Two-Dimensional Fourier Transform Near-Infrared Spectroscopy

Two-Dimensional Fourier Transform Near-Infrared Spectroscopy Study of Heat .... A series of dynamic NIR spectra modulated by the concentration at ...
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J. Phys. Chem. B 1998, 102, 6655-6662

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Two-Dimensional Fourier Transform Near-Infrared Spectroscopy Study of Heat Denaturation of Ovalbumin in Aqueous Solutions Yan Wang,*,† Koichi Murayama,† Yoshiki Myojo,† Roumiana Tsenkova,‡ Nobuyuki Hayashi,§ and Yukihiro Ozaki*,† Department of Chemistry, School of Science, Kwansei-Gakuin UniVersity, Uegahara, Nishinomiya 662-8501, Japan, Department of EnVironment Information and Bio-production Engineering, Faculty of Agriculture, Kobe UniVersity, Rokkodai, Nada-ku, Kobe 657-8501, Japan, and Department of Applied Biological Sciences, Faculty of Agriculture, Saga UniVersity, Honjo, Saga 840-8502, Japan ReceiVed: March 24, 1998; In Final Form: June 3, 1998

Heat-induced denaturation of ovalbumin in aqueous solutions has been investigated by generalized twodimensional (2D) Fourier transform near-infrared (FT-NIR) correlation spectroscopy. New insight has been gained into hydration and the unfolding process of secondary structures of ovalbumin by studying temperaturedependent correlation patterns in 2D synchronous and asynchronous spectra, which are constructed from concentration-perturbed NIR spectra at different temperatures. The correlation patterns have provided information about the correlation and phase relationships between different absorption bands of ovalbumin and water. The hydration of ovalbumin is almost unchanged from 45 to 67 °C, where ovalbumin molecules are in a natively folded state. A sudden change in the hydration is detected in a narrow temperature range of 67-69 °C, where the unfolding of the ordered secondary structures starts. The hydration, again, remains nearly unchanged upon further heating to 80 °C, even though the unfolding process develops progressively until the denatured state. The sudden change in the hydration around 68 °C seems to be caused by the stabilization of slightly unstable hydrogen bonds in the folded state. The change may make the intramolecular hydrophobic cores of ovalbumin less condensed and more accessible to the solvent molecules. On the other hand, the development of unfolding from 69 to 80 °C results in band shifts for combination bands involving free NH stretching-amide II (amide A/II), intramolecular hydrogen-bonded NH stretching-amide II (amide B/II) of ovalbumin. The present experiment demonstrates that the generalized 2D NIR correlation spectroscopy is powerful in detecting subtle but valuable structural information about the protein denaturation in the aqueous solution.

Introduction Structural studies of proteins have been extensively carried out by means of various kinds of spectroscopies,1-8 such as circular dichroism (CD), infrared (IR), and Raman spectroscopies. Near-infrared (NIR) spectroscopy, which has emerged as a successful tool in practical applications for qualitative and quantitative analyses of proteins,9-15 has seldom been employed to investigate the structure of proteins. However, NIR spectroscopy does have several advantages over IR spectroscopy in explaining protein structure. First, water gives much less intense bands in the NIR region, so that one can measure spectra of protein aqueous solutions more easily. Second, NIR spectroscopy is useful in studying hydration and hydrogen bonds in protein in the NIR spectra of protein aqueous solutions; there several bands are observed due to overtones and combination modes of water and amide groups of protein. They are all more sensitive to changes in hydration and hydrogen bonds than IR bands arising from water and the amide groups.16,17 In fact, it is very difficult to investigate the hydration of proteins by IR and Raman spectroscopy. Third, a more convenient path length * To whom correspondence should be addressed: Fax +81-798-51-0914; E-mail [email protected]. † Kwansei-Gakuin University. ‡ Kobe University. § Saga University.

can be used and the exact volume of the sample can be evaluated. The basic research on the protein structures by NIR spectroscopy has been concerned mainly with proteins in solid states,12,13 and the structural investigations of proteins in aqueous solutions have been far behind. The difficulty comes from the fact that absorption bands arising from proteins in the aqueous solutions are much weaker than those due to water. Therefore, it is rather difficult to obtain solid information about the protein structures from conventional one-dimensional NIR spectra. In the present study, generalized two-dimensional (2D) correlation spectroscopy, a recently developed analytical method, is applied to the NIR region to extract out structural information about protein in aqueous solutions during a heat denaturation process. The theory of generalized 2D correlation spectroscopy was developed by Noda18 in 1993, as an extension of original 2D IR correlation spectroscopy proposed by himself.19 In the 2D analysis two kinds of correlation maps, synchronous and asynchronous ones, are generated based upon a set of dynamic spectra calculated from perturbation-induced dynamic fluctuations of spectroscopic signals.19 The new 2D method can handle signals fluctuating as an arbitrary function of time or any other physical variable such as temperature, pressure, or even concentration.20-23 The extension to various kinds of spectroscopies is also quite straightforward for the generalized 2D correlation analysis method.

S1089-5647(98)01611-3 CCC: $15.00 © 1998 American Chemical Society Published on Web 08/05/1998

6656 J. Phys. Chem. B, Vol. 102, No. 34, 1998 Correlation peaks appearing in the synchronous and asynchronous maps represent in-phase and out-of-phase variation tendencies of corresponding band intensities, respectively. The advantages of the 2D correlation analysis lie in several aspects: 18,19 (i) It has a powerful deconvolution ability for highly overlapped bands. (ii) It provides information about inter- and intramolecular interactions by correlating two absorption band intensities from different functional groups. (iii) One can probe the specific order of the spectral intensity changes taking place during the measurement from the analysis of the asynchronous spectra. Following the pioneering work by Noda, many research groups24-28 have adopted 2D IR or NIR correlation spectroscopy to study the structure of biological molecules, such as peptides, proteins, and lipids. It has been proven that the 2D correlation analysis holds a considerable promise in exploring structural changes in the biological molecules induces by perturbation. Our group,21-23,29-32 in collaboration with Noda, has been employing generalized 2D correlation spectroscopy to probe temperature-dependent structural variations of a variety of materials from basic compounds to polymers. Some new insights were provided by the 2D correlation studies into the dissociation mechanism of polymeric oleyl alcohol and a premelting process of nylon-12. This has encouraged us to explore the potential of the 2D NIR correlation spectroscopy in investigating protein structural changes in an aqueous solution. Denaturation of protein has long been a matter of keen interest.33-35 Hen egg white ovalbumin, a medium-sized globular protein with a molecular mass of 45 kDa, has been studied in aqueous solutions in the present study. The crystal structure of native ovalbumin reported by Stein et al.36 shows that, as a degenerated functional unit, a five-stranded β-sheet runs parallel to the long axis of the molecule and R-helices protrude as a loop that forms the reactive center. Recently, Tani et al.8 has carried out a detailed study on the heat-induced unfolding and refolding process of ovalbumin in aqueous solutions by means of size-exclusion chromatography, CD and other biochemical methods. A temperature-dependent unfolding transition curve for ovalbumin was drawn by measuring the ellipticity at 222 nm (θ222 nm) in the CD experiment. On the basis of the transition curve describing the unfolding process, they proposed a possible mechanism for the temperaturedependent conformational change of ovalbumin, which was explained as a two-state transition between the folded and unfolded states with a midpoint temperature of 76 °C. The measured θ222 nm of intact ovalbumin under nonreducing conditions at pH 7.5 changes from -12.0 × 10-3 to -7.0 × 10-3 deg‚cm2‚dmol-1 when the temperature of the ovalbumin solution was raised from 45 to 80 °C. At the midpoint temperature of 76 °C, the θ222 nm was -9.5 × 10-3 deg‚cm2‚dmol-1. The heat-denatured ovalbumin molecules at 80 °C were found to have retained secondary structures to some extent because the θ222 nm of completely unfolded ovalbumin in the presence of 6 M GdmCl (guanidinium chloride) was about -1.0 × 10-3 deg‚cm2‚dmol-1. It was, thus, concluded that the ovalbumin molecules are in the natively folded state below 65 °C and the partially unfolded denatured state above 80 °C.8 The purpose of this study is to investigate the heat denaturation and unfolding process of ovalbumin in the aqueous solutions by use of the 2D NIR correlation spectroscopy, which enables us to explore subtle differences in NIR spectral responses between the folded and unfolded states. Temperaturedependent NIR spectra were measured for ovalbumin solutions

Wang et al. in a temperature range of 45-80 °C, which covers the whole heat denaturation process. A series of dynamic NIR spectra modulated by the concentration at representative temperatures, such as 45, 76, and 80 °C, were utilized to construct 2D synchronous and asynchronous correlation spectra. The results for the 2D analysis have indicated, for the first time, that the NIR region is highly sensitive to the inter- and intramolecular interaction and conformational changes in protein in the aqueous solution. Experimental Section Sample Preparation. Hen egg-white ovalbumin (grade VI with purity of approximately 99%) was purchased from Sigma Chemical Co. and used without further purification. The protein was dissolved in doubly distilled water to prepare ovalbumin solutions with a concentration of 2%, 5%, and 8% by weight. Instrumentation and Sampling. NIR spectra of the ovalbumin solutions were measured for two regions (7500-5800 and 4900-4550 cm-1) at an 8 cm-1 resolution with a Jeol JRS 6500N FT-NIR/Raman spectrometer equipped with a TGS (triglycine sulfate) detector. Temperature-dependent spectra were collected from 45 to 80 °C at increments of 2 °C. Two hundred scans were coadded to ensure an acceptable signal-tonoise ratio. The solutions were contained in a quartz cuvette cell having a path length of 1.0 mm. The cell was inserted into a homemade thermostated cell holder and temperature was controlled by circulating thermostated water. Temperature was monitored by a digital thermometer (Anritsu HFT-50) dipped into the cell. The system gave a temperature stability of better than (0.2 °C. Background spectra of the empty cell were also taken at the corresponding temperatures. 2D Correlation Spectroscopic Analysis. A powerful yet easily executable software for constructing a generalized 2D correlation spectrum has been composed by one of the authors (Y. Wang) with the Array Basic programming language (The Galactic Industries Corp.).37 Our 2D software was programmed on the basis of the newly developed algorithm of generalized 2D correlation spectroscopy, which is capable of creating a 2D heterospectrum by combining different electromagnetic wave regions.38 One of the advantages of the 2D software is the ability of demonstrating three-dimensional (3D) representation of a 2D spectrum, enabling one to observe subtle correlation peaks by rotating the 3D picture.37 Results Raw and Second-Derivative NIR Spectra of Ovalbumin Solutions. Figure 1 shows a representative NIR spectrum in the spectral regions of (a) 8000-5400 and (b) 4950-4450 cm-1 of ovalbumin solution with a concentration of 5 wt % measured at 45 °C. The spectral region of 5300-5000 cm-1 was not measured because of an extremely strong absorption due to the combination mode of OH stretching and bending vibrations of water. A broad band centered at 7000 cm-1 consists mainly of the combination of OH stretching modes of water, but there are also contributions from bands due to amide groups of ovalbumin. As we reported in our previous study of temperature-dependent NIR spectral changes of water,39 even for pure water, this region contains several OH combination bands from various water species, water with 0-4 hydrogen bonds. Figure 2 shows NIR spectra in the 4900-4550 cm-1 region of ovalbumin solutions with concentrations of 0, 2, 5, and 8 wt % measured at (a) 45 and (b) 80 °C, where the protein is in the natively folded and partially unfolded states, respectively. Except for a strong absorption band of water dominating the

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Figure 1. Representative NIR spectrum in the two regions of (a) 80005400 cm-1 and (b) 4950-4450 cm-1 of ovalbumin solution with a concentration of 5 wt % measured at 45 °C.

Figure 3. Corresponding second derivative NIR spectra in the 49004550 cm-1 region of ovalbumin solutions with the concentration of 0, 2, 5, and 8 wt %, respectively. (a) In the natively folded state at 45 °C; (b) in the denatured unfolded state at 80 °C.

Figure 2. NIR spectra in the 4900-4550 cm-1 region of ovalbumin solutions with a concentration of 0, 2, 5, and 8 wt %, respectively. (a) In a natively folded state at 45 °C; (b) in a denatured unfolded state at 80 °C.

spectra, no peak arising from the protein can be observed. Thus, it is impossible to find any spectral difference between the folded and unfolded states. The corresponding second derivatives of the spectra in Figure 2 are depicted in Figure 3. Note that the calculation of the second derivative makes some bands observable. Two bands at 4850 and 4600 cm-1 in Figure 3 are assigned to combinations of free NH stretching-amide II (amide A/II) and intramolecular hydrogen-bonded NH stretching-amide II (amide B/II) of ovalbumin, respectively.15 (Here, we tentatively assign the intramolecular hydrogen-bonded NH stretching band to the amide B mode. A convincing evidence is provided by the 2D correlation analysis in the present study and will be explained

in detail in the Discussion section.) The intensities of the amide A/II and amide B/II increase with the concentration. However, it is still rather difficult to find any correlation between the two bands and any spectral difference between the folded and unfolded states. The weak NIR spectroscopic signals of biological fluids, like this, are a commonly encountered problem. To pick out useful information, we have applied the 2D technique to the above concentration-perturbed NIR spectra of ovalbumin solutions measured at different temperatures. 2D NIR Correlation Spectra in the 4900-4550 cm-1 Region. Figure 4 shows contour map representation of (a) synchronous and (b) asynchronous 2D correlation spectra, which are constructed from the concentration-perturbed NIR raw spectra in the 4900-4550 cm-1 region at 45 °C. Throughout this paper, solid and dashed lines in the contour maps denote positive and negative correlation peaks, respectively. Note that two strong autopeaks are clearly observed at 4850 and 4800 cm-1 in the synchronous spectrum. These peaks are hardly identified in the raw spectra (Figure 2a). The two peaks probably correspond to the combination band of the amide A/II and second overtone of OH bending (3ν2) of water, respectively. The appearances of the autopeaks mean that the intensities of these two bands vary most significantly with increasing the concentration in the folded state. In addition, positive crosspeaks (4850 vs 4800 cm-1) are seen in the synchronous spectrum, indicating their band intensities increase in-phase (simultaneously). Asymmetric cross-peaks (4850 vs 4600 cm-1 and 4800 vs 4600 cm-1) dominate the asynchronous map of Figure 4b. The band at 4600 cm-1 arises from the amide B/II combination of ovalbumin. The asynchronous spectrum reveals that the amide

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Figure 4. 2D NIR correlation spectra in the 4900-4550 cm-1 region, constructed from concentration-perturbed spectra of ovalbumin solutions in the natively folded state at 45 °C; (a) synchronous and (b) asynchronous contour maps.

Figure 5. 2D NIR correlation spectra in the 4900-4550 cm-1 region, constructed from concentration-perturbed spectra of ovalbumin solutions in the denatured unfolded state at 80 °C; (a) synchronous and (b) asynchronous contour maps.

B/II shows an out-of-phase variation with the amide A/II and 3ν2 bands. It is complementary to the band correlation observed in the synchronous map. The sign (positive or negative) of the cross-peaks in the asynchronous 2D correlation spectrum gives additional useful information about relative temporal relationships for different bands. According to the rule proposed by Noda40 for determining the sequential relationships between different bands, the negative peaks (4850 and 4800 vs 4600 cm-1) in Figure 4b indicate that the band intensity of the amide B/II varies at a lower concentration change than those of the amide A/II and 3ν2 bands. The above correlations verify that the band intensities of the amide A/II of ovalbumin and of the 3ν2 of water change inphase while they vary out-of-phase with the amide B/II band. A series of synchronous and asynchronous 2D NIR correlation spectra have been also constructed and examined at different temperatures from 45 to 65 °C. The results give correlation patterns very similar to those in Figure 4. It is probably because the secondary structures of ovalbumin remain almost unchanged below 65 °C, as indicated by the CD experiment.8 Thus, the correlation patterns in Figure 4 represent a typical 2D NIR spectral response of ovalbumin solutions in the natively folded state. In Figure 5 are shown (a) synchronous and (b) asynchronous 2D NIR correlation spectra of the denatured unfolded state at 80 °C. Both the synchronous and asynchronous 2D contour

maps give distinctly different correlation peaks from those of the folded state at 45 °C shown in Figure 4. A strong autopeak at 4610 cm-1 in the synchronous spectrum means that the intensity of the amide B/II changes most significantly in the spectra of the unfolded state. The two autopeaks at 4850 and 4800 cm-1 in the synchronous spectrum of Figure 4a become weak in Figure 5a. Instead, two new positive cross-peaks (4860 vs 4610 cm-1) are developed in the synchronous map at 80 °C, indicating the in-phase intensity variation of the amide A/II with the amide B/II. The in-phase intensity variation between the amide A/II and the 3ν2 in the folded state vanishes completely. In addition, the 3ν2 band changes out-of-phase with the amide A/II and amide B/II as verified below by the corresponding asynchronous spectrum (Figure 5b). The asynchronous map of Figure 5b shows two pairs of new cross-peaks (4860 vs 4780 cm-1 and 4780 vs 4610 cm-1). These peaks confirm the out-of-phase intensity variation of the 3ν2 band with the amide A/II and amide B/II. The signs of the two pairs of cross-peaks indicate that the 3ν2 band of water changes after the amide A/II and amide B/II of ovalbumin do. Also, of note is that the amide A/II and amide B/II bands shift upward by about 10 cm-1 while the 3ν2 band shifts downward by some 20 cm-1 in the 2D spectra with the temperature increase from 45 to 80 °C. These shifts may be due to the thermally induced unfolding of the secondary structures of ovalbumin.

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Figure 6. 2D NIR correlation spectra in the 4900-4550 cm-1 region, constructed from concentration-perturbed spectra of ovalbumin solutions at a midpoint temperature of 76 °C during the unfolding process; (a) synchronous and (b) asynchronous contour maps.

Figure 7. 2D NIR correlation spectra in the 4900-4550 cm-1 region, constructed from concentration-perturbed spectra of ovalbumin solutions at 67 °C during the unfolding process; (a) synchronous and (b) asynchronous contour maps.

Figure 6 shows the corresponding (a) synchronous and (b) asynchronous 2D correlation maps generated from the NIR spectra at 76 °C, which is the midpoint temperature during the unfolding process of ovalbumin.8 Interestingly, correlation patterns in Figure 6 are similar to those at 80 °C shown in Figure 5, indicating that the intermolecular interaction between ovalbumin and water, i.e., hydration of ovalbumin, is changed substantially even in the intermediate state of the unfolding process. Moreover, the 3ν2 and the amide A/II and amide B/II bands already shift to some extent at 76 °C. It is of essential importance to explore how the correlation patterns, reflecting the hydration of ovalbumin, change with temperature during the heat denaturation process. For this purpose, a series of the 2D spectra have been generated over a temperature range from 45 (the native state) to 80 °C (the denatured state) at an interval of 2 °C. The results show that the correlation patterns in the 2D spectra below 65 °C and above 71 °C resemble those of 45 and 80 °C, respectively. The correlation patterns change suddenly from the native to the denatured state in a narrow temperature range of 67-69 °C. The intermediate correlation patterns in the 2D spectra measured at 67 °C are presented in Figure 7. It can be concluded from the temperature-dependent changes in the correlation patterns that the hydration of ovalbumin remains in the native state below 65 °C, then undergoes the sudden change from the native to the denatured state in the region of 67-69

°C, and finally is unchanged again above 71 °C. It should be pointed out that the unfolding of secondary structures starts at about 69 °C and develops progressively until 80 °C.8 Apparently, the change in the hydration does not follow the unfolding process. We infer that it is the change in the hydration in the critical temperature region of 67-69 °C that leads to the start of the unfolding process from 69 to 80 °C. The conformational change in ovalbumin gives rise to the band shifts of amide groups during the unfolding. Note that the amide A/II and amide B/II bands shift progressively from 4850 to 4860 cm-1 and from 4600 to 4610 cm-1, respectively, in the temperature range of 69-80 °C (see Figures 4a and 5b). 2D NIR Correlation Spectra in the 7500-5800 cm-1 Region. Figure 8 illustrates (a) synchronous and (b) asynchronous contour maps in the 7500-5800 cm-1 region at 45 °C. In the synchronous map, an intense autopeak centered at 7000 cm-1 is observed with elongated cross-peaks from 6800 to 6000 cm-1. The band around 7000 cm-1 arises from a combination of OH symmetric and antisymmetric stretching modes (ν1 + ν3) of water.9-11,39 Several water species (free and bonded water with one, two, three, and four hydrogen bonds, etc.) coexist in the ovalbumin solution, so that the band near 7000 cm-1 is broad. This band shows the most significant variation with the concentration in the natively folded state. In the asynchronous map, cross-peaks (7000 vs 6250 cm-1, assigned to amide I + 3 amide II of ovalbumin41) show the out-of-phase correlation

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Figure 8. 2D NIR correlation spectra in the 7500-5800 cm-1 region, constructed from concentration-perturbed spectra of ovalbumin solutions in the natively folded state at 45 °C; (a) synchronous and (b) asynchronous contour maps.

Figure 9. 2D NIR correlation spectra in the 7500-5800 cm-1 region, constructed from concentration-perturbed spectra of ovalbumin solutions in the denatured unfolded state at 80 °C; (a) synchronous and (b) asynchronous contour maps.

of the ν1 + ν3 band due to the free and hydrogen-bonded water with the amide I + 3 amide II band. Figure 9 shows (a) synchronous and (b) asynchronous 2D correlation spectra constructed from NIR spectra at 80 °C. Remarkable differences can be found when comparing them with the corresponding 2D maps at 45 °C shown in Figure 8. A new autopeak appears around 6450 cm-1 while the autopeak at 7000 cm-1 almost disappears. It means that the major spectral variation in the partially unfolded state occurs in the spectral region containing several bands, bands due to the OH first overtone from hydrogen-bonded water and those due to a combination of the stretching mode of hydrogen-bonded NH (νNH-bonded) + amide I + amide II, and νNH-bonded + 2 amide II of ovalbumin.41 These bands vary ahead of the free and hydrogen bonded ν1 + ν3 OH of water, as is evident from the asynchronous map. The ν1 + ν3 band shifts from 7000 to 7020 cm-1 with the unfolding of ovalbumin (Figures 8b and 9b). The shift suggests that the amount of water species with zero or a small number of hydrogen bonds increases with temperature while the amount with three or four hydrogen bonds decreases. The 2D correlation maps in this spectral region constructed from the NIR spectra below 65 and above 71 °C resemble those at 45 and 80 °C, respectively. This is consistent with the results obtained from the 4900-4550 cm-1 region. Again, the correlation pattern of the 2D spectra changes suddenly in the temperature region of 67-69 °C, confirming that the intermo-

lecular interactions between ovalbumin and water experiences a sudden change from the native to the denatured state. 2D NIR Correlation Spectra between the 4900-4550 and 7500-5800 cm-1 Regions. Two-dimensional correlation spectra have also been constructed by combining the above two regions, which have abundant absorption bands of ovalbumin and water, respectively. Figure 10 shows the (a) synchronous and (b) asynchronous spectra at 45 °C. A strong positive crosspeak (7000 vs 4800 cm-1) is observed in the synchronous spectrum, revealing an in-phase variation relationship between the ν1 + ν3 and 3ν2 of water. In the asynchronous map, two negative cross-peaks (7000 vs 4600 cm-1 and 7000 vs 4850 cm-1) show an out-of-phase variation of the ν1 + ν3 with the amide A/II and amide B/II, respectively. The ν1 + ν3 water band varies behind those of the amide group. Figure 11 depicts the corresponding (a) synchronous and (b) asynchronous spectra at 80 °C. The synchronous map shows quite different correlation peaks from that at 45 °C. A strong positive peak (6450 vs 4610 cm-1) reveals the in-phase correlation of the bands due to the OH first overtone from hydrogen-bonded water, νNH-bonded + amide I + amide II, and νNH-bonded + 2 amide II with the amide B/II band. In the asynchronous map, two negative peaks (7020 vs 4860 cm-1 and 7020 vs 4610 cm-1) indicate an out-of-phase correlation of the ν1 + ν3 of water with the amide A/II and amide B/II. The ν1 + ν3 varies later. Another positive peak (6450 vs 4780

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Figure 10. 2D NIR correlation spectra between the 7500-5800 cm-1 and 4900-4550 cm-1 regions, constructed from concentration-perturbed spectra of ovalbumin solutions in the natively folded state at 45 °C; (a) synchronous and (b) asynchronous contour maps.

Figure 11. 2D NIR correlation spectra between the 7500-5800 cm-1 and 4900-4550 cm-1 regions, constructed from concentration-perturbed spectra of ovalbumin solutions in the denatured unfolded state at 80 °C; (a) synchronous, (b) asynchronous contour maps.

cm-1) shows that the 3ν2 band varies behind the several bands arising from the OH first overtone from hydrogen-bonded water, νNH-bonded + amide I + amide II, and νNH-bonded + 2 amide II. The band shifts are obvious from Figure 11 for the amide A/II and amide B/II of ovalbumin, as well as for the ν1 + ν3 and 3ν2 of water. As expected, the 2D correlation maps between the two spectral regions below 65 and above 71 °C bear a close resemblance to those in the native and denatured states of 45 and 80 °C, respectively. In the critical temperature range of 67-69 °C, the 2D correlation pattern is something between. Since the two spectral regions, 4900-4550 and 7500-5800 cm-1, contain rich structural information about both ovalbumin and water, the temperature-dependent 2D spectral correlation pattern provides the overall information about the change in the intermolecular interaction during the heat denaturation process.

coil (proteins have marginal stability due to effects such as the hydrophobic effect). The markedly different correlation patterns in the 2D synchronous and asynchronous spectra between the natively folded and denatured unfolded states of ovalbumin indicate that the hydration of ovalbumin is changed markedly upon heating. The sudden change in the hydration occurred at 67-69 °C may be brought about by the stabilization of the slightly unstable hydrogen bonds in the folded state. The change may lead to that the intramolecular hydrophobic cores of ovalbumin become less condensed and more accessible to the solvent molecules. The unfolding of secondary structures, including R-helices and β-sheets, develops progressively in the temperature range from 69 to 80 °C, but the hydration of ovalbumin does not change correspondingly. Most probably, it is the sudden change in the hydration that initiates the unfolding of secondary structures of ovalbumin. Another spectral response of the unfolding process in the NIR region, detectable only by the 2D analysis, is the phase relationships of different bands between water and amide groups. The phase relationships (in-phase or out-of-phase) of amide bands changed by heating may be attributed partly to the change in the hydrogen bonds. Re-formation of the hydrogen bonds between main-chain carbonyl and amino groups with water may slightly affect the peptide groups, resulting in the different phase relationships between the amide and water bands with the increased concentration. One more point of interest is that NIR

Discussion Heat-Induced Denaturation Process of Ovalbumin Solutions. Under aqueous conditions the amide groups of ovalbumin are stabilized by hydrogen bonds to water even in the unfolded (random coil) state. In the folded state these groups are almost invariably involved in the hydrogen bonds but overall there is normally a slight destabilization in comparison with the random

6662 J. Phys. Chem. B, Vol. 102, No. 34, 1998 band frequencies arising from amide groups, like those in the IR region, are also sensitive to the conformational changes. As described above, the unfolding of secondary structures is reflected by the progressively developed band shifts of amide groups. Assignment of Amide B Band. The assignment of the amide B mode has been the subject of some controversy.15 Initially, the amide B band was assigned to an intramolecular hydrogenbonded NH stretching vibration.42 Recently, the band has been assigned to Fermi resonance between the overtone of amide II or the amide I-amide II combination and the intense NH stretching vibration.43 The latter assignment leads to the conclusion that the phase relationship between the amide B and amide A should not be changed by temperature. In the Results section of this paper, we have tentatively assigned the amide B to the intramolecular hydrogen-bonded NH stretching band. This assignment can be verified by taking into account the temperature-dependent phase relationship between the amide A/II and amide B/II bands. As shown in the 2D correlation maps of Figures 4 and 5, the phase relationship between the two bands varies from out-of-phase to in-phase when the ovalbumin molecules are thermally denatured. It is contradictory to the assignment of amide B to the Fermi resonance of the overtone of amide II or the amide I-amide II combination with the NH stretching mode. In contrast, if the amide B is assigned to the intramolecular hydrogen-bonded NH stretching mode, the thermally induced change in the phase relationship may be explained because the hydrogen bonds can be changed greatly by temperature. Conclusions The application of 2D correlation spectroscopy to the concentration-perturbed NIR spectra of ovalbumin solutions has succeeded in detecting the extremely weak but important spectral variations during the heat-induced denaturation process. The correlation patterns, consisting of the peaks arising from ovalbumin and water, change remarkably in the critical temperature range of 67-69 °C in both the synchronous and asynchronous spectra. The hydration of ovalbumin remains unchanged in the native state below 67 °C, but it experiences an abrupt change at 67-69 °C. This sudden change in the hydration may induce the unfolding process of ordered secondary structures. The stabilized intermolecular interaction is unaltered again above 69 °C, although the unfolding develops progressively until 80 °C. The unfolding of secondary structures brings about the progressive band shifts for the amide bands of ovalbumin rather than the change in correlation patterns. Comparing with IR spectroscopy, which is an extensively used powerful method for studying conformational changes in protein structures, NIR spectroscopy has a unique advantage for analyzing protein structures in solution. In the NIR region the absorption of water becomes relatively weak, and therefore, structural information about protein and solvent can be obtained simultaneously. This makes it possible to directly study the hydration of protein. However, relatively weak absorptions of protein in an aqueous solution often reduce the usefulness of NIR spectroscopy. The generalized 2D correlation approach, reported in the present paper, has proven to be a sensitive and effective tool for overcoming this difficulty. A wide range of applications of 2D correlation analysis to biological fluids in NIR spectroscopy can be anticipated. Moreover, the 2D heterospectral analysis, which is capable of correlating spectral variations detected by different electromagnetic methods, may lead to more comprehensive understanding of structures and dynamics of biological molecules.

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