Characterization of molten globule state of cytochrome c at alkaline, native and acidic pH induced by butanol and SDS

In our earlier communications, we had studied the acid induced unfolding of stem bromelain, glucose oxidase and fetuin [Eur. J. Biochem. 269 (2002) 47; Biochem. Biophys. Res. Comm. 303 (2003) 685; Biochim. Biophys. Acta 1649 (2003) 164] and effect of salts and alcohols on the acid unfolded state of alpha-chymotrypsinogen and stem bromelain [Biochim. Biophy. Acta 1481 (2000) 229; Arch. Biochem. Biophys. 413 (2) (2003) 199]. Here, we report the presence of molten globule like equilibrium intermediate state under alkaline, native and acid conditions in the presence of SDS and butanol. A systematic investigation of sodium dodecyl sulphate and butanol induced conformational alterations in alkaline (U(1)) and acidic (U(2)) unfolded states of horse heart ferricytochrome c was examined by circular dichroism (CD), tryptophan fluorescence and 1-anilino-8-napthalene sulfonate (ANS) binding. The cytochrome c (cyt c) at pH 9 and 2 shows the loss of approximately 61% and 65% helical secondary structure. Addition of increasing concentrations of butanol (0-7.2 M) and sodium dodecyl sulphate (0-5 mM) led to an increase in ellipticity value at 208 and 222 nm, which is the characteristic of formation of alpha-helical structure. Cyt c is a heme protein in which the tryptophan fluorescence is quenched in the native state by resonance energy transfer to the heme group attached to cystines at positions 14 and 17. At alkaline and acidic pH protein shows enhancement in tryptophan fluorescence and quenched ANS fluorescence. Addition of increasing concentration of butanol and SDS to alkaline or acid unfolded state leads to decrease in tryptophan and increase in ANS fluorescence with a blue shift in lambda(max), respectively. In the presence of 7.2 M butanol and 5 mM SDS two different intermediate states I(1) and I(2) were obtained at alkaline and acidic pH, respectively. States I(1) and I(2) have native like secondary structure with disordered side chains (loss of tertiary structure) as predicted from tryptophan fluorescence and high ANS binding. These results altogether imply that the butanol and SDS induced intermediate states at alkaline and acid pH lies between the unfolded and native state. At pH 6, in the presence of 7.2 M butanol or 5 mM SDS leads to the loss of CD bands at 208 and 222 nm with the appearance of trough at 228 nm also with increase in tryptophan and ANS fluorescence in contrast to native protein. This partially unfolded intermediate state obtained represents the folding pathway from native to unfolded structure. To summarize; the 7.2 M butanol and 5 mM SDS stabilizes the intermediate state (I(1) and I(2)) obtained at low and alkaline pH. While the same destabilizes the native structure of protein at pH 6, suggesting a difference in the mechanism of conformational stability.     

Thermodynamic characterization of cytochrome c at low pH. Observation of the molten globule state and of the cold denaturation process.

Several reports have pointed out the existence of intermediate states (both kinetic and equilibrium intermediate) between the native and the denatured states. The molten globule state, a compact intermediate state in which the secondary structure is formed but the tertiary structure fluctuates considerably, is currently being studied intensively because of its possible implication in the folding process of several proteins. We have examined the thermal stability of horse cytochrome c at low pH between 2.0 and 3.2 and different potassium chloride concentrations by absorbance of the Soret band, far and near-ultraviolet circular dichroism (u.v. c.d.) and tryptophan fluorescence using a multidimensional spectrophotometer. The concentration of potassium chloride ranged from 0 M to 0.5 M. The experimental thermal denaturation curves show that: (1) the helical content of cytochrome c remains stable at higher temperature when the concentration of salt is increased; whereas (2) the extent of ordering of the tertiary structure is weakly dependent on salt concentration; and (3) for cytochrome c, the stabilization of the molten globule state is induced by the binding of anions. Other salts such as NaCl, LiCl, potassium ferricyanide (K3Fe(CN)6) and Na2SO4 may also be used to stabilize the molten globule state. The thermodynamic analysis of the denaturation curves of c.d. at 222 nm and c.d. at 282 nm shows that, whereas a two-state (native and denatured) transition is observed at low-salt concentration, the far and near-u.v. c.d. melting curves of cytochrome c do not coincide with each other at high-salt concentration, and a minimum of three different thermodynamic states (IIb, intermediate or IIc, and denatured) is necessary to achieve a sufficient analysis. The intermediate state (called IIc) is attributed to the molten globule state because of its high secondary structure content and the absence of tertiary structure. Therefore, at low pH, cytochrome c is present in at least four states (native, IIb, IIc and denatured) depending on the salt concentration and temperature. The thermodynamic parameters, i.e. the Gibbs free energy differences (delta G), the enthalpy differences (delta H), the midpoint temperatures (Tm) of the transition (IIb in equilibrium intermediate (IIc in equilibrium denatured) are determined. We also give estimates of the heat capacity differences (delta Cp) from the temperature dependence of the enthalpy differences. The enthalpy change and the heat capacity difference of the IIc in equilibrium denatured transition are non-zero. The number of charges (protons or chloride anions) released upon transitions are determined by analysing the pH and chloride anion concentration dependence of the Gibbs free energy.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of the stable, acid-induced, molten globule-like state of staphylococcal nuclease.

Titration of a salt-free solution of native staphylococcal nuclease by HCl leads to an unfolding transition in the vicinity of pH 4, as determined by near- and far-UV circular dichroism. At pH 2-3, the protein is substantially unfolded. The addition of further HCl results in a second transition, this one to a more structured species (the A state) with the properties of an expanded molten globule, namely substantial secondary structure, little or no tertiary structure, relatively compact size as determined by hydrodynamic radius, and the ability to bind the hydrophobic dye 1-anilino-8-naphthalene sulfonic acid. The addition of anions, in the form of neutral salts, to the acid-unfolded state at pH 2 also causes a transition leading to the A state. Fourier transform infrared analysis of the amide I band was used to compare the amount and type of secondary structure in the native and A states. A significant decrease in alpha-helix structure, with a corresponding increase in beta or extended structure, was observed in the A state, compared to the native state. A model to account for such compact denatured states is proposed.     

Optical spectroscopic differentiation of various equilibrium denatured states of horse cytochrome c

Thermal unfolding of cytochrome c (cyt c) from several states has been studied using equilibrium spectroscopic techniques. CD in the uv, vibrational circular dichroism, infrared, and uv-vis absorption spectra measured at various temperatures, pHs, salt concentrations, and GuHCl concentrations are used to show the conformational as well as heme structural differences between native and various denatured states. The difference in thermal denaturation behaviors of cyt c starting from acid denatured, molten globule (MG), and the A and native states are explored. Different final high temperature states were observed for cytochrome c unfolding from four different initial states (native, MG, A, and acid denatured state) by electronic CD, Fourier transform infrared (FTIR), and vibrational CD (VCD). Consistent with this, different thermal unfolding pathways for the MG and A states are suggested by the FTIR and VCD data for this process.     

Structural characterization of the pH-denatured states of ferricytochrome-c by synchrotron small angle X-ray scattering.

The ferricytochrome-c (cyt-c) shows a complex unfolding pathway characterized by a series of stable partially folded states. When titrated with HCl at low ionic strength, two transitions are detected. At pH 2, cyt-c assumes the U1 unfolded state, whereas the successive addition of Cl(-) ion from either HCl or NaCl induces the recompaction to a molten globule conformation (A1 and A2 states, respectively). A second unfolded state (U2) is also observed at pH 12. Recent data evidence different features for the local structure of the heme in the different states. To derive relationships between local and overall conformations, we analyzed the structural characteristics of the different states by synchrotron small angle X-ray scattering. The results show that in the acidic-unfolded U1 form the protein assumes a worm-like conformation, whereas in the alkaline-unfolded U2 state, the cyt-c is globular. Moreover, the molten globule states induced by adding HCl or NaCl to U1 appear structurally different: in the A1 state cyt-c is dimeric and less compact, whereas in the A2 form the protein reverts to a globular-like conformation. According to the local heme structure, a molecular model for the different forms is derived.     

Different molten globule-like folding intermediates of hen egg white lysozyme induced by high pH and tertiary butanol

We have provided evidence that hen egg white lysozyme (HEWL) existed in alpha helical and beta structure dominated molten globule (MG) states at high pH and in the presence of tertiary butanol, respectively. Circular dichroism (CD), intrinsic fluorescence, ANS binding and acrylamide-induced fluorescence quenching techniques have been used to investigate alkali-induced unfolding of HEWL and the effect of tertiary butanol on the alkaline-induced state. At pH 12.75, HEWL existed as molten globule like intermediate. The observed MG-like intermediate was characterized by (i) retention of 77% of the native secondary structure, (ii) enhanced binding of ANS (approximately 5 times) compared to native and completely unfolded state, (iii) loss of the tertiary structure as indicated by the tertiary structural probes (near-UV, CD and Intrinsic fluorescence) and (iv) acrylamide quenching studies showed that MG state has compactness intermediate between native and completely unfolded states. Moreover, structural properties of the protein at isoelectric point (pI) and denatured states have also been described. We have also shown that in the presence of 45% tertiary butanol (t-butanol), HEWL at pH 7.0 and 11.0 (pI 11.0) existed in helical structure without much affecting tertiary structure. Interestingly, MG state of HEWL at pH 12.7 transformed into another MG state (MG2) at 20% t-butanol (v/v), in which secondary structure is mainly beta sheets. On further increasing the t-butanol concentration alpha helix was found to reform. We have proposed that formation of both alpha helical and beta sheet dominated intermediate may be possible in the folding pathway of alpha + beta protein.     

Unfolding kinetics of beta-lactoglobulin induced by surfactant and denaturant: a stopped-flow/fluorescence study

The beta-->alpha transition of beta-lactoglobulin, a globular protein abundant in the milk of several mammals, is investigated in this work. This transition, induced by the cationic surfactant dodecyltrimethylammonium chloride (DTAC), is accompanied by partial unfolding of the protein. In this work, unfolding of bovine beta-lactoglobulin in DTAC is compared with its unfolding induced by the chemical denaturant guanidine hydrochloride (GnHCl). The final protein states attained in the two media have quite different secondary structure: in DTAC the alpha-helical content increases, leading to the so-called alpha-state; in GnHCl the amount of ordered secondary-structure decreases, resulting in a random coil-rich final state (denatured, or D, state). To obtain information on both mechanistic routes, in DTAC and GnHCl, and to characterize intermediates, the kinetics of unfolding were investigated in the two media. Equilibrium and kinetic data show the partial accumulation of an on-pathway intermediate in each unfolding route: in DTAC, an intermediate (I(1)) with mostly native secondary structure but loose tertiary structure appears between the native (beta) and alpha-states; in GnHCl, another intermediate (I(2)) appears between states beta and D. Kinetic rate constants follow a linear Chevron-plot representation in GnHCl, but show a more complex mechanism in DTAC, which acts like a stronger binding species.     

The compact and expanded denatured conformations of apomyoglobin in the methanol-water solvent

We have performed a detailed study of methanol-induced conformational transitions of horse heart apomyoglobin (apoMb) to investigate the existence of the compact and expanded denatured states. A combination of far- and near-ultraviolet circular dichroism, NMR spectroscopy, and small-angle X-ray scattering (SAXS) was used, allowing a phase diagram to be constructed as a function of pH and the methanol concentration. The phase diagram contains four conformational states, the native (N), acid-denatured (U(A)), compact denatured (I(M)), and expanded helical denatured (H) states, and indicates that the compact denatured state (I(M)) is stable under relatively mild denaturing conditions, whereas the expanded denatured states (U(A) and H) are realized under extreme conditions of pH (strong electric repulsion) or alcohol concentration (weak hydrophobic interaction). The results of this study, together with many previous studies in the literature, indicate the general existence of the compact denatured states not only in the salt-pH plane but also in the alcohol-pH plane. Furthermore, to determine the general feature of the H conformation we used several proteins including ubiquitin, ribonuclease A, alpha-lactalbumin, beta-lactoglobulin, and Streptomyces subtilisin inhibitor (SSI) in addition to apoMb. SAXS studies of these proteins in 60% methanol showed that the H states of these all proteins have expanded and nonglobular conformations. The qualitative agreement of the experimental data with computer-simulated Kratky profiles also supports this structural feature of the H state.     

Native-like beta-hairpin retained in the cold-denatured state of bovine beta-lactoglobulin.

Bovine beta-lactoglobulin is denatured by increased temperature (heat denaturation) and by decreased temperature (cold-denaturation) in the presence of 4 M urea at pH 2.5. We characterized the structure of the cold-denatured state of beta-lactoglobulin using circular dichroism (CD), small-angle X-ray scattering (SAXS) and heteronuclear nuclear magnetic resonance (NMR). CD and SAXS indicated that the cold-denatured state, in comparison with the highly denatured state induced by urea, is rather compact, retaining some secondary structure, but no tertiary structure. The location of the residual structures in the cold-denatured state and their stability were characterized by 1H/2H exchange combined with heteronuclear NMR. The results indicated that the residues adjacent to the disulfide bond (C106-C119) connecting beta-strands G and H had markedly high protection factors, suggesting the presence of a native-like beta-hairpin stabilized by the disulfide bond. Since this beta-hairpin is conserved between different conformational states, including the kinetic refolding intermediate, it should be of paramount importance for the folding and stability of beta-lactoglobulin. On the other hand, the non-native alpha-helix suggested for the folding intermediate was not detected in the cold-denatured state. The 1H/2H exchange experiments showed that the protection factors of a mixture of the native and cold-denatured states is strongly biased by that of the labile cold-denatured state, consistent with a two-process model of the exchange.     

Characterization of equilibrium intermediates in denaturant-induced unfolding of ferrous and ferric cytochromes c using magnetic circular dichroism, circular dichroism, and optical absorption spectroscopies

Protein unfolding during guanidine HCl denaturant titration of the reduced and oxidized forms of cytochrome c is monitored with magnetic circular dichroism (MCD), natural CD, and absorption of the heme bands and far-UV CD of the amide bands. Direct MCD spectral evidence is presented for bis-histidinyl heme ligation in the unfolded states of both the reduced and oxidized protein. For both redox states, the unfolding midpoints measured with MCD, which is an indicator of tertiary structure, are significantly lower than those measured with far-UV CD, an indicator of secondary structure. The disparate titration curves are interpreted in terms of a compound mechanism for denaturant-induced folding and unfolding involving a molten globulelike intermediate state (MG) with near-native secondary structure and nonnative tertiary structure and heme ligation. A comparison of the dependence of the free energy of formation of the MG intermediate on the redox state with the known contributions from heme ligation and solvation suggests that the heme is significantly more accessible to solvent in the MG intermediate than it is in the native state.     

Polyol osmolytes stabilize native-like cooperative intermediate state of yeast hexokinase A at low pH

Osmolytes produced under stress in animal and plant systems have been shown to increase thermal stability of the native state of a number of proteins as well as induce the formation of molten globule (MG) in acid denatured states and compact conformations in natively unfolded proteins. However, it is not clear whether these solutes stabilize native state relative to the MG state under partially denaturing conditions. Yeast hexokinase A exists as a MG state at pH 2.5 that does not show any cooperative transition upon heating. Does the presence of some of these osmolytes at pH 2.5 help in the retention of structure that is typical of native state? To answer this question, the effect of ethylene glycol (EG), glycerol, xylitol, sorbitol, trehalose and glucose at pH 2.5 on the structure and stability of yeast hexokinase A was investigated using spectroscopy and calorimetry. In presence of the above osmolytes, except EG, yeast hexokinase at pH 2.5 retains native secondary structure and hydrophobic core and unfolds with excessive heat absorption upon thermal denaturation. However, the cooperative structure binds to ANS suggesting that it is an intermediate between MG and the native state. Further, we show that at high concentration of polyols at pH 2.5, except EG, which populates a non-native ensemble, ΔH_cal/ΔH_van approaches unity indicative of two-state unfolding. The results suggest that osmolytes stabilize cooperative protein structure relative to non-cooperative ensemble. These findings have implications toward the structure formation, folding and stability of proteins produced under stress in cellular systems.     

Thermodynamic stability of the C-terminal domain of the human inducible heat shock protein 70

The stability of the substrate-binding region of human inducible Hsp70 was studied by a combination of spectroscopic and calorimetric methods. Thermal denaturation of the protein involves four accessible states: the native state, two largely populated intermediates, and the denatured state, with transition temperatures of 52.8, 56.2 and 71.2 degrees C, respectively, at pH 6.5. The intermediate spectroscopic properties resemble those of molten globules but they still retain substantial enthalpy and heat capacity of unfolding. Moreover, the similar heat capacities of the first intermediate and the native state suggests that the hydrophobic core of the intermediate would be highly native-like and that its formation would involve an increased disorder in localized portions of the structure rather than formation of a globally disordered state. The structure of the C-terminal of Hsp70 is destabilized as the pH separates from neutrality. The intermediates become populated under heat shock conditions at acidic and basic pHs. Denaturation by guanidine chloride also indicated that the protein undergoes a sequential unfolding process. The free energy change associated to the loss of secondary structure at 20 degrees C (pH 6.5) is 3.1 kcal.mol(-1) at high salt conditions. These values agree with the free energy changes estimated from differential scanning calorimetry for the transition between the second intermediate and the final denatured state.     

Effect of trifluoroethanol on native and acid-induced states of glucose oxidase from <Emphasis Type="Italic">Aspergillus niger</Emphasis>

We have studied the effect of trifluoroethanol (TFE) on the native (pH 7.0), acid unfolded (pH 2.6), and molten globule (pH 1.4) states of glucose oxidase (GOX) by circular dichroism and fluorescence spectroscopy. In the presence of 50% TFE, at pH 7.0 and 2.6, GOX exhibited a transition from native coiled-coil and acid unfolded state to non-associating α-helical state. Interestingly, at pH 1.4, 15% TFE induced the formation of β-structured intermediate by loss of 1-anilino-8-naphthalenesulfonate binding site and almost all tertiary contacts. The β-structured intermediate converted into open helical conformation on further addition of TFE.     

Detection and characterization of the intermediate on the folding pathway of human alpha-lactalbumin

To discuss the relation between the folding mechanism and the chemical structure of proteins, the reversible unfolding reactions of human alpha-lactalbumin by acidification and by guanidine hydrochloride at 25 degrees C are studied by means of circular dichroism, difference spectra and pH-jump measurements and are compared with those for bovine alpha-lactalbumin. As shown previously for bovine alpha-lactalbumin, the folding process at neutral pH is not explained by a simple two-state mechanism but involves an intermediate form that has the same amount of helical structures as the native form. The transition between the intermediate and the fully denatured states is too rapid to be measured and corresponds to the helix-coil transition of the backbone. One of the differences of human alpha-lactalbumin from the bovine protein is the remarkable stability of the intermediate at neutral pH, which can be explained by differences in the primary chemical structure. Another difference is the existence at acid pH of an additional helical form, which is more helical than the native form. The transition from this to the intermediate or to the fully denatured one also is shown to resemble the helix-coil transition. The following folding scheme of human alpha-lactalbumin is proposed: formula: (see text). Here N is the native form, and the intermediate is a macroscopic state distributed around the state A3 at neutral pH, while the distribution in the acid and fully denautured states shifts toward Am and A-n, respectively.     

A unique molten globule state occurs during unfolding of cytochrome c by LiClO4 near physiological pH and temperature: structural and thermodynamic characterization

We have carried out denaturation studies of bovine cytochrome c (cyt c) by LiClO4 at pH 6.0 and 25 degrees C by observing changes in difference molar absorbance at 400 nm (Deltaepsilon400), mean residue ellipticities at 222 nm ([theta]222) and difference mean residue ellipticity at 409 nm (Delta[theta]409). The denaturation is a three-step process when measured by Deltaepsilon400 and Delta[theta]409, and it is a two-step process when monitored by [theta]222. The stable folding intermediate state has been characterized by near- and far-UV circular dichroism, tryptophan fluorescence, 8-anilino-1-naphthalene sulfonic acid (ANS) binding, and intrinsic viscosity measurements. A comparison of the conformational and thermodynamic properties of the LiClO4-induced molten globule (MG) state with those induced by other solvent conditions (e.g., low pH, LiCl, and CaCl2) suggests that LiClO4 induces a unique MG state, i.e., (i) the core in the LiClO4-induced state retains less secondary and tertiary structure than that in the MG states obtained in other solvent conditions, and (ii) the thermodynamic stability associated with the LiClO4-induced process, native state <--> MG state, is the same as that observed for each transition between native and MG states induced by other solvent conditions.     

A molten globule-like intermediate state detected in the thermal transition of cytochrome c under low salt concentration

To understand the stabilization mechanism of the transient intermediate state in protein folding, it is very important to understand the structure and stability of the molten globule state under a native condition, in which the native state exists stably. The thermal transitions of horse cytochrome c were thermodynamically evaluated by highly precise differential scanning calorimetry (DSC) at pH 3.8-5.0. The heat capacity functions were analyzed using double deconvolution and the nonlinear least-squares method. An intermediate (I) state is clearly confirmed in the thermal native (N)-to-denatured (D) transition of horse cytochrome c. The mole fraction of the intermediate state shows the largest value, 0.4, at nearly 70 degrees C at pH 4.1. This intermediate state was also detected by the circular dichroism (CD) method and was found to have the properties of the molten globule-like structure by three-state analysis of the CD data. The Gibbs free-energy change between N and I, DeltaG(NI), and that between N and D, DeltaG(ND), were evaluated to be 9-22 kJ mol(-1) and 41-45 kJ mol(-1), respectively at 15( ) degrees C and pH 4.1.     

Acid-induced denaturation of Escherichia coli ribonuclease HI analyzed by CD and NMR spectroscopies

Acid-induced denaturation of the ribonuclease HI protein from Escherichia coli was analyzed by CD and NMR spectroscopies. The CD measurement revealed that the acid denaturation at 10 degrees C proceeds from the native state (N-state) to a molten globule-like state (A-state), through an apparently more unfolded state (U(A)-state). In (1)H-(15)N heteronuclear single-quantum coherence (HSQC) spectra, cross peaks from the N-state and those from the other two states are distinctively observed, while the U(A)-state and A-state are not distinguished from each other. Cross peaks from the U(A)/A-states showed a small pH dependence, which suggests a similarity in the backbone structure between the two states. The direct hydrogen-deuterium (H-D) exchange measurement at pH with the largest population of U(A)-state revealed that at least alpha-helix I is highly protected in the structure of the U(A)-state. A pH-jump H-D exchange analysis showed that the protection of alpha-helix I is highest also in the A-state. The profile of hydrogen-bond protection indicated that the structure of the A-state is closely related to that of the kinetic folding intermediate.     

Acid-induced partly folded conformation resembling a molten globule state of xylanase from an alkalothermophilic Bacillus sp.

Nonnative protein structures having a compact secondary, but not rigid tertiary structure, have been increasingly observed as intermediate states in protein folding. We have shown for the first time during acid-induced unfolding of xylanase (Xyl II) the presence of a partially structured intermediate form resembling a molten globule state. The conformation and stability of Xyl II at acidic pH was investigated by equilibrium unfolding methods. Using intrinsic fluorescence and CD spectroscopic studies, we have established that Xyl II at pH 1.8 (A-state) retains the helical secondary structure of the native protein at pH 7.0, while the tertiary interactions are much weaker. At variance, from the native species (N-state), Xyl II in the A-state binds 1-anilino-8-sulfonic acid (ANS) indicating a considerable exposure of aromatic side chains. Lower concentration of Gdn HCl are required to unfold the A-state. For denaturation by Gdn HCl, the midpoint of the cooperative unfolding transition measured by fluorescence for the N-state is 3.5 +/- 0.1 M, which is higher than the value (2.2 +/- 0.1 M) observed for the A-state at pH 1.8. This alternatively folded state exhibits certain characteristics of the molten globule but differs distinctly from it by its structural stability that is characteristic for native proteins.     

Structural and drug-binding properties of alpha(1)-acid glycoprotein in reverse micelles

Alpha(1)-acid glycoprotein (AGP) is a glycoprotein that consists of 183 amino acid residues and five carbohydrate chains and binds to neutral and basic drugs. We examined the structural properties and ligand-binding capacity of AGP in interactions with reverse micelles. Also, detailed information was obtained by comparing several different states of AGP. Interaction with reverse micelles induced a unique conformational transition (beta-sheet to alpha-helices) in AGP and decreased the binding capacity for the basic drug, chlorpromazine and the steroid hormone, progesterone to AGP. These structural conformations are very similar to those observed under conditions of acidity and high ionic strength (pH 2.0, 1.5 M NaCl). This structure seems to be an intermediate between the native state and the denatured state, possibly a molten globule. The present results suggest that when AGP interacts with the biomembrane, it undergoes a structural transition to a unique structure that differs from the native and denatured states and has a reduced ligand-binding capacity.