Unfolding and aggregation of a multi-domain protein, The

thermal stability of immunoglobulin: Unfolding and aggregation of a multi-domain protein, The

Vermeer, Arnoldus W P

ABSTRACT The denaturation of immunoglobulin G was studied by different calorimetric methods and circular dichroism spectroscopy. The thermogram of the immunoglobulin showed two main transitions that are a superimposition of distinct denaturation steps. It was shown that the two transitions have different sensitivities to changes in temperature and pH. The two peaks represent the Fab and F. fragments of the IgG molecule. The Fab fragment is most sensitive to heat treatment, whereas the Fo fragment is most sensitive to decreasing pH. The transitions were independent, and the unfolding was immediately followed by an irreversible aggregation step. Below the unfolding temperature, the unfolding is the rate– determining step in the overall denaturation process. At higher temperatures where a relatively high concentration of (partially) unfolded IgG molecules is present, the rate of aggregation is so fast that IgG molecules become locked in aggregates before they are completely denatured. Furthermore, the structure of the aggregates formed depends on the denaturation method. The circular dichroism spectrum of the IgG is also strongly affected by both heat treatment and low pH treatment. It was shown that a strong correlation exists between the denaturation transitions as observed by calorimetry and the changes in secondary structure derived from circular dichroism. After both heat- and low-pH-induced denaturation, a significant fraction of the secondary structure remains.

INTRODUCTION

The concept that proteins may comprise of a number of independent, compact globular regions, called domains, has become widely accepted (Hardie and Coggins, 1986). Sometimes these domains can be isolated as stable fragments. These fractions, or subunits, are generally associated with different functions. Interactions between the domains may provide the intra-protein communication that is necessary to coordinate the various protein functions. In some cases the functions of the protein are found to be connected to different sequences within a single polypeptide chain. Goto and Harnaguchi (1982) demonstrated that a polypeptide chain segment corresponding to a single domain can be refolded independently of the rest of the protein. However, independent unfolding of different domains is not a general phenomenon. Several proteins show cooperativity in the unfolding transition due to heat or pH treatment (Takahashi and Fukada, 1985; Solis-Mendiola et al., 1993; Protasevich et al., 1997).

An important class of proteins that conform to a common subunit structure are the immunoglobulin Gs (IgGs). These molecules have domains that are structurally independent, compact globular regions consisting of continuous stretches of the polypeptide chain approximately 100 amino acids long, with a characteristic fold (Chothia et al., 1995; Edmundson and Ely, 1986; Padlan, 1997). This fold contains two P-sheets and essentially no a-helices (Amzel and Poljak, 1979). A predominant feature of these globular protein structures is that nonpolar residues are sequestered into a core, where they largely avoid contact with water. Immunoglobulins, or antibodies, show a strong structure-function relation in the different domains, which makes these molecules excellent systems for various diagnostic tests. The domains of the antibodies with a high specificity to bind analytes (antigens) assure that these immunoglobulins can be used for a reliable and fast determination of low concentrations of analyte, whereas other domains of the immunoglobulin promote protein binding to a surface in the proper orientation, i.e., with its binding sites accessible to the antigen.

Studying the structure of immunoglobulins in more detail reveals that these proteins are composed of four polypeptide chains that are connected by disulphide bonds and noncovalent forces. The four polypeptide chains are grouped together in different fragments, two identical Fab segments and one Fc segment, thus forming a Y-shaped conformation. The antigen binding sites are located on the far ends of the Fab segments. The Fab segments are linked to the F,, by the hinge region, which varies in length and flexibility in the different antibody classes and isotypes. Data reported by Oi et al. (1984) suggested that immunoglobulin G (IgG) of isotype 2b exhibits considerable segmental flexibility, whereas, for example, IgG of isotype 1 is rather rigid. Both Fab and F. fragments consist of four of the above mentioned globular regions.

Changes in the secondary structure of IgG as a function of temperature and, e.g., pH, can be studied by circular dichroism (CD) spectroscopy (Fasman, 1996). The major advantages of this technique are that the spectroscopic signal is not affected by the presence of the surrounding solution and that well-defined procedures are available to elucidate the secondary structure based on reference spectra of the different structure elements (de Jongh et al., 1994). The fractions of the secondary structural elements can be obtained from the CD spectra. CD has been used previously to study the secondary structure of IgG (Rousseaux et al., 1982; Kars et al., 1995; Tetin and Linthicum, 1996; Kats et al., 1997); however, these authors did not calculate the fractions of the structural elements.

The stability of multi-domain proteins is commonly investigated using differential scanning calorimetry (DSQ. One of the great advantages of DSC is that it can detect fine-tuning of interactions between the individual domains of a protein (Privalov and Putekhm, 1986). Numerous studies on the thermal denaturation of immunoglobulins are available, reporting on the effects of pH (Tischenko et al., 1982; Buchner et al., 1991; Martsev et al., 1995; Vlasov et al., 1996), pre-heating (Lindstrom et al., 1994; Vlasov et al., 1996), and, for example, the cooperativity between the IgG fragments (Tischenko et al., 1982; Shimba et al., 1995). However, based on the literature mentioned above, it is far from clear to what extent the denaturation of the different isotypes of immunoglobulin G is reversible, how it depends on the solution conditions, and whether the denaturation of the domains is cooperative.

Goto et al. (1988) mentioned that the constant domain of isolated Fab denatures more easily than the variable domain and that the denaturation of the isolated domains can be described as a two-state process. This supports the idea that the denaturation of a multi-domain protein can be described by the denaturation of the individual domains. If we assume for the moment that the various domains in a multi-domain protein denature independently, different denaturation routes may exist, whereby the different domains are affected in different orders. Depending on the conditions, either of the routes may be preferred.

The possibility that partly denatured intermediates of immunoglobulins exist was first suggested by Rowe and Tanford (1973). There is indirect evidence for the existence of such intermediates (Tischenko et al., 1982; Buchner et al., 1991; Martsev et al., 1995; Shimba et al., 1995; Vlasov et al., 1996). DSC allows individual domains to be seen as they undergo thermal unfolding. If the domain transitions are well separated along the temperature axis, the thermodynamic parameters of each transition can be derived. Although some immunoglobulins show several transitions in their DSC thermogram (Tischenko et al., 1982; Shimba et al., 1995), the overlap of the corresponding peaks is, generally, too strong to allow for a rigorous thermodynamic analysis of the individual transitions. However, even when these transitions overlap considerably, available software allows rapid deconvolution of the transition envelope to obtain estimates of the individual domains’ transitions (Buchner et al., 1991; Martsev et al., 1995; Vlasov et al., 1996). The existence of denaturation intermediates has also been reported by Brody (1997), who detected a ladder of denaturation intermediates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after exposure of the IgG to sodium dodecyl sulfate, urea, or beat, and by Hughes and coworkers (Hughes and Richberg, 1993; Alexander and Hughes, 1995), who described IgG ladder formation by capillary electrophoresis. The different intermediates were formed, as proposed by Brody, due to differences in sensitivity of the disulphide bonds in the different IgG domains. However, stable IgG intermediates have not yet been obtained.

From this short overview it may be concluded that the presence of different domains in immunoglobulins has a strong impact on the overall behavior of these proteins. It was suggested that, at least to some extent, the domains denature independently and that changes in the solution conditions may effect the individual domains differently. In this paper, the unfolding process of immunoglobulin G, induced by heat, will be investigated in detail by differential scanning calorimetry, the kinetics of the process will be studied by isothermal calorimetry, and the effect of the denaturation process on the secondary structure will be monitored by circular dichroism. In addition to the heat– induced transitions, the effect of pH on the secondary structure will be studied. The results obtained by DSC and CD are consistent and they indicate different domains that denature independently and irreversibly. Furthermore, it will be shown that the denaturation method affects the structure of the aggregates formed.

MATERIALS AND METHODS

All chemicals were of analytical grade and were used without further purification. The water was purified by percolation through a mixed bed of ion exchangers followed by an activated carbon column and a microfilter. Immunoglobulin The immunoglobulin is a monoclonal mouse anti-rat antibody of isotype 2b, specific for the glycosylated N-terminal part of the P-chain of human hemoglobin Alc.

The degree of aggregation of the immunoglobulin in a 10-mM phosphate buffer, pH 8.1, was measured by Gel Permeation Chromatography using a Sephadex 200 HR 10/30 column on an Akta explorer (Pharmacia Biotech, Uppsala, Sweden). The protein concentration was measured by UV-spectroscopy at a wavelength of 280 mn. The IgG was monomeric; only one peak was observed, representing molecules with a molecular weight of 150 kd. The isoelectric point (iep) of the molecules was measured on a PhastSystem (Pharmacia LKB, Uppsala, Sweden), using a Phastgel with a pH range of 3.5 to 8.65. The iep of the IgG molecules ranges from pH 6.0 to 7.0.

Differential scanning calorimetry

The DSC experiments at low heating rate (

The DSC experiments at a heating rate of 5 deg C /min were performed on a Perkin-Elmer DSC7, which is preferably used at faster heating rates because of the low thermal inertia. The samples were measured in aluminium cells containing 20 (mu)l of a 20 mg/ml IgG solution. As a reference the same amount of the buffer solution was used.

Isothermal calorimetry

Isothermal calorimetry (IQ experiments were done in a 2277 Thermal Activity Monitor (LKB, Bromma, Sweden). The calorimeter contains a 4-ml reference cell and a 4-ml sample cell. The reference cell was filled with 2.000 +/- 0.001 g nano-pure water and sealed using a Teflon interface. The other cell was filled with about 2 g of the sample (mass determination within +/-0.001 g). The cells were brought to the experimental temperature by equilibrating for 20 min (Hoffmann and van Mil, 1997) in the temperature equilibration position. Immediately after the cells were brought into their final position the difference in heat flow between the sample and reference cell was monitored. This difference corresponds to the heat generated in, or consumed by, the sample. The heat flow of the samples is always compared with the heat flow of a reference-reference measurement at comparable temperature. The reproducibility of the heat flow in the first hour of the experiment was very poor and depended strongly on the sample treatment before the IC experiment. The reproducibility in the absolute heat flow values was about ItW and the baseline stability, at longer time scales, was within 0.2 AW.

Circular dichroism measurements

The CD spectra were measured with a JASCO spectropolarimeter, model J-715 (JASCO International Co., Tokyo, Japan). For the far-UV and near-UV measurements quartz cuvettes having light path lengths of 0. 1 and 1.0 cm, respectively, were used. Temperature regulation was carried out using a PTC-348WI thermocouple (JASCO). Comparison of the actual temperature in the cell with the temperature set by the Peltier element showed that the deviation of the actual temperature was

Circular dichroism spectral analysis

The CD curves of poly-L-lysine containing varying amounts of a-helix, P-sheet, P-turn, and randomly coiled conformations have been applied for determining the content of the structural components of the immunoglobulin. The measured CD curves for the IgG are a superposition of these four structure elements. The poly-L-lysine reference spectra were measured (de Jongh et al., 1994) as described by Greenfield and Fasman (1969) and Chang et al. (1978). Fitting of the spectra was performed by a nonlinear regression procedure, making use of the Gauss-Newton algorithm (de Jongh et al., 1994). The reference spectra (data from Chang et al., 1978) were fitted independently from 190 to 240 nm with 1-nin resolution. No constraints were used in the fit procedure. The quality of the fit was expressed using the definition of the normalized root-mean-square (RMS) error as described by Brahms and Brahms (1990). A fit was considered as reliable only if the RMS error was

RESULTS AND DISCUSSION

Heat-induced denaturation of immunoglobulin G Thermal denaturation of immunoglobulin G was studied by differential scanning calorimetry. A typical thermogram of IgG in a 10-mM phosphate buffer, pH 8. 1, is shown in Fig. 1. The IgG concentration of the sample was 6 mg/mI and the heating rate was 0.5 deg C/min. The curve shows two transitions, one with a denaturation temperature, Tn, of 61 deg C, and with a denaturation enthalpy, Delta^sub d^H, of 12.5 J/g, and a second at 71 deg C with an enthalpy of 4.5 J/g. As has already been discussed in the introduction of this paper, IgG can be described as a multi-domain protein. The presence of these two peaks may therefore indicate that at least two domains, or groups of domains, exist that denature under distinct conditions. In a previous paper (Vermeer et al., 1998) we reported the thermogram of IgG of isotype 1, which showed only one transition peak. This difference between the two IgG isotopes may arise from the higher flexibility of the hinge region of the IgG used in this investigation, as has been proposed by Oi et al. (1984). As reported in a separate paper (Vermeer, Norde, and van Amerongen, unpublished manuscript), we isolated the Fab and F,, fragments of the present IgG as major products from papain digests. These authors report that the isolated Fab fragment showed only one transition at 61 deg C. The F^subc^ fragment showed a single transition at 71 deg C. Moreover, the denaturation enthalpies of both fragments were comparable to the enthalpies of the two peaks observed in Fig. 1. These results show that the transitions at 61 deg C and 71 deg C for whole IgG represent the denaturation of the F^subab^ and F^subc^ domains, respectively. After cooling the IgG sample, the thermogram of a subsequent cycle did not show any peak, indicating irreversible denaturation. Although under the experimental conditions no aggregation peak was observed in the thermogram, aggregates were observed to be formed after the heat treatment in DSC and a gel-like structure resulted.

A simple model that is consistent with irreversible protein denaturation is a reversible unfolding step followed by an irreversible process that locks the unfolded protein in a state from which it does not refold (Lepock et al., 1992; Castronouvo, 1991). It seems reasonable that the observed aggregation is the step that induces the irreversibility of the overall denaturation process; this has also been suggested by several other authors (Augener and Grey, 1970; Oreskes and Mandel, 1983; McCarthy and Drake, 1989). Thus, the denaturation of the IgG domains should be described as a three-state process rather than a two-state process.

It has been demonstrated (Hu and Sturtevant, 1987; Sanchez-Ruiz et al., 1988) that irreversible DSC thermograms can be interpreted in terms of reversible thermodynamics, provided that Tn and AdH are independent of the heating rate. However, the data in Table I show a heating rate dependency, indicating that the protein denaturation is kinetically controlled. To explain the heating rate dependence of Tm, we follow the theory of Sanchez-Ruiz et al. (1988) and assume that the irreversible denaturation reaction can be represented by

In Fig. 2 the apparent heat capacities curves for IgG measured at various heating rates are shown. From the data given in Table 1, three major effects are inferred: (1) the peak temperature of both peaks increases with increasing heating rate, the effect being most pronounced for the first transition; (2) the enthalpy of, especially, the first transition decreases with increasing heating rate; and (3) an exothermic gelation peak appears at higher heating rates. The latter phenomenon has also been found for other proteins (Barone et al., 1992), such as bovine serum albumin and human serum albumin.

The fact that the denaturation of IgG has to be described by a three-state process follows also from the change in the shape of the thermogram when varying the heating rate. For a two-state irreversible denaturation process an increase in heating rate is expected to shift Tm to a higher value, whereas the influence on AdH is expected to be small (Lepock et al., 1992). For a three-state process the effects of increasing the heating rate strongly depend on both the rate of the unfolding step and the rate of the irreversible aggregation step.

The values calculated for the activation energy, Ea for the two transitions are 456 kJ/mol (9.1 J/g) for the first transition, and 692 kJ/mol (13.8 J/g) for the second. These values are reasonable for the denaturation of globular proteins (Haynes and Norde, 1995, and references therein).

In order to investigate whether the two endothermic transitions are related to each other, a thermogram of an IgG sample, with a concentration of 20 mg/ml, was determined after incubation for 40 h at 55 deg C. As a result of this incubation the least thermostable domains will be affected. The result is shown in Fig. 3. It is observed that the peak at 61 deg C has disappeared, whereas the peak at 71 deg C is still present. It indicates that the F^subab^ and F^subc^ fragments denature independently of each other. Further, it can be seen that, at this high IgG concentration, a shoulder appears in the peak at 71 deg C. It suggests that, although only two major peaks are observed in the thermogram shown in Fig. 1, several transitions take place that are likely to be related to different domains of the IgG molecule. It has been discussed by Tischenko et al. (1982) that in the pH range 2.5 to 5.5, the Fab and Fc fragments of rabbit IgG are thermodynamically independent subunits. These authors further showed that the thermogram of the intact IgG is the sum of those of its individual fragments. Using a deconvolution procedure it was revealed that the Fab fragment exhibits three cooperative melting transitions, whereas the F,, fragment contains four transitions. These transitions were ascribed to different globular domains (or parts of a domain in the case of the Fc fragment). This picture corresponds well with the results of the thermograms described above.

Rate of denaturation of immunoglobulin G

As mentioned above, the rates of both the unfolding and the subsequent aggregation step affect the overall denaturation process. It may well be that unfolding and aggregation of IgG take place on a much longer time scale than that in the DSC experiment discussed above. Then, it is virtually impossible to derive a detailed picture of the kinetics of these processes based on DSC experiments only. To study the complex reactions involved in IgG denaturation, IC may be more useful because it gives more insight into the mechanism, especially in the kinetics of the reaction (Hoffmann and van Mil, 1997). With IC the heat flow at constant temperature is monitored as a function of time.

Isothermal calorimetry data at 41, 55, 60, and 70 deg C are shown in Fig. 4. The heat flow at 4PC did not deviate from the blank curve, indicating that neither an endothermic nor an exothermic process occurred. A DSC thermogram of the sample that was determined directly after the IC experiment was identical to the one shown in Fig. 1. At 55 deg C an endothermic process occurs which indicates that native IgG is transferred into an unfolded conformation. At t = 0 (this is after 20 min of thermal equilibration) this endothermic effect is very strong, implying that the concentration of the native IgG molecules is still relatively high. After about 25 h, the heat flow has returned to the blank value, indicating that the IgG is essentially completely denatured. After this time period the sample had formed a gel. The formation of this gel may be explained as follows: upon unfolding the hydrophobic patches become exposed to the solution (Arntfield et al., 1989); this triggers the formation of aggregates through intermolecular hydrophobic binding (Augener and Grey, 1970; Rosenqvist et al., 1987; Lopez-Bote et al., 1993; McCarthy and Drake, 1989; Oreskes and Mandel, 1983). In the DSC thermogram determined after 40 h at 55 deg C (Fig. 3) the peak at 61 deg C has vanished, whereas the peak at 71 C representing the Fc fragment is still present. This picture is in good agreement with the results of McCarthy and Drake (1989), who mentioned that in IgG aggregates produced by heating at 63 deg C the Fab fragments are associated, leaving the F^subc^ fragments exposed. Light microscopy revealed that the resulting gel is composed of closely packed aggregates of about 1 (mu)m diameter that could easily be disrupted into fragments, which demonstrates that attraction between the aggregates is only weak.

The IC thermogram at 60 deg C shows that the endothermic heat flow decreases strongly over the first hour, after which the signal is almost equal to the blank. Because the enthalpy of the denaturation process at 55 deg C and 60 deg C should be equal, we must conclude that a significant fraction of the IgG is already denatured within 20 min equilibration time. Hence, at a temperature this close to T, the kinetics of denaturation are fast as compared to 55 deg C. The DSC thermogram. measured after incubation at 60 deg C was comparable to that after incubation at 55 deg C, i.e., only the second peak was present. Light microscopy showed closely packed aggregates of about 1(mu)m diameter. However, under these conditions the gel could not easily be broken into smaller fragments, indicating that the attraction between the aggregates is strong. Clearly, when the rate of unfolding relative to the rate of aggregation becomes higher, a more coarse and strong aggregate results.

Finally, at 70 deg C the measured heat flow in the IC experiment was again comparable to the blank. The DSC thermogram did not show any peak, indicating that the IgG was completely denatured within 20 min equilibration time.

Based on the calorimetric experiments, the following denaturation scheme is proposed. At low heating rate and/or incubation at a constant temperature considerably below TI, the concentration of (partially) unfolded IgG molecules is low, so that aggregation of the unfolded IgG molecules will be slow. The result is a relatively open aggregate structure. At higher heating rate and/or incubation around or above Tm the unfolding occurs at a high speed, leading to a high concentration of (partially) unfolded IgG molecules. The rate of aggregation is correspondingly faster, and it could well be that at such high aggregation rates IgG molecules are incorporated in the aggregate before they have had sufficient time for complete unfolding. The decrease in the transition enthalpy with increasing heating rate as shown in Fig. 2 is probably caused by native domains being locked in aggregates.

Effect of solution conditions on the heat-induced denaturation of IgG

DSC thermograms were determined at various pH values (buffered at pH 2.0, 3.5, 6.0, 7.0, and 8.1), without buffer, and as a function of salt concentrations 0.01 M Na^sub 2^HPO^sub 4^ and 0. 1 M (0.01 M Na^sub 2^HPO^sub 4^ + NaCl), at a constant heating rate of 0.5 deg C/min. At pH 6.0, 7.0, and 8.1 the thermograms showed, within experimental error, two peaks at the same temperatures and with comparable enthalpies, as indicated in Table 1. The thermograms were identical to the one shown in Fig. 1. However, at more extreme pH values the appearance of the thermogram was significantly altered. In a glycine buffer, pH 3.5, the thermogram of the IgG contains only the peak characterized by a Tm of 61 deg C and a Delta^subd^H of about 12 J/g (Fig. 5). The second peak at 7PC has disappeared. As discussed above, the denaturation of the Fab and F, fragments is independent. The isolated Fab fragment showed only a transition at 61 deg C. Under these conditions an aggregation peak was observed at 61.5 deg C. After heat treatment in the DSC the IgG aggregated as judged by the presence of large flocs. At pH 2.0 both peaks were no longer present. After heat treatment at this pH the protein solution was still clear, implying that if aggregation has occurred, it has formed small units that are invisible to the naked eye.

From these results it is inferred that pH and heat treatment influence the IgG structure in different ways. Temperature-induced denaturation primarily affects the domains that cause the transition at 61 deg C, whereas the domains corresponding to the second thermal transition are more sensitive to low pH. The exothermic transition at pH 3.5 and the variation in the appearance of aggregates at different heating rates may be explained in terms of differences in the aggregation mechanism following the unfolding of the IgG. This supports the conclusions drawn from the experiments where the heating rate was varied.

Heat-induced changes in the secondary structure

The endothermic enthalpy change observed in a DSC experiment is ascribed to unfolding of a part of the protein molecule. However, DSC does not give detailed information, i.e., on a molecular scale, about the change in protein structure. In a previous paper (Vermeer et al., 1998) it was shown that a combination of DSC and CD spectroscopy results in a better understanding of the processes that are involved in the denaturation of IgG. The most common CD technique is to measure the ellipticity as a function of the wavelength at a given temperature. In addition to these wavelength scans the ellipticity as a function of temperature at a given wavelength may also be measured. With these temperature scans the heating rate can be varied and spectra can be obtained under conditions that are comparable to those of the DSC thermograms.

The CD reference spectra (Chang et al., 1978) show that at -a wavelength of 206.5- mn the intensity due to the beta-sheets is essentially zero, whereas the other structural elements significantly contribute. Thus, by measuring the ellipticity at 206.5 nm as a function of temperature, one monitors the changes in beta-turn, alpha-helix, and random coil contents. At this wavelength, an increase in beta-turns shifts the ellipticity in a positive direction, whereas increased alpha-helix and random coil contents cause a negative shift.

The CD temperature scans of IgG in a 10-mM phosphate buffer, pH 8. 1, at heating rates the same as those in the DSC experiments, are given in Fig. 6. All ellipticity-temperature profiles display two steps at which the intensity strongly decreases, indicating two distinct temperatures where changes in secondary structure occur. These temperatures are summarized in Table 2. The major changes at, for example, a heating rate of 0.5 deg C/min occur at 60 deg C, followed by a second step at about 70 deg C. These temperatures correspond unambiguously with those observed for the structural transitions observed in the DSC experiment (Fig. 2 and Table 1). The decrease in ellipticity at 206.5 nm suggests that the fraction of random coil structures increases with increasing temperature; however, the contributions from a variation in alpha-helix and beta-turn may not be neglected. Thus, by evaluating the far-UV wavelength spectra, changes in the secondary structure involved in heat denaturation can be assessed.

CD spectra of intact and heat-denatured IgG in a 10-mM phosphate buffer, pH 8.1, were examined for both the far-LJV and near-UV regions (Figs. 7 and 8). For reasons of clarity, the results obtained at some temperatures (i.e., 35, 50, 55, 65, and 70 deg C) are represented by smooth curves only. It is observed that at temperatures >55 deg C the smoothed curves match the experimental data very well (60 deg C and 75 deg C). The smoothed curve, representing the data at 20 deg C, however, deviates somewhat from the experimental data in the wavelength range of 200 to 210 nm. Nevertheless, the effect of temperature is evident. The CD spectra of the intact IgG (T+/- 55 deg C) are that of a typical immunoglobulin, with a negative band at 217 nm and a zero intensity at a wavelength of 206 nm, representing a high content of beta-sheet, and several smaller positive and negative bands in the near-UV region between 260 and 300 nin. The ellipticity in the far-UV region is affected only slightly by temperature changes between 20 and 55 deg C, indicating that the IgG secondary structure is stable within this temperature range. At higher temperatures the spectra change gradually with increasing temperature. The minimum at 217 mn broadens and shifts to a lower wavelength, and a shoulder appears at about 208 nm. The wavelength corresponding to zero intensity also shifts to a lower value. The IgG sample measured at 75 deg C was cooled and again a wavelength scan was measured. This scan is also shown in Fig. 7 (open symbols). The irreversibility of the denaturation indicated by the DSC measurements is confirmed by the CD data.

The structural changes reflected in the near-UV region of the CD spectra are usually associated with reorientation of the aromatic amino acids tyrosine and tryptophan, and also from the asymmetric environment of disulphide linkages. It is observed that the negative band at 270 nrn gradually decreases with increasing temperature. This may be explained by an increasing degree of freedom of the side chains of the aromatic amino acids. Around 300 nm (see CD temperature scan shown in inset, Fig. 8) a strong decrease in the ellipticity occurs at a temperature of about 550C. It suggests rupture of disulphide bonds due to the denaturation process.

Comparison of the CD results with the DSC thermograms shown in Fig. I helps to relate the changes in the secondary structure to the overall denaturation of the F^subab^ and F^subc^ fragments. Thus, the differences between the spectra at 55 deg C and 60 deg C are ascribed to the denaturation of the Fab segment, whereas the gradual change between 60 deg C and 75 deg C is caused by denaturation of the F^subc^ fragment.

To estimate the content of the various structural elements above, a described fitting procedure was applied. Table 3 summarizes the trends that were observed with increasing temperature. The structural composition calculated for temperatures=50 deg C) the fraction of alpha-helices and random coils increases, whereas the fraction of beta-sheets and beta-turns decreases.

The alpha-helix induction observed with increasing temperature may be ascribed to peptide units arriving in a nonaqueous environment upon aggregation of the IgG. For dissolved, native proteins it is more favorable for the peptide units at the aqueous periphery to form hydrogen bonds with water molecules than among each other. As hydrophobic interaction is a major force of protein folding (Dill, 1990), it may be expected that upon denaturation part of the hydrophobic interior of the IgG will be exposed to the solution, which, in turn, causes the formation of aggregates. Now, at the interfaces between the building blocks of the aggregate, hydrogen bonds between peptide units in the polypeptide chain may be formed, inducing the formation of alpha-helixes. This explanation is supported by the experimental observation that a-helixes are induced in proteins that adsorb at hydrophobic surfaces (Maste et al., 1996; Zoungrana and Norde, 1997; Vermeer et al., 1998).

Although the IgG is completely denatured at 75 deg C, a significant fraction of ordered secondary structural elements remain, i.e., about 50% of the polypeptide chain is present in beta-sheets, alpha-helix, and beta-turn conformations. This is in contrast to the denaturation of IgG induced by guanidine hydrochloride, where only the random coiled conformation was observed (Buchner et al., 1991; Goto et al., 1988).

pH-induced changes in the secondary structure

It was already concluded from the DSC thermograms that the low-pH-induced denaturation of IgG has a different effect on the domains of the protein as compared to heat– induced denaturation. Upon decreasing the pH, the Fc fragment is primarily affected followed by the denaturation of the Fab fragment at even more extreme conditions. To study the effect of pH on the secondary structure of IgG, CD experiments were performed. Fig. 9 shows the results of IgG in a glycine buffer at pH 3.5, for different temperatures. The spectra are significantly altered relative to those for the native IgG at pH 8. L The secondary structure obtained by the fitting procedure is summarized in Table 4. Even at 20’C a significant fraction of the IgG is denatured, which is reflected by the low beta-sheet content. This observation is supported by Buchner et al. (1991), who concluded in their paper that the formation of a new, well-defined IgG structure occurs at low pH values. The protonation of amino acid side chains was supposed to cause the reorganization of the native state into the so-called A-state. This A-state was found to be relatively compact, which supports our observation that the IgG, after heat treatment at pH 2.0, is not strongly aggregated. This structural rearrangement may also explain the presence of an exothermic aggregation peak in the thermogram at pH 3.5, whereas it was not present in the phosphate buffer. As for the thermal denaturation, the pH– induced structural changes in IgG observed with CD are consistent with the calorimetric data.

CONCLUSIONS

The unfolding of immunoglobulin G is a complex process, in which the denatured state is obtained from the native protein through several, at least partly independent, intermediate states. Moreover, depending on the type of denaturing stress the denaturation process follows different paths: the Fb fragment is most sensitive to heat treatment, whereas the Fc fragment is most sensitive to lowering the pH. At temperatures lower than the temperature of the first peak observed in DSC (and for low heating rates), the concentration of unfolded IgG is low; consequently, so is the rate of aggregation. At those conditions, most IgG molecules will be completely unfolded before they are incorporated in the aggregates. At higher temperatures (high heating rates) the unfolding occurs at a high speed, leading to a high concentration of (partially) unfolded IgG molecules. The rate of aggregation is correspondingly faster and it could well be that at such high aggregation rates IgG molecules are incorporated in the aggregate before they had sufficient time for complete unfolding. Furthermore, the route and method of denaturation affect the structure of the aggregates formed.

This work has been made possible by the support of Bayer AG (Leverkusen, Germany), who provided us with the materials. We thank Dr. H. H. J. de Jongh of the Centre for Protein Technology (Wageningen, The Netherlands) for his assistance with carrying out the CD experiments and the data processing.

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Arnoldus W. P. Vermeer and Willem Norde

Laboratory for Physical Chemistry and Colloid Science, Wageningen Agricultural University, Dreijenplein 6, 6703 HB Wageningen, The Netherlands

Address reprint requests to Dr. Vermeer’s present address: Bayer AG, ZF-FP Biophysik (Building E41), D-51368 Leverkusen, Germany. Tel.: 49-214-3023988; Fax: 49-214-3050698; E-mail: Ronald.Vermeer.RV@ BAYER-AG.DE.

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