (15) Arnaudov, L. N.; de Vries, R.; Ippel, H.; van Mierlo, C. P. M. Biomacromolecules 2003, 4, 1614-1622. (16) Bromley, E. H. C.; Krebs, M. R. H.; Donald, A. M. Faraday Discuss. 2004, 128, 13-27. (17) Gosal, W. S.; Clark, A. H.; Pudney, P. D. A.; Ross-Murphy, S. B. Langmuir 2002, 18, 7174-7181. (18) Hamada, D.; Dobson, C. M. Protein Sci. 2002, 11, 2417-2426. (19) Hill, E. K.; Krebs, B.; Goodall, D. G.; Howlett, G. J.; Dunstan, D. E. Biomacromolecules 2006, 7, 10-13. (20) Ikeda, S.; Morris, V. J. Biomacromolecules 2002, 3, 382-389. (21) Kavanagh, G. M.; Clark, A. H.; Ross-Murphy, S. B. Int. J. Biol. Macromol. 2000, 28, 41-50. (22) Klunk, W. E. In Methods Enzymology: Amyloids, Prions and other Protein Aggregates; Wetzel, R., Ed.; Academic Press: London, 1999; Vol. 309, pp 285305. (23) LeVine, H. In Methods Enzymology: Amyloids, Prions and other Protein Aggregates; Wetzel, R., Ed.; Academic Press: London, 1999; Vol. 309, pp 274284. (24) Naiki, H.; Nakakuki, K. Lab. InVest. 1996, 74, 374-383.
however, do not give a quantitative measure for the concentration of fibrils in a sample.25 Recently, the formation of fibrils from food proteins has received considerable attention20,27-30 because of the potential use as functional ingredients in food products, such as thickening or gelling agents or stabilizers of foams and emulsions. Whey proteins, and specifically β-lg, are known to form fibrils upon heating at pH 2 and low ionic strength. These fibrils are semiflexible, polydisperse in length (∼1 µm), and monodisperse in cross section (∼4 nm).15,25,26,31 In this study, we have focused on whey protein isolate (WPI). WPI is a commercial product with β-lg as the main component. For industry, isolated whey proteins are not attractive because of relatively high costs of purification and implementation. Previous work has shown that WPI can be used for fundamental studies on fibril formation and that β-lg is the only whey protein involved in fibril formation in WPI under the conditions used.28,29,32 For designing and optimizing a process for fibril formation, it is desired to be able to follow the effect of various process parameters on the yield of fibril material. In previous work, we developed a method to determine the conversion of proteins into fibrils using centrifugal filtration with repeated washing and centrifugation steps.32 In this work, we determined the conversion of whey protein monomers into fibrils. The conversion of WPI into fibrils was found to increase with concentration and heating time. A drawback of the centrifugal filtration method is that it is a time-consuming and laborious assay. It would be advantageous to have faster assays to determine the fibril concentration in samples. Two possible assays are based on steady-shear birefringence measurements and Thioflavin T (ThT) fluorescence experiments. (25) Rogers, S. S.; Venema, P.; Sagis, L. M. C.; van der Linden, E.; Donald, A. M. Macromolecules 2005, 38, 2948-2958. (26) Veerman, C.; Ruis, H.; Sagis, L. M. C.; van der Linden, E. Biomacromolecules 2002, 3, 869-873. (27) Akkermans, C.; Venema, P.; Rogers, S. S.; van der Goot, A. J.; Boom, R. M.; van der Linden, E. Food Biophys. 2006, 1, 144-150. (28) Bolder, S. G.; Hendrickx, H.; Sagis, L. M. C.; van der Linden, E. Appl. Rheol. 2006, 16, 258-264. (29) Bolder, S. G.; Hendrickx, H.; Sagis, L. M. C.; van der Linden, E. J. Agric. Food Chem. 2006, 54, 4229-4234. (30) Sagis, L. M. C.; Veerman, C.; Ganzevles, R.; Ramaekers, M.; Bolder, S. G.; van der Linden, E. Food Hydrocolloids 2002, 16, 207-213. (31) Goers, J.; Permyakov, S. E.; Permyakov, E. A.; Uversky, V. N.; Fink, A. L. Biochemistry 2002, 41, 12546-12551. (32) Bolder, S. G.; Vasbinder, A. J.; Sagis, L. M. C.; van der Linden, E. Int. Dairy J. 2006, accepted for publication, doi:10.1016/j.idairyj.2006.10.002.
10.1021/la063048k CCC: $37.00 © 2007 American Chemical Society Published on Web 03/07/2007
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Table 1. Overview of Sample Treatment of the Various Sample Series Studied series R0 S0 S10 S30
WPI concentration [wt %] 2 2 2 2
treatment rest stirred stirred stirred
seedsa [wt %] 0 0 10 30
a Seeds were prepared by heating a 2 wt % WPI solution for 10 h at pH 2 at 80 °C while stirring constantly. The seeds concentration is [wt %] of the total WPI concentration.
With steady-shear birefringence measurements, the concentration of fibril material can be measured quantitatively, after calibration.25,33 In this paper, we describe ThT fluorescence experiments that were performed on the same WPI fibril solutions as used for steady-shear birefringence measurements in a previous study.33 We calibrated the steady-shear birefringence and ThT fluorescence results with the conversion as determined with the centrifugal filtration method. We were able to extract a correlation between the ThT fluorescence, the steady-shear birefringence, and the fibril concentration in our samples, resulting in two fast assays to determine the fibril concentration quantitatively. With these assays, time-consuming experiments with repeated washing and centrifugation steps can be avoided once they have been calibrated for a certain protein system, using the centrifugal filtration method. Experimental Section Sample Preparation. BiPRO whey protein isolate (WPI) was obtained from Davisco Foods International, Inc. (Le Sueur, MN). All the other chemicals used were of analytical grade. The WPI powder was dissolved and was left to stir overnight at 4 °C. The WPI stock solution was purified using the method described by Weinbreck et al.34 to remove whey protein aggregates and undissolved protein. After purification, the WPI solution was set to pH 2 using 6 M HCl. The protein concentration was determined using a UV spectrophotometer (Cary 50 Bio, Varian) and calibration curves of known protein concentrations at a wavelength of 278 nm. Absorption spectra that were determined before and after heating overlapped, indicating that the heating step caused no shift in the UV absorption. The stock solution was diluted to 2 wt % WPI with HCl solution of pH 2. Samples of 20 mL WPI solution were heated in sealed glass vessels in a metal heating plate, in combination with a magnetic stirrer, at 80 °C ((2 °C). In Table 1, an overview is given of the treatment of the various sample series studied. Samples were heated at rest (R0) or while being stirred constantly (at a rate of about 200 rpm) with a magnetic stirring bar (S0). Samples S10 and S30 were prepared with seeds added to the sample prior to heating. Seeds were prepared by heating a 2 wt % WPI solution for 10 h at pH 2 and 80 °C while stirring constantly. After heating, the sample was cooled on icewater and was kept at 0 °C overnight. Seeds solutions and unheated whey protein solutions were mixed in various ratios. Both samples (WPI seeds and unheated WPI) had the same overall WPI concentration of 2 wt %. Therefore, the total WPI concentration was constant, independent of the concentration of seeds added. The concentrations of seeds added were 0% seeds (series S0), 10% seeds (S10), or 30% seeds (S30). The seeded samples were heated at pH 2 and 80 °C while being stirred constantly. After various heating times between 0 and 24 h, samples were taken out of the heating plate and were immediately cooled on icewater. Conversion Experiments. The conversion is the percentage of aggregated protein material in the sample, that is, the percentage of (33) Bolder, S. G.; Sagis, L. M. C.; Venema, P.; van der Linden, E. Submitted for publication. (34) Weinbreck, F.; de Vries, R.; Schrooyen, P.; de Kruif, C. G. Biomacromolecules 2003, 4, 293-303.
the total protein concentration that has converted into fibrils. To determine the degree of conversion of whey protein monomers into aggregates, the centrifugal filtration method as described in Bolder et al.32 was used. The heated samples with a WPI concentration of 2 wt % were diluted to 0.1 wt % in pH 2 solution. Diluted samples (2 mL) were brought into Centricon YM-100 centrifugal filters (100 kDa molecular weight cutoff, Millipore Corporation, Bedford, MA) and were filtered by centrifuging at 1000g for 30 min. The concentration of nonaggregated protein present in the filtrate was determined with a UV spectrophotometer at a wavelength of 278 nm. The retentate was diluted with pH 2 solution to a total of 2 mL. The sample was gently mixed by turning the total centrifuge filter device upside down a few times and was subsequently centrifuged as before. This step was used to remove nonaggregated material left in the retentate after the first filtration step. Having repeated the washing of the fibrils, the retentate was recovered. The total amount of protein in the various fractions was quantified by weighing the fractions and determining the protein concentrations with UV spectrophotometry.32 An unheated whey protein sample was included as a control in every experiment, and the total protein recovery (filtrate + retentate) was in a range of 95-100%. The experiments were repeated three times for all samples. Rheo-Optical Measurements. Birefringence experiments on the WPI fibrils were performed using a strain-controlled ARES rheometer (Rheometrics Scientific) equipped with a modified optical analysis module.35 Measurements were performed in a Couette geometry with a static inner bob of diameter 30 mm and rotating outer cup (with a quartz bottom plate) of diameter 33.8 mm. A laser beam of wavelength 670 nm (5 mW) passes vertically between the cup and bob through the sample. In this setup, the apparatus was capable of measuring the birefringence, ∆n′, to values as low as 10-8, at a sampling frequency of 24 Hz. The optical signal from the detector was digitized using an analogue-to-digital converter and was analyzed using Labview (National Instruments). The rheo-optical experiments were performed in an air-conditioned room at 20 °C ((1 °C). WPI fibril solutions were subjected to steady-shear flow at a shear rate of 5 s-1 for 60 s. We assume here that fibrils are completely aligned at this shear rate.25,27,33 Samples were measured both in clockwise and counterclockwise rotation during the shear step. Several dilutions were made for most samples to avoid complications related to multiple orders of birefringence. At the shear rate applied, the steady-shear flow did not damage the fibrils, as was reported by Rogers et al.25 and Akkermans et al.27 for pure β-lg fibrils. The steady-state value for the flow birefringence, ∆n′, was measured. This value is related to the length and concentration of the fibrils via ν∆n′ ) M
∫ c‚L‚dL
(1)
where ν is the dilution factor of the sample, M is a constant equal to the birefringence per unit length of the fibrils,25 c is the concentration of fibrils with a length between L and L + dL. The integral in eq 1 is referred to as the total length concentration.25 Thioflavin T Fluorescence Assay. The fluorescence of the dye Thioflavin T (ThT) is enhanced by its specific binding to amyloid fibrils. Krebs et al.36 proposed that ThT molecules bind to fibrils in the grooves on the face of the β-sheets that form the backbone of the fibril structure. The concentration of bound ThT molecules is then proportional to the total fibril length. In a solution of excess ThT, the change in fluorescence intensity is commonly used as a relative measure of the total length concentration of fibrils.13,14,22 We used ThT fluorescence to investigate the total length concentration of WPI fibrils. The increase in fluorescence is attributed to the binding of ThT to fibrils and is proportional to the length and concentration of fibrils. A ThT stock solution was made by dissolving 3.0 mM (35) Klein, C.; Venema, P.; Sagis, L. M. C.; van Dusschoten, D.; Wilhelm, M.; Spiess, H. W.; van der Linden, E.; Rogers, S. S.; Donald, A. M. Appl. Rheol. 2007, 17, 45210. (36) Krebs, M. R. H.; Bromley, E. H. C.; Donald, A. M. J. Struct. Biol. 2005, 149, 30-37.
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Figure 1. (A) Conversion determined with the centrifugal filtration method as a function of the heating time for various sample series. (B) Steady-shear birefringence corrected for the dilution factor of the sample, ν∆n′, as a function of the heating time. (C) Normalized ThT fluorescence intensity, IThT*, as a function of the heating time for various sample series: R0 (9); S0 (2); S10 ([); S30 (f). Samples were heated at 2 wt % WPI at pH 2 at 80 °C. Error bars indicate the maximum deviation from the mean values.
Figure 2. (A) The correlation between the measured steady-shear birefringence, corrected for the dilution factor of the samples, ν∆n′, and the average fibril concentration on the basis of the conversion of WPI (corrected for the β-lg concentration) as determined with the centrifugal filtration method, Cfibril. The equation of the trend line is given by Cfibril ) K1‚ν∆n′, with K1 ) 0.26 × 105 wt % and R2 ) 0.91. (B) The correlation between the normalized ThT fluorescence intensity, IThT*, and Cfibril. The equation of the trendline is given by Cfibril ) K2‚IThT*, with K2 ) 0.35 wt % and R2 ) 0.93. (C) The correlation between ν∆n′ and IThT*. The equation of the trend line is given by ν∆n′ ) K3‚IThT*, with K3 ) K2/K1 ) 1.39 × 10-5 and R2 ) 0.94. Thioflavin T (Merck-Schuchardt) in phosphate buffer (10 mM phosphate, 150 mM NaCl, pH 7.0) and filtering through a 0.2-µm filter (Schleicher & Schuell). This stock solution was diluted 50fold in the phosphate buffer to generate the working solution. Small samples of 48 µL were taken from the WPI samples after heating and cooling on ice-water. Each of these samples was added to 4 mL of the working solution, was mixed, and was allowed to bind the ThT for 1 min. The fluorescence was measured using a Varian Cary Eclipse fluorescence spectrophotometer (Varian, Walton-onThames, United Kingdom). The samples were excited at a wavelength of 440 nm (slit width 10 nm), and the subsequent emission was detected between 460 and 500 nm (slit width 10 nm) at medium speed. The fluorescence intensity peak was determined at 484 nm. The ThT fluorescence intensity, IThT, was measured as a function of the heating time of the samples. The ThT fluorescence intensity of the working solution, I0, was used as a background value for the fluorescence. IThT was also normalized by I0, and IThT* ()(IThT I0)/I0) was plotted versus the heating time. Samples were measured in triplicate, and the sample series were measured in duplicate. By calibrating this assay with the centrifugal filtration method, an absolute value for the fibril concentration can be extracted and a value for M (see eq 1) can be obtained.
Results and Discussion The conversion, the steady-shear birefringence, and the ThT fluorescence intensity were measured as a function of the heating time for the WPI sample series as indicated in Table 1. In Figure 1, the conversion (A), the shear birefringence corrected for the dilution factor of the sample, ν∆n′ (B), and the normalized ThT fluorescence intensity, IThT* (C), are plotted as a function of the heating time.
WPI samples that were being stirred constantly during heating (S0) showed an increase in fibril material relative to those that were heated at rest (R0). The addition of seeds did not result in an additional increase in the amount of fibril material. The curves for samples heated without added seeds (S0) and for samples heated in the presence of seeds (S10 and S30) are almost identical. Seeded samples (S10 and S30) only show a slightly higher amount of fibril material in the initial sample compared to samples without added seeds (S0). This is caused by the presence of preformed fibrils in the initial S10 and S30 samples.33 On the basis of these results, we can determine the quantitative relation between birefringence or ThT fluorescence and the fibril concentration in the samples. In Figure 1A, the results for the conversion of WPI monomers into fibrils are shown as a function of the heating time of the samples. From previous studies, we know that β-lg is the only whey protein involved in fibril formation in WPI under these conditions.28,29,32 Our purified WPI sample contained about 65% β-lg.29 We corrected the WPI concentration for the β-lg concentration in the samples and calculated the average fibril concentration that was obtained during heating, Cfibril (i.e., the amount of protein monomers that was converted into fibrils in wt %), using the centrifugal filtration method. Cfibril was plotted versus ν∆n′ for all four samples, as shown in Figure 2A. There is a good correlation (R2 ) 0.91) between the measured birefringence signal and the average fibril concentration. We can now, by combining the results for the centrifugal filtration method and the steady-shear birefringence measurements, easily deduct a quantitative fibril concentration from our birefringence
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measurements. We also plotted Cfibril versus IThT*, as shown in Figure 2B. Here also, a good correlation (R2 ) 0.93) was found between the ThT fluorescence and the average fibril concentration. Therefore, ThT fluorescence can be used as a fast assay to quantitatively determine the fibril concentration in heated WPI samples. In Figure 2C, ν∆n′ is plotted versus IThT*, which also shows a good correlation (R2 ) 0.94). The three figures in Figure 2 show linear fits with correlations close to 1. This indicates that the three methods (centrifugal filtration, flow birefringence, and ThT fluorescence) are consistent. They are linearly related via
Cfibril ) K1‚ν∆n′ )
K2 ‚I * K1 ThT
(2)
The three measured values (Cfibril, ν∆n′, and IThT*) are proportional to the total length of the fibrils per m3. The measured birefringence is related to the length concentration of the fibrils by eq 1. Rogers et al.25 determined a value for M on the basis of the conversion as derived using a precipitation method,25,26,37 and a fibril line density (number of monomers per unit length), as measured by neutron scattering measurements for β-lg fibrils.37 Their value for M was calculated to be M ) 1.74 × 10-20 m2 for pure β-lg fibrils. With the precipitation method, the conversion of monomers into fibrils is overestimated.25,32 Because of the overestimation of the conversion value used to determine M, Rogers et al.25 find overestimated fibril concentrations. Using the centrifugal filtration method, a more accurate value for the conversion of WPI monomers into fibrils was determined. Knowing that β-lg is the only whey protein involved in fibril formation in WPI under these conditions,28,29,32 we are able to calculate a corrected value for M. The expression for the line in Figure 2A is given by
ν∆n′ )
Cfibril K1
(3)
and the value of K1 is 0.26 × 105 wt %. The value of K1 is related to M by
M)
R‚Mmono 10‚K1‚NAv
(4)
(37) Aymard, P.; Nicolai, T.; Durand, D.; Clark, A. Macromolecules 1999, 32, 2542-2552.
where R is the fibril line density of 0.28 nm-1,37 Mmono is the molecular weight of the protein monomers (18.3 kg‚mol-1), and Nav is Avogadro’s number of 6.02 × 1023 mol-1. Substituting these values in eq 4 gives a value for M equal to 3.30 × 10-20 m2. This corrected value for M allows us to calculate a corrected value for β, the prefactor in the Doi-Edwards expression for the rotational diffusion coefficient of the fibrils.25 This prefactor is a measure for the stiffness of the fibrils. It is of order 1 for rodlike fibrils and much higher for semiflexible fibrils. The value of β is related to M by (M2β)1/7 ) 1.1 × 10-5 m4/7.25 Substituting the value of M in this expression, we find for the corrected value β ) 1.8 × 104. Rogers et al.25 found a value of 6.5 × 104 for this parameter. The corrections of M and β cancel each other so the results for the size distributions in Rogers et al.25 remain unaffected.
Conclusion The conversion of protein monomers into fibrils can be determined using the centrifugal filtration method. Other methods that can be used to determine the concentration of fibril material qualitatively are based on steady-shear birefringence and Thioflavin T (ThT) fluorescence measurements. Centrifugal filtration, shear birefringence, and ThT fluorescence experiments were performed to study the effect of stirring and seeding on the amount of fibril material formed in WPI samples as a function of heating time and sample treatment. The three methods are consistent and linearly correlated. After calibrating with the centrifugal filtration method, steady-shear birefringence and ThT fluorescence can both be used as an assay to determine the fibril concentration quantitatively. Birefringence and ThT fluorescence provide faster alternatives for determining the fibril concentration than the more time-consuming and laborious centrifugal filtration method. Possible uses of these fast assays are in large-scale processes where the yield of fibril formation can be determined in-line. After calibration with the centrifugal filtration method, these assays can also be used for studying the kinetics of protein assembly into fibrils in general, as long as the fibrils are based on a cross β-sheet structure. From birefringence measurements and the conversion determined using the centrifugal filtration method, we were able to calculate corrected values for both M and β. LA063048K
