Spectroscopic Characterization of Cytochrome C Entrapped in AOT Reverse Micelles. Kristen Grinstead
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1 Spectroscopic Characterization of Cytochrome C Entrapped in AOT Reverse Micelles Kristen Grinstead Chemistry Department Marshall University One John Marshall Drive Huntington, WV grinstead1@marshall.edu Abstract: The interactions of cytochrome c and the aqueous environment of the AOT reverse micelle were investigated in this study. The effects of micellar properties such as the water/surfactant ratio, the ph of the water pool, and temperature were also investigated. Our data show the destabilizing effect of the interfacial layer of the reverse micelle on the structure and stability of the protein. Cytochrome c absorption and emission spectra showed a dependency of protein conformation on the size of the reverse micelle. The absorption values of the heme in the cytochrome c under different ph values reveal the vulnerability of the protein structure to destabilization. The differences in the fluorescence emission of cytochrome c within reverse micelles of several sizes with a variety of solvent conditions provide additional support for the destabilizing effect of the micellar interfacial layer on cytochrome c. Keywords: Protein stability, cytochrome c, reverse micelles 1. Introduction Reverse micelles consist of a small amount of a polar solvent such as water and a suitable amount of amphiphilic surfactant molecules dispersed in a nonpolar organic solvent. The components then self-assemble into a spherical aggregate with an inner water pool. The surfactant polar head groups are in contact with the water pool whereas
2 the hydrophobic tails extend outward into the nonpolar solvent 1,2. Figure 1. Structure of an AOT/isooctane reverse micelle. The red circles represent the surfactant heads in contact with the water represented by the blue. The branched hydrophobic tails are in the isooctane solution. The polar cores of the reverse micelles are of nanometer size and are therefore able to solubilize significant amounts of water and proteins. Previous studies have shown that properties of water molecules at the interface with surfactant polar groups vary significantly from those of bulk water 1, 3,4. The water molecules contained within reverse micelles may be bound or free. Approximately five molecules of water are bound tightly to one AOT surfactant head 5. The bound water has restricted mobility, increasing viscosity and increasing polarity 4. An increase in the amount of water molecules results in an increase of the size of the water pool and partial recovering of bulk water properties 3. In biochemistry, reverse micelles have been used to mimic properties of biological membranes and for catalyses of enzymatic reactions 1,6. Studies have been conducted previously concerning the impact of the unique environment of reverse micelles on the structure of proteins solubilized within their aqueous phase, but little is known about the reverse micelles influence on fundamental protein processes such as protein folding and unfolding transitions, protein ligand binding and protein-protein interactions 2, 4-8. To characterize the impact of reverse micelles on the stability of proteins we have investigated the stability of cytochrome c when solubilized within reverse micelles. The folding and unfolding of the globular heme protein cytochrome c has been extensively studied using many different methods including steady-state absorption and emission spectroscopy by virtue of its spectroscopically visible heme group and single tryptophan residue 4,8,9. The changes in protein structure are often associated with changes in heme ligation. In the native state, the heme is low-spin six-coordinated, having His18 in the position of proximal ligand and Met80 in the position of distal ligand 9,10. Under denaturing conditions, the axial ligand His18 remains attached to the central iron atom
3 whereas the Met80 dissociates from the heme and can be replaced by a lysine or histidine (His26 or His33) under neutral ph 10,11. Acidic ph stabilizes a 5-coordinated heme with a histidine residue being a proximal ligand. A single tryptophan residue (Trp59) in the cytochrome c can be used as a fluorescent probe to study tertiary structural changes in protein conformation. The fluorescence of the single tryptophan is quenched by the heme group in the native structure of cytochrome c, and emission intensity increases during protein unfolding 4,9,12. Previous studies on the properties of cytochrome c in reverse micelles are incomplete. It has been shown that electrostatic interactions between surfactant heads and positively charged cytochrome c results in the disruption of Met80 ligation to iron heme likely due to the opening of the protein structure. Brochette et al. 4 have reported that cytochrome c conformation is strongly influenced by micellar size. From the fluorescence and CD studies, they reported that two different structures of cytochrome c exist below and above W 0 = In both cases, the protein conformation is not identical to that of native cytochrome c. Therefore, to understand the effect of the reverse micelle on the structure of cytochrome c, we chose to use CTAB and AOT reverse micelles to compare the interactions of the protein with both positively and negatively charged surfactants. We expect electrostatic interactions between the negatively charged AOT and cytochrome c and that the protein would be localized in the central water pool of the positively CTAB reverse micelles. The impact of the size of the reverse micelles on protein-membrane and protein-solvent interactions for both surfactants was also studied. To investigate the conformational changes in the protein caused by unfolding, we observed the effects of temperature changes, ph changes, and the denaturant urea on the cytochrome c encapsulated within the reverse micelles. 2. Materials and Methods Surfactant hexyldecyltrimethyl ammonium bromide (CTAB), co-surfactant hexanol, solvent isooctane, and equine heart cytochrome c were acquired from Sigma and used as received. Sodium bis(2-ethylhexyl)sulfosuccinate (AOT) was acquired from Fluka and also used as received. Distilled water (18 mω) was used to prepare buffer solutions. Following buffers were used to prepare reverse micelles: 0.1M and 0.05M Tris ph=7, 0.1M CAPS ph=10, and 0.1M Acetate ph=4. An aqueous solution of 5M urea was also prepared. The reverse micelles were prepared by injecting an aqueous solution of the protein into a 0.1M AOT in isooctane solution to have a final volume of 2 ml. The CTAB reverse micelles were prepared a similar way. Aqueous solutions of cytochrome c were added to a mixture of 0.1M CTAB and isooctane: hexanol (9:1 v/v). The solution was vortexed until it became transparent. The sizes of the reverse micelles (W 0 ) were easily controlled by manipulating the ratio of the water to surfactant as given by, W 0 = [water]/ [AOT] The reverse micelles were prepared to have W 0 = 5, 10, and 20. Samples were measured on the same day they were made.
4 Steady-state absorption spectra were acquired using a UV visible spectrophotometer (Cary 50, Varian) and steady-state emission spectra were acquired using an PC1 Photon Counting Spectrofluorometer (ISS). Absorbance spectra were performed from nm. Emissions spectra were taken at an excitation wavelength of 295 nm to selectively excite the tryptophan residue and emission was detected from nm. Samples were held in a 1 cm quartz cuvette. Figure 1 shows the lack of the band in the cytochrome c with the reverse micelle compared to the cytochrome c in bulk solution.. The absence of 695 nm bands indicates the absence of the Met80 in the position of distal ligand. 3. Results and Discussion The absorbance spectra of the cytochrome c solubilized within the reverse micelle did not show the band at 695 nm in agreement with previously published data. Figure1: Cytochrome c in AOT reverse micelles W 0 =15 at ph=7. Abs Cytochrome C in AOTM Wavelength (nm) 10 ph4 10 ph7 10 ph10 20 ph4 20 ph7 20 ph10 Figure2: Cytochrome c in AOT reverse micelles W 0 =10 and W 0 =20 at ph= 4, 7, and 10. Figure 2 shows that at neutral ph, in reverse micelles W 0 =10 and W 0 =20 (amount of cytochrome c normalized), the Soret band underwent a small blue shift (~ 1 nm)
5 relative to the cytochrome c in bulk water. This suggests that the heme group in cytochrome c is in slightly less polar environment compared to native protein. At ph=4, the spectrum of the heme in reverse micelles of W 0 = 10 and 20 is shifted towards a shorter wavelength compared to the spectrum of cytochrome c in aqueous solution of ph =4. The more dramatic shift was observed in case of reverse micelles of W 0 =20. At ph=10 the Soret displayed a red shift, again with the W 0 =20 having a larger shift. This suggests the protein is located in a more polar environment at W 0 =20. This provides evidence that the protein is localized in different parts of the reverse micelle and interacting with different types of water. As previously mentioned, the water bound to the polar heads of the surfactant is more polar than unbound water. Therefore the data supports the conclusion that the protein is more associated with the membrane bound water at higher ph. Also, the high intensity of the fluorescence in W 0 =20 suggests that the protein is more destabilized in that size of reverse micelle than at W 0 = 10. References: 1. Carrvalho, C. M. L., Cabral, J. M. S. (2000) Biochimic 82, Naoe, K., Noda, K., Kawagoe, M. Imai, M., (2004) Colloids and Surfaces B: Biointerfaces 38, Faeder, J., Ladanyi, B. M. (1999) American Chemical Society, /jp993076u 4. Brochette, P., Petit, C., Pileni, M.P. (1987) J. Phys. Chem., 92, Rodgers, M. A. I., Becker, J.C., (1980) J. Phys. Chem. 84, Das, P. K., Srilakshmi, G. V., Chaudhuri, A. (1999) Langmuir 2000, 16, Airoldi, M., Boicelli, A., Gennaro, G., Giomini, M., Giuliani, A. M., Giustani, M., Scibetta, L. (2002) Phys. Chem. Chem. Phys., 4, Valdez, D., Le Huérou, J-Y., Gindre, M., Urbach, W., Waks, M., (2001) Biophysical Journal 80, Latypov, R. F., Cheng, H., Roder, N. A., Zhang, J., Roder, H. (2006) J. Mol. Biol., 357, Battistuzzi, G., Borsari, M., Sola, M. (2001) Eur. J. Chem., Abbruzzetti, S., Viappiani, C., Small, R., Libertini, L. J., Small, E. W., (2001) J. Am. Chem. Soc. 123, Vanderkooi, J. M.,, Erecinska, M. (1975) Eur. J. Biochem. 60, 199.
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