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Virtual Journal for Biomedical Optics

Virtual Journal for Biomedical Optics

| EXPLORING THE INTERFACE OF LIGHT AND BIOMEDICINE

  • Editor: Gregory W. Faris
  • Vol. 2, Iss. 3 — Mar. 7, 2007
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Intrinsic fluorescence changes associated with the conformational state of silk fibroin in biomaterial matrices

Irene Georgakoudi, Irene Tsai, Cherry Greiner, Cheryl Wong, Jordy DeFelice, and David Kaplan  »View Author Affiliations


Optics Express, Vol. 15, Issue 3, pp. 1043-1053 (2007)
http://dx.doi.org/10.1364/OE.15.001043


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Abstract

Silk fibroin is emerging as an important biomaterial for tissue engineering applications. The ability to monitor non-invasively the structural conformation of silk matrices prior to and following cell seeding could provide important insights with regards to matrix remodeling and cell-matrix interactions that are critical for the functional development of silk-based engineered tissues. Thus, we examined the potential of intrinsic fluorescence as a tool for assessing the structural conformation of silk proteins. Specifically, we characterized the intrinsic fluorescence spectra of silk in solution, gel and scaffold configurations for excitation in the 250 to 335 nm range and emission from 265 to 600 nm. We have identified spectral components that are attributed to tyrosine, tryptophan and crosslinks based on their excitation-emission profiles. We have discovered significant spectral shifts in the emission profiles and relative contributions of these components among the silk solution, gel and scaffold samples that represent enhancements in the levels of crosslinking, hydrophobic and intermolecular interactions that are consistent with an increase in the levels of β-sheet formation and stacking. This information can be easily utilized for the development of simple, non-invasive, ratiometric methods to assess and monitor the structural conformation of silk in engineered tissues.

© 2007 Optical Society of America

1. Introduction

A variety of degradable polymeric biomaterial devices, such as collagen and poly-DL-lactic-glycolic acid (PLGA)-based sutures and sponges, are used in clinical settings and are designed to match biological, chemical and physical requirements in specific tissue restorative applications [1

1. B. D. Ratner, A. S. Hoffman, F. J. Schoen, and J. E. Lemons, Biomaterials Science, 2nd ed. (Elsevier Academic Press, San Diego, 2004).

]. In recent years, interest in these types of biomaterial matrices has expanded into scaffold designs for cell and tissue growth in vitro and in vivo for tissue engineering [1

1. B. D. Ratner, A. S. Hoffman, F. J. Schoen, and J. E. Lemons, Biomaterials Science, 2nd ed. (Elsevier Academic Press, San Diego, 2004).

]. With these expanding uses, it has become more important to understand the structure of the biomaterials used and their remodeling in vitro and in vivo, such that the rates and extent of remodeling can be factored into tissue integration in the form of new extracellular matrix formation and function.

Intrinsic fluorescence of proteins has been used in numerous basic biochemical and biophysical studies to probe protein structure and dynamics [20

20. Y. K. Reshetnyak and E. A. Burstein, “Decomposition of protein tryptophan fluorescence spectra into log-normal components. II. The statistical proof of discreteness of tryptophan classes in proteins,” Biophys J 81(3),1710–1734 (2001). [CrossRef] [PubMed]

, 21

21. Y. K. Reshetnyak, Y. Koshevnik, and E. A. Burstein, “Decomposition of protein tryptophan fluorescence spectra into log-normal components. III. Correlation between fluorescence and microenvironment parameters of individual tryptophan residues,” Biophys J 81(3),1735–1758 (2001). [CrossRef] [PubMed]

]. Spectral signatures are influenced by the characteristics of the microenvironment and the location (chemical sequence) of the fluorophores in the protein macromolecule [20

20. Y. K. Reshetnyak and E. A. Burstein, “Decomposition of protein tryptophan fluorescence spectra into log-normal components. II. The statistical proof of discreteness of tryptophan classes in proteins,” Biophys J 81(3),1710–1734 (2001). [CrossRef] [PubMed]

, 21

21. Y. K. Reshetnyak, Y. Koshevnik, and E. A. Burstein, “Decomposition of protein tryptophan fluorescence spectra into log-normal components. III. Correlation between fluorescence and microenvironment parameters of individual tryptophan residues,” Biophys J 81(3),1735–1758 (2001). [CrossRef] [PubMed]

]. Tryptophan has the highest quantum yield and extinction coefficient of the three aromatic amino acids, and therefore is often used to assess protein structure [22

22. C. Cantor and P. Schimmel, Biophysical Chemistry Part II:Techniques for the study of biological structure and function, 1st ed. (W.H. Freeman and Company, New York, 1980).

]. For example, tryptophan fluorescence spectra of actin, a cytoskeletal protein, and the contribution of each individual tryptophan was assessed using intrinsic fluorescence spectroscopy [23

23. T. C. Doyle, J. E. Hansen, and E. Reisler, “Tryptophan fluorescence of yeast actin resolved via conserved mutations,” Biophysical Journal 80(1),427–434 (2001). [CrossRef] [PubMed]

, 24

24. I. M. Kuznetsova, T. A. Yakusheva, and K. K. Turoverov, “Contribution of separate tryptophan residues to intrinsic fluorescence of actin. Analysis of 3D structure,” Febs Letters 452(3),205–210 (1999). [CrossRef] [PubMed]

]. Since the complete primary sequence of silk fibroin protein is known [25

25. C. Z. Zhou, F. Confalonieri, N. Medina, Y. Zivanovic, C. Esnault, T. Yang, M. Jacquet, J. Janin, M. Duguet, R. Perasso, and Z. G. Li, “Fine organization of Bombyx mori fibroin heavy chain gene,” Nucleic Acids Res 28(12),2413–2419 (2000). [CrossRef] [PubMed]

], and the mechanism of protein folding and assembly into functional materials has also been described [26

26. H. J. Jin and D. L. Kaplan, “Mechanism of silk processing in insects and spiders,” Nature 424(6952),1057–1061 (2003). [CrossRef] [PubMed]

], interpretations of spectral changes are feasible.

To begin to map tissue remodeling in the context of silk biomaterials and cell interactions, a baseline understanding of silk protein matrix structure and changes in this structure in vitro or in vivo is critical. Toward this goal, detailed fluorescence spectroscopic assessments of silk fibroin protein matrices prepared in solution, gel and solid state forms (3D scaffolds) are described in the present work. The results provide an initial window into options to track the silk fibroin matrix structure and remodeling using nondestructive optical imaging and spectroscopy tools.

2. Materials and methods

2.1 Preparation of silk biomaterials

White Japanese raw Bombyx mori silkworm cocoons were boiled for 20 minutes in an aqueous solution of 0.02 M Na2CO3 and rinsed with cold de-ionized water to remove the gluelike sericin proteins using methods we have previously reported [27

27. S. Sofia, M. B. McCarthy, G. Gronowicz, and D. L. Kaplan, “Functionalized silk-based biomaterials for bone formation,” J Biomed Mater Res 54(1),139–148 (2001). [CrossRef]

]. The silk fibroin fibers were dissolved in 9 M LiBr solution at 60°C for 4 hours. After complete dissolution, the concentration of the silk solution was about 20 w/v %. The solution was subsequently dialyzed for 2 days (Pierce, MWCO 3500) and after dialysis the concentration was about 8 w/v %. This solution was diluted to 1 w/v % for spectroscopic measurements. All solutions were stored at 7°C to avoid premature gelation. The 8 w/v % silk fibroin solution gelled after about 1 month at 7°C. The preparation of the solid state 3D porous silk fibroin scaffolds has also been previously reported [28

28. U. J. Kim, J. Park, H. J. Kim, M. Wada, and D. L. Kaplan, “Three-dimensional aqueous-derived biomaterial scaffolds from silk fibroin,” Biomaterials 26(15),2775–2785 (2005). [CrossRef]

]. Briefly, granular NaCl about 500 μm in diameter (4 g) was added to 2 ml of ~8 % silk aqueous solution in disk-shaped vials. The vials were covered at room temperature for 24 hours and then submerged in de-ionized water for 2 days at room temperature to remove the NaCl. Detailed structural and morphological features of the above materials have been previously reported [7

7. R. Nazarov, H. J. Jin, and D. L. Kaplan, “Porous 3-D scaffolds from regenerated silk fibroin,” Biomacromolecules 5(3),718–726 (2004). [CrossRef] [PubMed]

, 10

10. U. J. Kim, J. Park, H. J. Kim, M. Wada, and D. L. Kaplan, “Three-dimensional aqueous-derived biomaterial scaffolds from silk fibroin,” Biomaterials 26(15),2775–2785 (2005). [CrossRef]

].

2.2 Preparation of pure component solutions

Pure L-tryptophan and L-tyrosine were purchased from Sigma (St. Louis, MO) and diluted in PBS at a concentration of 0.0186 and 1 mM, respectively. Di-tyrosine was prepared from L-tyrosine as described previously [29

29. D. Malencik, J. Sprouse, C. Swanson, and S. Anderson, “Dityrosine: Preparation, Isolation and Analysis,” Analytical Biochemistry 242,202–213 (1996). [CrossRef] [PubMed]

] and further purified by high performance liquid chromatography. The fluorescent fractions monitored with excitation and emission wavelengths set to 320nm and 400nm, respectively, were collected, pooled, and lyophilized to dryness. Fluorescence EEMs were acquired from a 0.28 mM sample of di-tyrosine dissolved in water.

2.3 Intrinsic fluorescence spectroscopy of silk solution, gel and scaffold samples

A Hitachi fluorescence spectrophotometer (F4500, 450 W Xenon arc lamp; San Jose, CA) was used to characterize the fluorescence properties of silk solutions, gels and scaffolds. Spectra were collected in the front-face geometry to reduce the effects of scattering, especially in the case of the gels and scaffolds. Samples in solution were placed in a 1 cm path length quartz cuvette; while silk gels were sandwiched between two quartz slides. Since silk scaffolds are a spongy solid, no supporting plates were used for these measurements. Before the start of each experiment, a fluorescence excitation-emission matrx of a rhodamine standard (2.14 μM in ethylene glecol) and a diffuser were acquired and used to account for day to day variations and the spectral response of the instrument. Water was employed to check sensitivity of the instrument, as specified by the manufacturer. Typically, the signal to noise ratio was greater than 100 and drift was less than 1%. Fluorescence excitation-emission matrices were measured incrementally for excitation wavelengths in the 250–335 nm range and corresponding emission in the 250 – 600 nm range in 5 nm increments. The scan speed was 1200 nm/min with excitation and emission slits set to yield a 2.5 nm resolution. The photomultiplier tube detector gain was set to 700 V. These wavelength ranges allowed for intrinsic fluorescence measurements of tyrosine, tryptophan and other endogenous fluorophores in the UV-VIS range.

2.4 Data analysis

To quantify and understand the biochemical origins of the fluorescence EEMs acquired from the different silk samples, we used the alternating least squares (ALS) algorithm of the Matlab PLS Toolbox (Mathworks Inc, Natick MA). The ALS algorithm was developed to extract the spectral line shapes and corresponding concentrations of the components that describe a set of spectra [30

30. R. Tauler, A. Smilde, J. Henshaw, L. Burgess, and B. Kowalski, “Multicomponent determination of chlorinated hydrocarbons using a reaction-based chemical sensor. 1. Chemical speciation using multivatiate curve resolution,” Analytical Chemistry 66,3337–3344 (1994). [CrossRef]

]. Inputs to the algorithm include the data set to be analyzed, the number of components that are expected to describe the spectral features of the data and an initial guess for either the components’ concentrations for each sample or their spectral line shape. To ensure that physiologically meaningful parameters would be extracted from the fits, we imposed non-negativity constraints on both the concentration and spectral profiles of the components. Our goal was to identify the smallest number of components that could be used to describe the data for the range of excitation and emission wavelengths that we examined.

Fig. 1. Schematic flow of data analysis steps for silk solution and gel samples (left panel). The slightly modified approach to extract the spectral components contributing to the silk scaffold fluorescence is shown on the right panel.

3. Results and discussion

3.1 Silk fibroin protein composition and structure

Fig. 2. Conformational transitions of silk fibroin protein from solution to gel to 3D scaffold; and relationship between the folding of a single chain of B. mori silkworm silk heavy chain fibroin and its primary sequence. The tyrosine (y) and tryptophan (w) residues are highlighted.

3.2 Intrinsic fluorescence spectra of silk solution, gel and scaffold samples

Fig. 3. (A) Fluorescence excitation-emission matrix of silk in solution. Arrows indicate major contributions from tyrosine, tryptophan and cross-links. Representative fluorescence emission spectra acquired at 265 nm (panel B) and 310 nm (panel C) from silk in solution, gel and scaffold configurations are shown as solid lines. The corresponding fits achieved using the ALS scheme outlined in Fig. 1 are shown as dashed lines.

3.3 Spectral decomposition of silk solution, gel and scaffold samples

To model and understand the origins of the spectral differences depicted in Fig. 3, we used an alternating least squares (ALS) fitting algorithm to extract the spectral line shapes and relative intensity contributions of the minimum number of components that could describe the recorded fluorescence excitation-emission matrices (Fig. 1). This analysis identified five spectral components that were needed to describe the silk solution, gel and scaffold spectra. The corresponding fits that we achieved for each one of the spectra in Figs. 3(B) and 3(C) are shown as dotted lines and demonstrate excellent agreement between the analysis model and the data. More detailed information with respect to the range and the mean values for the root mean square error (RMSE) and the % of unmodeled variance for each type of silk sample is included in Table 1.

Table 1. Measures of fit quality of silk spectra using the ALS-based algorithm

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Figures 4 and 5 show the fluorescence emission spectra of each one of the components extracted from ALS analysis of the data and used to achieve the fits of Fig. 3. To describe the fluorescence recorded from silk solution, gel and scaffold samples for 250–335 nm excitation, we needed to include five distinct spectral components. The wavelength of maximum emission (λ max), the corresponding full width at half maximum (FWHM) for each component and its relative contribution to the overall fluorescence from each sample type are included in Table 2.

The spectral profiles of each of these components required for the fits of silk in solution, gel and scaffold conformation were similar but not identical, as shown in Figs 4 and 5 and Table 2. For example, the spectrum of the dominant tryptophan component, identified as Tryptophan 1, extracted from analysis of the gel and scaffold data becomes progressively narrower and blue-shifted, when compared to the corresponding spectrum extracted from the silk solution samples [Fig. 5(A)]. The latter spectrum is in turn blue shifted with respect to the pure tryptophan solution spectrum [Fig. 5(A)]. These spectral changes reflect an increasingly hydrophobic milieu for the tryptophan residues [20

20. Y. K. Reshetnyak and E. A. Burstein, “Decomposition of protein tryptophan fluorescence spectra into log-normal components. II. The statistical proof of discreteness of tryptophan classes in proteins,” Biophys J 81(3),1710–1734 (2001). [CrossRef] [PubMed]

, 21

21. Y. K. Reshetnyak, Y. Koshevnik, and E. A. Burstein, “Decomposition of protein tryptophan fluorescence spectra into log-normal components. III. Correlation between fluorescence and microenvironment parameters of individual tryptophan residues,” Biophys J 81(3),1735–1758 (2001). [CrossRef] [PubMed]

, 35

35. E. A. Burstein, S. M. Abornev, and Y. K. Reshetnyak, “Decomposition of protein tryptophan fluorescence spectra into log-normal components. I. Decomposition algorithms,” Biophys J 81(3),1699–1709 (2001). [CrossRef] [PubMed]

], consistent with the protein forming more extensive β-sheets and excluding water in the process (Fig. 2; [26

26. H. J. Jin and D. L. Kaplan, “Mechanism of silk processing in insects and spiders,” Nature 424(6952),1057–1061 (2003). [CrossRef] [PubMed]

]). However, the location of the emission maximum of the tryptophan spectrum at 345 nm is characteristic of tryptophan residues that are exposed to water [20

20. Y. K. Reshetnyak and E. A. Burstein, “Decomposition of protein tryptophan fluorescence spectra into log-normal components. II. The statistical proof of discreteness of tryptophan classes in proteins,” Biophys J 81(3),1710–1734 (2001). [CrossRef] [PubMed]

, 21

21. Y. K. Reshetnyak, Y. Koshevnik, and E. A. Burstein, “Decomposition of protein tryptophan fluorescence spectra into log-normal components. III. Correlation between fluorescence and microenvironment parameters of individual tryptophan residues,” Biophys J 81(3),1735–1758 (2001). [CrossRef] [PubMed]

] and it is consistent with the sequence data shown in Fig. 2 indicating that tryptophan is present along the hydrophilic chains of the silk protein. Emission from the third tryptophan component is also consistent with tryptophan present in a highly hydrophilic environment that is similar for all types of silk samples.

Table 2. Summary of spectral components of the components used to describe silk samples

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Fig. 4. Fluorescence emission spectra of the components extracted from ALS analysis of silk in solution (A), gel (B) and scaffold (C) configuration.

The tryptophan component identified as tryptophan 2 is significantly blue-shifted with respect to the dominant tryptophan component with the emission peak at 310 nm. The spectrum of Trp2 from the silk solution samples includes a prominent feature in its tail region, indicating that it may be actually a composite spectrum from two chemical species. The spectrum of Trp2 is broader for the silk gel and scaffold samples than the silk solution samples, may be as a result of a change in the relative contributions of these two components. Unfortunately, we were not able to identify two distinct spectral features in this wavelength regime using the ALS algorithm. Tryptophan emission exhibiting a maximum at 308 nm is consistent with tryptophan residues that do not form hydrogen-bound complexes in the excited state [20

20. Y. K. Reshetnyak and E. A. Burstein, “Decomposition of protein tryptophan fluorescence spectra into log-normal components. II. The statistical proof of discreteness of tryptophan classes in proteins,” Biophys J 81(3),1710–1734 (2001). [CrossRef] [PubMed]

, 21

21. Y. K. Reshetnyak, Y. Koshevnik, and E. A. Burstein, “Decomposition of protein tryptophan fluorescence spectra into log-normal components. III. Correlation between fluorescence and microenvironment parameters of individual tryptophan residues,” Biophys J 81(3),1735–1758 (2001). [CrossRef] [PubMed]

, 35

35. E. A. Burstein, S. M. Abornev, and Y. K. Reshetnyak, “Decomposition of protein tryptophan fluorescence spectra into log-normal components. I. Decomposition algorithms,” Biophys J 81(3),1699–1709 (2001). [CrossRef] [PubMed]

]. Peak emission in the 316–332 nm region is typically associated with buried tryptophan residues that do form hydrogen-bound complexes [20

20. Y. K. Reshetnyak and E. A. Burstein, “Decomposition of protein tryptophan fluorescence spectra into log-normal components. II. The statistical proof of discreteness of tryptophan classes in proteins,” Biophys J 81(3),1710–1734 (2001). [CrossRef] [PubMed]

, 21

21. Y. K. Reshetnyak, Y. Koshevnik, and E. A. Burstein, “Decomposition of protein tryptophan fluorescence spectra into log-normal components. III. Correlation between fluorescence and microenvironment parameters of individual tryptophan residues,” Biophys J 81(3),1735–1758 (2001). [CrossRef] [PubMed]

, 35

35. E. A. Burstein, S. M. Abornev, and Y. K. Reshetnyak, “Decomposition of protein tryptophan fluorescence spectra into log-normal components. I. Decomposition algorithms,” Biophys J 81(3),1699–1709 (2001). [CrossRef] [PubMed]

]. Thus, this component likely represents emission from a subset of tryptophan residues that have limited access to water as silk β-sheets acquire an increasingly stacked conformation within the scaffold structure. This hypothesis is also supported by the fact that the contribution of this component to the overall silk sample fluorescence, increases gradually from 12% to 23% and to 32% for the silk solution, gel and scaffold samples, respectively. Another possibility for a species contributing to fluorescence emission in the 320–330 nm region is tyrosinate. However, tyrosinate is highly unstable and would likely appear as crosslinked products [31

31. J. R. Lakowicz, Principles of Fluorescence Spectroscopy, Second ed. (Kluwer Academic/Plenum Publishers, New York, NY, 1999).

]. Since crosslinking in silk has not been found to any large degree to date, despite the high content of tyrosine, tyrosinate is not expected to be a major contributor to the observed spectra.

Fig. 5. Comparison of the spectral features of the components attributed to (A) tryptophan 1, (B) tryptophan 2, (C) tryptophan 3, and (D) crosslinks from each type of silk sample. Measured spectra from a tryptophan (A), tyrosine and dityrosine (D) solution are also included.

The tyrosine emission spectrum that we employed for analysis of all silk samples was fixed to the measured tyrosine solution spectrum, since tyrosine emission is not very sensitive to its local environment [31

31. J. R. Lakowicz, Principles of Fluorescence Spectroscopy, Second ed. (Kluwer Academic/Plenum Publishers, New York, NY, 1999).

]. While the relative concentration of tyrosine to tryptophan doesn’t change as the silk changes conformations from its silk to its gel and scaffold conformations, we observe a significant decrease in relative contribution of tyrosine fluorescence to the overall sample fluorescencence. Specifically, while tyrosine contributes significantly (32%) to the overall fluorescence emission from silk in solution, it comprises only 12 % and 1% of the silk gel and scaffold samples, respectively. This is likely the result of enhanced resonance energy transfer (RET) between tyrosine and tryptophan [31

31. J. R. Lakowicz, Principles of Fluorescence Spectroscopy, Second ed. (Kluwer Academic/Plenum Publishers, New York, NY, 1999).

]. Specifically, as the tyrosine and tryptophan molecules are found in closer proximity to each other when the silk acquires its two- and three-dimensional conformation, it becomes more probable for non-radiative energy transfer to occur between an excited tyrosine and one of the tryptophan molecules, which then fluoresces as it decays to its ground state.

Thus, from examination of the differences in the emission spectral properties of the fluorescent components that yield the measured excitation-emission matrices of different silk samples we can acquire insight on the structural and conformational changes that characterize the state of the silk protein in solution, gels and scaffolds. In addition, this spectral decomposition yields information about the contribution of each one of the components to the measured fluorescence intensity. For example, we can see in Fig. 6(A) that the ratio of tryptophan to tyrosine fluorescence at 275 nm excitation increases gradually as we compare the solution, gel and scaffold samples. As mentioned above, this increase represents the gradually increasing levels of RET that occur as the protein acquires a tighter three-dimensional conformation that brings the tyrosine and tryptophan molecules closer together.

Fig. 6. (A). The ratio of tryptophan to tyrosine fluorescence detected at 275 nm excitation and (B) the level of fluorescence attributed to crosslinks relevant to the overall amino acid (tyr and trp) fluorescence increase as silk achieves increasing levels of β-sheet conformation in its solution, gel and scaffold configurations.

4. Summary

Acknowledgments

This work was supported by NIH grant P41EB002520 (DK) and NSF grant BES0547292 (IG). We would also like to acknowledge Heyon-Joo Kim for assistance with sample preparation.

References and links

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2.

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20.

Y. K. Reshetnyak and E. A. Burstein, “Decomposition of protein tryptophan fluorescence spectra into log-normal components. II. The statistical proof of discreteness of tryptophan classes in proteins,” Biophys J 81(3),1710–1734 (2001). [CrossRef] [PubMed]

21.

Y. K. Reshetnyak, Y. Koshevnik, and E. A. Burstein, “Decomposition of protein tryptophan fluorescence spectra into log-normal components. III. Correlation between fluorescence and microenvironment parameters of individual tryptophan residues,” Biophys J 81(3),1735–1758 (2001). [CrossRef] [PubMed]

22.

C. Cantor and P. Schimmel, Biophysical Chemistry Part II:Techniques for the study of biological structure and function, 1st ed. (W.H. Freeman and Company, New York, 1980).

23.

T. C. Doyle, J. E. Hansen, and E. Reisler, “Tryptophan fluorescence of yeast actin resolved via conserved mutations,” Biophysical Journal 80(1),427–434 (2001). [CrossRef] [PubMed]

24.

I. M. Kuznetsova, T. A. Yakusheva, and K. K. Turoverov, “Contribution of separate tryptophan residues to intrinsic fluorescence of actin. Analysis of 3D structure,” Febs Letters 452(3),205–210 (1999). [CrossRef] [PubMed]

25.

C. Z. Zhou, F. Confalonieri, N. Medina, Y. Zivanovic, C. Esnault, T. Yang, M. Jacquet, J. Janin, M. Duguet, R. Perasso, and Z. G. Li, “Fine organization of Bombyx mori fibroin heavy chain gene,” Nucleic Acids Res 28(12),2413–2419 (2000). [CrossRef] [PubMed]

26.

H. J. Jin and D. L. Kaplan, “Mechanism of silk processing in insects and spiders,” Nature 424(6952),1057–1061 (2003). [CrossRef] [PubMed]

27.

S. Sofia, M. B. McCarthy, G. Gronowicz, and D. L. Kaplan, “Functionalized silk-based biomaterials for bone formation,” J Biomed Mater Res 54(1),139–148 (2001). [CrossRef]

28.

U. J. Kim, J. Park, H. J. Kim, M. Wada, and D. L. Kaplan, “Three-dimensional aqueous-derived biomaterial scaffolds from silk fibroin,” Biomaterials 26(15),2775–2785 (2005). [CrossRef]

29.

D. Malencik, J. Sprouse, C. Swanson, and S. Anderson, “Dityrosine: Preparation, Isolation and Analysis,” Analytical Biochemistry 242,202–213 (1996). [CrossRef] [PubMed]

30.

R. Tauler, A. Smilde, J. Henshaw, L. Burgess, and B. Kowalski, “Multicomponent determination of chlorinated hydrocarbons using a reaction-based chemical sensor. 1. Chemical speciation using multivatiate curve resolution,” Analytical Chemistry 66,3337–3344 (1994). [CrossRef]

31.

J. R. Lakowicz, Principles of Fluorescence Spectroscopy, Second ed. (Kluwer Academic/Plenum Publishers, New York, NY, 1999).

32.

B. Lotz, “Crystal structure of polyglycine I,” J Mol Biol 87(2),169–180 (1974). [CrossRef] [PubMed]

33.

R. E. Marsh, R. B. Corey, and L. Pauling, “An investigation of the structure of silk fibroin,” Biochim Biophys Acta 16(1),1–34 (1955). [CrossRef] [PubMed]

34.

D. L. Kaplan, W. Adams, B. Farmer, and C. Viney, eds., Silk Polymers: Science and Biotechnology, (American Chemical Society Symposium Series1994), Vol.544.

35.

E. A. Burstein, S. M. Abornev, and Y. K. Reshetnyak, “Decomposition of protein tryptophan fluorescence spectra into log-normal components. I. Decomposition algorithms,” Biophys J 81(3),1699–1709 (2001). [CrossRef] [PubMed]

36.

D. Malencik and S. Anderson, “Dityrosine as a product of oxidative stress and fluorescent probe,” Amino Acids 25,233–247 (2003). [CrossRef] [PubMed]

OCIS Codes
(170.1580) Medical optics and biotechnology : Chemometrics
(170.6280) Medical optics and biotechnology : Spectroscopy, fluorescence and luminescence

ToC Category:
Medical Optics and Biotechnology

History
Original Manuscript: January 16, 2007
Manuscript Accepted: January 29, 2007
Published: February 5, 2007

Virtual Issues
Vol. 2, Iss. 3 Virtual Journal for Biomedical Optics

Citation
Irene Georgakoudi, Irene Tsai, Cherry Greiner, Cheryl Wong, Jordy DeFelice, and David Kaplan, "Intrinsic fluorescence changes associated with the conformational state of silk fibroin in biomaterial matrices," Opt. Express 15, 1043-1053 (2007)
http://www.opticsinfobase.org/vjbo/abstract.cfm?URI=oe-15-3-1043


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