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Department of Chemistry, School of Science, Kwansei-Gakuin UniVersity, Nishinomiya 662-8501, Japan. ReceiVed: February 10, 2000; In Final Form: April ...
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J. Phys. Chem. B 2000, 104, 5840-5847

Two-Dimensional Near-Infrared Spectroscopy Study of Human Serum Albumin in Aqueous Solutions: Using Overtones and Combination Modes to Monitor Temperature-Dependent Changes in the Secondary Structure Yuqing Wu,† Bogusława Czarnik-Matusewicz,‡ Koichi Murayama,§ and Yukihiro Ozaki* Department of Chemistry, School of Science, Kwansei-Gakuin UniVersity, Nishinomiya 662-8501, Japan ReceiVed: February 10, 2000; In Final Form: April 10, 2000

FT-NIR spectra were measured for human serum albumin (HSA) in aqueous solutions with concentrations of 1.0, 2.0, 3.0, 4.0, and 5.0 wt % over a temperature range of 45-80 °C. Concentration-perturbed two-dimensional (2D) correlation spectra were calculated for the spectra in the 7500-5500 cm-1 and 4900-4200 cm-1 regions at different temperatures. To investigate temperature-induced changes in the secondary structure and hydration, power spectra and slice spectra were calculated from the synchronous and asynchronous spectra, respectively. In the power spectra, a band near 4600 cm-1 due to the combination mode of amide B and amide II (amide B/II) shows an abrupt shift by 5 cm-1 between 58 and 60 °C, indicating that the secondary structure of HSA changes suddenly near 60 °C. Both the power and slice spectra in the 7500-5500 cm-1 region provide explicit evidence that the hydration changes markedly near 60 °C. A comparison of the temperature-dependent frequency shifts between the band near 4600 cm-1 due to amide B/II and that near 7000 cm-1 due to the combination mode of water reveals that the protein unfolding occurs almost in parallel with the change in the hydration. The present study demonstrates that the overtone and combination modes are very useful in monitoring subtle changes in protein dynamics.

Introduction Vibrational spectroscopy has been used extensively to investigate the structure and dynamics of proteins in aqueous solutions, because the vibrational frequencies are very sensitive not only to changes in the bonding and geometrical arrangement of atoms in molecules but also to inter- and intramolecular interactions.1-4 Among vibrational spectroscopy techniques, infrared (IR) spectroscopy is very useful in studying the secondary structure of proteins, whereas Raman spectroscopy is powerful in exploring the secondary structure, the microenvironment of amino acid residues, and the structure of the prosthetic groups (resonance Raman). Recently, time-resolved vibrational spectroscopy and ultraviolet excited Raman spectroscopy have been employed to investigate protein dynamics.5,6 In addition to IR and Raman spectroscopy, near-IR (NIR) spectroscopy7-9 has also proved useful for protein research.10-14 NIR spectroscopy has several advantages over IR spectroscopy in studying protein structure. First, one can use a cell having a thickness of 0.1-1 mm, and thus, the exact concentration of a protein solution can be estimated. In the case of IR spectroscopy, one must use a very thin cell (on the order of micrometers) or an attenuated total reflection (ATR) prism for the IR measurement of the protein solution. Therefore, there always remains a * Author to whom all correspondence should be addressed. Mailing address: Yukihiro Ozaki, Department of Chemistry, School of Science, Kwansei-Gakuin University, Nishinomiya 662-8501, Japan. Fax: +81-79851-0914. E-mail: [email protected]. † Present address: Key Laboratory of Supramolecular Structure and Spectroscopy, Jilin University, Changchun, 130023, P. R. China. ‡ Present address: Faculty of Chemistry, University of Wrocław, F. JoliotCurie 14, 50-383 Wrocław, Poland. § Present address: Department of Environment Information and Bioproduction Engineering, Faculty of Agriculture, Kobe University, Rokkodai, Nada-ku, Kobe 657-8501, Japan.

problem of adsorption of protein molecules on the cell. In contrast, NIR spectroscopy is almost free from such problems. Another advantage of NIR spectroscopy is that the effects of bands due to water vapor are much weaker in the NIR region. In the IR region, bands arising from water vapor appear in the amide I band region, often adding noise to the amide I bands, which are key bands for exploring the secondary structure. Yet another advantage is that one can probe hydration of proteins by means of NIR spectroscopy because bands due to proteins and those arising from water have comparable intensities in the NIR region.14 On the other hand, in the IR region, bands due to water are so strong compared with those arising from proteins that it is very difficult to investigate hydrogen-bonded water by IR spectroscopy. It is also very important to point out that bands due to various water species (i.e. free and bonded water) are better separated in the NIR region.15 The disadvantage of NIR spectroscopy is that a number of bands due to combination and overtone modes overlap each other in the NIR region.7-9 Therefore, it is not always straightforward to extract information about the protein structure and hydration from conventional one-dimensional NIR spectra. Recently, generalized two-dimensional (2D) correlation spectroscopy16,17 has been applied to the analysis of NIR spectra of proteins in aqueous solutions.11,12,14 It has the following advantages in the analysis of NIR spectra:16,17 (i) It sorts out complex or overlapped spectral features by spreading peaks along the second dimension. (ii) It provides the specific order of the spectral intensity changes taking place during the measurement or the value of the controlling variable affecting the spectra. (iii) It assists in the identification of various interand intramolecular interactions through the selective correlation of bands. (iv) It permits studies of correlations between NIR bands and bands in other spectra such as Raman and IR spectra.

10.1021/jp000537z CCC: $19.00 © 2000 American Chemical Society Published on Web 05/31/2000

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Figure 1. NIR spectra in the (a) 7500-5500 and (b) 4900-4200 cm-1 regions of HSA in aqueous solutions with concentrations of 1.0, 2.0, 3.0, 4.0, and 5.0 wt %.

In our previous study, heat-induced denaturation of ovalbumin in aqueous solutions was explored by 2D NIR correlation spectroscopy.14 New insight was gained into the hydration and unfolding process of ovalbumin by investigating temperaturedependent correlation patterns in 2D synchronous and asynchronous spectra generated from concentration-perturbed NIR spectra of the ovalbumin solutions measured over a temperature range of 45-80 °C. The purpose of the present study is to further develop generalized 2D NIR correlation spectroscopy studies of protein dynamics. To do that, we have attempted two new approaches. One is to expand the spectral region analyzed from the 49004550 cm-1 region to the 4900-4250 cm-1 region. By expanding the spectral region, we may be able to obtain information about the side chains, because bands due to the CHn combination modes appear near 4370 cm-1. Another is to use power spectra in the synchronous spectra and slice spectra in the asynchronous spectra. By plotting peak top frequencies of power and slice spectra versus temperature, one can monitor changes in the secondary structure, hydration, and hydrogen bonding. The present study has opened an avenue for investigating protein hydration by means of NIR spectroscopy. We demonstrate that one can explore simultaneously changes in the secondary structure and hydration from a series of temperature-dependent NIR spectra of protein with the aid of 2D correlation spectroscopy. The protein we have investigated is human serum albumin (HSA), which is a midsized protein with a molecular weight of approximately 66.4 kDa built from 585 residues.18 The crystal structure and peptide sequence of HSA were reported by Brown in 1977.19 Recently, a high-resolution X-ray crystallographic study was performed by Curry et al.20 The coordinates and structure factors for HSA lattice X obtained by Curry et al.20 are deposited in the Brookhaven Protein Data Bank with a access code 1BJ5.21 Denaturation temperatures and enthalpies of HSA measured by DSC at different pH values and protein concentrations were reported by Barone et al.22 The DSC method has provided direct values of the denaturation enthalpy (∆dH), the temperature of the midpoint of denaturation corresponding to the maximum of the heat emission plot (Td), and the change of

the molar specific heat (∆dCp) for the transition from the native to the unfolding state.22 Structural changes of HSA in aqueous solutions upon pH and heat denaturation were investigated by use of vibrational spectroscopy.23-26 Hvidt24 studied the conformational changes in HSA as revealed by hydrogendeuterium exchange. Bramanti et al.23 explored quantitative determination of the secondary structure from IR spectra of HSA in aqueous solutions in the native state and in the denatured states induced by heat and acid treatment. Each of these perturbations induces variations of the secondary structure of HSA in aqueous solutions.18 However, the more detailed structural basis, the order of secondary structure changes, and the changes in hydration that take place during the the folding or unfolding procedure still must be elucidated. Experimental Section 1. Sample Preparation. Human serum albumin (HSA, Grade V with purity of approximately 99%) was purchased from Sigma Chemical Co. and used without further purification. We confirmed the high purity of HSA by standard gel electrophoresis. The protein was dissolved in doubly distilled water to prepare the HSA solutions (pH 6.6) with concentrations of 1.0, 2.0, 3.0, 4.0, and 5.0 wt %. 2. Instrumentation. NIR spectra of the HSA solutions were measured with 4 cm-1 resolution by use of a Nicolet Magna 760 FTIR/NIR spectrometer equipped with a PbSe detector. To yield a high signal-to-noise ratio, 512 interferograms were coadded. For investigations of the temperature-dependent unfolding and folding procedure, the spectra were collected with an increment of 5 °C between 45 and 58 °C and with an increment of 2 °C between 58 and 80 °C, and an equilibration time of 15 min was used at each temperature. A quartz cell having a length of 1 mm was employed for the present experiments. The cell was put into a cell holder whose temperature was controlled by circulating water. To measure the temperature, a digital thermometer (Anritsu HFT-50) was dipped into the cell before and after each spectral measurement. The system gave a temperature stability of better than (0.2 °C. Background spectra of the empty cell were also obtained at each selected temperature.

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Figure 2. 2D NIR correlation spectra in the 4900-4200 cm-1 region, constructed from concentration-perturbed spectra of HSA in aqueous solutions in the natively folded state at 45 °C. (a) Synchronous and (b) asynchronous contour maps.

Figure 3. 2D NIR correlation spectra in the 4900-4250 cm-1 region, constructed from concentration-perturbed spectra of HSA in aqueous solutions in the denatured unfolded state at 80 °C. (a) Synchronous and (b) asynchronous contour maps.

3. 2D Correlation Analysis. Powerful, yet easily executable, software named KG2D for constructing generalized 2D correlation spectra has been composed by Y. Wang (Kwansei Gakuin University) with the Array Basic programming language (The Galactic Industries Corp.).14 This 2D software was programmed on the basis of the newly developed algorithm of generalized 2D correlation spectroscopy.16,17 Results 1. NIR Spectra of HSA in Aqueous Solutions. Figure 1 shows NIR spectra in the (a) 7500-5500 cm-1 and (b) 4900-

4250 cm-1 regions of HSA in aqueous solutions with concentrations of 1.0, 2.0, 3.0, 4.0, and 5.0 wt % at 45 °C (pH 6.6). Strong bands due to water dominate these two regions. No peak arising from the protein can be detected visually, and it is very difficult to find any significant spectral changes due to the folding and unfolding of HSA in aqueous solutions, even if we compare the spectra shown in Figure 1 with those measured at 80°. Thus, we have carried out 2D correlation analysis for the two spectral regions. 2. 2D NIR Correlation Spectra in the Range of 49004250 cm-1. 2.1. Band Assignment in the Range of 4900-4250

Human Serum Albumin in Aqueous Solutions

Figure 4. Power spectra along the diagonal line in the range of 49004250 cm-1 in the synchronous contour maps, constructed from concentration-perturbed spectra of HSA in aqueous solutions at 45, 58, 60, and 80 °C.

cm-1. Figure 2 shows (a) synchronous and (b) asynchronous 2D correlation spectra constructed from the concentrationperturbed NIR spectra in the 4900-4250 cm-1 region of the HSA solutions. Throughout this paper, solid and dashed lines in the contour maps represent positive and negative peaks, respectively. Compared with our previous work,14 we have investigated a wider spectral region of 4900-4250 cm-1 because bands due to CHn combination modes of the side chains appear near 4370 cm-1. Two strong autopeaks are observed at 4600 and 4367 cm-1 along the diagonal line in the synchronous spectrum. These peaks probably come from the combination mode of amide B and amide II (amide B/II) and that of the antisymmetric CH2 stretching mode and HCH bending mode (νa,CH2/δCH) in the side chains, respectively.13 As for the assignment of another autopeak near 4830 cm-1, there is some controversy. In our previous paper,14 we assigned it to the combination mode of amide A and amide II (amide A/II) of ovalbumin in aqueous solutions. However, in the present study, we propose a new assignment for the band near 4830 cm-1; it arises from the combination mode of water or the second overtone of an OH bending vibration of water.7-9 The amide A/II band may be overlapped by the strong water band near 5000 cm-1. These assignments will be confirmed below. The appearance of the autopeaks means that the intensities of these peaks vary significantly with increases in the concentration of the folded state of HSA. In the synchronous spectrum, a positive cross-peak is observed at 4600 vs 4367 cm-1, and negative cross-peaks develop at 4830 vs 4600 cm-1 and at 4830 vs 4367 cm-1. The positive peak that developed at 4600 vs 4367 cm-1 suggests that the intensities of these two bands change simultaneously in the same direction with an increase in the concentration. On the other hand, the two negative cross-peaks

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Figure 5. Slice spectra at 4600 cm-1 extracted from concentrationperturbed asynchronous contour maps of HSA in aqueous solutions at 45, 58, 60, and 80 °C.

at 4830 vs 4600 cm-1 and 4830 vs 4367 cm-1 indicate that the intensity of the band at 4830 cm-1 and those of the other two bands at 4600 and 4367 cm-1 change in phase but in the opposite direction. The directions of the intensity changes in these three bands support the above assignments that the band at 4830 cm-1 is due to water, while those at 4600 and 4367 cm-1 arise from the protein. An asynchronous cross-peak develops only if the intensities of two dynamic spectral peaks vary out of phase, i.e., delayed or accelerated, with respect to each other.16,17 Several crosspeaks are observed in the asynchronous map shown in Figure 2b. The sign (positive or negative) of the cross-peaks in an asynchronous spectrum provides additional information about the order of the intensity changes in different bands.16,17 Crosspeaks appear at 4830 vs 4600 cm-1 and at 4830 vs 4367 cm-1 in the asynchronous spectrum. According to the rule proposed by Noda16 for determining the order of sequential events, the intensity of the band at 4830 cm-1 varies at a lower concentration than the intensities of the bands at 4600 and 4367 cm-1, assigned to the amide B/II and CHn combination modes, respectively. Of particular note in the asynchronous spectrum is the appearance of a new band at 4487 cm-1. However, this band may be due to an artifact. This conclusion has very recently been reached by use of filtering 2D cross-correlation functions.27 In fact, the feature at 4487 cm-1 corresponds to a valley in the one-dimensional spectra. The concentration-dependent spectral changes of the HSA solutions may be caused by the following factors: (1) a simple change in the concentration, and thus the population density, of HSA; (2) conformational changes in the secondary structure and microenvironmental changes in side chains of HSA; and (3) changes in hydration. If the concentration change produced strictly proportional changes in band intensities, the correspond-

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Figure 6. 2D NIR correlation spectra in the 7500-5500 cm-1 region, constructed from concentration-perturbed spectra of HSA in aqueous solutions in the natively folded state at 45 °C. (a) Synchronous and (b) asynchronous contour maps.

ing asynchronous correlation intensity would be zero. This is not the case in the present study. We do observe a number of peaks in the asynchronous spectrum. The appearance of the asynchronous cross-peaks indicates the possibilities of factors 2 and 3. There is no doubt that the protein hydration changes significantly with the increase in the concentration; the number of bonded water molecules increases, while that of free water molecules decreases. These changes in hydration should lead to the development of cross-peaks in the asynchronous spectrum. However, it is very unlikely that solely the concentrationdependent variation in the hydration causes the secondary structural changes. Thus, the protein aggregations or associations are probably formed at a higher concentration,22 inducing slight but significant secondary structural changes and changes in the microenvironment of the side chains. The sign of the crosspeaks in the asynchronous spectrum (Figure 2b) shows that the band due to water varies at a lower concentration than the bands arising from the protein. 2.2. Denaturation of HSA in Aqueous Solutions. A series of the concentration-perturbed 2D spectra were calculated over a temperature range from 45 to 80 °C to explore how the correlation patterns vary with temperature during the heat denaturation process. The 2D correlation patterns, both synchronous and asynchronous, below 58 °C are very close to those at 45 °C. This is probably because the secondary structure of HSA remains almost unchanged below 58 °C. This result is consistent with that of the DSC study.21 Therefore, the correlation patterns in Figure 2a and b represent a typical 2D NIR spectral response of HSA in aqueous solutions in the natively folded state. Figure 3 illustrates the concentration-perturbed (a) synchronous and (b) asynchronous 2D correlation spectra of HSA in aqueous solutions at 80 °C. The synchronous map at 80 °C bears a close resemblance to that at 45 °C. However, note that a new autopeak appears at 4850 cm-1. This peak probably corresponds to a band due to the combination mode of amide A and amide II (amide A/II). More detailed temperature-dependent changes in the 2D synchronous spectra can be illustrated in power spectra along the diagonal line in Figure 3a. Figure 4 exhibits the power

spectra at 45, 58, 60, and 80 °C. Of note in Figure 4 is a significant change in the band near 4600 cm-1 due to amide B/II between 58 and 60 °C. The temperature-induced highfrequency shift of this band suggests that the secondary structure of HSA changes between 58 and 60 °C. There is one important clear variation in the asynchronous spectra between 45 and 80 °C; the signs of all asynchronous peaks reverse. To investigate temperature-dependent changes in the asynchronous spectra more clearly, we calculated slice spectra along the line at 4600 cm-1. Figure 5 displays these spectra at 45, 58, 60, and 80 °C. In addition to the reversal of the signs of the asynchronous peaks, it is noted that the band near 4600 cm-1 again shows a significant shift between 58 and 60 °C. This observation is in good agreement with that in the power spectra. Coincident with the band shift near 4600 cm-1 due to amide B/II, the band near 4850 cm-1 arising from amide A/II shows a shift. In contrast to these two bands, the band at 4367 cm-1 assigned to the combination mode of the CHn vibrations does not show a significant shift with temperature. We will discuss the heat-induced unfolding process in more detail in the Discussion. 3. 2D NIR Correlation Spectra in the Range of 75005700 cm-1. 3.1. Temperature-Dependent Change in the Hydration of HSA in Aqueous Solutions. Figure 6 shows (a) synchronous and (b) asynchronous 2D contour maps in the range of 7500-5700 cm-1 at 45 °C, generated from the concentrationdependent spectral variations of HSA in aqueous solutions. In the synchronous map, an intense autopeak centered at 7000 cm-1 arises primarily from a combination of the OH symmetric and antisymmetric stretching modes (ν1 + ν3) of water.7-9,14 Several water species (free and bonded water with one, two, three, or four hydrogen bonds) coexist in the HSA solutions, so the band near 7000 cm-1 is very broad.14,15 The first overtone of the NH stretching mode of the amide groups in the protein should be located in the range of 68006100 cm-1,13 but it is overlaid strongly by the intense water band. The autopeak near 7000 cm-1 is so strong and broad that it is difficult to monitor the intensity changes in each of the bands assigned to various water species. The negative cross-

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Figure 7. 2D NIR correlation spectra in the 7500-5500 cm-1 region, constructed from concentration-perturbed spectra of HSA in aqueous solutions in the denatured unfolded state at 80 °C. (a) Synchronous and (b) asynchronous contour maps.

Figure 8. Power spectra along the diagonal line in the 7500-5500 cm-1 region in the synchronous contour maps, constructed from concentration-perturbed spectra of HSA in aqueous solutions at 45, 58, 60, and 80 °C.

peaks appear at 5892 vs 7000 cm-1 and at 5750 vs 7000 cm-1, indicating that the bands at 5892 and 5750 cm-1 due to the first overtones of the CH stretching modes of HSA change in an opposite direction to the band near 7000 cm-1 due to water.

Of note in the asynchronous spectrum (Figure 6b) is the development of asynchronous cross-peaks at 5892 vs 7000 cm-1 and 5750 vs 7000 cm-1. The signs of the peaks indicate that the intensity of the bands at 7000 cm-1 due to water changes at a lower concentration than those of the bands at 5892 and 5750 cm-1 arising from HSA. This result is consistent with that obtained from the 4900-4250 cm-1 region. With the temperature increase, remarkable changes take place in both the synchronous and the asynchronous spectra of HSA. Figure 7 shows the (a) synchronous and (b) asynchronous 2D contour maps in the 7500-5500 cm-1 region at 80 °C. Drastic changes are observed in the synchronous spectra between 45 and 80 °C, showing that the hydration varies dramatically with temperature. At 80 °C, the peak top frequency of the autopeak near 7000 cm-1 shows a high frequency shift. This is recognized more clearly in the power spectra along the diagonal line in the synchronous spectra. Figure 8 shows the power spectra at 45, 58, 60, and 80 °C. Particularly striking is that the peak top frequency of the band near 7000 cm-1 shows a large shift between 58 and 60 °C; a 13 cm-1 shift only for 2 °C. It seems that there is a marked change in the hydration between 58 and 60 °C. It is very likely that the unfolding process starts at 58 °C, leading to the abrupt change in hydration. The most prominent spectral change between the asynchronous spectra at 45 and 80 °C (Figures 6b and 7b) is the reversal of the signs of asynchronous cross-peaks. Figure 9 shows slice spectra at 45, 58, 60, and 80 °C along the line at 7500 cm-1 in the asynchronous spectra. The correlation pattern changes even between 45 and 58 °C. This may be because of the temperaturedependent changes in the hydrogen bonds in water. In the slice spectrum at 58 °C, a downward peak at 7130 cm-1 probably corresponds to free water, and a broad upward peak near 6900 cm-1 arises from bonded water.15 Discussion To explore the unfolding process of HSA in more detail, we plotted the frequency of the band at 4600 cm-1 assigned to the amide B/II mode and that of the band near 7000 cm-1 due to

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Figure 9. Slice spectra near 7500 cm-1 extracted from concentrationperturbed asynchronous contour maps of HSA solutions at 45, 58, 60, and 80 °C.

the combination mode (ν1 + ν3) of water as a function of temperature. Figure 10a and b shows the plots for the bands near 4600 and 7000 cm-1, respectively. It can be seen from Figure 10a that the band near 4600 cm-1 shows an upward shift in the very narrow temperature range of 58-60 °C. It can be concluded from the plot that the unfolding of HSA occurs suddenly in this temperature range. This conclusion is consistent with the previous conclusion reached by Barone et al.22 on the basis of the DSC study of HSA. According to them, the midpoint temperature of thermal denaturation of HSA in an aqueous solution at pH ) 6.5 is 60 °C.22 The amide B/II mode is the combination of the amide B and amide II modes. Amide B is an NH stretching vibration, and amide II is a vibrational mode involving an NH bending motion coupled with a CN stretching vibration. Thus, both modes are sensitive to the hydrogen bonds of the amide groups of the protein. However, amide B shows a low-frequency shift with the increase in the strength of hydrogen bonds, while amide II shows a high-frequency shift. Thus, although it is possible to monitor changes in the strength of the hydrogen bonds by the frequency of amide B/II, it is rather difficult to conclude what kinds of secondary structural changes take place by its frequency. One can see, in Figure 10b, that the band near 7000 cm-1 also shows an upward shift in the same temperature range as the band near 4600 cm-1. This shift suggests that the protein hydration changes drastically between 58 and 60 °C. The drastic change in the hydration is also clearly recognized by the inverse of the sign of the asynchronous cross-peaks (Figures 6b, 7b, and 9). A comparison of the two plots in Figure 10 reveals that the water band shows a more gradual shift than the amide band

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Figure 10. (a) Plot of the frequency of the band near 4600 cm-1 in the power spectra versus temperature. (b) Plot of the frequency of the band near 7000 cm-1 in the power spectra versus temperature.

and that the former starts shifting at a lower temperature than the latter. However, it must be kept in mind that the frequency shift of the water band occurs because of two factors. One is the change in protein hydration, and another is the temperaturedependent change in the hydrogen bonds of water. The band near 7000 cm-1 due to the ν1 + ν3 mode of pure water shows a gradual upward shift with temperature because the hydrogen bonds of water are broken. Therefore, it seems likely that the temperature-dependent change in the water structure makes the shift in Figure 10b gradual. Thus, the intrinsic shift caused by the changes in the protein hydration may be steeper. This consideration leads us to conclude that both the unfolding of HSA and the change in protein hydration happen almost in parallel in the narrow temperature range of 58-60 °C. It is of note in Figure 10b that the band near 7000 cm-1 shows an upward shift, and not a downward shift, upon the unfolding of the protein. This result suggests that the number of water species with no or a small number of hydrogen bonds increases. Upon the unfolding, more hydrophobic residues, originally at the protein’s interior, are exposed to water, and they change the amount of ordered solvent in the system. This, in turn, easily perturbs the vibrational spectrum of water. It is likely that that the unfolding process breaks the cluster structure of free water, increasing water species with no or a small number of hydrogen bonds. Conclusions This paper has demonstrated that the frequencies of the overtone and combination bands in the NIR region are very sensitive to changes in the hydration and the secondary structure of HSA. The application of generalized 2D correlation spectroscopy to the concentration-perturbed NIR spectral variations of the HSA solution has enabled us to detect subtle spectral

Human Serum Albumin in Aqueous Solutions variations in the NIR regions. The following conclusions can be achieved from the present study: 1. The calculation of the power and slice spectra is very useful for monitoring the process of protein unfolding. The plot of the band at 4600 cm-1 due to amide B/II versus temperature shows that HSA undergoes rather sudden changes in the secondary structure near 60 °C. The similar plot for the band near 7000 cm-1 assigned to the combination mode of water reveals that the protein hydration also varies markedly near 60 °C. 2. NIR spectroscopy is a powerful method for studying protein hydration in solutions. Structural information about the protein and the solvent can be obtained at the same time. This makes it possible to directly monitor changes in the hydration of the protein during the unfolding process. Acknowledgment. The present study was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences. References and Notes (1) Clark, R. J. H., Hester, R. E., Eds. Biomolecular Spectroscopy; John Wiley & Sons: New York, 1993; Vol. 20, Part A, and Vol. 21, Part B. (2) Havel, H. A. Spectroscopic Methods for Determining Protein Structure in Solution; John Wiley & Sons: New York, 1995. (3) Jackson, M.; Mantsch, H. H. Crit. ReV. Biochem. Mol. Biol. 1995, 30, 95. (4) Mantsch, H. H., Chapman, D., Eds. Infrared Spectroscopy of Biomolecules; John Wiley & Sons: New York, 1996. (5) Siebert, F. In Infrared Spectroscopy of Biomolecules; Mantsch, H. H., Chapman, D., Eds.; John Wiley & Sons: New York, 1996; p 83. (6) Xu, X.; Rodgers, K. R.; Mukerji, I.; Spiro, T. G. Biochemistry 1999, 38, 3462. (7) Williams, P., Norris, K., Eds. Near-Infrared Technology in the Agricultural and Food Industries, 2nd ed.; American Association of Cereal Chemists: St. Paul, MN, 1990.

J. Phys. Chem. B, Vol. 104, No. 24, 2000 5847 (8) Burns, D. A., Ciurczak, E. W., Eds. Handbook of Near-Infrared Analysis; Marcel Dekker: New York, 1992. (9) Osborne, B. G.; Fearn, T.; Hindle, P. H. Practical Near Infrared Spectroscopy with Applications in Food and BeVerage Analysis; Longman Scientific &Technical: Essex, England, 1993. (10) Holly, S.; Rgyed, O.; Jalsovszky, G. Spectrochim. Acta 1992, 48A, 101. (11) Sefara, N. L.; Magtoto, N. P.; Richardson, H. H. Appl. Spectrosc. 1997, 51, 536. (12) Schultz, C. P.; Fabian, H.; Mantsch, H. H. Biospectrosc. 1998, 4, S19. (13) Wang, J.; Sowa, M. G.; Ahmed, M. K.; Mantsch, H. H. J. Phys. Chem. 1994, 98, 4748. (14) Wang, Y.; Murayama, K.; Myojo, Y.; Tsenkova, R.; Hayashi, N.; Ozaki, Y. J. Phys. Chem. 1998, 102, 6655. (15) Murayama, K.; Czarnik-Matusewicz, B.; Wu, Y.; Ozaki, Y. Appl. Spectrosc., in press. (16) Noda, I. Appl. Spectrosc. 1993, 47, 550. (17) Noda, I. Appl. Spectrosc., in press. (18) Peters, T., Jr. All About Albumin; Academic Press: New York, 1994 and references therein. (19) Brown, J. R. In Albumin Structure, Function and Uses; Rosenoer, V. M., Oraz, M., Rotshild, M. A., Eds.; Pergamon Press: Oxford, 1977; pp 27-51. (20) Curry, S.; Mandelkow, H.; Brick, P.; Franks, N. Nat. Struct. Biol. 1998, 5, 827. (21) Protein Data Bank, Department of Chemistry, Brookhaven National Laboratory, Upton, NY 11973, http://www.pdb.bnl.gov/index. html. (22) Barone, G.; Giancola, C.; Verdoliva, A. Thermochim. Acta 1992, 199, 197. (23) Bramanti, E.; Benedetti, E. Biopolymers 1996, 38, 639. (24) Hvidt, A.; Wallevik, K. J. Biol. Chem. 1972, 247, 1530. (25) Lin, V. J. C.; Koenig, J. L. Biopolymers 1976, 15, 203. (26) Wetzel, R.; Becker, M.; Behlke, J.; Billwitz, H.; Bohn, S,; Ebert, B.; Hamann, H.; Krumbiegel, J. Lassmann, G. Eur. J. Biochem. 1980, 104, 469. (27) Buchet, B.; Wu, Y.; Lachenal, G.; Ozaki, Y., submitted for publication. (28) Maeda, H.; Ozaki, Y.; Tanaka, M.; Hayashi, N.; Kojima, T. J. Near Infrared Spectrosc. 1995, 3, 191.