Attenuated Total Reflection Infrared Correlation

Apr 26, 2001 - The synchronous and asynchronous 2D spectra were generated from ... to a C O stretching mode of free and hydrogen bonded (weak, medium,...
3 downloads 0 Views 161KB Size
Download PDF
J. Phys. Chem. B 2001, 105, 4763-4769

4763

Two-Dimensional/Attenuated Total Reflection Infrared Correlation Spectroscopy Studies on Secondary Structural Changes in Human Serum Albumin in Aqueous Solutions: pH-Dependent Structural Changes in the Secondary Structures and in the Hydrogen Bondings of Side Chains Koichi Murayama,†,‡ Yuqing Wu,†,§ Bogusława Czarnik-Matusewicz,†,| 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, Nada-ku, Kobe 657-8501, Japan. ReceiVed: December 20, 2000; In Final Form: March 2, 2001

Attenuated total reflection (ATR)/infrared (IR) spectra were measured for human serum albumin (HSA) in aqueous solutions over a pH range of 5.0-3.2. Generalized two-dimensional (2D) correlation analysis was applied to the amide I region of the spectra to investigate pH-dependent changes in secondary structures and in hydrogen bondings of side chains of HSA. The synchronous and asynchronous 2D spectra were generated from the pH-dependent spectral variations for the three states of HSA, the N isomeric form (pH 5.0-4.4), the N-F transition (pH 4.6-3.8), and the F isomeric form (pH 3.8-3.0). The most interesting finding in the 2D spectra is identification of four bands at 1740, 1715, 1705, and 1696 cm-1 due to a CdO stretching mode of free and hydrogen bonded (weak, medium, and strong) COOH groups of HSA. The 2D correlation analysis provides unambiguous evidence for the existence at least the four CdO bands, demonstrating its powerful deconvolution ability. The asynchronous spectrum of the N form is characterized by a rather broad crosspeak centered at (1715, 1654) cm-1. The sign of the cross-peak indicates that protonation of COO- groups of glutamic (Glu) and aspartic (Asp) acid residues occurs at pH’s higher than those of structural changes in the R-helices. In the N-F transition, it seems that the formation of free COOH groups and those with the hydrogen bonds of the medium strength occurs is linked with changes in secondary structures of HSA. The asynchronous spectrum indicates that the formation of the strongly hydrogen-bonded COOH groups upon the protonation of COO- groups plays a key role in the initiation of the N-F transition, where mainly R-helices undergo the conformational changes. The synchronous and asynchronous spectra of the F form show that β-strands and β-turns of HSA change significantly in this pH region.

Introduction Fourier transform infrared (FT-IR) spectroscopy has been used for protein research for many years.1-16 It is a powerful technique not only for investigating secondary structures of proteins but also for exploring their dynamics.10-12 One of the significant advantages of IR spectroscopy for the studies of proteins is that spectra of proteins with a high signal-to-noise ratio can be obtained easily under various conditions irrespective of the size of proteins.1-5 One can use FT-IR spectroscopy to study even structure of water molecules in proteins.13,14 However, even nowadays, the analysis of amide I and II regions of protein spectra is not always straightforward. Moreover, information about the side chains obtained from IR spectra of proteins is limited.15,16 In recent years, generalized two-dimensional (2D) correlation spectroscopy17-21 has been applied extensively to analyze IR spectra of proteins for two major reasons.22-32 One reason is * Corresponding author. FAX: +81-798-51-0914. E-mail; ozaki@ kwansei.ac.jp. † Kwansei-Gakuin University. ‡ Kobe University. § Present address: Key Laboratory of Supramolecular Structure and Spectroscopy, Jilin University, No. 119, Jiefang Road, Changchun, 130023, China. | Present address: Faculty of Chemistry, University of Wrocław, F. JoliotCurie 14, PL-50-383, Wrocław, Poland.

that 2D correlation spectroscopy enhances apparent spectral resolution, deconvoluting the amide I and II regions into component amide bands. Another reason is that it gives information about specific order of protein secondary structure changes. Several research groups reported interesting examples of 2D IR correlation spectroscopy studies of proteins. For example, Nabet and Pezolet22 demonstrated the potential of 2D IR correlation spectroscopy in investigating the conformation of proteins by use of hydrogen-deuterium (H-D) exchange. Sefara and Richardson23 studied thermal transitions in β-lactoglobulin (BLG) using both 2D IR and 2D NIR spectroscopy. Czarnik-Matusewicz et al.24 showed the usefulness of the 2D correlation analysis in unraveling adsorption-induced and concentration-dependent ATR/IR spectral variations of BLG in aqueous solutions. It was found from their study that the intensity changes observed for the series of aqueous solutions of BLG with different concentrations are predominantly due to concentration-induced secondary structure changes even in the presence of the adsorption of protein molecules on the ATR crystal.24 However, still there are many basic things to do to establish 2D IR correlation spectral analysis for protein research. For example, one must investigate if 2D correlation analysis enables one to monitor intensity changes even in weak bands such as those due to the COOH and COO- groups of glutamic (Glu)

10.1021/jp004537a CCC: $20.00 © 2001 American Chemical Society Published on Web 04/26/2001

4764 J. Phys. Chem. B, Vol. 105, No. 20, 2001

Murayama et al. of HSA molecule. Recently, Dockal et al.44 suggested from fluorescence and CD studies of recombinant HSA domains that the loosening of the HSA structure in the N-F transition occurs primarily in domain III and that domain I undergoes a structural rearrangement with only minor modifications in secondary structure, whereas domain II transforms to a molten globulelike state as the pH is reduced. Although the acid-induced structural changes in bovine serum albumin (BSA) and HSA have been investigated in detail by use of a variety of methods, there are still dispute as to the secondary structural changes, roles in microenvironmental changes in side chains, and functions of the three domains.33,34,37-46 The present study provides evidence for the alterations in the secondary structure and hydrogen bondings of side chains, particularly of COOH and COO- groups and the relation between the changes in the secondary structures and the hydrogen bonds.

Figure 1. Ribbon diagram of the structure of HSA drawn from the X-ray data by Carter and Ho.36

and aspartic (Asp) acid residues and aromatic amino acid residues. It is also important to investigate whether 2D correlation analysis allows correlating amide I, II, and III bands. Comparison among 2D correlation analysis, second derivative (SD), and Fourier self-deconvolution (FSD) in the spectral analysis of the amide I and II regions is also interesting. The purpose of the present study is to give some answer to the above first questions. This paper reports a 2D correlation spectroscopy study of pH-dependent ATR/IR spectral changes of human serum albumin (HSA). This paper has two purposes; one is to investigate the usefulness of the 2D correlation spectroscopy in the spectral analysis of the CdO stretching region, and another is to provide new insight into the acid-induced structural changes in HSA. It has been found that protonation of COOgroups in various environments play important roles in the pHdependent transitions of HSA. We measured ATR/IR spectra of HSA in buffer solutions over a pH range of 5.0 to 3.0. Then, synchronous and asynchronous spectra were calculated from the pH-dependent spectral changes in three pH ranges, pH 5.0-4.4, pH 4.6-3.8, and pH 3.8-3.0. The 2D correlation spectra of the pH-dependent spectral changes have clearly identified four bands at 1740, 1715, 1705, and 1696 cm-1, assignable to the CdO stretching modes of free COOH groups and those with weak, medium, and strong hydrogen bonds, respectively. Human serum albumin (HSA),33-47 one of the most important proteins in blood, is composed of three homologous domains I, II, and III built from 585 amino acid residues (a molecular weight of approximately 66.4 kDa). It contains 98 acidic side chains with 36 Asp and 62 Glu residues and 100 basic side chains with 24 Arg, 59 Lys, and 17 His. Figure 1 shows a ribbon diagram of the HSA structure derived from X-ray crystallography by Carter and Ho in 1994.35,36 HSA molecule has 67% helix, no β-sheet, 10% turn, and 23% extended chain.35,36 Under acidic and basic conditions, HSA molecule undergoes several transitions in dependence of pH, assuming E form (below pH 2.7), F form (pH 2.7-4.3), N form (pH 4.3-8), B form (pH 8-10), and A form (over pH 10).33,34,37-39 The acid-induced structural changes of HSA are characterized by modifications in secondary as well as tertiary structure.37-46 Era et al.40 interpreted pH-dependent spectral variations of far-ultraviolet (UV) circular dichroism (CD) of HSA by assuming that R-helix f β and R-helix f random coil transitions occur during the N-F transition. As the pH of a HSA solution is lowered below 3.5, further unfolding takes place, leading to the full expansion

Experimental Section HSA (Fraction V) was purchased from Sigma Chemical Co. and used without further purification. Purity of HSA sample was proved by electrophoresis to be approximately 96-99%. HSA was dissolved in McIlvine type of buffer system.48 The buffer solutions were prepared by dissolving Na2HPO4, citric acid, and KCl in distilled water (R ) 18.2 MΩ) at the same time. This type of buffer solution is usable in the pH range of 2.2-7.0. In the present experiment, the ionic strength was kept at 0.5 M. The protein was dissolved in the buffer solutions in the pH range of 3.0-5.0 (2.0 wt %). To prepare water for the buffer solutions, we passed city water through activated charcoal and reverse osmosis filters and then distilled it. Finally, it was purified by an Ultrapure Water System model CPW-101 (Advantec, Japan). ATR/FT-IR spectra of the HSA solutions and buffers were measured at a spectral resolution of 2 cm-1 with a Nicolet Magna 760 FT-IR spectrophotometer with a MCT detector. For each measurement, 256 scans were coadded to ensure a good signal-to-noise ratio. Double-sided interferograms were apodized with a Happ-Genzel function prior to further transformation of the data. An ATR cell was made of a horizontal Ge crystal with an incidence angle of 45° (Spectra-Tech, Inc.). Each solution was kept on the ATR cell for 15 min for equilibration before the IR measurement. The temperature of the protein solutions (25.0 °C) was monitored by a digital thermometer (ANRITSU HFT-50) dipped into the cell before and after each measurement. The temperature stability was better than (0.2 °C during the IR measurements. Grams software (The Galactic Industries Corp.) was used for the data processing. The ATR/IR spectra obtained were subjected to the ATR depth correction formula.24 The refractive index of HSA solutions were measured at 25 °C by a refractive index meter (Atago No. 00601, Tokyo, Japan). The IR spectra of the buffer solutions and minor noise components were eliminated by the following way. First, the spectrum of the buffer solution with particular pH was subtracted from the spectrum of protein solution with the same pH by the method proposed by Dousseau et al.49 Next, the spectra obtained were subjected to maximum entropy smoothing of normal noise and Bayesian derivative with Lorentian function of 20 cm-1 by RAZOR program (Spectrum Square Associates, Inc., Ithaca, NY). For the generalized 2D correlation analysis, we used a macro program named KG2D for Grams composed by Y. Wang (Kwansei Gakuin University) with Array Basic programming

Human Serum Albumin in Aqueous Solutions

J. Phys. Chem. B, Vol. 105, No. 20, 2001 4765 TABLE 1: Frequencies (cm-1) and Assignments of IR Bands of the HSA Solutions Observed in the Second Derivative Spectra and 2D Correlation Maps second derivative

N form (2D)

1740 1715

1715

N-F transition (2D)

F form (2D)

1740 1715 1705 1696

1681 1654

1667 1654 1640

1696 1678 1667

1654 1640 1632

1647 1638 1635

1628 1620 Figure 2. Representative difference IR spectra in the 1750-1580 cm-1 region of HSA in buffer solutions (2.0 wt %) of pH 3.2, 3.6, 4.2, 4.6, and 5.0. At each pH, the spectrum of the buffer solution was subtracted from the corresponding spectrum of the HSA solution.

Figure 3. Second derivatives of the spectra shown in Figure 2.

language.50 The software was programmed on the newly developed algorithm of generalized 2D correlation spectroscopy.21 Results and Discussion IR Spectra of HSA Solutions. Figure 2 displays representative ATR/IR spectra in the 1750-1580 cm-1 region of the HSA solutions (2.0 wt %) with pH of 3.2, 3.6, 4.2, 4.6, and 5.0. The spectra are those after the spectral pretreatments described in Experimental procedure. The spectra in Figure 2 are characterized by a major band at 1654 cm-1 due to amide I vibration of HSA. Figure 3 presents the second derivatives of the spectra shown in Figure 2. It can be seen from Figure 3 that the amide I band is composed of a strong band at 1654 cm-1 and weak satellite components. The amide I bands at 1681, 1654, and 1628 cm-1 are assigned to the β-turn, R-helix, and β-strand structures, respectively (for the detailed discussion on the band assignment in the amide I region, we report in a separate paper31). 1-4 The bands at 1740 and 1598 cm-1 are assigned to a CdO stretching mode of COOH groups and COO- antisymmetric stretching mode, respectively, of Glu and Asp acid

1614 1598

1614 1598

1622 1616

assignment free COOH group hydrogen bonded (weak) COOH hydrogen bonded (medium) COOH hydrogen bonded (strong) COOH β-turn β-turn R-helix random coil β-strand β-strand β-strand β-sheet side chains COO-

residues in HSA. The positions of the bands observed in the second derivative spectra and their assignments are summarized in Table 1. In ATR/IR studies of proteins, one must consider the influence of the protein adsorption on an ATR prism on the secondary structure of proteins. However, in our previous study on 2D ATR/IR correlation spectroscopy of adsorption-induced and concentration-dependent spectral variations of BLG in aqueous solutions,24 we revealed that the signals from the adsorbed layer of protein molecules is 1 order of magnitude less intense than those from the bulk protein solution. Hence, in the present studies, we assume that the adsorption affect little the overall band shape in the amide I range. The generalized 2D IR correlation analysis was carried out for two isomeric configurations of HSA (N and F forms) in the pH region of 5.0-3.0 in order to investigate the pH-dependent spectral variations in the amide I region. N Isomeric Form of HSA. Figure 4a,b shows synchronous and asynchronous 2D IR correlation maps of the N isomeric form of HSA in the buffer solutions, constructed from the pHdependent (pH 5.0, 4.8, 4.6, and 4.4) spectral variations in the 1750-1580 cm-1 region. The synchronous correlation map is characterized by a prominent autopeak near 1654 cm-1. The autopeak is largely due to the amide I band of the R-helix structure of HSA.1-5 New information is obtained from the analysis of the asynchronous correlation map. The asynchronous correlation map can be used to locate different bands in the amide I region. A number of cross-peaks can be identified in the asynchronous correlation map shown in Figure 4b. From the analysis of asynchronous cross-peaks, one can identify bands at least at 1715, 1667, 1654, 1641, and 1614 cm-1. Of note is an appearance of a broad feature around 1715 cm-1. This band is not identified in the second derivative spectra at pH 5.0 and 4.6. Thus, the clear observation of the band at 1715 cm-1 demonstrates the usefulness of 2D correlation spectroscopy. Judging from its wavenumber, it may be due to a CdO stretching mode of the hydrogen bonded COOH groups of Glu and Asp residues of HSA. It was reported that about half of the carboxyls of Asp and Glu residues are considered to ionize with an intrinsic pK of 4.3.33 Thus, probably some carboxylate groups undergo protonation even in this pH range (pH 4.0-5.0), giving the CdO stretching mode of the COOH groups at 1715 cm-1. The broad feature of the CdO stretching band indicates that the COOH groups with the hydrogen bonds of different strength

4766 J. Phys. Chem. B, Vol. 105, No. 20, 2001

Murayama et al.

Figure 4. (a) Synchronous 2D IR correlation spectrum in the 17501580 cm-1 region constructed from the pH-dependent (pH 5.0, 4.8, 4.6, and 4.4) spectral variations of the HSA solutions with a concentration of 2.0 wt %. (b) The corresponding asynchronous correlation spectrum.

Figure 5. (a) Synchronous 2D IR correlation spectrum in the 17501580 cm-1 region constructed from the pH-dependent (pH 4.6, 4.4, 4.2, 4.0, and 3.8) spectral variations of the HSA solutions with a concentration of 2.0 wt %. (b) The corresponding asynchronous correlation spectrum.

are formed upon the protonation. HSA contains 36 Asp and 62 Glu residues, and thus, it is very important to monitor the protonation and to explore pH-dependent microenvironmental changes in the carboxyls. The assignment of the bands observed in the 2D correlation spectra is summarized in Table 1. The band at 1654 cm-1 due to R-helix is asynchronously correlated with a band at 1614 cm-1 assigned to side chains.1-5 On the basis of the signs of asynchronous cross-peaks, we can discuss sequence of band intensity changes; according to Noda,17 the sign of an asynchronous cross-peak becomes positive if the intensity change at ν1 occurs predominantly before ν2 in the sequential order of t. It becomes negative, on the other hand, if the change occurs after ν2. This rule, however, is reversed if the corresponding synchronous peak at (ν1,ν2) has negative sign, φ(ν1,ν2) < 0. On the basis of this rule, we deduce the following sequences of the intensity variations:

Therefore, the asynchronous spectrum of HSA in the N isomeric form indicates that the protonation of the COO- groups and microenvironmental changes in the side chains precede the secondary structural changes, which begin with the R-helices, followed by β-strands and β-turns. The above sequence implies that the protonation of some carboxylic groups at relatively low pH (4.5-5.0) is a trigger for the secondary structural changes in the N form. It was reported by several groups that the N-F transition takes place primarily in domain III.42-44 Thus, it seems that the 2D correlation maps largely reflect the structural variations in domain III. N-F Transition Region of HSA. The loosening of the HSA structure starts around pH 5.0.37-39 In the N-F transition the major structural changes arise from domain III and domain I undergoes only minor changes in secondary structure.42-44 To investigate the N-F transition of HSA, we generated 2D IR correlation maps from the IR spectra of HSA solutions of pH 4.6, 4.4, 4.2, 4.0, and 3.8. Panels a and b of Figure 5 show the synchronous and asynchronous correlation maps of the N-F transition, respectively. Besides a prominent autopeak at 1654 cm-1, the synchronous map develops negative cross-peaks at (1705, 1632) cm-1 and (1740, 1640) cm-1. The bands at 1740

hydrogen-bonded COOH (1715 cm-1), side chains (1614 cm-1) f R-helix (1654 cm-1) f β-strand (1641 cm-1) f β-turn (1667 cm-1)

Human Serum Albumin in Aqueous Solutions

J. Phys. Chem. B, Vol. 105, No. 20, 2001 4767

and 1705 cm-1 are assigned to a CdO stretching vibration of free and hydrogen bonded COOH groups, respectively, of Asp and Glu side chains of HSA. In this pH range, many carboxylate groups are protonated, giving the bands at 1740 and 1705 cm-1. The existence of the free COOH groups is clear also from the second derivative spectra (Figure 3). It is very likely that the free COOH groups are formed with the unfolding or expansion of domain III in the N-F transition. The bands at 1640 and 1632 cm-1 are most likely due to β-strand structures of HSA.1-5,31 The cross-peak at (1705, 1632) cm-1 suggests that the formation of the hydrogen bonded COOH groups upon the protonation and the secondary structure change in β-strand structures are cooperative in the N-F transition. Moreover, the long cross-peak between the band at 1740 cm-1 and other bands indicates that both the R-helix and β-strand structures change in-phase with the formation of the free COOH groups. In the asynchronous spectrum (Figure 5b), cross-peaks appear at (1696, 1654) and (1620, 1654) cm-1. The bands at 1696, 1654, and 1620 cm-1 may be due to hydrogen bonded COOH groups, R-helices, and β-sheets, respectively. It is noted that there are two kinds of hydrogen bonded COOH groups in the N-F transition that show the CdO stretching mode at 1705 and 1696 cm-1. It seems that they have hydrogen bonds of different strengths. The CdO groups can form hydrogen bonds with NH groups of the main chains or side chains or with OH groups of side chains or water molecules. The OH group of COOH also can form a hydrogen bond with various parts of the protein. Therefore, the CdO stretching band of the hydrogen-bonded COOH groups may appear in a rather wide range. The detection of the CdO stretching bands arising from COOH groups located in a variety of environments is very difficult in one-dimensional spectra. Thus, this shows potential of 2D correlation spectroscopy in detecting weak features in IR spectra of proteins. The detailed analysis of the signs of the asynchronous crosspeaks in Figure 5b suggests the following sequence of the structural changes in HSA during the N-F transition

hydrogen-bonded COOH (1696 cm-1) f R-helix (1654 cm-1) f β-sheet (1620 cm-1), β-turn (1667 cm-1) f β-strand (1632 cm-1)

Figure 6. (a) Synchronous 2D IR correlation spectrum in the 17501580 cm-1 region constructed from the pH-dependent (pH 3.8, 3.6, 3.4, 3.2, and 3.0) spectral variations of the HSA solutions with a concentration of 2.0 wt %. (b) The corresponding asynchronous correlation spectrum.

The asynchronous 2D IR correlation map allows us to explore the mechanism of the N-F transition. As shown in the above sequence, it seems that the protonation of COO- groups starts the N-F transition, followed by the unfolding of the R-helices in domain III. In the last step, the β-turns, β-sheets, and β-strands undergo the secondary structure change, leading to the F isomeric form of HSA. The sequence in the N-F transition resembles that in the N isomeric form, but the last part of the sequence is different. Both in the N isomeric form and in the N-F transition, the formation of hydrogen bonded COOH groups upon the protonation of COO- groups play important roles in the conformational changes of HSA. However, probably the COO- (COOH) groups of Asp and Glu side chains in the different microenvironments induce the secondary structural changes because the bands due to the COOH groups appear at 1715 cm-1 in the N form but they appear at 1705 and 1696 cm-1 in the N-F transition. It seems that the COOH groups with the stronger hydrogen bonds are formed in the N-F transition. There are much evidence showing that the protonation of carboxylate groups plays a key role in the initiation of the N-F transition.44-46 Most of the evidences come from the Trp and Tyr fluorescence studies.44-46 For example, Dockal et al.44

reported that pH profiles of the fluorescence intensity of Trp residues show a two-step change, one corresponding to the N-F transition and the other to the acid expansion. They attributed the pH-dependent decrease of the Trp fluorescence of HSA at the onset of the N-F transition to quenching by protonated imidazole and carboxylate groups of Asp and Glu residues. Cowgill45 reported that loss of helical conformation is correlated with the decrease in the Trp fluorescence intensity, which is the case in the N-F transition. As for pH-dependent intensity changes in Tyr fluorescence of HSA, Dockal et al.44 observed a significant increase in Tyr fluorescence intensity of domain I in the pH region from 6.0 to 3.5. They ascribed the intensity increase to protonation of the Asp side chains accompanied by structural changes that lead to disruption of hydrogen bondings. F Isomeric Form of HSA. Panels a and b of Figure 6 show the synchronous and asynchronous 2D IR correlation maps, respectively, constructed from the pH-dependent (pH 3.8, 3.6, 3.4, 3.2, and 3.0) IR spectral variations in the 1750-1580 cm-1 region of the F isomeric form. The synchronous correlation spectrum is complicated for the F form, and the almost all the cross-peaks in the map exhibit positive sign. It develops at least four autopeaks at 1715 (hydrogen bonded COOH), 1667 (β-

4768 J. Phys. Chem. B, Vol. 105, No. 20, 2001 turn), 1635 (β-strand), and 1622 (β-sheet) cm-1. In addition, a number of cross-peaks between them are observed in Figure 6a. These cross-peaks indicate that cooperative changes in the secondary structures, and the COOH groups occur in the F isomeric form. In Figure 6a, the bands due to β-strand and β-turn structures appear, but a peak assignable to R-helix structures is missing. Therefore, it seems that in the F isometric form of HSA, the conformational changes taking place in the β-strands and β-turns are much more significant than those in the R-helices. The asynchronous correlation map shows a number of crosspeaks (Figure 6b). The band at 1667 cm-1 (β-turn) is asynchronously correlated with the bands at 1695 (strongly hydrogen bonded COOH), 1647 (random coil), and 1616 (side chains) cm-1, indicating that changes in the β-turn structures occur outof-phase with changes in the strongly hydrogen bonded COOH groups, in the random coil structures, and in the side chains. The frequencies of band at 1616 cm-1 share an asynchronous cross-peak with the bands at 1715 (hydrogen bonded COOH), 1667 (β-turn), and 1638 cm-1 (β-strand). The frequencies of bands observed in Figure 6a and b and their assignments are also summarized in Table 1. The close inspection of the signs of asynchronous cross-peaks in Figure 6b reveals the following sequences of spectral changes in the pH range of 3.8 to 3.0:

Murayama et al. following sequence of the structural changes in the N form:

hydrogen-bonded COOH, side chain f R-helix f β-strand f β-turn Probably, the protonation of some COO- groups and the microenvironmental changes in the side chains induce the conformational changes in the R-helices in the N form. (2) The N-F transition (pH 4.6-3.8): In this pH region, both the 2D and the second derivative spectra show clear evidence for the existence of the free COOH groups. It seems that some free COOH groups are formed by the expansion of domain III through separation of its subunits during the N-F transition. In addition to the free COOH groups, there are at least two kinds of COOH groups with the medium (1705 cm-1) and strong (1696 cm-1) hydrogen bonds. The asynchronous map indicates the following sequence of conformational changes during the N-F transition:

strongly hydrogen-bonded exposed COOH (1696 cm-1), f R-helix f β-turn, β-sheet fβ-strand

(b) β-turn (1667 cm-1) f hydrogen bonded COOH (1715 cm-1) f side chain (1616 cm-1)

It is suggested from the present study that the formation of the COOH groups with strong hydrogen bonds plays key roles in the secondary structural changes in the N-F transitions. (3) The F form (pH 3.8-3.0): The characteristic features of the synchronous spectrum of the F form are the simultaneous changes of the COOH groups with the medium hydrogen bonds, β-turns, and β-strands and the missing of the peak arising from the R-helices. The asynchronous map has suggested three different sequences

(c) β-turn (1667 cm-1) f random coil (1647 cm-1)

(a) β-strand f side chain

(a) β-strand (1638 cm-1) f side chain (1616 cm-1)

It seems very likely that in the F form the conformational changes occur in the β-strands and β-turns first and then they induce changes in the random coil, side chains, and hydrogenbonded COOH groups. Conclusions We have investigated the acidic unfolding process of HSA by use of generalized 2D ATR/IR correlation spectroscopy. The synchronous and asynchronous correlation spectra have provided more detailed pH-dependent spectral changes of the HSA solutions than the second derivative spectra. The 2D correlation spectra have yielded information not only about the secondary structural changes but also about the changes in the COO- and COOH groups and other side chains. At least four kinds of COOH groups with hydrogen bonds of different strength have been identified by the present 2D correlation analysis. It is very difficult to explore the microenvironmental changes and the strength of hydrogen bonds of COOH groups in proteins by other techniques. Thus, 2D IR correlation spectroscopy has unique advantage for protein research. Moreover, the signs of the asynchronous cross-peaks have enabled us to discuss the order of structural changes in HSA in the N form, N-F transition, and F form. The following conclusions can be reached from the 2D correlation analysis of the pH-dependent spectral variations of the HSA solutions. (1) N form (pH 5.0-4.4): The broad cross-peak centered at (1715, 1654) cm-1 indicates that COOH groups with different strengths of hydrogen bonds are formed upon the protonation of COO- groups even in this pH range. The asynchronous 2D correlation map suggests the

(b) β-turn f hydrogen bonded exposed COOH f side chain (c) β-turn f random coil Probably, the conformational changes in the β-strands and β-turns are important in the F form. Moreover, 2D results in the acidic unfolding process of HSA suggest the appearance of the band of hydrogen bonded exposed COOH groups may be Asp and Glu of peptide chain between each domain. Acknowledgment. The study reported in this paper was supported by a grant-in-aid to Y. Ozaki (11640516) from the Ministry of Education, Science, and Culture, Japan. The authors acknowledge Dr. R. Tsenkova (Kobe University) for continuous encouragement. References and Notes (1) Clark, R. J. H., Hester, R. E., Eds. In Biomolecular Spectroscopy; John Wiley & Sons: Chichester, 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: Chichester, 1995. (3) Jackson, M.; Mantsch, H. H. Crit. ReV. Biochem. Mol. Biol. 1995, 30, 95. (4) Torii, H.; Tasumi, M. In Infrared Spectroscopy of Biomolecules; Mantsch, H. H., Chapman, D., Eds.; John Wiley & Sons: New York, 1996; p 1. (5) Haris, P. I.; Chapman, D. In Infrared Spectroscopy of Biomolecules; Mantsch, H. H., Chapman, D., Eds.; John Wiley & Sons: New York, 1996; p 239. (6) Krimm, S.; Bandekar, J. AdV. Protein Chem. 1986, 38, 181. (7) Cooper, E. A.; Knutson, K. In Physical Methods To Charaterize Pharmaceutical Proteins; Heaven, Z, N., Ed.; Plenum Press, New York, 1995; p S101.

Human Serum Albumin in Aqueous Solutions (8) Jackson, M.; Mantsch, H. H. In Infrared Spectroscopy of Biomolecules; Mantsch, H. H., Chapman, D., Eds.; John Wiley & Sons: New York, 1996; p 311. (9) Gremlich, H.-U.; Yan, B. Infrared and Raman Spectroscopy of Biological Materials; Marcel Dekker: New York, 2000. (10) Raimbaut, C.; Clottes, E.; Leydier, C.; Vial, C.; Buchet, R. Eur. J. Biochem. 1997, 247, 1197. (11) Siebert, F. Infrared Spectroscopy of Biomolecules; John Wiley & Sons: New York, 1996; p 83. (12) Gerwert, K. Infrared and Raman Spectroscopy of Biological Materials, Marcel Dekker: New York, 2000; p 193. (13) Maeda, A.; Tomson, F. L.; Gennis, R. B.; Ebrey, T. G.; Balashov, S. P. Biochemistry 1999, 27, 8800. (14) Kandori, H. Biochim. Biophys. Acta 2000, 1460, 177. (15) Nara, M.; Tasumi, M.; Tanokura, M.; Hiroaki, T.; Yazawa, M.; Tastumi, A.; FEBS Lett. 1994, 349, 84. (16) Mizuguchi, M.; Nara, M.; Kawano, K.; Nitta, K. FEBS Lett. 1997, 417, 153. (17) Noda, I. Appl. Spectrosc. 1993, 47, 1329. (18) Ozaki, Y., and Noda, I. Two-Dimensional Correlation Spectroscopy; AIP Conference Proceedings; American Institute of Physics: New York, 2000; Vol. 503. (19) Noda, I.; Dowrey, A. E.; Marcott, C.; Ozaki, Y.; Story, G. M. Appl. Spectrosc. 2000, 54, 236A. (20) Ozaki, Y.; Noda, I. In Encyclopedia of Analytical Chemistry; John Wiley & Sons: Chichester, 2000; p 322. (21) Noda, I. Appl. Spectrosc. 2000, 54, 994. (22) Nabet, A.; Pezolet, M. Appl. Spectrosc. 1997, 51, 466. (23) Schultz, C. P.; Fabian, H.; Mantsch, H. H. Biospectroscopy 1998, 4, S19. (24) Czarnik-Matusewicz, B.; Murayama, K.; Wu, Y.; Ozaki, Y. J. Phys. Chem. B 2000, 104, 7803. (25) Sefara, N. L.; Richardson, H. H. Appl. Spectrosc. 1997, 51, 536. (26) Graff, D. G.; Pastrana-Rios, B.; Venyaminor, S. Yu.; Predergast, F. G. J. Am. Chem. Soc. 1997, 119, 11282. (27) Smeller, L.; Heremans, K. Vib. Spectrosc. 1999, 19, 375. (28) Schultz, C. P.; Barzu, O.; Mantsch, H. H. Appl. Spectrosc. 2000, 54, 931. (29) Ismoyo, F.; Wang, Y.; Esmail, A. A. Appl. Spectrosc. 2000, 54, 939.

J. Phys. Chem. B, Vol. 105, No. 20, 2001 4769 (30) Dzwolak, W.; Kato, M.; Shimizu, A.; Taniguchi, Y. Appl. Spectrosc. 2000, 54, 963. (31) Wu, Y.; Murayama, K.; Ozaki, Y. J. Phys. Chem. B, submitted for publication. (32) Jung, Y.; Czarinik-Matusewicz, B.; Ozaki, Y. J. Phys. Chem. B 2000, 104, 7812. (33) Peters. T. Jr. All About Albumin, Biochemistry, Genetics, and Medical Applications; Academic Press: New York, 1994; p 35. (34) Brown, J. R. In Albumin; Structure, Function and Uses; Rosenoer, V. M., Oraz, M., Rotshild, M. A., Eds.; Pergamon Press: Oxford, 1977; p 27. (35) He, X. M.; Carter, D. C. Nature 1992, 358, 209. (36) Structure Explore-1AO6. Protein Data Bank, Department of Chemistry, Brookhaven National Laboratory, Upton, NY 11973, USA (http://www.rcsb.org/pdp). (37) Carter, D. C.; Ho, J. X. AdV. Protein Chem. 1994, 45, 153. (38) Dockal, M.; Carter, D. C.; Ruker, F. J. Biol. Chem. 1999, 274, 29303. (39) Era, S.; Ashida, H.; Nagaoka, S.; Inouye, M.; Sogami, M. Int. J. Pept. Protein Res. 1983, 22, 333. (40) Era, S.; Sogami, M. J. Pept. Res. 1998, 52, 431. (41) Lee, J. Y.; Hirose, M. J. Biol. Chem. 1992, 267, 14753. (42) Khan, Y. Biochem. J. 1986, 236, 307. (43) Bos, O. J.; Labro, J. F.; Fischer, M. J.; Wilting, J.; Janssen, L. H. J. Biol. Chem. 1989, 264, 953. (44) Dockal, M.; Carter, D. C.; Ruker, F. J. Biol. Chem. 2000, 275, 3042. (45) Cowgill, R. W. Biochim. Biophys. Acta 1968, 168, 417. (46) Sugio, S.; Kashima, A.; Mochizuki, S.; Noda, M.; Kobayashi, K. Protein Eng. 1999, 12, 439. (47) Qing, H.; Yanlin, He; Fenlin, S.; Zuyi, T. Spectrochim. Acta 1996, A52, 1795. (48) Perrin, D. D.; Demsey, B. In Buffers for pH and Metal Ion Control; Hilderson, H. J., Ralston, G. B., Eds.; Chapmann & Hall: London, 1974. (49) Dousseau, F.; Therrien, M.; Pezolet, M. Appl. Spectrosc. 1989, 43, 538. (50) Wang, Y.; Murayama, K.; Myojo, Y.; Tsenkova, R.; Hayashi, N.; Ozaki, Y. J. Phys. Chem. B 1998, 102, 11282.