Secondary Structural Changes of Homologous Proteins, Lysozyme

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Secondary Structural Changes of Homologous Proteins, Lysozyme and #-Lactalbumin, in Thermal Denaturation up to 130 °C and SDS Effects on these Changes: Comparison of Thermal Stabilities of SDS-Induced Helical Structures in these Proteins Yoshiko Moriyama, Naoaki Kondo, and Kunio Takeda Langmuir, Just Accepted Manuscript • Publication Date (Web): 30 Oct 2012 Downloaded from http://pubs.acs.org on November 6, 2012

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Secondary Structural Changes of Homologous Proteins, Lysozyme and α-Lactalbumin, in Thermal Denaturation up to 130 °C and SDS Effects on these Changes: Comparison of Thermal Stabilities of SDS-Induced Helical Structures in these Proteins Yoshiko Moriyama, Naoaki Kondo, and Kunio Takeda* Department of Applied Chemistry and Biotechnology, Okayama University of Science 1-1 Ridai-cho, Kita-ku, Okayama 700-0005, Japan Corresponding author: Kunio Takeda* Department of Applied Chemistry and Biotechnology, Okayama University of Science 1-1 Ridai-cho, Kita-ku, Okayama 700-0005, Japan Telephone/fax number: +81-86-256-9553, e-mail address: [email protected] running title: SDS Effect on Thermal Denaturations of Lysozyme and α-Lactalbumin at high temperatures

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ABSTRACT

The thermal stability of two homologous proteins, lysozyme and α-lactalbumin, were examined by circular dichroism. The present study clearly showed two different aspects between the homologous proteins; (1) the original helices of lysozyme and α-lactalbumin were unchanged at heat treatments up to 60 and 40 °C, respectively, indicating a higher thermal stability of lysozyme and (2) upon cooling to 25 °C, the original helices of lysozyme were never reformed after they were once disrupted, while those of α-lactalbumin, disrupted at a particular temperature range between 40 and 60 °C, were completely reformed. In addition, the structural changes were also examined in the coexistence of sodium dodecyl sulfate (SDS) which induced the formation of helical structures in these proteins at 25 °C. A distinct difference appeared in the thermal stabilities of the SDS-induced helices. All of the SDS-induced helices of lysozyme were disrupted below 60 °C, while those of α-lactalbumin at 10 mM SDS were unchanged up to 130 °C. A similarity was also fixed. Not only the SDS-induced helices but also the original helices of the two proteins were reformed upon cooling to 25 °C after the thermal denaturation below 100 °C in the coexistence of 10 mM SDS.

KEYWORDS: sodium dodecyl sulfate, lysozyme, α-lactalbumin, thermal denaturation, secondary structure, circular dichroism


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Introduction Many studies of the protein-surfactant interaction have been carried out for more than half a century.1-4 These studies have received attention in view of their biochemical importance as well as their physicochemical interest. In the studies of the interactions, sodium dodecyl sulfate (SDS) has been most frequently adopted as a representative ionic surfactant. As is well known, this surfactant and many other surfactants have been used as tools to isolate, solubilize, and manipulate many proteins for subsequent biophysical and biochemical characterization.5,6 These applications skillfully depend on the unique nature of surfactant as an amphiphilic material. The surfactants more or less affect the original structures of proteins. In the ordinal SDS denaturation at room temperature, helical structures are partially formed in non-helical proteins and proteins with lower helicity, whereas they are disrupted in proteins with higher helicity.4,7-10 (The term "helicity" will be used throughout this paper as a short-hand notation for the cumbersome phrase "extent of α-helical structure".) On the other hand, very little is known about the effects of surfactants on proteins which are denatured or affected by any other factor. The interaction with a denatured protein was first investigated by Duggan and Luck.11 They observed that the addition of SDS prevents the rise in viscosity of bovine serum albumin (BSA) in urea solutions, supposing the protective effect of the surfactant on the protein structure. Unfortunately, however, the interaction of a surfactant with a denatured protein has been much less intensively studied. Thereafter, only Markus and Karush12 and Markus et al.13 have studied the protective effect of surfactants on the urea denaturation of human serum albumin by means of optical rotatory dispersion methods. Recently, the present authors’ group has studied the effect of the SDS addition on the helicity changes of


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urea-denatured14,15 and thermally denatured16-18 proteins by applying the circular dichroism (CD) method. Lysozyme19-25 and α-lactalbumin25-35 are homologous proteins. These proteins, which have similar low helicities, adopt more helical structures in the SDS solutions and then they have two types of helices at 25 °C: the original helices and the SDS-induced helices. We hope to gain some insight into the thermal stabilities of the original helices and the SDS-induced helices of the two homologous proteins at high temperatures. Of particular interest are differences and similarities between the secondary structural changes of the proteins in the absence and the presence of SDS.

Materials and Methods Lysozyme (hen-egg) and α-lactalbumin (bovine) were purchased from Seikagaku Co. and Sigma Chemical Co., respectively. SDS14-18,36,37 was obtained from Fluka Chemie AG. A sodium phosphate buffer of pH 7.0 and ionic strength 0.01416-18,36,37 was exclusively used to prepare each solution. The CMC of SDS in the buffer was 5.6 mM at 25 °C.16-18,36,37 The concentrations of lysozyme and α-lactalbumin were determined spectrophotometrically using ε280 = 3.80 X 104 M-1·cm-1 and ε280 = 2.85 X 104 M-1·cm-1, respectively.38,39 The final concentrations of both the proteins were adjusted to 10 µM in all the measurements. CD measurements were carried out with a Jasco J-720 spectropolarimeter using a 1.0 mm path length cell at various temperatures up to 130 °C. Absorbance was measured with Jasco V-550 spectrophotometer at 25 °C. In the CD measurements, we used a special high temperature cell holder system ordered from Japan Spectroscopic Co. to heat an aqueous solution up to temperatures more than 100 °C.17,18 It was confirmed by NMR measurement that a degradation


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of dodecyl sulfate (DS) ion was not induced even at 130 °C.17 A real temperature of the solution in the CD cell was determined with a thermocouple detector in each measurement. The CD spectrum was measured at a desired temperature after keeping the protein solution at the temperature for 15 min. When the temperature was raised or cooled to another temperature through the measurements or when the protein solution was kept at a certain temperature, the cell containing the protein solution was protected from the ultraviolet beam.40 The helicity was estimated by the curve-fitting method of the CD spectrum, using the reference spectra as determined by Chen et al.41 In this method, the optical activities of α-helix, β-structure, and random coil are assumed to be additive at any wavelength and the helicity is calculated by the least-squares method within some particular wavelength region.41 Since the calculation itself is simple, the accuracy depends only on the reproducibility of the spectrum. The size of the circular mark in the following figures was adjusted to be comparable with a maximum error range of obtained helicity. The present simulation was carried out in the wavelength region 200-240 nm at 1-nm intervals.14-18,36,37

Results Thermal Effects on Structures of Lysozyme and α-Lactalbumin Lysozyme, a small monomeric protein of 129 residues, is one of the earliest characterized and most studied globular proteins.19-25 On the other hand, α-lactalbumin, with 123 residues, has been targeted because of the striking similarity to lysozyme. Indeed, α-lactalbumin has high sequence homology and closely similar folded structure to lysozyme.25-35 The CD spectra of lysozyme and α-lactalbumin were measured at various temperatures up to 130 °C. Figure 1 shows the CD spectra of lysozyme at 25 and 90 °C together with the spectrum


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at 25 °C obtained by curve-fitting. Although lysozyme was a protein with low helicity, the CD spectrum at 25 °C showed a double minimum characteristic of α-helix. When the temperature was raised, the α-helical type spectrum was unchanged in a temperature range from 25 °C to a rather high temperature, 60 °C. The spectrum began to change above 60 °C, but still maintained the α-helical shape at 90 °C (Fig. 1). The spectrum did not change so much above 90 °C. In order to explain such a change of CD spectrum, the helicity was estimated from the spectrum by the curve-fitting method using the reference spectra as determined by Chen et al.41 Then, the helicity change was investigated in order to determine the change in secondary structure. The helicity of 27 % was estimated from the spectrum of lysozyme at 25 °C by the curve-fitting (dotted line in the range 200-240 nm in Fig. 1). Lysozyme has been found to consist of four helical segments on the basis of many X-ray analyses. One of the early X-ray studies reported that helices A, B, C, and D are residues 5-15, 24-34, 80-85, and 88-96, respectively,19 indicating that 37 of the 129 residues (29 %) are included in these helices. In the present work, a value of 27 %, which was substantially consistent with those deduced from the X-ray studies, was used as the original helicity of lysozyme at 25 °C. On the other hand, even though α-lactalbumin is generally known to have a very similar structure to lysozyme, a detailed X-ray analysis has not been made, as yet, on the present bovine α-lactalbumin in a monomer state. According to the X-ray study of bovine α-lactalbumin in a hexamer state, this protein contains three major helices per molecule.35 The residue number of the major helices accounts for 31-32 % of the 123 residues. The curve-fitting of the CD spectrum of α-lactalbumin gave the helicity of 34 % at 25 °C. This helicity was used as the original helicity of this protein. Figure 2 shows the helicity change of lysozyme with the rise of temperature and upon cooling to 25 °C from each temperature (temperature on the abscissa). The original helicity of lysozyme


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was maintained up to a particular temperature of 60 °C (this is termed as Critical Temperature below). This indicates that this protein has a heat-resistant characteristic. Above this Critical Temperature, the helicity began to decrease with the rise of temperature. The decrease in the helicity was prominent between 70 and 90 °C. On the other hand, upon cooling to 25 °C after heating up to temperatures below Critical Temperature, no change was observed in the helicity, because the original helices were maintained in this temperature range. Upon cooling to 25 °C after heating up to temperatures of 75-100 °C, the helicity was partially recovered, but this partial recovery was not observed upon cooling after the heat treatments at 65 and 70 °C as well as above 110 °C. Figure 3 shows the helicity change of α-lactalbumin with the rise of temperature and upon cooling to 25 °C from each temperature. The original helicity of α-lactalbumin was maintained up to Critical Temperature of 40 °C. Critical Temperature of α-lactalbumin is appreciably lower than that of lysozyme, indicating that this protein is susceptible to the thermal effect. Above Critical Temperature, the helicity of α-lactalbumin gradually decreased with the rise of temperature up to 100 °C and abruptly decreased above 100 °C. Although the original helicities of lysozyme and α-lactalbumin were 27 and 34 %, respectively, at 25 °C, they finally decreased to a similar extent at 130 °C. As can be seen in Fig. 3, the helicity of α-lactalbumin was completely recovered upon cooling to 25 °C after heating up to a temperature range between 40 °C and 60 °C. On the other hand, upon cooling after heating between 65 and 100 °C, only partial recovery of helicity was observed and no recovery was observed upon cooling after the heat treatments above 110 °C.

SDS Effects on Structural Changes in Thermal Denaturation


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In the SDS denaturation at 25 °C, the helical structure is partially formed in proteins with lower helicity, whereas it is disrupted in proteins with higher helicity, as mentioned in Introduction.4,7-10 Lysozyme36 and α-lactalbumin37 belong to the former, while proteins such as BSA14-17 and myoglobin18 belong to the latter. Then the helical structures are partially formed in lysozyme and α-lactalbumin in the SDS denaturation around room temperature.36,37,42 In the present work, the structural changes of these proteins at high temperatures were examined in the coexistence of SDS of three concentrations, 3, 5, and 10 mM. The total concentration of 3 mM SDS is substantially high enough to saturate the binding of DS ion to these two proteins (10 µM) around room temperature.37,42,43 However, the interaction of protein with surfactant ion such as DS ion becomes weak with the rise of temperature16-18 and then the rise of temperature helps to shift a binding equilibrium between protein and DS ion toward the direction of dissociation. Therefore, a difference appears in the results at 3, 5, and 10 mM SDS with the rise of temperature. Figure 4 shows the SDS effects on the helicity changes of lysozyme with the rise of temperature up to 130 °C and upon cooling to 25 °C from each temperature on the abscissa. In the coexistence of 3, 5, and 10 mM SDS, the helicity of lysozyme increased to 34 % at 25 °C. The helicities of lysozyme-SDS complexes at 3 and 5 mM SDS began to decrease above 35 °C. The helicity at 10 mM SDS also slightly decreased at 40 °C. The stability of lysozyme structure appeared to be lost below 60 °C in the complex state with SDS. However, the decreasing extent of helicity by heating above 75 °C was restrained independently of the SDS concentration as compared with the decreasing extent of helicity in the absence of the surfactant (dotted line in Fig. 4). By the way, the lysozyme solutions containing 1-2 mM SDS are known to become turbid around room temperature.36,42,43 In the present work, the lysozyme solutions containing 3 and 5


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mM SDS became insoluble above 100 and 110 °C, respectively. On the other hand, the helical structures, disrupted by heating, were reformed upon cooling to 25 °C in the coexistence of SDS. The complete recovery of helices was observed upon cooling to 25 °C after the thermal denaturation up to 60, 85, and 95 °C in the coexistence of 3, 5, and 10 mM SDS, respectively. Figure 5 shows the SDS effects on the helicity changes of α-lactalbumin with the rise of temperature up to 130 °C and upon cooling to 25 °C from each temperature. In the coexistence of 3, 5, and 10 mM SDS, the helicity of α-lactalbumin increased to 44 % at 25 °C. This helicity of α-lactalbumin-SDS complex began to decrease above 35 °C. Above 90 °C, the disruption of helices at 10 mM SDS was more restrained than at 3 and 5 mM SDS. On the other hand, upon cooling to 25 °C in the coexistence of 10 mM SDS, the helicity of α-lactalbumin, lost by heating up to 100 °C, was completely recovered. In 3 and 5 mM SDS, the helicities, lost by heating up to 80 °C, were completely recovered upon cooling to 25 °C. The upper limit of temperature for the complete recovery substantially became higher with an increase of the SDS concentration. This was also observed in the lysozyme-SDS complex. The SDS concentration dependence of the upper limit of temperature suggests that the structures of protein complexes with more DS ions tend to recover. This is probably related to an anticipation that not only 3 mM but also 5 mM SDS is not enough at high temperatures to saturate the binding of DS ion to the proteins.

Discussion Comparison of Original Helices of Lysozyme and α-Lactalbumin The present study clearly shows two different aspects between the homologous proteins; (1) the original helices of lysozyme with Critical Temperature of 60 °C is proof against the heat treatment, while those of α-lactalbumin are susceptible to the thermal effect and (2) only the


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structural changes of the original helices of α-lactalbumin are reversible in some temperature range. Related to the first difference (1), lysozyme has been found to have greater resistance against guanidine denaturation44 and trypsin digestion45 compared with α-lactalbumin. The thermodynamic studies reported that lysozyme has more stability than α-lactalbumin with higher thermal transition temperature, higher denaturation enthalpy, higher heat capacity change, and higher Gibbs free-energy change.46,47 The structural stability has also been discussed focusing on the four disulfide bonds which are located at similar positions in both the proteins.36,44,48-56 The disulfide bonds of lysozyme has a resistance against reduction compared with those of α-lactalbumin.44 The reduction of two disulfide bonds in the four bonds causes significant changes of helicity and fluorescence behavior in α-lactalbumin, but not in lysozyme.36,56 These suggest that lysozyme polypeptide itself more tends to fold than α-lactalbumin and then the secondary and tertiary structures of the former are more stable than those of the latter. The other difference (2) between the two proteins is that only α-lactalbumin has the particular temperature range between 40 and 60 °C where the helicity, decreased by heating, completely recovers to the original extent upon cooling to 25 °C. Similar particular temperature ranges have been found in BSA16,17 and myoglobin.18 On the other hand, the original helicity of lysozyme, lost by heating above Critical Temperature, never recovers upon cooling. In this point, lysozyme is contrastive especially with myoglobin which maintains the structural reversibility up to 75 °C.18

Comparison of SDS-Induced Helices of Lysozyme and α-Lactalbumin


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The complexes of lysozyme and α-lactalbumin with SDS adopt higher helicities than the proteins alone at 25 °C.9,10,36,37,42 Then the SDS complexes of these proteins have two types of helices in the SDS solution at 25 °C; the original helices and the SDS-induced helices. Although the total helicities of both the complexes begin to decrease at low temperatures, a distinct difference can be found in the thermal stabilities of the SDS-induced helices in the two proteins. In the case of lysozyme-SDS complexes, only the SDS-induced helices might be disrupted at the heat treatments up to 60 °C, because the original helices are not affected by heating up to this temperature. Indeed, the helicity of lysozyme in the coexistence of SDS decreases to a level at 60 °C which is maintained in the absence of the surfactant at the same temperature. This indicates that the original helices of this protein are still proof against the thermal denaturation in the complex state with SDS. Therefore, the decrement of helicity above 60 °C must be due to the disruption of the originally helical moieties of this protein. By contrast, the SDS-induced helices of α-lactalbumin appear to be so stable in the heat treatment. In the thermal denaturation at any temperature up to 90 °C, the helicities of α-lactalbumin in the coexistence of SDS are approximately 10 % higher than in the absence of the surfactant. In the coexistence of 10 mM SDS, an approximately 10 % higher helicity of the protein is almost maintained up to 130 °C. This difference of 10 % helicity is interpreted as being due to the helicity increment of this protein in SDS (from 34 % to 44 % at 25 °C), that is, the SDS-induced helices. This suggests that, even at high temperatures, the SDS-induced helices remain unchanged and only the original-helices are disrupted in the case of α-lactalbumin-SDS complexes.

Effect of SDS on Original Helices of Lysozyme and α-Lactalbumin


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There is another difference in the decreasing profiles of helicities of the SDS complexes of the two proteins. In the temperature range of 75-120 °C in Fig. 4, the helicities of the lysozyme-SDS complexes are appreciably higher than those of the protein alone (dotted line). This indicates that the binding of DS ions protects the original helices of lysozyme to some extent against the thermal denaturation in this temperature range. On the other hand, the original helices of α-lactalbumin are not protected by the coexistence of SDS at any temperature, taking into account that an approximately 10 % higher helicity is maintained up to 90 °C in the coexistence of the surfactant (Fig. 5). As mentioned above, in the case of α-lactalbumin, only the original helices are disrupted with rise of temperature and the SDS-induced helices remain unchanged even at 130 °C in the coexistence of 10 mM SDS. Thus the binding of DS ions protects or strengthens only the original helices of lysozyme against the thermal denaturation. It is noteworthy that the binding of SDS to lysozyme functions to protect the original helices at high temperatures as well as to induce new helices at 25 °C. This might relate to a characteristic of lysozyme (a typical basic protein) with many positively charged residues which can electrostatically interact with DS ions.43

SDS-induced Reversibility of Helical Structures Upon cooling after the heat treatments at high temperatures, the helical structures of lysozyme-SDS complex and α-lactalbumin-SDS complex are almost completely reformed to each level of SDS denaturation at 25 °C before heating (these denatured states are more helical than the proteins alone at 25 °C). The reversibility can be observed in the two proteins , especially at 10 mM SDS, near a temperature as high as 100 °C. It seems that, upon cooling after the thermal denaturation, each protein-SDS complex has a nature to return to the state at the


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corresponding surfactant concentrations at 25 °C before heating, that is, the cooling treatment tends to rearrange the state of each protein-SDS complex at a high temperature to the state formed at the corresponding SDS concentration at 25 °C. It should be noted that not only SDS-induced helices but also the original helices of each protein are reformed in these recoveries. This means that the SDS-complex states of these proteins at 25 °C are so stable that the DS ions, dissociated from protein at high temperatures, tend to re-bind to protein and reform each complex structure upon cooling to 25 °C. Such a profile has also been observed in the SDS-complexes of BSA and myoglobin, the helicities of which are lower than the original helicities of the proteins alone. Upon cooling to 25 °C after the heat treatments below 90 °C, the helicities of the complexes of BSA and myoglobin, which are further lowered by heating, increase up to each level of SDS denaturation of the corresponding surfactant concentration at 25 °C.16-18 Then, some parts of original helices, which are unsusceptible to the SDS denaturation at 25 °C but are disrupted by heating, might be completely reformed upon cooling after the heat treatment at high temperatures.16-18

In conclusion, the present study of the interaction of SDS with the thermally denatured lysozyme and α-lactalbumin has clarified some important aspects of the structures of the homologous proteins. Remarkable is that the SDS-induced helices of lysozyme are easily susceptible to the thermal effect, while those of α-lactalbumin remain unchanged even at high temperatures. In the thermal denaturations in the absence and the presence of SDS, different and similar behaviors have also been observed between the group of lysozyme and α-lactalbumin with lower original helicities and the group of BSA and myoglobin with higher original helicities. Hopefully, further studies of these and analogous interactions will lead to clarification


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of the mechanism in which the amphiphilic materials, including natural lipids, work on proteins in biological systems as well.

References (1) Steinhardt, J.; Reynolds, J. A. Binding of Organic Ions by Proteins: Multiple Equilibria in Proteins; Academic Press: New York, 1969; pp 234-350. (2) Jones, M. N. Protein-Surfactant Interaction: Biological Interfaces; Elsevier: Amsterdam, 1975; pp 114-134. (3) Lapanje, S. Denaturation by Detergents in Physicochemical Aspects of Protein Denaturation; Physicochemical Aspects of Protein Denaturation; Wiley-Interscience: New York, 1978; pp 156-179. (4) Takeda, K.; Moriyama, Y.; Hachiya, K. Interaction of Protein with Ionic Surfactants: Part 1. Binding of Surfactant to Protein and Protein Fragments, and Comformational Changes Induced by Binding In Encyclopedia of Surfactant and Colloid Science; Hubbard, A. T., Ed.; Marcel Dekker: New York, 2002; pp 2558-2574. (5) Garavito, R. M.; Ferguson-Miller, S. Detergents as Tools in Membrane Biochemistry. J. Biol. Chem. 2001, 276, 32403-32406. (6) Palladino, P.; Ragone, R. Ionic Strength Effects on Critical Micellar Concentration of Ionic and Nonionic Surfactants: The Biding Model. Langmuir. 2011, 27, 14065-14070.


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(7) Jirgensons, B. Effect of n-Propyl Alcohol and Detergents on the Optical Rotatory Dispersion of α-Chymotrypsinogen, β-Casein, Histone Fraction F1, and Soybean Trypsin Inhibitor. J. Biol. Chem. 1967, 242, 912-918. (8) Hunt, A. H.; Jirgensons, B. Effect of Sodium Dodecyl Sulfate and Its Homologs on Circular Dichroism of α-Chymotrypsin. Biochemistry 1973, 12, 4435-4441. (9) Jirgensons, B. Factors Determining the Reconstructive Denaturation of Proteins in Sodium Dodecyl Sulfate Solutions. Further Circular Dichroism Studies on Structural Reorganization of Seven Proteins. J. Protein Chem. 1982, 1, 71-84. (10) Takeda, K.; Moriyama, Y. Circular Dichroism Studies on Helical Structure Preferences of Amino Acid Residues of Proteins Caused by Sodium Dodecyl Sulfate. J. Protein Chem. 1990, 9, 573-582. (11) Duggan, E. L.; Luck, F. M. The Combination of Organic Anions with Serum Albumin. IV. Stabilization against Urea Denaturation. J. Biol. Chem. 1948, 172, 205-220. (12) Murkus, G.; Karush, F. Structural Effects of the Interaction of Human Serum Albumin with Sodium Decyl Sulfate. J. Am. Chem. Soc. 1957, 79, 3264-3269. (13) Murkus, G.; Love, R. L.; Wissler, F. C. Mechanism of Protection by Anionic Detergents against Denaturation of Serum Albumin. J. Biol. Chem. 1964, 239, 3687-3693. (14) Moriyama, Y.; Sato, Y.; Takeda, K. Reformation of the Helical Structure of Bovine Serum Albumin by the Addition of Small Amounts of Sodium Dodecyl Sulfate after the Disruption of the Structure by Urea. J. Colloid Interface Sci. 1993, 156, 420-424.


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(15) Moriyama, Y.; Takeda, K. Re-formation of the Helical Structure of Human Serum Albumin by the Addition of Small Amounts of Sodium Dodecyl Sulfate after the Disruption of the Structure by Urea. A Comparison with Bovine Serum Albumin. Langmuir 1999, 15, 2003-2008. (16) Moriyama, Y.; Kawasaka, Y.; Takeda, K. Protective Effect of Small Amounts of Sodium Dodecyl Sulfate on the Helical Structure of Bovine Serum Albumin in Thermal Denaturation. J. Colloid Interface Sci. 2003, 257, 41-46. (17) Moriyama, Y.; Watanabe, E.; Kobayashi, K.; Harano, H.; Inui, E; Takeda, K. Secondary Structural Changes of Bovine Serum Albumin in Thermal Denaturation up to 130°C and Protective Effect of Sodium Dodecyl Sulfate on the Change. J. Phys. Chem. B 2008, 112, 16585-16589. (18) Moriyama, Y.; Takeda, K. Critical Temperature of Secondary Structural Change of Myoglobin in Thermal Denaturation up to 130°C and Effect of Sodium Dodecyl Sulfate on the Change. J. Phys. Chem. B 2010, 114, 2430-2434. (19) Blake, C. C. F.; Koenig, D. F.; Air, G. A.; North, A. C. T.; Phillips, D. C.; Sarma, V. R. Structure of Hen Egg-White Lysozyme. A Three Dimensional Fourier Synthesis at 2 angstrom Resolution. Nature 1965, 206, 757-761. (20) Tanford, C.; Pain, R. H.; Octin, N. S. Equilibrium and Kinetics of the Unfolding of Lysozyme (Muramidase) by Guanidine Hydrochloride. J. Mol. Biol. 1966, 15, 489-504. (21) Aune, K. C.; Tanford, C. Thermodynamics of the Denaturation of Lysozyme by Guanidine Hydrochloride. I Dependence on pH at 25°. Biochemistry 1969, 8, 4579-4585.


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(22) Aune, K. C.; Tanford, C. Thermodynamics of the Denaturation of Lysozyme by Guanidine Hydrochloride. II Dependence on Denaturant Concentration at 25°C. Biochemistry 1969, 8, 4586-4590. (23) Diamond, R. Real-space Refinement of the Structure of Hen-egg White Lysozyme, J. Mol. Biol. 1974, 82, 371-374. (24) Radford, S. E.; Dobson, C. M.; Evans, P. A. The Folding of Hen Lysozyme Involves Partially Structured Intermediates and Multiple Pathways. Nature, 1992, 358, 302-307. (25) Mckenzie, H. A.; White, F. H. Jr. Lysozyme and α-Lactalbumin: Structure, Function, and Interrelationships. Adv. Protein Chem. 1991, 41, 173-315. (26) Browne, W. J.; North, A. C. T.; Phillips, D. C.; Brew, K.; Vanaman, T. C.; Hill, R. L. A Possible Three-Dimensional Structure of Bovine α-Lactalbumin Based on That of Hen's Egg-White Lysozyme. J. Mol. Biol. 1969, 42, 65-86. (27) Cowburn, D. A.; Brew, K.; Gratzer, W. B. Analysis of the Circular Dichroism of the Lysozyme-α-Lactalbumin Group of Proteins. Biochemistry 1972, 11, 1228-1234. (28) Shechter, Y.; Patchornik, A.; Burstein, Y. Selective Reduction of Cystine I-VIII in α-Lactalbumin of Bovine Milk. Biochemistry 1973, 12, 3407-3413. (29) Warme, P. K.; Nomany, F. A.; Rumball, S. V.; Tuttle, R. W.; Scheraga, H. A. Computation of Structures of Homologous Proteins. α-Lactalbumin from Lysozyme. Biochemistry 1974, 13, 768-782.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(30) Calorimetric Study on the Conformational Transition of α-Lactalbumin Induced by Guanidine Hydrochloride. J. Biochem. 1977, 81, 1051-1056. (31) Kuwajima, K.; Hiraoka, K.; Ikeguchi, M.; Sugai, S. Comparison of the Transient Folding Intermediates in Lysozyme and α-Lactalbumin. Biochemistry 1985, 24, 874-881. (32) Acharya, K. R.; Stuart, D. I.; Walker, N. P. C.; Lewis, M.; Phillips, D. C. Refined Structure of Baboon α-Lactalbumin at 1.7 angstrom Resolution. Comparison with C-type Lysozyme. J. Mol. Biol. 1989, 208, 99-127. (33) Acharya, K. R.; Ren, J.; Stuart, D. I.; Phillips, D. C. Crystal Structure of Human α-Lactalbumin at 1.7 angstrom Resolution. J. Mol. Biol. 1991, 221, 571-581. (34) Kuwajima, K.; Semisotnov, G. V.; Finkelstein, A. V.; Sugai, S.; Ptitsyn, O. B. Seconfdary Structure of Globular Proteins at the Early and the Final Stages in Protein Folding. FEBS Lett. 1993, 334, 265-268. (35) Chrysina, E. D.; Brew, K.; Acharya, K. R. Crystal Structures of apo- and holo-Bovine α-Lactalbumin at 2.2 Angstrom Resolution Reveal an Effect of Calcium on Inter-lobe Interactions. J. Biol. Chem. 2000, 275, 37021-37029. (36) Moriyama, Y.; Hirao, K.; Takeda, K. Conformational Changes of Lysozymes with Different Numbers of Disulfide Bridges in Sodium Dodecyl Sulfate Solutions. Colloid Polym. Sci. 2000, 278, 979-985. (37) Hamada, S.; Takeda, K. Conformational Changes of α-Lactalbumin and Its Fragment, Phe31-Ile59, Induced by Sodium Dodecyl Sulfate. J. Protein Chem. 1993, 12, 477-482.


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(38) Saxena, V. P.; Wetlaufer, D. B. Formation of Three-Dimensional Structure in Proteins. I. Rapid Nonenzymic Reactivation of Reduced Lysozyme. Biochemistry 1970, 9, 5015-5023. (39) Kronman, M. J.; Andreotti, R. E. Inter- and Intramolecular Interactions of α-Lactalbumin. I. The Apparent Heterogeneity at Acid pH. Biochemistry 1964, 3, 1145-1151. (40) Takeda, K.; Moriyama, Y. Unavoidable Time-Dependent Ellipticity Changes of Proteins in the Current CD Measurements. J. Am. Chem. Soc. 1991, 113, 6700-6701. (41) Chen, Y. H.; Yang, J. T.; Chau, K. H. Determination of the Helix and β Form of Proteins in Aqueous Solution by Circular Dichroism. Biochemistry 1974, 13, 3350-3359. (42) Fukushima, K; Murata, Y.; Nishikido, N.; Sugihara, G.; Tanaka, M. The Binding of Sodium Dodecyl Sulfate to Lysozyme in Aqueous Solutions. Bull. Chem. Soc. Japan 1981, 54, 3122-3127. (43) Fukushima, K; Murata, Y.; Sugihara, G.; Tanaka, M. The Biding of Sodium Dodecyl Sulfate to Lysozyme in Aqueous Solutions. II The Effect of Added NaCl. Bull. Chem. Soc. Japan 1982, 55, 1376-1378. (44) Tamburro, A. M.; Jori, G.; Vidali, G.; Scatturin, A.; Saccomani, G. Studies on the Structure in Solution of α-Lactalbumin. Biochim. Biophys. Acta 1972, 263, 704-713. (45) Barman, T. E.; Bagshaw, W. The Modification of the Tryptophan Residues of Bovine α-Lactabumin with 2-Hydroxy-5-Nitrobenzyl Bromide and with Dimethyl(2-Hydroxy-5-Nitrobenzyl)Sulphonium Bromide II. Effect on the Specifier Protein Activity. Biochim. Biophys. Acta 1972, 278, 491-500.


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(46) Habeeb, A. F. S. A.; Atassi, M. Z. Enzymic and Immunochemical Properties of Lysozyme: IV. Demonstration of Conformational Differences between α-Lactalbumin and Lysozyme, Biochim. Biophys. Acta 1971, 236, 131-141. (47) Pfeil, W. Thermodynamics of α-Lactalbumin Unfolding. Biophys. Chem. 1981, 13, 181-186. (48) Lee, C-L.; Atassi, M. Z. Conformational Studies on Modified Proteins and Peptides. VI. Conformation and Immunochemistry of Methylated and Carboxymethylated Derivatives of Lysozyme. Biochemistry 1973, 12, 2690-2695. (49) Acharya, A. S.; Taniuchi, H. Preparation of a Two-Disulfide Bonded Enzymically Active Derivative from Hen Egg Lysozyme. Int. J. Pept. Protein Res. 1980, 15, 503-509. (50) White, F. H. Jr. Studies on the Relationship of Disulfide Bonds to the Formation and Maintenance of Secondary Structure in Chicken Egg White Lysozyme. Biochemistry 1982, 21, 967-977. (51) Denton, M. E.; Sheraga, H. A. Spectroscopic, Immunochemical, and Thermodynamic Properties of Carboxymethyl(Cys6, Cys127)-Hen Egg White Lysozyme. J. Protein Chem. 1991, 10, 213-232. (52) Eyles, S. J.; Radford, S. E.; Robinson, C. V.; Dobson, C. M. Kinetic Consequences of the Removal of a Disulfide Bridge on the Folding of Hen Lysozyme. Biochemistry 1994, 33, 13038-13048.


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(53) Ikeguchi, M.; Sugai, S.; Fujino, M.; Sugahara, T.; Kuwajima, K. Contribution of the 6-120 Disulfide Bond of α-Lactalbumin to the Stabilities of Its Native and Molten Globule States. Biochemistry 1992, 31, 12695-12700. (54) Ewbank, J. J.; Creighton, T. E. Pathway of Disulfide-Coupled Unfolding and Refolding of Bovine α-Lactalbumin. Biochemistry 1993, 32, 3677-3693. (55) Ewbank, J. J.; Creighton, T. E. Structural Characterization of the Disulfide Folding Intermediates of Bovine α-Lactalbumin. Biochemistry 1993, 32, 3694-3707. (56) Takeda, K.; Ogawa, K.; Ohara, M.; Hamada, S.; Moriyama, Y. Conformational Changes of α-Lactalbumin Induced by the Stepwise Reduction of Its Disulfide Bridges: The Effect of the Disulfide Bridges on the Structural Stability of the Protein in Sodium Dodecyl Sulfate Solution. J. Protein Chem. 1995, 14, 679-684.

Figure Captions Figure 1. CD spectra of lysozyme at 25 (solid line) and 90 °C (broken line). The curve-fitting gave the simulated spectrum (dotted line) in the region 200-240 nm to the experimentally obtained spectrum at 25 °C.

Figure 2. Helicity change of lysozyme in the thermal denaturation up to 130 °C. ○: helicity upon keeping at each temperature of abscissa, ●: helicity upon cooling to 25 °C from each temperature of abscissa. The data upon cooling from temperatures below 60 °C were omitted because the helicity did not change in the temperature range.


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Figure 3. Helicity change of α-lactalbumin in the thermal denaturation up to 130 °C. ○: helicity upon keeping at each temperature of abscissa, ●: helicity upon cooling to 25 °C from each temperature of abscissa. The data upon cooling from temperatures below 40 °C were omitted because the helicity did not change in the temperature range.

Figure 4. Helicity change of lysozyme-SDS complex in the thermal denaturation up to 130 °C. ○; at 3 mM SDS, □; at 5 mM SDS, and △; at 10 mM SDS upon keeping at each temperature of abscissa, ●; at 3 mM SDS, ■; at 5 mM SDS, and ▲; at 10 mM SDS upon cooling to 25 °C from each temperature of abscissa. There are no data at 3 and 5 mM SDS above 105 and 115 °C, respectively. Some of data upon cooling were omitted because they were piled up on the same points. The dotted line indicates the helicity upon keeping at each temperature in the absence of SDS.

Figure 5. Helicity change of α-lactalbumin-SDS complex in the thermal denaturation up to 130 °C. ○; at 3 mM SDS, □; at 5 mM SDS, and △; at 10 mM SDS upon keeping at each temperature of abscissa, ●; at 3 mM SDS, ■; at 5 mM SDS, and ▲; at 10 mM SDS upon cooling to 25 °C from each temperature of abscissa. Some of data upon cooling were omitted because they were piled up on the same points. The dotted line indicates the helicity upon keeping at each temperature in the absence of SDS.


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Table of Contents Graphic


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20 [θ] / 103 deg cm2 dmol-1

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Secondary Structural Changes of Homologous Proteins, Lysozyme

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