Unfolding of Rabbit Muscle Creatine Kinase Induced by Acid A STUDY USING ELECTROSPRAY IONIZATION MASS SPECTROMETRY, ISOTHERMAL TITRATION CALORIMETRY, AND FLUORESCENCE SPECTROSCOPY*

, Electrospray ionization mass spectrometry, isothermal titration calorimetry (ITC), fluorescence spectroscopy, and glutaraldehyde cross-linking SDS-PAGE have been used to study the unfolding of rabbit muscle creatine kinase (MM-CK) induced by acid. The mass spectrometric experiments show that MM-CK is unfolded gradually when titrated with acid. MM-CK is a dimer (the native state) at pH 7.0 and becomes an equilibrium mixture of the dimer and a partially folded monomer (the intermediate) between pH 6.7 and 5.0. The dimeric protein becomes an equilibrium mixture of the intermediate and an unfolded monomer (the unfolded state) between pH 5.0 and 3.0 and is almost fully unfolded at pH 3.0 reached. The results from a “phase diagram” method of fluorescence show that the conformational TOF MS and ITC experiments and in 0.1 M citric acid-Na 2 HPO 4 buffers (pH 3.0–7.0) for fluorescence and glutaraldehyde cross-linking experiments. Electrospray Ionization Mass Spectrometry— ESI-TOF MS experiments were conducted using a hybrid quadrupole time-of-flight mass spectrometer (Q-TOF II, Micromass, Altrincham, UK) equipped with a Z-Spray ESI source at room temperature. Calibration of the instrument is performed by using the multiply charged ions produced by hen egg white lysozyme diluted to 10 (cid:1) M in pure water. Prior to mass spectrom- etry analysis, an additional desalting procedure was performed with Centricon (Millipore Corp.) using 10 m M ammonium acetate buffer (pH 7.0) as a reconstitution solution. Samples are prepared by adding 6 (cid:1) M MM-CK (final concentration) to 10 m M ammonium acetate buffers of different pH. After a short incubation time at room temperature, they are continuously infused into the ESI ion source at a flow rate of 5 (cid:1) l/min. The accelerating voltage was equal to 200 V, and the pressure in the interface region was 6.5 millibars. All spectra were recorded in the positive ion mode in the 500–6500 m / z mass range. Isothermal Titration and All each sample sample and cell The were varied were square with each other. The above results indicated that the molar heat capacity change of the unfolding was independ-ent of temperature in the range studied. A plot of (cid:5) U H m0 versus T (cid:5) U S m0 for the unfolding at different temperatures showed a slope of 1.00 with a linear correlation coefficient of 1.00 and an enthalpy intercept of (cid:2) 3.54 kcal mol (cid:2) 1 , indicating strong en-thalpy-entropy compensation. hydrophilic

Because most of the CK in the body normally exists in muscle, the elevated level of CK in human blood is an important diagnostic indicator for diseases of the nervous system and the heart muscle, for malignant hypothermia and for certain tumors. Cytosolic creatine kinase from rabbit muscle (MM-CK) is a dimer of two identical 43-kDa subunits of known sequence (5). The crystal structure of the enzyme at 2.35-Å resolution has revealed that the dimeric interface of the enzyme is held together by a small number of hydrogen bonds (6). Among its 380 amino acids, each monomer contains four tryptophans located in a restricted area at positions 210, 217, 228, and 272, in which tryptophan 228 is crucial for the activity of the enzyme (6) and tryptophan 210 is important for dimer cohesion (7). Tryptophan is a very useful intrinsic probe, because its emission fluorescence spectrum varied with the molecular environment of the side chain; disruption of the native structure leads to changes in the exposure of the tryptophan side chains to solvent that can readily be monitored by recording the protein fluorescence emission spectrum (7).
Chemical denaturants, such as guanidine hydrochloride and urea, have been widely used in the investigations of the unfolding of CK (7)(8)(9)(10)(11)(12)(13)(14)(15)(16). Because there is a direct interaction between a denaturant and a protein, some thermodynamic parameters may reflect protein-denaturant interaction rather than intrinsic parameters of the protein (15). On the other hand, the heat of dilution of a denaturant may disturb seriously the measurement of heat accompanying the conformational change of a protein by the denaturant (15,17). Acid-induced unfolding of proteins, however, obviates the above inconveniences, because the unfolding agent, H ϩ , is itself a part of the buffer used. Although some experimental approaches have been used to elucidate the mechanism of the unfolding of CK induced by acid in the past decade (18), thermodynamic information for the unfolding, which is necessary for a thorough understanding of the mechanism, is eagerly awaited. One of the purposes of this investigation is to provide detailed thermodynamic data for the acid-induced unfolding of MM-CK to furnish insights into the mechanism for the unfolding of this dimeric protein.
In late 1980s and early 1990s, the emergence of two revolutionary techniques, electrospray ionization time-of-flight (ESI-TOF) mass spectrometry (MS) and matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, has greatly enhanced the role of MS in the study of biological macromolecules instead of volatile small molecules (19 -21). These techniques have achieved good mass resolution at higher masses for biological macromolecules and are now becoming increasingly popular for probing protein higher order structure and dynamics under a variety of conditions. ESI-TOF MS is a versatile and highly sensitive method for the detection, quantitation, and structural analysis of a wide variety of analytes, including large biomolecules such as proteins and nucleic acids. At present, this method has been widely used to study noncovalent biological complexes (22)(23)(24)(25) and protein folding/unfolding (26 -34). Changes in the protein conformation can be detected by alterations of the ESI charge state distribution. An unfolded protein in solution leads to the formation of higher charge states than the same protein in a tightly folded conformation (26 -30).
Isothermal titration calorimetry (ITC) is an important tool for the study of both thermodynamic and kinetic properties of biological macromolecules by virtue of its general applicability and high precision, as shown by recent developments (35)(36)(37)(38). This method has yielded a large amount of useful thermodynamic data on protein folding/unfolding (17, 39 -48). Only a limited number of authors have, however, paid attention to the isothermal titration calorimetric investigations of the protein unfolding induced by acid (40,46).
In a previous publication from this laboratory (15), the unfolding of MM-CK induced by guanidine hydrochloride was investigated by isothermal calorimetry. In this study, ESI-TOF MS, ITC, fluorescence spectroscopy, and glutaraldehyde crosslinking SDS-PAGE were combined to study the unfolding of MM-CK induced by acid. The intrinsic molar enthalpy changes for formation of the unfolded state of this protein induced by acid at different temperatures are reported for the first time. Combining the results from these four methods, we conclude that hydrophobic interaction is the dominant driving force stabilizing the native structure of MM-CK and the acid-induced unfolding of this protein follows a "three-state" model.

EXPERIMENTAL PROCEDURES
Materials-Rabbit MM-CK (Sigma Chemical Co., St. Louis, MO) and hen egg-white lysozyme (Amresco Chemical Co., Solon, OH) were used without further purification. The A 1 cm 1% values of 8.8 (11) and 26.5 (49) at 280 nm were used for the concentration measurements of MM-CK and lysozyme, respectively. 8-Anilino-1-naphthalene-sulfonic acid (ANS) was purchased from Sigma. Creatine was obtained from Shanghai Chemical Reagent Factory (Shanghai, China) with purity of 99%, and the disodium salt of ATP was a Boehringer Ingelheim Bioproducts product (Heidelberg, Germany) with purity of Ͼ99%. All chemicals used were made in China and of analytical grade. All reagent solutions were prepared in 10 mM ammonium acetate buffers (pH 3.0 -7.0) for ESI- Electrospray Ionization Mass Spectrometry-ESI-TOF MS experiments were conducted using a hybrid quadrupole time-of-flight mass spectrometer (Q-TOF II, Micromass, Altrincham, UK) equipped with a Z-Spray ESI source at room temperature. Calibration of the instrument is performed by using the multiply charged ions produced by hen egg white lysozyme diluted to 10 M in pure water. Prior to mass spectrometry analysis, an additional desalting procedure was performed with Centricon (Millipore Corp.) using 10 mM ammonium acetate buffer (pH 7.0) as a reconstitution solution. Samples are prepared by adding 6 M MM-CK (final concentration) to 10 mM ammonium acetate buffers of different pH. After a short incubation time at room temperature, they are continuously infused into the ESI ion source at a flow rate of 5 l/min. The accelerating voltage was equal to 200 V, and the pressure in the interface region was 6.5 millibars. All spectra were recorded in the positive ion mode in the 500 -6500 m/z mass range.
Isothermal Titration Calorimetry-ITC measurements were carried out at 15.0, 25.0, 30.0, and 37.0°C using a VP-ITC titration calorimeter (MicroCal, Northampton, MA). All solutions were thoroughly degassed before use by stirring under vacuum. Before each experiment, the ITC sample cell was washed several times with acetate buffer. The sample cell was loaded with 1.43 ml of acetate buffer (pH 3.0 -6.7), and the reference cell contained doubly distilled water. Titration was carried out using a 250-l syringe filled with the native MM-CK solution (pH 7.0), with stirring at 300 rpm. The concentrations of MM-CK were varied between 15 and 30 M. Injections were started after baseline stability had been achieved. A titration experiment consisted of 28 consecutive injections of 10-l volume and 20-s duration each, with a 6-min interval between injections. To correct for the heat effects of dilution and mixing, control experiments were performed in which an identical solution but without MM-CK (pH 7.0) was injected into acetate buffer (pH 3.0 -6.7). The heat released by dilution of MM-CK was negligible. Calorimetric data were analyzed using MicroCal ORIGIN software supplied with the instrument. The enthalpy change for each injection was calculated by integrating the area under the peaks of recorded time course of change of power and then subtracting that for the control titration. The molar enthalpy change accompanying the conformational change of MM-CK induced by acid, ⌬ conf H m , was the average value of the enthalpy change for each injection, and the intrinsic molar enthalpy change for formation of the unfolded state of MM-CK induced by acid, ⌬ U H m 0 , was the average value of the molar enthalpy changes at pH 3.0, 3.5, and 4.0 at each temperature. The heats of ionization for the carboxylic groups of the protein during the acidinduced unfolding at different pH values were calculated using n Glu ϭ 54, n Asp ϭ 58 (56Asp ϩ 2C-terminal) and the reported thermodynamic parameters for the ionization of the side chains of aspartate and glutamate (46). The intrinsic molar free energy change, ⌬ U G m 0 , was deter-mined by a three-state model as described previously (8,11) using H ϩ as an unfolding agent. The intrinsic molar entropy change, ⌬ U S m 0 , and the molar heat capacity change associated with the unfolding of MM-CK, ⌬ U C P,m , were thus obtained. After the calorimetric experiment on the acid-induced unfolding, the pH value of the residual solution taken from the sample cell was almost the same as that of the acetate buffer used.
The residual activities of MM-CK in 10 mM ammonium acetate buffers at different pH values were also determined by ITC at 25.0°C. The specific activity of the enzyme under the native condition (pH 7.0) was 159 Ϯ 10 units mg Ϫ1 (n ϭ 3). The sample cell was loaded with 1.43 ml of MM-CK-creatine-mixed solution (pH 3.0 -7.0), and the reference cell contained doubly distilled water. Titration was carried out using a 250-l syringe filled with ATP-MgAc 2 mixture (pH 3.0 -7.0), with stirring at 300 rpm. A titration experiment consisted of one injection of 1 l of the titrant and nine consecutive injections of 20-l volume and 40-s duration each, with a 4-min interval between injections. The control experiment was performed in which ATP-MgAc 2 mixture was injected into creatine solution in the absence of the enzyme at the same pH. The specific activity and the remaining activity of the enzyme are thus calculated from the thermal power and the apparent molar enthalpy change of the enzymatic reaction using the reported equations (2, 50).
Intrinsic Fluorescence Spectroscopy-Intrinsic fluorescence spectro- scopic experiments on the unfolding of MM-CK induced by acid in citric acid-Na 2 HPO 4 buffers at different pH values were carried out at 25°C using an LS-55 luminescence spectrometer (PerkinElmer Life Sciences, Shelton, CT). Each MM-CK solution, at a final concentration of 0.29 M, was incubated for 30 -60 min at room temperature. The excitation wavelength at 295 nm was used for the intrinsic fluorescence measurements, and the fluorescence spectra were recorded between 300 and 390 nm. The excitation and emission slits were both 10 nm, and the scan speed was 1000 nm min Ϫ1 .
ANS Binding Measurements-For the binding studies using the hydrophobic dye ANS, the samples from the acid-induced MM-CK (final concentration of 0.11 M) unfolding series in citric acid-Na 2 HPO 4 buffers at different pH values were incubated with a 150-fold molar excess of ANS for 30 -60 min at room temperature in the dark. The fluorescence emission spectra were then recorded between 400 and 650 nm at 25°C with excitation at 390 nm. The excitation and emission slits were both 10 nm, and the scan speed was 100 nm min Ϫ1 . Assays in the absence of the protein were performed to correct for unbound ANS emission fluorescence intensities.
"Phase Diagram" Method of Fluorescence-The phase diagram method of fluorescence is a sensitive approach for the detection of unfolding/refolding intermediates of proteins (16,51,52). The essence of this method is to build up the diagram of I( 1 ) versus I( 2 ), where I( 1 ) and I( 2 ) are the fluorescence intensity values measured on wavelengths 1 and 2 under different experimental conditions for a protein under going structural transformations. Because fluorescence intensity is the extensive parameter, it will describe any two-component system by a simple relationship (16,51,52), where a and b are the slope and intercept of a plot of I( 1 ) against I( 2 ), respectively, and are defined by Equations 2 and 3, where, I 1 ( 1 ) and I 2 ( 1 ) are the fluorescence intensities of the first and second components measured on wavelength 1 , respectively, and I 1 ( 2 ) and I 2 ( 2 ) are those of the first and second components measured on wavelength 2 , respectively. As stated in Equation 1, with the change of denaturing factor, such as denaturant concentration, temperature, and pH of the solution, the transition from the initial to the final state follows a "two-state" or "all-or-none" model without formation of the intermediate states, the dependence I( 1 ) ϭ f(I( 2 )) must be linear. If the transition from the initial to the final state follows a "three-state" or "multi-state" model with the formation of one or several intermediate states, the dependence I( 1 ) ϭ f(I( 2 )) must be nonlinear and contains two or more linear portions. Moreover, each linear portion of the I( 1 ) ϭ f(I( 2 )) dependence will describe an individual all-or-none transition. In principle, 1 and 2 are arbitrary wavelengths of the fluorescence spectrum, but in practice such diagrams will be more informative if 1 and 2 will be on different slopes of the spectrum such as 320 and 365 nm (16,51,52).
Cross-linking Using Glutaraldehyde-For native and acid-treated MM-CK (5.33 M, in buffers at pH 7.0, 6.0, 5.0, and 3.0 for 60 min at 25°C), an aliquot of 25% (m/v) glutaraldehyde was added so as to make a final concentration of 1% glutaraldehyde (53). This sample was incubated at 25°C for 20 min followed by quenching the cross-linking reaction by adding 200 mM sodium borohydride. After 20-min incubation, 5 l of 10% aqueous sodium deoxycholate was added. The pH of the reaction mixture was lowered to 2-2.5 by addition of orthophosphoric acid, which resulted in precipitation of the cross-linked protein. After centrifugation (10,000 rpm, 4°C), the precipitate was re-dissolved in loading buffer containing 2-mercaptoethanol and heated at 100°C for 5 min. SDS-PAGE was run with 10% gels. Fig. 1, MM-CK unfolded gradually when titrated with acid. MM-CK is a dimer (the native state) at pH 7.0 (data not shown) and becomes an equilibrium mixture of the dimer and a partially folded monomer (the intermediate) between pH 6.7 and 5.0. At pH 6.7, the major signals observed in the mass spectrum were due to multiply charged ions of the dimer. Minor signals reveal the co-existence of small amounts of the intermediate. An equilibrium mixture of the intermediate and an unfolded monomer (the unfolded state) was detected between pH 5.0 and 3.0, and decreasing the pH to 4.0 resulted in disappearance of the dimer. The protein was almost fully unfolded at pH 3.0 reached, and only a trace of the intermediate was observed.

Mass Spectrometry of Acid-induced Unfolding of MM-CK-As shown in
Comparison of Inactivation with Quaternary Structure Changes of MM-CK at Different pH-ITC curves of the reversible phosphoryl transfer from ATP to creatine catalyzed by MM-CK at pH 7.0 and 5.0 and at 25.0°C are shown in Fig. 2, from which (in part, the ITC curves at other pH values were not shown) the residual activities of the enzyme at different pH values were determined using the given method. The contents of the dimer at different pH values were estimated by ESI-TOF MS from Fig. 1 (in part, the mass spectra at other pH values were not shown). Fig. 3 shows a comparison of inactivation with quaternary structure (represented by the contents of the dimer) changes of MM-CK in acetate buffers of different pH. As can be seen from Fig. 3, the residual activity of MM-CK measured by ITC decreased with decreasing the pH value. When the pH value is 5.0, the activity of MM-CK was completely lost, whereas a relatively large content of the dimer (40%) was observed. These results indicate that during, denaturation by acid, the inactivation of MM-CK precedes the quaternary structure change of this enzyme, providing a mass spectrometric evidence for the proposition that the active site of an enzyme is more easily disrupted than the enzyme molecule as a whole (54). However, whether the loss in enzymatic activity with decreasing pH correlates with the protonation of an essential residue near the catalytic or binding site of MM-CK (for example, His 296 ) (6) cannot be determined in this study. Similarly, a conclusion that the active form of MM-CK is the dimer but not the partially folded monomer of the protein (4) cannot be reached from Fig. 3 alone. Fig. 4 shows the ITC curves of the unfolding of MM-CK induced by acid at 25.0°C in acetate buffers at pH 5.0 and 3.0. As can be seen from Fig. 4, even though the apparent molar enthalpy change accompanying the conformational change of MM-CK induced by acid at pH 3.0 is negative, the conformational change during the forma-tion of the unfolded state of the protein is endothermic in practice. Fig. 5A shows the molar enthalpy changes accompanying the conformational changes of MM-CK in acetate buffers between pH 7.0 and 3.0 and at 15.0, 25.0, 30.0, and 37.0°C, measured by ITC, and Fig. 5B displays the predicted heats of ionization for the carboxylic groups of the protein under such conditions. As shown in Fig. 5, the molar enthalpy change for the unfolding of MM-CK induced by acid, which includes contributions of the heats of ionization for the carboxylic groups of the protein, depends on the temperature at which unfolding occurs, which is varied by adjusting pH. The intrinsic unfolding enthalpy is negative at lower temperature (15.0°C) but increases rapidly with temperature, becoming positive at higher temperatures (25.0, 30.0, and 37.0°C). Fig. 6 shows the temperature dependence of the intrinsic thermodynamic parameters for formation of the unfolded state of MM-CK induced by acid. As shown in Fig. 6, the molar heat capacity changes associated with the unfolding of MM-CK, ⌬ U C P,m , were 8.78 kcal mol Ϫ1 K Ϫ1 for the plot of ⌬ U H m 0 versus T and 8.77 kcal mol Ϫ1 K Ϫ1 for the plot of ⌬ U S m 0 versus ln T with linear correlation coefficients of 0.9942 and 0.9915, respectively. The values of ⌬ U C P,m obtained from the above two plots FIG. 6. Temperature dependence of the intrinsic thermodynamic parameters for formation of the unfolded state of MM-CK induced by acid. The intrinsic molar enthalpy change (f) was the average value of the apparent molar enthalpy changes at pH 3.0, 3.5, and 4.0 at each temperature. The intrinsic molar free energy change (q) was determined by a three-state model as described (8,11) using H ϩ as an unfolding agent, and the intrinsic molar entropy change (Ⅺ) was calculated using ⌬ U S m 0 ϭ (⌬ U H m 0 Ϫ ⌬ U G m 0 )/T. The molar heat capacity change associated with the unfolding of MM-CK, ⌬ U C P,m , was determined by linear regression analysis of the plot by ⌬ U H m 0 ϭ ⌬ U C P,m T ϩ ⌬H 0 (A) and ⌬ U S m 0 ϭ ⌬ U C P,m ln T ϩ ⌬S 0 (B), respectively, using the data in A and B. The data with error bars were expressed as mean Ϯ S.D. (n ϭ 3).

Molar Enthalpy Changes Accompanying the Conformational Changes of MM-CK Induced by Acid-
were square with each other. The above results indicated that the molar heat capacity change of the unfolding was independent of temperature in the range studied. A plot of ⌬ U H m 0 versus T⌬ U S m 0 for the unfolding at different temperatures showed a slope of 1.00 with a linear correlation coefficient of 1.00 and an enthalpy intercept of Ϫ3.54 kcal mol Ϫ1 , indicating strong enthalpy-entropy compensation.
Acid-induced Unfolding of MM-CK Monitored by Fluorescence max Shift and ANS Binding-As shown in Fig. 7A, native MM-CK (pH 7.0) had an intrinsic fluorescence emission maximum of about 333 nm when excited at 295 nm. The tryptophan emission maximum was not affected in the pH range of 7.0 -5.4. With further decreasing the pH values, the tryptophan emission maximum of the protein was red-shifted, reaching 344 nm at pH 3.0. This indicates that with the decrease of the pH values, the tryptophan residues are gradually exposed to a more hydrophilic environment and MM-CK is gradually unfolded.
Changes in ANS fluorescence are frequently used to detect partially folded intermediates of globular proteins. This is because such intermediates are characterized by the presence of solvent-exposed hydrophobic clusters to which ANS binds, resulting in a considerable increase in the ANS fluorescence intensity and in a pronounced blue shift of the fluorescence emission maximum (16). As shown in Fig. 7B, a steep increase in ANS fluorescence intensity together with a blue shift of ANS emission maximum takes place upon decreasing the pH values, reaching a maximum at pH 4.0, at which there is a 4.4-fold increase in ANS fluorescence intensity. With a further decrease of the pH values, however, the ANS fluorescence enhancement declines. These results indicate that the partially folded monomer of MM-CK has a significant amount of exposed hydrophobic surface area.
Phase also studied by glutaraldehyde cross-linking. MM-CKs treated with varying pH values were cross-linked using glutaraldehyde, and the products obtained were analyzed on SDS-PAGE followed by Coomassie Blue staining of protein bands. As shown in Fig. 9, for MM-CK at pH 7.0, a cross-linked protein band corresponding to only the dimer (the native state) was observed. For MM-CK at pH 6.0 and 5.0, however, cross-linked protein bands corresponding to both the dimer and a partially folded monomer (the intermediate, combining the results from ESI-TOF MS) were observed, and the band corresponding to the monomer at pH 5.0 was thicker than that at pH 6.0, indicating that MM-CK was gradually unfolded induced by acid. For MM-CK at pH 3.0, the protein band corresponding only to an unfolded monomer (the unfolded state) was observed. At pH 6.0 and 5.0, there was also a band of high molecular mass at about 129 kDa, corresponding to a trimer of MM-CK, which is formed by intermolecular disulfide linkage of the dimer and the intermediate. DISCUSSION Electrolytic oxidation of water can lead to changes in the pH during ESI, and this might complicate the relationship the charge state distribution observed in ESI-TOF MS and the protein conformation in solution (26). However, this work indicates that, under the experimental conditions used, such electrolytic processes are negligible, because the major conformational transitions for the unfolding induced by acid observed by ESI-TOF MS are almost the same as those observed by the phase diagram method of fluorescence.
It remains unclear whether some of MM-CK is aggregated during the unfolding of the protein induced by acid, especially at higher temperatures (25.0, 30.0, and 37.0°C). Aggregation of MM-CK, measured by the absorbance of the protein at 320 nm, was not observed at these three temperatures between pH 7.0 and 3.0 (data not shown). The results showed that aggregation did not take place even when the temperature was increased to 37.0°C during the acid-induced unfolding of MM-CK.
The unfolding of MM-CK induced by acid is driven by a favorable enthalpy change but with an unfavorable entropy decrease at lower temperature (15.0°C), becoming entropydriven at higher temperatures (25.0, 30.0, and 37.0°C). The increase in ⌬ conf H m with increasing temperature at pH 3.5 or 4.0 indicates that thermal unfolding occurs. The changes of enthalpy and entropy for the unfolding strongly depend on the temperature, whereas the Gibbs free energy change of the unfolding is almost temperature-independent. The enthalpy change for the unfolding is almost compensated for by a corresponding change in entropy resulting in a smaller net Gibbs free energy increase. That is, remarkable enthalpy-entropy compensation occurs in the unfolding of the protein induced by acid, suggesting that the water reorganization (56) is involved in the unfolding reaction. It is noticed that the value of ⌬ U G m 0 or the unfolding of MM-CK induced by guanidine hydrochloride (6.24 kcal mol Ϫ1 ) (11) is 2-fold of that induced by acid (3.37 kcal mol Ϫ1 ), indicating that the protein is not completely unfolded induced by acid compared with that induced by guanidine hydrochloride.
It is well known that the unfolding reactions of different proteins display certain common properties (57,58). The positive molar heat capacity change associated with the unfolding of a protein, for example, is commonly attributed to the hydrophobic interaction, although other factors may contribute to ⌬ U C P,m (41,57). A large positive molar heat capacity change of the unfolding of MM-CK induced by acid, 8.78 kcal mol Ϫ1 K Ϫ1 , at all temperatures examined indicates that hydrophobic interaction is the dominant driving force stabilizing the native structure of this protein. ⌬ U C P,m is the thermodynamic term that has been most often scaled to structural feature of a protein (58). The value of ⌬ U C P,m per amino acid residue for MM-CK, 11.6 Ϯ 0.9 cal K Ϫ1 (mol residue) Ϫ1 , is the same order of magnitude as those for monomeric proteins (13.9 Ϯ 0.5 cal K Ϫ1 (mol residue) Ϫ1 (58)) and homodimeric pea lectin (11.4 cal K Ϫ1 (mol residue) Ϫ1 (59)), suggesting that ⌬ U C P,m is approximately proportional to the size of a protein. Combining the results from ESI-TOF MS, ITC, fluorescence spectroscopy, and glutaraldehyde cross-linking SDS-PAGE, we conclude that the acid-induced unfolding of MM-CK follows a three-state model and that the intermediate state of the protein is a partially folded monomer.