• cytochrome c;
  • molten-globule-like state;
  • protein folding;
  • SDS;
  • thermal denaturation
  • BSA, bovine serum albumin;
  • CD, circular dichroism;
  • CMC, critical micelle concentration;
  • cyt c, cytochrome c;
  • DTA, dodecyltrimethylammonium chloride;
  • FTIR, Fourier transform Infrared;
  • FTIR-ATR, FTIR attenuated total reflection;
  • MG, molten globule;
  • NMR, nuclear magnetic resonance;
  • SDS, sodium dodecyl sulfate;
  • UV-Vis, ultraviolet and visible


  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and methods
  6. Acknowledgements
  7. References

The molten globule (MG) state can be an intermediate in the protein folding pathway; thus, its detailed description can help understanding protein folding. Sodium dodecyl sulfate (SDS), an anionic surfactant that is commonly used to mimic hydrophobic binding environments such as cell membranes, is known to denature some native state proteins, including horse cytochrome c (cyt c). In this article, refolding of acid denatured cyt c is studied under the influence of SDS to form MG-like states at both low concentration and above the critical micelle concentration using Fourier transform Infrared (FTIR) and ultraviolet and visible absorption as well as fluorescence and circular dichroism (CD). Thermal denaturation monitored with FTIR and CD shows distinct final high temperature states starting from MG-like states formed with different SDS/protein ratios. The results suggest that the SDS/protein ratio as well as the actual SDS (or protein) concentration affects structure and its thermal stability. Thermal denaturation monitored with CD and FTIR for cyt c at neutral pH but denatured with SDS showed that at a high SDS/protein ratio, the thermal behavior of MG-like states formed at low and neutral pH are quite similar. Based on the results obtained, the merits of two models of the protein–surfactant structure are discussed for different SDS concentrations.

Cytochrome c (cyt c) plays an important role in the biological electron transfer system and has been extensively studied (Moore and Pettigrew 1990; Diaz-Quintana et al. 2003; Grealis and Magner 2003), including having its fully resolved three-dimensional structure determined by X-ray and nuclear magnetic resonance (NMR) (Feng et al. 1989, 1990; Bushnell et al. 1990). For cyt c, several probes (IR, UV-Vis, CD, fluorescence) can be used to monitor the structural changes needed to obtain the variety of states accessible under different solution perturbations (GuHCl, urea, pH, temperature, etc.) (Tsong 1976; Roder et al. 1988; Jeng et al. 1990; Pryse et al. 1992; Goto et al. 1993; Fink et al. 1994; Bai et al. 1995; Yeh et al. 1998; Yeh and Rousseau 1999). When acidified, cyt c is denatured to a primarily random coil structure, destabilized due to the electrostatic repulsion between positively charged residues. A molten globule (MG) state of cyt c can be achieved by adding salt to this acid-denatured state, whereby the electrostatic repulsion is reduced, which is believed to drive the protein to become more compact (Goto et al. 1990). That state is characterized by high helical content in its secondary structure, but with little evident tertiary structure (Goto and Nishikiori 1991; Xu and Keiderling 2004).

It is well known that ionic surfactants can interact very strongly with oppositely charged globular proteins (Jones and Manley 1980). Studies of such interactions between proteins and surfactants have been carried out for half a century; however, the mechanism by which the surfactants influence protein structure is still not well defined. Among these, bovine serum albumin (BSA) has been most frequently studied with sodium dodecyl sulfate (SDS), a representative anionic surfactant that has been widely used in the purification and characterization of proteins (Jones 1975; Lapanje 1978; Gelamo and Tabak 2000; Gelamo et al. 2002). Normally, under saturation binding conditions, 1 g of protein can be expected to bind as much as 1.5–2 g of the surfactant (Reynolds and Tanford 1970a; Tanford 1980). SDS is known to be a strong denaturant for many proteins even at the millimolar level (Turro et al. 1995; Gelamo and Tabak 2000). However, its ability to counteract the effect of other denaturants was first reported by Duggan and Luck (1948) by addition of SDS to BSA in urea to reduce the viscosity of the solution, and addressed more recently by the thermal denaturation experiments of Moriyama (Moriyama and Takeda 1999).

Information concerning the structure of protein–surfactant complexes has been derived from rheological (Reynolds and Tanford 1970b), spectroscopic (Das et al. 1998; Gelamo et al. 2002), electrophoretic (Gelamo et al. 2002), binding (Jones 1975; Tanford 1980), and scattering studies (Chen and Teixeira 1986; Guo et al. 1990). Several models have been proposed for the structure of protein–surfactant complexes in SDS over its critical micelle concentration (CMC): (1) a correlated “necklace and bead” model in which clusters with a micelle-like structure are stabilized by protein (Fig. 1; Shirahama et al. 1974; Guo et al. 1990; Turro et al. 1995); (2) “rod-like” prolate elliposidal surfactant aggregate with a semi-minor axis of ∼18 Å, corresponding to the surfactant chain length (Reynolds and Tanford 1970b; Tanford 1980); and (3) a flexible capped helical cylinder micelle with the protein wrapping around the micelle (Lundahl et al. 1986). A small-angle neutron scattering study of BSA and ovalbumin complexes with SDS led to the conclusion that a necklace and bead structure, composed of protein–surfactant aggregates, accounted for the scattering behavior of these systems (Chen and Teixeira 1986; Guo et al. 1990), which was confirmed by NMR experiments by Turro et al. (1995).

In this article, the interaction between positively charged acid-denatured cyt c and anionic SDS is studied. MG-like states can be achieved at very low concentration and above the CMC of SDS. Recently, other workers also showed the formation of a MG-like state of cyt c induced by low concentration n-alkyl sulfate (Moosavi-Movahedi et al. 2003). Those authors suggested that hydrophobic interactions play an important role in stabilizing the MG state. While this article was in preparation, another paper on cyt c in surfactant appeared (Chattopadhyay and Mazumdar 2003), which focused on urea unfolding and refolding with SDS, but also recognized some of the acid unfolding effects studied here. In the present work, by comparing the results of salt and cationic surfactant-induced MG-like states, we propose that the primary driving force for the formation of cyt-SDS-induced MG-like states is the reduction of electrostatic repulsion, although the hydrophobic effect does remain a factor. Thermal denaturation studies of several MG-like states at both neutral and low pH monitored by CD and IR indicate a protein concentration-dependent thermal behavior for the cyt-SDS complex. Finally, the results of thermal denaturation as well as fluorescence experiments suggest different structures for cyt-SDS complexes at different SDS concentrations as well as at various [SDS]/[cyt] ratios.


  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and methods
  6. Acknowledgements
  7. References

Far-UV CD equilibrium measurement

The far-UV CD spectra obtained in the titration of acid-denatured cyt c (16 μM) with SDS at pH ∼2.0 were recorded as shown in Figure 2. Three phases can be found in this titration process, starting with the initial acid-denatured state, which can be viewed as dominantly a random coil showing a negative peak at ∼195 nm and a broad negative band at 200–220 nm (Woody 1992; Sreerama and Woody 2000; Shi et al. 2002; Xu and Keiderling 2004).

For [SDS] < 0.25 mM (Fig. 2A), formation of secondary structure from the acid-denatured state was observed as an ellipticity (negative) gain at both 222 nm and 207 nm, which is typical of α-helix (Woody 1996; Sreerama and Woody 2000). This helix formation is almost linearly dependent on the concentration of SDS up to 0.25 mM, as can be seen from the ellipticity change at 222 nm (inset), with a stable MG-like state being formed at [SDS]/[cyt] ∼14. The inset curve marked in diamonds represents the titration of acid-denatured cyt c (16 μM) with a solution having the same protein concentration at the same pH but including 4 mM SDS. A sigmoidal curve can be fit to this ellipticity change, but the breadth of the transition calls into question the possibility of a two-state transition.

For [SDS] values from 3 mM to 60 mM (Fig. 2B), the low pH cyt c far-UV CD spectra also show native-like secondary structure content, suggesting another MG-like state. These spectra seem independent of SDS concentration once it is above 3 mM. A surface tension test (data not shown) shows that the critical micelle concentration (CMC) for SDS solution at pH = 2.0 is around 3 mM. The fact that the same cyt c spectra were obtained for this range of [SDS] values suggests that micelle formation may affect the stability of this complex, but the number (concentration) of micelles is less important.

The third cyt c form occurs for [SDS] from 0.3 to 2.5 mM, between the above noted ranges, where the cyt–SDS solution becomes opaque and visual precipitation occurs as more SDS is added. The corresponding CD spectra first become distorted with a sharp ellipticity loss at 207 nm, and then appear to have β-structure characteristics before losing most of the intensity upon precipitation. These samples by contrast are not stable. A time-dependent experiment for the cyt–SDS complex solution (16 μM cyt c with 0.36 mM SDS at pH = 2) was carried out at T = 25°C, as shown in Figure 3A. With time, a spectrum suggesting formation of a large fraction of β-sheet structure is obtained. The ellipticity change at 222 nm versus time is plotted as an inset in Figure 3A, and can be fit to a single exponential curve. A separate FTIR-ATR spectrum was recorded on the precipitate collected from cyt c in 1 mM SDS, as shown in Figure 3B, and compared to solution data for the acid-denatured and SDS-induced MG-like forms. The FTIR-ATR spectrum shows three amide I features, a broad band at 1652 cm−1 representing the residual helix and turns, and a more intense one at 1625 cm−1 with a weaker shoulder at 1695 cm−1, corresponding to contributions from the newly formed β-sheet structure that are not seen in the denatured or MG-like spectra.

As a comparison, a titration of 16 μM cyt c at pH ∼2.0 with a positively charged surfactant, dodecyltrimethylammonium chloride (DTA), was recorded at the same pH with far-UV CD, as shown in Figure 4. Over the whole surfactant concentration range from 0 to 50 mM, no precipitation is observed, which differs from what is observed with SDS. The ellipticity change at 222 nm is plotted as an inset in Figure 4. Although both surfactants provide hydrophobic tails to aid solubility of the core, they differ because with DTA there is no charge neutralization. In the DTA case, ellipticity starts to increase at 6 mM (inset in Fig. 4), which is the CMC for DTA at pH ∼2 (data not shown), but a MG-like state is not obtained until [DTA] > 16 mM, much higher than for SDS, which offers charge neutralization as well as hydrophobic interactions.

Soret absorbance measurement

The UV-Vis absorption in the Soret region of the same protein–surfactant complex as in Figure 2 is shown in Figure 5. Three phases can also be found in this titration process. The acid-denatured cyt c has a peak located at 394 nm, which agrees well with previous studies (Brems and Stellwagen 1983). At low [SDS] (<0.2 mM) (Fig. 5A), the absorbance at 394 nm decreases sharply with little peak shift, suggesting no fundamental heme change in this concentration range but the possibility of multiple heme micro-environments broadening the Soret band. When [SDS] is larger than 0.2 mM (data not shown), the baseline increases, due to precipitation, and a red peak shift is observed. For [SDS] in the 3–60 mM range (Fig. 5B), the precipitate redissolves, with only a minor peak intensity decrease; however, especially at high [SDS] (20 mM or above), a band shift is observed, suggesting that a structural change at the heme occurs within this region even though the secondary structure is constant, as shown from far-UV CD.

Fluorescence measurement

Fluorescence spectra (Fig. 6) arising from the single Trp in cyt c were also recorded for the same cyt–SDS samples used in UV CD and Soret absorption experiments. In the native state structure, Trp 59 is buried in the hydrophobic core and almost no fluorescence is observed, presumably being quenched by Forster energy transfer to the heme group (Pinheiro et al. 1997). However, when the protein is denatured, the Trp residue becomes solvated and intense fluorescence results. As seen in Figure 6A, even 0.05 mM of SDS induces a big decrease in the fluorescence of acid-denatured cyt c, and about 80% of the fluorescence was quenched for [SDS] = 0.2 mM, suggesting a hydrophobic collapse even at this very low SDS concentration. There is a blue shift of the peak (∼10 nm) accompanying this decreased fluorescence intensity when [SDS] is varied from 0 to 0.2 mM. Between [SDS] = 0.2 mM and 3 mM, the turbidity distorts the observed fluorescence. When [SDS] is above 3 mM (Fig. 6B), an increase in the fluorescence is observed, and a small red shift occurs, especially at high SDS concentration (>20 mM), indicating that SDS starts to unfold the protein. Factor analysis of the fluorescence spectra as a function of bandshape also shows that there is a distinct maximum in the frequency shift of the band shape at around 10 mM SDS (data not shown), which agrees well with UV-Vis spectral results.

Thermal denaturation monitored by far-UV CD

In the above titration experiments, MG-like states were observed at both low [SDS] and above the CMC by use of far-UV CD, UV-Vis, and fluorescence. Thermal denaturation experiments monitored with far-UV CD were carried out to differentiate the MG-like states found above. Figure 7, A–C, shows the thermal denaturation behaviors of three MG-like states that have the same protein concentration but with different [SDS]/[Cyt] ratios, ∼12 (MG1), 240 (MG2), and 1500 (MG3), respectively. At low temperature, all three states show a typical α-helical pattern, similar to that of the salt-induced MG state at low pH (Xu and Keiderling 2004). For the MG1 state (Fig. 7A), the ellipticity at 222 nm is almost constant up to 20°C (inset in Fig. 7A), and then a loss in ellipticity that is linear with temperature is observed up to T = 85°C. Upon cooling back to 25°C, the spectral shape still shows typical α-helical character, and ∼64% of the ellipticity change at 222 nm is recovered. The thermal denaturation behavior of MG2 (Fig. 7B) is different, wherein the ellipticity at 222 nm (inset in Fig. 7B) does not have any low-temperature stable region. Instead, a linear loss of ellipticity with temperature is observed from 5°C up to 55°C, followed by a major transition above 60°C. This is probably due to aggregation, which is consistent with the CD band shape seen at high temperature. Only 24% of the ellipticity is recovered on cooling and the pattern remains β-sheet-like, indicating an irreversible transition. For the MG3 state, which has a high concentration of SDS (25 mM) and a high [SDS]/[cyt] ratio of ∼1500 (Fig. 7C), a linear loss of ellipticity at 222 nm (inset in Fig. 7C) is observed from low temperature (5°C) without any thermally stable region. No band-shape disruption is observed for MG3 even up to 80°C, and when the sample is cooled down to 20°C, 82% of the CD signal change at 222 nm is recovered. Meanwhile maintenance of the [θ]222/[θ]207 ratio before and after heating indicates that this process is mostly reversible. In addition, isodichroic points at 202 nm are observed in the thermal denaturation experiments for both MG1 and MG3, suggesting a two-state process, probably from α-helical to random coil conformation.

For comparison, the thermal behavior of native state cyt c denatured by SDS solutions at neutral pH is shown in Figure 7, D–F, under varying conditions. When only 0.2 mM SDS is involved, CD spectra at low temperature are just like that of native state, suggesting almost no unfolding occurs (Fig. 7D), which agrees with previous studies that unfolding by SDS at neutral pH starts at [SDS]/[cyt] ≈40 (Das et al. 1998). At high temperature, aggregation is apparent, with the Tm being ∼10°C lower than that observed for the native state (inset in Fig. 7D), suggesting that even low concentration SDS (submicellar) destabilizes cyt c. For the other two solutions with [SDS]/[Cyt] at ∼235 and 1400, MG-like CD spectra typical of helical structure were observed at both low and high temperatures, and no aggregation was observed for the whole temperature range. An isodichroic point at ∼204 nm is found for these two samples, which could be consistent with a two-state transition with an increase of temperature, but only a broad transition with no sigmoidal behavior is evident in the inset curves, partially due to a lack of a low temperature stable region, much as seen at low pH. Upon cooling, CD intensity at 222 nm was fully recovered for both samples, and a similar (θ)222/(θ)207 ratio is obtained, indicating that these two high [SDS] thermal unfolding processes are fully reversible.

Thermal denaturation monitored by FTIR

The thermal behaviors of cyt/SDS samples having the same protein concentration (∼1.6 mM) at low pH (∼2.0) but with different amounts of SDS were also studied with FTIR, as shown in Figure 8, A–C, corresponding to [SDS]/[Cyt] = 2, 12, and 270, respectively. When T = 5°C, the FTIR spectrum for cyt c with 3.8 mM SDS shows an amide I′ peak at ∼1649 cm−1 (Fig. 7A), which is consistent with a high content of α-helical structure. By T = 45°C, a shoulder at ∼1620 cm−1 appeared, suggesting some β structure or aggregation, but it did not increase much even at 80°C, although it remained after cooling, indicating a partially irreversible change.

For cyt c in 22 mM SDS (Fig. 8B), a similar result is obtained at low temperature, but the intensity sharply decreases with temperature increase, and a new peak at ∼1620 cm−1 and a shoulder at ∼1687 cm−1 appear at 25°C, indicating formation of β structure due to aggregation. Both peaks are nearly constant in intensity for T > 30°C, with no precipitation, and remain after cooling, suggesting irreversibility.

For cyt c in 0.51 M SDS at pH ∼2 (Fig. 8C), low temperature second derivative analysis shows that the amide I′ band is initially composed of two bands centered at 1652 cm−1 and 1635 cm−1, which could be assigned to interior α-helical and solvated α-helical structure, respectively (Haris and Chapman 1995; Williams et al. 1996). With an increase of temperature, the peak at 1635 cm−1 disappears at ∼40°C, while the intensity at 1641 cm−1 grows, suggesting a helix to coil transition. By contrast to samples in 3.8 mM and 22 mM SDS, cyt c in 0.51 M SDS does not show any aggregation features up to 80°C, and its spectral changes reverse on cooling.

The changes of A1620/A1649 with temperature for the above samples are plotted in Figure 9A. There is no obvious transition when [SDS]/[Cyt] is 270 (triangle), and only a small transition is observed for [SDS]/[Cyt] ∼2 (diamond). By contrast, a large transition is observed for [SDS]/[Cyt] around 12 (filled circle).

Figure 9B indicates singular-value decomposition (SVD) analysis results for the transitions observed in Figures 8, A–C, and 9A, as well as for an additional sample having 80 μM cyt c and 0.98 mM SDS ([SDS]/[Cyt] = 12, open circle). The small transition observed in Figure 9A for the [SDS]/[cyt] ratio = 2 is clearer with SVD (diamonds), yielding a transition with Tm = 37°C. When the ratio = 12, for both samples having 80 μM or 1.6 mM cyt c, big transitions are observed. However, the temperatures for the transitions are different, with Tm ∼ 25°C for high [Cyt] (closed circles), whereas no complete denaturation was observed for low [Cyt], indicating that the protein concentration as well as the ratio of [SDS]/[Cyt] are determining factors for stability of the protein–surfactant complex.

For comparison, the thermal behavior of native state cyt c denatured with SDS at a neutral pH was also studied by FTIR under varying conditions, as shown in Figure 8, D–F. For [SDS]/[cyt] ∼2 (Fig. 8D), at low temperature the amide I′ peak is located at 1649 cm−1, and intensity loss begins above 35°C with the appearance of a shoulder at ∼1618 cm−1 suggesting partial aggregation. When the [SDS]/[Cyt] ratio is 13 (Fig. 8E), the spectra show a sharp peak at 1616 cm−1 at higher temperatures, which remains on cooling, and precipitation can be observed visually, indicating irreversible aggregation.

At high SDS concentration (Fig. 8F), when T = 5°C the amide I′ peak was located at ∼1645 cm−1, which is normally assigned to random coil structure. However, second derivative analysis showed this amide I′ peak to be composed of components at 1650 cm−1 and 1635 cm−1, much as seen at low pH, which could be assigned to interior and solvated α-helical structure (Haris and Chapman 1995; Williams et al. 1996), respectively, and indicated that a large amount of secondary structure still existed. The component at ∼1635 cm−1 disappears and the one at 1650 cm−1 stays almost constant with increasing temperature, suggesting a helix to coil transition that is reversible on cooling. The changes of A1616/A1649 versus temperature for the above samples are plotted in Figure 9C. Two transitions atTm1 ∼ 37°C and Tm2 ∼70°C are observed for [SDS]/[cyt] = 2, and only one with Tm ∼40°C is seen for this ratio at 13, but no transition is observed for this ratio at 270.


  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and methods
  6. Acknowledgements
  7. References

Formation of a low [SDS]-induced MG-like state

SDS, as well as other ionic surfactants, is known for its ability to denature some native proteins, including cyt c, by unordering its tertiary structure but leaving much secondary structure intact (Hiramatsu and Yang 1983; Takeda et al. 1985; Muga et al. 1991; Das et al. 1998; Gebicka and Gebicki 1999; Gelamo and Tabak 2000; Gelamo et al. 2002). It has been reported that SDS above its CMC stabilizes peptides in helical conformations (Mammi and Peggion 1990; Rizo et al. 1993), whereas below its CMC, SDS has also been found to stabilize β-strands (Zhong and Johnson 1992; Waterhous and Johnson 1994). Adding a small amount of SDS (negative) to aqueous lysozyme (net positive) solution has been reported to cause precipitation (Jones and Manley 1980; Moren and Khan 1995), but complete redissolution of the precipitate occurs when [SDS]/[protein] increases to >19. Duggan and Luck (1948) very early observed that the addition of SDS prevents the rise in viscosity of urea-denatured serum albumin. Recent research on cyt c denaturation by urea with SDS at physiological pH suggests that the existence of SDS will prevent the complete denaturation of cyt c by instead forming some stable partially folded states (Chattopadhyay and Mazumdar 2003). Another recent study reported the formation of a MG-like state of acid-denatured cyt c with n-alkyl sulfates at low concentrations (Moosavi-Movahedi et al. 2003); however, they focused on the effect of different chain lengths, and they concluded that hydrophobic interactions played an important role in stabilizing the MG state.

In this work, SDS is shown to refold acid-denatured cyt c into MG-like states that are obtained at both low and high (above its CMC) SDS concentrations, whereas aggregation and precipitation are observed (medium [SDS]) between these two states. We have used the term “MG-like” because we have judged the character of this state by its spectral (CD, IR, fluorescence) response. Although we have not established its compactness, the fluorescence quenching of the Trp (denatured) by the heme on adding SDS does demonstrate an increase in compactness. This structural nomenclature is consistent with the literature (Moosavi-Movahedi et al. 2003), but future light scattering studies could provide useful insight into the mechanism and structures of these “MG-like” states. Aggregation is accompanied by formation of intermolecular β-structure as demonstrated by the use of FTIR-ATR of the precipitate. Formation of the MG-like state with low [SDS] is found to be dependent on the [SDS]/[cyt] ratio, as almost double the SDS concentration was needed to reach a MG-like state when the protein concentration was increased from 16 μM to 32 μM.

Refolding experiments with the positively charged DTA surfactant also showed an ellipticity increase (MG-like helix formation) for 6 mM at pH ∼2 (its CMC). The full MG-like state is formed at ∼16 mM, a concentration that is much smaller than the ∼0.3 M required for NaCl (Goto et al. 1990; Goto and Nishikiori 1991; Xu and Keiderling 2004), indicating that the hydrophobic effect, enhanced by the lipid, is an important factor for this protein refolding. However, when this is compared with only 0.2 mM SDS needed to form the MG-like state, it becomes obvious that neutralization of the positive charges of acid cyt c is a critical factor for protein refolding in the SDS case and is the source of its higher efficiency over just the hydrophobic effects available with DTA. The huge difference between SDS- and NaCl-induced refolding is probably also affected by the higher SDS binding efficiency. The native conformation has been proposed to be stabilized by a cross-linking function of the anionic surfactant ion between a group of nonpolar residues and a positive charged residue located on different loops of the protein (Markus and Karush 1957; Moriyama et al. 2003). For lysozyme, this function of the DS anion has also been postulated to explain the efficiency of SDS in the precipitate (Moren and Khan 1995), and its orientation between charged and hydrophobic groups has been verified by X-ray crystallography (Yonath et al. 1977).

Thermal behaviors of MG-like states at different SDS/cyt ratios

Although MG-like states are observed at various [SDS]/[cyt] ratios at low pH, they show different thermal denaturation behaviors as monitored by CD and FTIR. Compared with the results for cyt–SDS complexes at neutral pH, we found that only at high [SDS] or [SDS]/[cyt] ratios are the thermal denaturation behaviors similar in both ECD (Fig. 7) and FTIR (Fig. 8). The above results suggest that the protein–surfactant structure with a high [SDS]/[cyt] ratio and high [SDS] are similar, regardless of the difference in pH. Adding SDS to urea-denatured serum albumin has been shown to disrupt the structure of BSA to an extent favorable for the interactions with surfactant ion independent of urea (Moriyama and Takeda 1999). This parallels our result for acid-denatured cyt c.

Structure of the cyt-SDS complex

Aggregation is observed in the thermal denaturation of the cyt–SDS complex at certain [SDS]/[cyt] ratios, which is assumed to be due to the local folding/unfolding of some of the α-helices. The process could be summarized as:

  • equation image

in which the opening of α-helices requires energy. Such an opening may go on to form more extended structure, such as left-handed 31-helices (or Pro II-like helices), which are major components of random coils because they are stabilized by hydrogen bonding to water (Dukor and Keiderling 1991; Keiderling et al. 1999; Keiderling and Xu 2002; Shi et al. 2002). These structures have been also postulated as intermediates in β-structure formation (McColl et al. 2003). When extended components or β-strands in different molecules come together, intermolecular β-sheets (aggregation) can form.

However, for cyt c in 0.5 M SDS, no aggregation was observed at both low and neutral pH for temperatures as high as 80°C. A revisit of the model for protein–surfactant interaction may give us some helpful information to understand the above difference. The “necklace and bead structure” of protein–surfactant complexes has two possibilities for a dominant mode of interaction (Fig. 1): (1) The protein wraps around the micelle and (2) the micelles nucleate on the protein hydrophobic sites (Shirahama et al. 1974; Chen and Teixeira 1986; Guo et al. 1990; Turro et al. 1995). Although we can not prove or disprove this model, we can evaluate the consistency of either model with our observed data. Our results suggest that both are active in that the behavior of the cyt–SDS complex at 1.6 mM cyt c with ∼20 mM SDS seems to follow structure A, whereas at high [SDS] (0.5 M) the cyt–SDS complex has, at least partially, structure B.

This assumption is also consistent with fluorescence results. The blue shift as observed in Figure 6A has been assigned to the change of the Trp environment from locally aqueous to hydrophobic, namely, from a polar to a nonpolar environment (Pinheiro et al. 1997). For example, they showed the fluorescence of a Trp tri-peptide, Lys-Trp-Lys, has a 14-nm blue shift upon adding DOPS vesicles but no intensity change. Thus, the blue shift at low [SDS] in Figure 6A suggests a change for Trp from a polar to a nonpolar environment whereas the small red shift occurring at 20 mM SDS indicates a change back to more polar. That the MG state with low [SDS]/[cyt] has smaller fluorescence intensity occurring at a lower wavelength is also consistent with structure A, because different segments of the protein will be more compact after binding to the same micelle, which could result in quenching the Trp fluorescence by the heme group. In structure B, the more extended protein conformation could further expose the Trp residue to water, resulting in a red shift compared with structure A. More importantly, it certainly would be less compact, resulting in less quenching due to the increased average separation between Trp and heme. Thus the characteristics of structure B are consistent with the experimental results at high [SDS]. Additionally, the absorbance peak shift to higher wavelength in the UV-Vis region at 20 mM SDS confirms some transition occurred in the micellar range.

Our thermal denaturation results also support the structure assignment above. For structure A, the SDS molecules occupy the hydrophilic sites on the polypeptide chain and allow the hydrophobic sites to be exposed to the solvent by expanding the molecule. This destabilizes the protein molecule and enhances the formation of intermolecular β-sheet, once the hydrophobic parts in different molecules meet. By contrast, for structure B, the hydrophobic segments, which are important initiators of β-sheet formation, are buried in the micelles, inhibiting their interactions. At the same time, the charged nature of the micelle will also prevent aggregation. This model can also explain the pH-independent thermal results obtained experimentally for high [SDS]. The structure B assumption seems to partially conflict with the NMR results from Turro et al. (1995), who reported that the BSA/SDS system is best explained with structure A, for SDS from 7 mM to 35 mM with a protein concentration of several millimolar. With cyt c, this is concentration dependent. Our FTIR results for protein–SDS mixtures within the same [SDS] range agree well with that protein–SDS structure in model A. However, at much higher SDS concentration (or [SDS]/[cyt] ∼0.5 M), structure B better explains our results.


The effect of the anionic surfactant (SDS) on acid-denatured cyt c was studied in this article, using spectral and thermal techniques. [SDS] at very low concentration and above the CMC is shown to induce acid-denatured cyt c to refold and form MG-like states. Comparison of SDS- and DTA-induced refolding showed that the SDS advantage arose from the reduction of electrostatic repulsion, although the hydrophobic effect may be important in the overall process. However, intermediate concentrations of [SDS] lead to precipitation, which is probably due to charge neutralization. The existence of intermolecular β-sheet structure in the precipitate indicates it results from aggregation following unfolding.

Thermal denaturation experiments show that cyt–SDS complexes with large [SDS]/[cyt] (and/or high [SDS]) have similar thermal behavior in both low and neutral pH, suggesting negligible pH effect under these conditions. However, the protein or SDS absolute concentration is an important factor in addition to the [SDS]/[cyt] ratio in determining the thermal behavior of the protein–surfactant complex, as monitored by far-UV CD and FTIR. Separate “necklace and bead” structures could explain the cyt–SDS complex behavior under different conditions. For ∼1.6 mM cyt c, the results suggest that the protein chain wraps around the micelle at about 20 mM SDS, whereas the micelle nucleates on the protein hydrophobic sites at higher [SDS] concentration (∼0.5 M).

Materials and methods

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and methods
  6. Acknowledgements
  7. References


Cytochrome c (horse heart, C-7752) was purchased from Sigma and used without further purification, and sodium dodecyl sulfate (SDS) was purchased from Fluka. D2O and DCl were purchased from Cambridge Isotope Laboratories, Inc., and NaOD was prepared by reacting solid Na with D2O. All the pH values represent apparent pH meter readings; no correction is made for solutions prepared in D2O.


Cyt c and SDS were dissolved directly into D2O to have a final protein concentration of ∼20 mg/mL with various SDS concentrations; DCl and/or NaOD were used to adjust the pH, and 10 mM phosphate buffer solution was used to prepare Cyt/SDS at neutral pH. Solutions were then transferred to a demountable homemade cell composed of two CaF2 windows separated by a 50 μ spacer held in a brass ring. The IR absorption spectra were recorded at 4 cm−1 nominal resolution with an average of 940 scans using a Bio-Rad FTS-60A (FTIR) spectrometer. For the temperature variation studies, the cells were tightly fit into a homemade double-walled brass jacket (Wang 1993) through which water was pumped from a thermostatically controlled Neslab RTE-7 water bath. Programmed heating was used to change the temperature of the cell, and a thermocouple placed in the outer jacket of the cell was used to regulate the bath to achieve a constant sample temperature.

Fluorescence measurements

Measurements of steady-state fluorescence were performed on a Fluoromax-2 spectrofluorimeter (Jobin Yvon). Cyt c samples (0.2 mg/mL) with various concentrations of SDS at pH = 2.0 were placed in a 4-mm-pathlength cylindrical cuvette, and the emission spectra in the range of 300–450 nm were recorded with a fixed excitation wavelength at 290 nm. All the spectra were an average of three scans and were corrected by subtraction of the blank (just SDS at pH = 2) spectrum.

CD measurements

Far-UV experiments were performed on a Jasco-810 spectropolarimeter equipped with a Jasco 2-syringe titrator. Spectra were recorded with protein concentrations of 16–32 μM in a 1-mm-path-length quartz cuvette. For temperature variation experiments, a Neslab RTE-111 water bath pumped through a variable temperature cell holder was used under control of Jasco software. A bandwidth of 1 nm and a response of 2 sec were used, with a scanning rate at 50 nm/min to obtain final spectra as an average of four scans. In autotitration experiments, 2 mL of 16 μM acid-denatured cyt c (16 μM cyt c with 0.8 mM SDS at pH = 2 for reverse titration) was placed into a 1-cm rectangular cell placed in a Jasco cell holder. A solution with the same protein concentration but containing 4 mM SDS at the same pH (acid-denatured cyt c solution only for reverse titration) was put into one of the two syringes of the titrator. The titration was carried out under the control of the Jasco titration program, and a magnetic stirring bar in the cell was used to make the mixing more efficient.

UV absorption

The UV-Vis absorption in the Soret region of the same cyt c samples in a 1-mm-pathlength quartz cuvette used in far-UV CD measurements was recorded with an Olis-Cary 14 spectrophotometer.

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Figure Figure 1.. The “necklace and bead” structure of protein–surfactant complexes and its two possibilities. (A) The protein wraps around the micelle. (B) The micelles nucleate on the protein hydrophobic sites.

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Figure Figure 2.. SDS titration of 16 μM acid-denatured cyt c (pH = 2). (A) [SDS] at 0, 0.05, 0.1, 0.15, 0.2, and 0.25 mM; inset shows the ellipticity change at 222 nm. (B) [SDS] from 3–60 mM.

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Figure Figure 3.. (A) Time-dependent CD change for 16 μM cyt c with 0.36 mM SDS at low pH under 25°C; inset shows the ellipticity change at 222 nm for which a single exponential curve can fit all the points. (B) FTIR-ATR spectra of cyt–SDS precipitate (dashed line), which resulted from mixing 16 μM cyt c with ∼1 mM SDS, and the FTIR transmission spectra for SDS-induced MG-like state (solid line) and acid-denatured state cyt c (dash-dot-dash line). All IR intensities are in arbitrary units.

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Figure Figure 4.. Titration of acid-denatured cyt c with cationic surfactant dodeltrimethylammonium chloride (DTA) from 0.05 mM to 50 mM. The acid denatured cyt c (no surfactant; triangle) is shown as a comparison. Inset shows the ellipticity change at 222 nm.

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Figure Figure 5.. UV-Vis spectra of titration of 16 μM acid-denatured cyt c with SDS at low pH. (A) [SDS] from 0 to 0.2 mM. (B) [SDS] from 3 to 60 mM. Arrows indicate the direction of the change.

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Figure Figure 6.. Fluorescence spectra of titration of 16 μM acid-denatured cyt c with SDS at low pH. (A) [SDS] from 0 to 0.2 mM. (B) [SDS] from 3 to 60 mM. The fluorescence spectrum of cyt c at native state (circles) is shown as a comparison. Arrows indicate the direction of change.

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Figure Figure 7.. Thermal denaturation of cyt-SDS complexes monitored by far-UV CD. Shown is 16 μM cyt c with a [SDS]/[cyt] ratio of 12 (A), 240 (B), and 1500 (C) at low pH and 12 (D), 235 (E), 1400 (F) at neutral pH.

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Figure Figure 8.. Thermal denaturation of cyt–SDS complexes monitored with FTIR. Shown is 1.6 mM cyt c with a [SDS]/[cyt] ratio at 2 (A), 12 (B), 270 (C) at low pH and 2 (D), 13 (E), 298 (F) at neutral pH.

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Figure Figure 9.. (A) The ratio of amide I intensity at 1620 over 1649 cm−1 for cyt c (∼1.6 mM) with SDS at pH ∼1.7 with [SDS]/[cyt] = 2 (♦), 12 (•), 270(▴). (B) Singular-value decomposition (SVD) results for the spectra shown in A with [SDS]/[cyt] = 2 (♦), 12 (•), and 12 (∼80 μM cyt, ○). (C) The ratio of amide I intensity at 1616 over 1647 cm−1 at pH ∼7 with [SDS]/[cyt] = 2 (♦), 13 (•), 298(▴).

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  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and methods
  6. Acknowledgements
  7. References

This research was supported in part by a grant from the Research Corp., and the CD and fluorescence instrument purchase was supported by the NSF (CHE-0091994).


  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and methods
  6. Acknowledgements
  7. References
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