Secondary Structural Change of Bovine Serum Albumin in Thermal

The secondary structure of bovine serum albumin (BSA) was first examined in the thermal denaturation up to 130 °C. The helicity (66%) of the protein ...
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J. Phys. Chem. B 2008, 112, 16585–16589

16585

Secondary Structural Change of Bovine Serum Albumin in Thermal Denaturation up to 130 °C and Protective Effect of Sodium Dodecyl Sulfate on the Change Yoshiko Moriyama, Emi Watanabe, Kentaro Kobayashi, Hironori Harano, Etsuo Inui, and Kunio Takeda* Department of Applied Chemistry and Biotechnology, Okayama UniVersity of Science, 1-1 Ridai-cho, Okayama 700-0005, Japan ReceiVed: July 30, 2008; ReVised Manuscript ReceiVed: September 29, 2008

The secondary structure of bovine serum albumin (BSA) was first examined in the thermal denaturation up to 130 °C. The helicity (66%) of the protein decreased with rise of temperature. Half of the original helicity was lost at 80 °C, but the helicity of 16% was still maintained even at 130 °C. When the BSA solution was cooled down to 25 °C after heating at temperatures above 50 °C, the helicity was not completely recovered. The higher the thermal denaturation temperature was, the lower was the recovered helicity. On the other hand, upon the addition of sodium dodecyl sulfate (SDS), the secondary structure of BSA was partially protected against the thermal denaturation above 50 °C where the structural change became irreversible. A particular protective effect was observed below 85 °C upon the coexistence of SDS of extremely low concentrations. For example, the helicity was 34% at 80 °C in the absence of SDS, but it was maintained at 58% at the same temperature upon the coexistence of 0.75 mM SDS. Upon cooling down from 80 to 25 °C, the helicity of BSA was recovered to 62% in the presence of 0.75 mM SDS. Such a protective effect of SDS was not observed above 95 °C. In the interaction with the surfactant, this protein structure appeared likely to have a critical temperature between 90 and 100 °C in addition to the critical temperature in the vicinity of 50 °C. This protective effect of SDS, characterized by the specific amphiphilic nature of this anionic surfactant, is considered to be attained by building cross-linking bridges between particular nonpolar residues and particular positively charged residues in the protein molecule. Introduction Thermal denaturation is one of the characteristic properties of protein. It has been understood in a qualitative sense that the original protein structure is disrupted in the thermal denaturation. On the other hand, many studies of protein-surfactant interaction have been carried out for more than half a century. In these studies, the combination of bovine serum albumin (BSA) and sodium dodecyl sulfate (SDS) has been most frequently adopted.1-7 Although many investigators have studied the interactions of surfactants with proteins, their objects have been almost always restricted to the interactions with proteins which are not affected by any other factor. The interaction between a surfactant and a protein, denatured by urea, was first investigated by Duggan and Luck.8 They observed that the addition of SDS prevents the rise in viscosity of serum albumins in urea solutions, supposing the protective effect of the surfactant on the protein structures. However, little attention has been paid to this protective effect of a surfactant on a protein denatured by another factor. Thereafter, only Markus and Karush and Markus et al. have studied the protective effect of surfactants on the urea denaturation of human serum albumin (HSA) by means of optical rotatory dispersion methods.9,10 Recently, the present authors’ group has studied the effect of the SDS addition on the helicities of urea-denatured BSA11 and HSA12 by applying the circular dichroism (CD) method. It has been found that, in the coexistence of small amounts of SDS, the helicities of the proteins are protected against the urea denaturations.11,12 More recently, we have also * Corresponding author. Tel./Fax: +81-86-256-9553. E-mail: takeda@ dac.ous.ac.jp.

reported that the coexistence of a small amount of SDS protects the BSA structure against the thermal denaturation up to 65 °C.13 The thermal denaturation of BSA has a characteristic aspect: the conformational change is reversible below 50 °C but is only partially reversible above this temperature.13-16 In the present study, the secondary structural change of BSA has been examined in high-temperature range up to 130 °C in the absence and the presence of SDS. The results show that the BSA structure can be protected in the thermal denaturation below 85 °C by the addition of a small amount of SDS, which is too low concentration to form aggregates on the protein polypeptide. It has been found through the protective effect of SDS on the structural change of BSA that the protein structure has a critical temperature region between 90 and 100 °C, which appears in the interaction with the surfactant, in addition to the critical temperature in the vicinity of 50 °C. Experimental Methods BSA (A1900) and SDS were purchased from Sigma Chemical Co. and Fluka Chemie AG, respectively. A sodium phosphate buffer of pH 7.0 and ionic strength 0.01417 was exclusively used to prepare each solution. The cmc of SDS in the buffer was 5.6 mM at 25 °C.17 The concentrations of BSA were determined spectrophotometrically using ε280 ) 4.4 × 104 M-1 cm-1.18 The final protein concentration was adjusted to 10 µM. CD measurements were carried out with a Jasco J-720W spectropolarimeter using a 1.0 mm path length cell at various temperatures up to 130 °C. We ordered a special hightemperature CD cell holder system from Japan Spectroscopic

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Figure 1. Comparison of CD spectra of BSA at 80 and 130 °C with that at 25 °C. The dotted line indicates the spectrum of the protein at 80 °C in the coexistence of 0.75 mM SDS.

Co. to heat an aqueous solution up to temperature more than 100 °C. Nitrogen gas of 1.0 MPa was applied to this cell holder. Temperature of the solution in the cell was measured with a thermocouple detector (Technol Seven Co., Ltd.) in each measurement. To check the effect of the exerted pressure on the CD signal, we compared the CD spectra of the protein at 1.0 MPa with those at atmospheric pressure. As a result, it was concluded that the effect of the pressure was negligible. The CD spectrum was measured at a desired temperature after keeping the protein solution at the temperature for 30 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. This is because the irradiation of such an ultraviolet light disrupts the structure of protein.19,20 The helicity was estimated by the curve-fitting method of the CD spectrum, using the reference spectra as determined by Chen et al.21 The simulation was carried out in the wavelength region 200-240 nm at 1 nm intervals. The CD spectrum of BSA can be well simulated5,13,22-26 by using the reference spectra of Chen et al. In the present study, the helicity, calculated by the curve-fitting, is used as a measure of helicity, although the helicity has frequently been discussed on the basis of change of [θ]222. On the other hand, it was confirmed by NMR measurement that a degradation of the dodecyl sulfate (DS) ion was not induced even at 130 °C. The NMR measurement was carried out with a Bruker ARX NMR spectrometer (400 MHz). Results The helicity of BSA has been estimated to be 66%.5,13,22-26 This helicity might be reasonable compared with values determined by other groups27-29 and especially with the helicity of 67% determined for HSA, which is closely homologous protein of BSA, by the X-ray crystallographic study.30,31 The helicity of 66% indicates that the helical structures are formed at 385 amino acid residues in the total residues, 583,32 of BSA molecule. Structural Change of BSA in the Thermal Denaturation at High Temperatures. In the present study, the structural change of BSA was examined in the thermal denaturation up to temperature as high as 130 °C. Figure 1 shows the CD spectra of the protein at three temperatures, 25, 80, and 130 °C. The spectrum showed a typical helical intensity at 25 °C. This spectrum drastically changed with rise of temperature. Figure 2 shows the helicity change of BSA as a function of temperature. The helicity sharply decreased between 50 and 100 °C. The helicity was 22% at 100 °C and 16% at 130 °C. In other words, most of the thermal disruption of helical structures occurred in

Moriyama et al.

Figure 2. Helicity change of BSA with rise of temperature up to 130 °C (O) and the recovered helicity upon cooling down to 25 °C from each temperature (b). The abscissa for the recovered helicity indicates a denaturation temperature before cooling down to 25 °C.

the temperature range up to 100 °C. The profile of the helicity change of BSA in the thermal denaturation below 65 °C has been described in our previous paper.13 Beyond 30 °C, the helicity of the protein began to decrease with rise of temperature and reached 16% at 130 °C. The helicity of 16% indicates that most of the helical structures are disrupted at 130 °C, but helical moieties, consisting of 93 amino acid residues in calculation (583 total residues × 0.16), are still maintained at this temperature. This figure also shows the recovery degree of the helicity upon cooling down to 25 °C after the heat treatment at each temperature. It is already known that the secondary structure of BSA completely recovers upon cooling down from temperatures below 50 °C, but it does not above this temperature.13 As shown in Figure 2, when the temperature was cooled down to 25 °C from high temperatures beyond 50 °C, the helicity did not attain the original value before the heat treatment. The higher the denaturation temperature was, the lower the recovered helicity was. When the temperature was cooled down from 130 to 25 °C, the helicity did not attain the original value before heating up to 130 °C, increasing only to 20%. The difference between 66% and 20% corresponds to the disruption of helices formed at approximately 270 amino acid residues (583 total residues × (0.66 - 0.20)). This means that about 70% (270/helical 385 residues) of the original helices are not recovered upon the descent of temperature. Protective Effect of SDS on BSA Structure at High Temperatures. One more purpose of this work is to examine the protective effect of SDS on the structure of the protein in the thermal denaturation at high temperatures up to 130 °C. Then, the CD spectra of BSA were measured under the coexistence of SDS of various concentrations at several temperatures up to 130 °C. We also examined the SDS effect on the recovery degree of the protein structure upon cooling down to 25 °C after the thermal denaturations at high temperatures, as mentioned later. Figure 3 shows the dependences of BSA helicity on the SDS concentration at several temperatures between 80 and 130 °C. Although the helicity decreased from 66% down to 34% at 80 °C in the absence of SDS, the decrement of it was restrained in coexistence of the surfactant of concentrations lower than 2 mM, as seen in Figure. 3. The CD spectrum of the protein in 0.75 mM SDS at 80 °C is indicated by the dotted line in Figure 1, as a typical coexistence effect of the surfactant. The helicity at 80 °C was most markedly maintained in the coexistence of 0.75 mM SDS. At this SDS concentration, the helicity slightly decreased to 55%, as shown in Figure 3. This protected helicity of 55% at 80 °C is not so different from that of 58% observed at 65 °C13 in the coexistence of 0.15 mM SDS. At 85 °C, the

SDS Effect on BSA Structure in Heat Denaturation

Figure 3. Dependences of BSA helicity on the SDS concentration at 80 (O), 85 (b), 90 (4), 100 (2), 110 (0), 120 (9), and 130 °C ()). The dotted line indicates the helicity change of the protein at 25 °C.

helicity was maximally protected at 1.0 mM SDS. The maximal protective effect at 85 °C was 42% in the coexistence of 1.0 mM SDS. The protective effect at low SDS concentrations became weak at 85 °C than at 80 °C. Beyond these particular SDS concentrations, the protected helicity decreased with an increase in the surfactant concentration at both temperatures of 80 and 85 °C. Above these SDS concentartions, DS ions appear likely to begin to form a micelle-like aggregate on the BSA polypeptide. This aggregation causes the SDS denaturation to decrease the helicity at 80 and 85 °C. Figure 3 also shows the helicity change (dotted line) of BSA as a function of SDS at 25 °C5,17,22 (the ordinal SDS denaturation). In the SDS denaturation, the helical structure is generally formed in nonhelical proteins and proteins with less helicity, while it is disrupted in proteins with high helicity5 (BSA belongs to the latter). Therefore, BSA suffers the SDS denaturation in addition to the thermal denaturations of high temperatures, both of which disrupt the helical structures, above the aforementioned particular SDS concentrations. The protective effect of the surfactant appears only below 2 mM SDS. An important finding is that the great protective effect appears below 1 mM SDS at temperatures below 85 °C. The protective effect is observed in low SDS concentration region at temperatures below 85 °C. This is the same trend as observed at temperatures between 50 and 65 °C.13 However, the protective effect in low SDS concentration region was not observed at temperatures above 90 °C, as seen in Figure 3. Above this temperature, the decrement of helicity by heating was gradually restrained with an increase in the SDS concentration. At 90 and 100 °C, the protected helicity gradually increased with an increase in the SDS concentration up to 2.0 mM and up to 4.0 mM, respectively. Above 4.0 mM SDS, the helicity of approximately 30-40% was maintained at temperatures below 100 °C. Then, at these high temperatures, the helicity of BSA at high SDS concentrations seems to be kept at a considerably high level compatible with that (50%) at 25 °C.5,17,22 However, the protected helicity in the coexistence of SDS of high concentrations decreased with rise of temperature above 110 °C. The presence of SDS hardly affected the helicity at 120 and 130 °C. It is another finding that, upon keeping at high temperatures, the profile of the effect of high SDS concentration on the BSA structure differs below and above the temperature range of 100 to 110 °C. Protective Effect of SDS on BSA Structure upon Cooling down to 25 °C from High Temperatures. A distinct protective effect of SDS on the BSA helicity was observed upon cooling down to 25 °C from high temperatures. Figure 4 shows the CD spectra of BSA upon cooling down to 25 °C from 80 °C in the

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Figure 4. CD spectra of BSA upon cooling down to 25 °C from 80 °C in the coexistence of SDS of several concentrations. The dotted line indicates the spectrum of the native protein at 25 °C.

Figure 5. Dependences of BSA helicity on the SDS concentration upon cooling down to 25 °C from 80 (O), 85 (b), 90 (4), 100 (2), 110 (0), 120 (9), and 130 °C ()).

coexistence of SDS of several concentrations. The presence of 0.75 mM SDS mostly restored the CD spectrum indicative of disordered form to the spectrum indicative of R-helical structure. However, a higher concentration of the surfactant caused the distortion of this R-helical type spectrum. Figure 5 shows the changes of BSA helicity as a function of SDS concentration upon cooling down to 25 °C from high temperatures. When the temperature was lowered from 80 °C, the recovered helicity increased with an increase of added SDS concentration up to 0.75 mM. Beyond this SDS concentration, the recovery extent decreased with an increase of SDS concentration. The maximal recovered helicity, 62%, is very close to the original extent, 66%,5,22 before heating up to 80 °C. In the absence of SDS, the helical structures are maintained only at 198 residues (583 total residues × 0.34) at 80 °C and they are additionally reformed only at about 50 residues upon cooling from 80 to 25 °C. Upon cooling from 80 to 25 °C in the coexistence of 0.75 mM SDS, however, the helical structures are formed at 360 residues (583 × 0.62) in the original 385 residues. Most of the original helices are practically protected by such a small amount of SDS. A similar recovery has been observed upon cooling to 25 °C from 65 °C.13 The maximal recovery of helicity was observed at low SDS concentrations upon cooling from 85 and 90 to 25 °C, but the maximal recovery extents apparently became lower than that from 80 °C. The protein concentration is 0.01 mM and then the molar ratio of [DS]/[BSA] of about 15 is enough to induce the maximal protective effect on the helicity of BSA at 65 °C.13 The SDS concentration required to induce the maximal protective effect increases with rise of temperature not only upon cooling down from high temperatures but also upon keeping at high temperatures (Figure 3). It should be noted that such a small amount of DS ion functionally protect the protein structure, since the molecular weight of DS ion is about 1/250 of that32 of BSA (66 400).

16588 J. Phys. Chem. B, Vol. 112, No. 51, 2008 In the presence of high SDS concentrations above 4 mM, the helicity was recovered to a similar extent upon cooling down from temperatures below 90 °C (Figure 5). The helicity of BSA decreases to 50% in the ordinal SDS denaturation at 25 °C.5,22 The similar helicity is attained upon cooling down from temperatures below 90 °C. This suggests that the BSA structure, denatured by heating up to temperatures below 90 °C, can be reconstructed to the state of ordinal BSA-SDS complex at 25 °C. However, upon cooling down from temperatures above 100 °C, the recovery extent became lower with rise of temperature. It was examined by the NMR measurement that DS ion did not decompose on heating up to 130 °C. It can therefore be presumed that BSA structure itself is incurably damaged by heating up to temperatures above 100 °C. Discussion As mentioned above, it is already known that the BSA structure has a critical temperature region around 50 °C.13-16 Beyond this region in the thermal denaturation, the structural change of the protein becomes irreversible. Since the helicity is 62% at 50 °C as against 66% at 25 °C, the structural change occurs on a small scale in the temperature range where it does reversibly. On the other hand, it has been proposed by Brown that BSA has three domains and that the helical moieties of the protein can be divided into 18 segments.5,17,33 The validity of his model is verified by the crystallographical analysis of HSA30,31 which is a homologous protein of BSA. The helicity of 62% is corresponding to a state that the helical structures are disrupted only at C-terminal end or only at two connecting segments between the three domains. Since these moieties are susceptible to various effects such as pepsin digestion,22,25,27,34-37 the helical structures are anticipated to be unstable there. Stable helical structures appear likely to be formed within the three domains.5,22,33 It is considered that BSA loses the reversibility of the structural change in the thermal denaturation when these stable helical structures begin to be disrupted. The binding of DS ion to BSA molecule saturates above 7 mM SDS (total concentration) in the present buffer and the saturated binding number is approximately 200 mol/mol.17 It is worth noting in the present study that a maximal protective effect appears below or around 1 mM SDS (total concentration) upon keeping at certain high temperatures and upon cooling down to 25 °C. When the total SDS concentration is 1 mM, the number of bound DS ion to BSA is estimated to be approximately 25 mol/mol (Figure 5 in ref 17). At 1 mM SDS where such a number of DS ions bind to BSA, the helical structure of BSA is hardly affected in the ordinal SDS denaturation at 25 °C5,22 (dotted line in Figure 3). However, upon cooling down to 25 °C from 80 °C in the presence of 0.75 mM SDS, the helicity, lost by heating, recovers to the similar extent to that at 25 °C5,13,22 before heating. This tendency remains up to 90 °C. On the other hand, upon keeping at high temperatures, the protective effect of SDS of low concentrations occurs up to 85 °C, but disappears at 90 °C. Upon keeping at high temperatures, a boundary temperature region of the effect exists at 85-90 °C, while a similar region appears at 95-100 °C upon cooling down to 25 °C. The boundary temperature region slightly shifts higher upon cooling down to 25 °C. This might be due to a decrease of binding number of DS ion to BSA with rise of temperature in addition to the disruption of helical structures by heating. In the protective profile of SDS, the BSA structure appears likely to show another critical temperature region around 90 °C. We have previously examined the removal of DS ions bound to the BSA molecule by adding surplus amount of potassium

Moriyama et al. ion (changing the sodium salt of DS ion to the potassium salt in order to use a low solubility of the latter in aqueous solution).38 The remaining number of DS ions on the protein is approximately 25 mol/mol at high potassium concentrations.38 Interestingly, this number agrees with the aforementioned binding number of DS ion estimated around 1 mM SDS (total concentration). It might be unreasonable to assume that this number of DS ion separately forms aggregates at a few sites on the BSA polypeptide.1,5,17 The present SDS concentrations, where the protective effects are observed, should be considered to be too low to form micelle-like aggregates on the protein. Upon cooling down to 25 °C from 65 °C,13 the maximal protective effect appears at 0.15 mM SDS (total concentration). An actual binding number of DS ion is expected in this case to be apparently smaller than the mixing ratio of 15 (0.15 mM DS ion/0.010 mM BSA). Indeed, when smaller amounts of helical structures of BSA are disrupted at temperatures below 65 °C, the SDS concentration range, where the protective effect appears, becomes lower.13 Such an extremely small amount of DS ion exhibits the protective effect of the protein structure. We have assumed that the protein structure is stabilized by a specific function of amphiphilic nature of DS ion.11-13 It is probably a cross-linking function of the anionic surfactant ion between a group of nonpolar residues and a positively charged residue in the protein. This concept was first introduced by Markus et al.9,10 Furthermore, such linkages of DS ions have been verified in the case of lysozyme by the X-ray crystallography: the hydrophobic chain makes hydrophobic contacts with particular residues, while the hydrophilic group is saltbridged to charged residues.39 We have previously examined in the thermal denaturation of BSA up to 65 °C that this crosslinking function is influenced by the amphiphilic nature of ionic surfactant, that is, the hydrophobic chain length and a positive or negative charge of hydrophilic group.13 The hydrophobic nature of the ionic surfactant plays an important role in the protective effect, on the one hand. On the other hand, the negative charge of the hydrophilic group also plays another role against the amino acid residues with a positive charge, such as Lys, since a cationic surfactant does not protect the protein structure at all.13 The present protective effect is due to both the hydrophobic nature and hydrophilic nature of the anionic surfactant ion. Such cross-linkages are anticipated to be formed at particular sites in the BSA molecule to protect its structure in the present thermal denaturation at high temperatures. More linkages appear likely to be required to protect the protein structure with rise of temperatue, that is, with the disrupted extent of the structure.13 A surfactant is generally recognized to fulfill its function in the aggregated state such as a micelle. It is also well discussed that a surfactant exhibits a characteristic function by forming a micelle-like aggregate on a protein or polymer.1-5 In connection with the formation of micelle-like aggregates, the necklace model was proposed more than 30 years ago.40 This model was originally proposed to insist that micelle-like aggregates were locally formed at some hydrophobic moieties of BSA polypeptide, all of the disulfide bridges of which were reduced and blocked. The intact BSA has 17 disulfide bridges, which are rather regularly located in the protein polypeptide consisting of 583 residues. Unfortunately, the polypeptide of the intact BSA is sometimes assumed to behave like a flexible string. However, it should be noted that the flexibility of BSA polypeptide is strongly restricted by these 17 disulfide bridges in addition to the general folding nature of protein polypeptide. Furthermore, it is doubtful that the long polypeptide chain of

SDS Effect on BSA Structure in Heat Denaturation the reduced BSA behaves like a flexible string. The present authors understand that the speculated sectional view of the aggregate looks like a necklace tied round a neck (a section of polypeptide). The authors consider that the polypeptide chain of the intact BSA neither plays a role of flexible string of long necklace nor wraps around micelles. The present recovery of the secondary structure upon cooling down from high temperatures also suggests that the protein polypeptide keeps its whole steric conformation to a considerable extent even in the thermal denaturations at high temperatures. The present paper demonstrates the novel effect of a small amount of SDS, which is too low concentration to form such aggregates. It has become clear through such a protective behavior of DS ion that BSA-SDS complex has a critical temperature around 90 °C. The DS ions must fulfill the present protective function in a monomer state in the thermal denaturations at high temperatures. The effect is due to the amphiphilic nature of the surfactant ion: the anionic charge of hydrophilic group and hydrophobicity of hydrophobic group. This is a new and unique function presented by the characteristic nature of ionic surfactant as an amphiphilic material. The present information might be useful to know the mechanism in which the amphiphilic materials, like a lipid, work on proteins in biological system as well. In other words, the amphiphilic materials such as a phospholipid might act not only to form vesicles but also to affect protein structures and functions as characteristic and strong amphiphilic monomers. Acknowledgment. The authors thank Professor Susumu Ohira, Department of Biochemistry, Okayama University of Science, for his helpful cooperation and discussion in the NMR measurement. References and Notes (1) Steinhardt, J.; Reynolds, J. A. In Multiple Equilibria in Proteins; Academic Press: New York, 1969; pp 239-302. (2) Jones, M. N. In Biological Interfaces; Elsevier: Amsterdam, 1975; pp 101-130. (3) Waehneldt, T. V. Biosystems 1975, 6, 176. (4) Lapanje, S. In Physicochemical Aspects of Protein Denaturation; Wiley-Interscience: New York, 1978; pp 156-179. (5) Takeda, K.; Moriyama, Y.; Hachiya, K. In Encyclopedia of Surfactant and Colloid Science; Hubbard, A. T., Ed.; Marcel Dekker: New York, 2002; pp 2558-2574. (6) Takeda, K.; Hachiya, K.; Moriyama, Y. In Encyclopedia of Surfactant and Colloid Science; Hubbard, A. T., Ed.; Marcel Dekker: New York, 2002; pp 2575-2592. (7) Takeda, K.; Moriyama, Y. J. Phys. Chem. B 2007, 111, 1244. (8) Duggan, E. L.; Luck, F. M. J. Biol. Chem. 1948, 172, 205. (9) Markus, G.; Karush, F. J. Am. Chem. Soc. 1957, 79, 3264.

J. Phys. Chem. B, Vol. 112, No. 51, 2008 16589 (10) Markus, G.; Love, R. L.; Wissler, F. C. J. Biol. Chem. 1964, 239, 3687. (11) Moriyama, Y.; Sato, Y.; Takeda, K. J. Colloid Interface Sci. 1993, 117, 420. (12) Moriyama, Y.; Takeda, K. Langmuir 1999, 15, 2003. (13) Moriyama, Y.; Kawasaka, Y.; Takeda, K. J. Colloid Interface Sci. 2003, 257, 41. (14) Imahori, K. Biochim. Biophys. Acta 1960, 37, 336. (15) Anderle, G.; Mendelsohn, R. Biophys. J. 1987, 52, 69. (16) Lin, V. J. C.; Koenig, J. L. Biopolymers 1976, 15, 203. (17) Takeda, K.; Miura, M.; Takagi, T. J. Colloid Interface Sci. 1981, 82, 38. (18) Sober, H. A., Harte, R. A., Eds.; Handbook of Biochemistry (Selected Data for Molecular Biology), 2nd ed.; CRC Press: Cleveland, OH, 1973; p, C-71. (19) Takeda, K.; Moriyama, Y. J. Am. Chem. Soc. 1991, 113, 6700. (20) Moriyama, Y.; Kakehi, T.; Takeda, K. Anal. Biochem. 1994, 219, 378. (21) Chen, Y. H.; Yang, J. T.; Chau, K. H. Biochemistry 1974, 13, 3350. (22) Takeda, K.; Shigeta, M.; Aoki, K. J. Colloid Interface Sci. 1987, 117, 120. (23) Takeda, K.; Sasa, K.; Nagao, M.; Batra, P. P. Biochim. Biophys. Acta 1988, 957, 340. (24) Takeda, K.; Sasa, K.; Kawamoto, K.; Wada, A.; Aoki, K. J. Colloid Interface Sci. 1988, 124, 284. (25) Takeda, K.; Wada, A.; Nishimura, T.; Ueki, T.; Aoki, K. J. Colloid Interface Sci. 1989, 133, 497. (26) Batra, P. P.; Sasa, K.; Ueki, T.; Takeda, K. Int. J. Biochem. 1989, 8, 857. (27) Reed, R. G.; Feldhoff, R. C.; Clute, O. L.; Peters, T., Jr. Biochemistry 1975, 14, 4578. (28) Geisow, M. J.; Beaven, G. H Biochem. J. 1979, 163, 477. (29) Wetzel, R.; Becker, M.; Behlke, J.; Billwitz, H.; Bohm, S.; Ebert, B.; Hamann, H.; Krumbiegel, J.; Lassmann, G. Eur. J. Biochem. 1980, 104, 469. (30) He, X. M.; Carter, D. C. Nature 1992, 358, 209. (31) Carter, D. C.; Ho, J. X. AdVan. Protein Chem. 1994, 45, 153. (32) Hirayama, K.; Akashi, S.; Furuya, M.; Fukuhara, K. Biochem. Biophys. Res. Commun. 1990, 173, 639 This reference shows a new tyrosine residue at 156th position in the old sequence of 582 residues. Now, the total residue number of BSA is 583 and its molecular weight can be calculated to be 66 428, although there are still wrong descriptions of the total residue number 582 or the molecular weight 69 000 even in recent publications. (33) Brown, J. R. In Albumin Structure, Function, and Uses; Rosenoer, V. M., Oratz, M., Rothschild, M. A., Eds.; Pergamon Press: Oxford, UK, 1977; pp 27-51. (34) Braam, W. G. M.; Hilak, M. C.; Harmsen, B. J. M.; Van Os, G. A. J. Int. J. Peptide Protein Res. 1974, 6, 21. (35) Hilak, M. C.; Harmsen, B. J. M.; Braam, W. G. M.; Joordens, J. J. M.; Van Os, G. A. J. Int. J. Peptide Protein Res. 1974, 6, 95. (36) Feldhoff, R. C.; Peters, T., Jr. Biochemistry 1975, 14, 4508. (37) Reed, R. G.; Feldhoff, R. C.; Peters, T., Jr. Biochemistry 1976, 15, 5394. (38) Takeda, K.; Wada, A.; Sato, Y.; Hamada, S. J. Colloid Interface Sci. 1991, 147, 51. (39) Yonath, A.; Prodjarny, A.; Honig, B.; Sielecki, A.; Traub, W. Biochemistry 1977, 16, 1418. (40) Shirahama, K.; Tsujii, K.; Takagi, T. J. Biochem. 1974, 75, 309.

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