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Measurement of protein stability and protein denaturation in cells using differential scanning calorimetry
Methods 35 (2005) 117–125 www.elsevier.com/locate/ymeth
Measurement of protein stability and protein denaturation in cells using differential scanning calorimetry James R. Lepock Department of Medical Biophysics and Ontario Cancer Institute, University of Toronto, 610 University Ave., Toronto, Ont., Canada M5G 2M9 Accepted 20 August 2004 Available online 19 December 2004
Abstract Many methods exist for measuring and studying protein denaturation in vitro. However, measuring protein denaturation in cells under conditions relevant to heat shock presents problems due to cellular complexity and high levels of light scattering that interfere with optical techniques. A general method for measuring protein denaturation in cells using high sensitivity differential scanning calorimetry (DSC) is given. Profiles of specific heat (cp vs. temperature) are obtained providing information about transitions in cellular components including the denaturation of proteins. The specific approaches employed with erythrocytes, bacteria, and mammalian cells are described, and an identification of several features of the DSC profiles is given. Protein denaturation on the level of roughly 7–20% occurs for commonly used heat shocks in mammalian cells. 2004 Elsevier Inc. All rights reserved.
1. Introduction Cells and organisms respond to supra-optimal temperatures, referred to as hyperthermia or heat shock, in several ways. Cytotoxicity results if the heat shock is severe enough [1]. Milder heat shocks produce sublethal damage that can result in sensitization to other stressors. Thermal radiosensitization is of particular interest because of its potential in cancer therapy [2]. Heat shock also results in the induction of heat shock proteins, which correlate well, in general, with thermotolerance [3]. Each of these responses appear related and in large part due to a single initiating event. For nucleated cells, especially mammalian cells, temperatures only 3–5 C above normal growth temperature are needed to induce a response to heat shock. There is considerable evidence that this initiating event, occurring after a small increase in temperature, is the denaturation of thermolabile cellular proteins. Some of the earliest evidence implicating protein dena-
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turation followed from the thermodynamics of cell killing. A high activation energy, usually in the range of 600–800 kJ/mol, is associated with hyperthermic cell killing [1]. In the simplest interpretation, the activation energy is just the temperature dependence of killing. However, the Arrhenius model predicts that the underlying mechanism of killing should have the same activation energy. Most enzymatic processes have activation energies in the range of 20–100 kJ/mol, while molecular transitions have much higher activation energies in the range of 400–800 kJ/mol. In addition, the thermal inactivation of enzymes also requires a high activation energy [4]. These observations are consistent with a temperature-induced transition such as protein denaturation being the initiating event during heat shock. The implication that the high Arrhenius activation energy of killing suggests that protein denaturation is the initial event in thermal damage was recognized independently by two groups as early as 1971 [1,5]. A number of chemical agents, referred to as sensitizers or protectors, respectively, either sensitize cells to heat shock or protect when present during heating. Table 1 is a partial listing of the known cellular sensitizers and
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Table 1 Hyperthermic sensitizers and protectors Sensitizers
Protectors
Alcohols (methanol to octanol) H+ (low pH) Solvents (e.g., DMSO) Sodium arsenite Sulfhydryl reagents (e.g., diamide) Amino acid analogs Local anesthetics Amphotericin B Alkylating agents
Polyhydroxyl alcohols (e.g., glycerol) D2O [Thermotolerance] [Cycloheximide]
protectors. In addition to their effects on cell survival during heat shock, nearly all the sensitizers have been shown to induce heat shock protein (Hsp) synthesis at normal growth temperatures, while the protectors inhibit Hsp synthesis, and reduce the level of thermotolerance when present during heating (for a review see [6]). The agents given in Table 1 have little in common except that most, if not all, of the sensitizers destabilize or perturb protein structure. The protectors glycerol and D2O are protein stabilizers. The other two protective conditions, acquired thermotolerance and the tolerance induced by cycloheximide, are associated with protein stabilization in cells [6]. That numerous chemical agents sensitize cells to heat shock and induce Hsp synthesis and thermotolerance suggests that common initiating signals are responsible for these responses. Similar observations led Hightower to suggest in 1981 that Hsp induction is due to damaged or denatured protein [7]. Since then it has been shown that injection of denatured protein into cells is sufficient to induce Hsp synthesis [8,9]. The correlation between Hsp synthesis and thermotolerance also suggests that protein denaturation is the initiating signal for both and also the initiating event for thermal damage resulting in death.
2. Assays for protein denaturation If protein denaturation is the initial event occurring during heat shock that is ultimately responsible for thermal damage, then it is important to directly measure protein denaturation in cells during heat shock. Protein denaturation refers to a conformational transition resulting in a partial or total unfolding, depending on denaturing conditions, of a protein from its native state to a more disordered conformation. This transition is usually of first-order, and for thermal denaturation usually it results in a molten globule-like structure [10]. Thermal denaturation is accompanied by enzymatic inactivation, but thermal inactivation can occur without the unfolding characteristic of denaturation. Thus, the
most direct and reliable way of detecting protein denaturation is to monitor a parameter that is sensitive to protein conformation. The basic concept is that if a parameter y is sensitive to a conformation so that its value differs for a protein in the native state (yN) compared to the denatured state (yD) then the fraction of denatured protein (fD) is given by fD ¼ ðy y N Þ=ðy D y N Þ; where y is the value of the parameter under particular denaturing conditions. This approach has been extensively used to monitor the denaturation of proteins in vitro using various spectroscopic techniques such as UV/ Vis absorbance, intrinsic (trp and tyr) fluorescence, extrinsic fluorescent probes, IR absorbance, circular dichroism (both near and far UV), Raman spectroscopy, and electron paramagnetic spectroscopy (EPR) using spin labels. For example, protein denaturation during heating in membranes isolated from normal and thermotolerant HeLa cells has been monitored using EPR spectroscopy [11]. It was found that proteins are more stable in membranes isolated from thermotolerant cells, suggesting that protein stabilization may play a role in thermotolerance. The optical spectroscopic techniques listed above are applicable to isolated proteins and membranes but are not suitable for studying protein denaturation in intact cells due to light scattering and the complexity of cellular systems. Two other techniques have been applied to cells: differential scanning calorimetry (DSC) and protein insolubilization. The principle underlying protein insolubilization is that irreversible protein denaturation nearly always results in aggregation. These aggregates are resistant to solubilization by mild, non-ionic detergents. Thus, a measure of protein denaturation is the difference between the amounts of detergent-insoluble protein in control compared to heated cells. The detergents most commonly used for this assay are Triton X-100 and NP-40. For unheated cells, these detergents extract all cytoplasmic proteins and the more soluble fraction of nuclear proteins [12]. Protein aggregation in the nucleus results in a reduction in protein extraction and an increase in the protein associated with the nucleus. This approach was first used to detect protein aggregation in nuclei of HeLa cells, measured as an increase in the nuclear protein/DNA ratio of isolated nuclei following heat shock [13]. This increase in insoluble protein is due to heat-induced protein aggregation resulting in a decrease in the amount of soluble nuclear protein extracted by the detergent treatment used during isolation of nuclei [12]. Several enzymes involved in DNA replication and repair and several nuclear oncoproteins and proto-oncoproteins have reduced extractability following heat shock [14]. The thermal inactivation of a number of reporter enzymes in mouse and Drosophila cells
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has been shown to be due to the insolubilization of these proteins [15]. Protein aggregation and insolubilization occurs not only in the nucleus. Detergent extraction using the plasma membrane permeabilizer digitonin has shown that aggregation also occurs in the cytoplasm, although cytoplasmic aggregates are more easily dispersed than nuclear aggregates [12]. Protein aggregation influences the response of a cell to heat shock since the state of thermotolerance is associated with either a reduced level of protein insolubilization, indicative of reduced thermal damage, or an increased rate of resolubilization during recovery, indicative of faster repair [16]. Protein insolubilization is only an indirect measure of protein denaturation as defined above. The problem is that the insoluble aggregates consist of both denatured and native protein. For example, the majority of the aggregated, insoluble protein in nuclei isolated from L929 and CHL V79 cells following heat shock is native [12,17]. Denatured protein in the cell appears to act as a nucleation site for further aggregation of native, non-denatured protein [6,17]. Thus, while protein insolubilization is indicative of protein denaturation, the amount of insoluble protein is not an accurate measure of the amount of protein actually denatured.
3. Protein denaturation by differential scanning calorimetry Differential scanning calorimetry (DSC) is a powerful technique for studying the thermodynamics of transitions in biological macromolecules [18]. The specific heat (cp) as a function of temperature can be determined for a protein solution or more complex biological structure. Differential scanning calorimeters ordinarily consist of two cells: one containing sample and the other the reference solvent. The heat flow into the cells is monitored as the temperature is increased at a constant rate. The heat absorbed by the sample due to temperature increase and any conformational transitions is monitored. From the differential heat flow, the specific heat can be determined. Fig. 1A shows the plot of cp vs. temperature for citrate synthase, which undergoes a reversible unfolding near 52 C represented by an endothermic transition. There is an intrinsic curvature in the baseline due to instrumental effects which can be corrected by subtracting the rescan. The intrinsic curvature is not visible in the rescan of Fig. 1A because of the limited temperature range shown (20 C), but it is visible in the rescans in Fig. 3A and 4B which cover a range of 100 C. Below the denaturation transition, the specific heat of the native state increases with temperature while that of the denatured state is usually independent of temperature (not enough of the scan is shown clearly demonstrate this). There is an increase in the cp upon denaturation (Dcp), which is shown by the higher value of cp of the scan above
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Fig. 1. DSC analysis of the denaturation of citrate synthase. (A) Uncorrected DSC scan of cp vs. T (solid line) and the rescan obtained after scanning to 100 C at a scan rate of 1 C/min (broken line). (B) Scan corrected for intrinsic baseline curvature by subtraction of rescan (solid line) and the generated baseline used to correct for Dcp (broken line). (C) Corrected plot of cp(excess) vs. T. (D) Plot of fractional denaturation (fD vs. T) obtained by integration of scan shown in (C).
60 C compared to the rescan value. This must be corrected for to determine the excess cp due to the transition itself. A baseline is generated under the assumption that Dcp at any point through the transition is given by the difference between cp for the denatured and native forms of the protein. The method of Hemminger and Hohne [19] was used to generate the baseline shown in Fig. 1B. Subtraction of the baseline gives the plot for cp(excess) due to the transition itself (Fig. 1C). The fractional denaturation (fD) upon heating to any temperature is given by the fractional area under the cp vs. T curve to that temperature (Fig. 1D). The transition temperature (Tm) is defined as the temperature at which one-half of the protein has denatured, which is approximately 52 C for citrate synthase. The fractional denaturation on heating to any temperature can be determined from fD vs. T curves such as that shown in Fig. 1D. For reversible denaturation, the thermodynamic parameters for protein unfolding such as the transition temperature Tm, DHVH (vanÕt Hoff enthalpy), DHcal (calorimetric enthalpy), and DS (entropy) can be determined from curves such as those shown in Figs. 1B and C. The simplest case of reversible denaturation follows the two-state model: N $ U; where N and U represent the native and unfolded denatured states, respectively. The presence of multiple domains in a protein leads to a more complex
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denaturation profile and is indicated by the presence of multiple transitions, often superimposed. However, very few proteins denature reversibly, especially in the extremely crowded interior of the cell. Irreversible denaturation alters the shape of the cp vs. temperature profile such that it is ordinarily impossible to extract the reversible thermodynamic parameters. Irreversible denaturation of a protein can be modeled by the three-state transition N $ U ! D; where D represents an irreversibly unfolded state due to a second, irreversible step following the initial reversible unfolding [20]. For proteins denaturing at lower temperatures (e.g., below 60 C) and neutral pH, irreversibility is usually due to aggregation [21]. The three-state model can be approximated by N ! D: This is the simplest model for an irreversible process and is identical to the model assumed for cell killing during heating (i.e., Cell(live) fi Cell(dead)) that was used to calculate the Arrhenius activation energy of cell killing [1]. The critical parameter in this model is the apparent rate constant for the transition N to D. The temperature dependence of this rate constant is the Arrhenius activation energy. The activation energy of denaturation can be determined from curves of cp vs. T for irreversible denaturation [6]. This allows a direct comparison between the thermodynamics of cell killing due to heat shock and protein denaturation as determined by DSC [22]. In addition to protein denaturation being largely irreversible in cells, the denaturation profile of whole cells would be expected to consist of multiple transitions since cells contain large numbers of proteins. This should lead to a very complicated profile of cp vs. T. In the simplest case, one would expect the profile to be the sum of the transitions of all major proteins in the cell, possibly altered by protein–protein interactions occurring in supramolecular structures and organelles. Expected DSC profiles for cells were modeled by summing various numbers of independent transitions with the Tm, peak amplitude (DHcal), and peak width (activation energy) of each transition chosen randomly as described in the legend for Fig. 2. The overall profile and individual transitions are shown in Fig. 2 for a DSC scan consisting of 20 independent transitions. These 20 transitions yield a profile consisting of three resolvable peaks, which is roughly similar to what is observed for the cell scans shown in Figs. 3–7. For a very large number of independent transitions (e.g., 1000), the transitions overlap to such an extent that a smooth profile results (Fig. 2). This is very different than what is observed for cells and suggests there are a limited number of independent denaturation transitions in complex cellular systems.
Fig. 2. Simulation of a multi-component DSC profile (cp vs. T) of a cellular system consisting of multiple protein denaturation transitions. Twenty transitions of random amplitude (DHcal) with Tm selected from a Poisson distribution with an average Tm of 55 C and a minimum of 40 C (small peaks). The sum of these 20 transitions (solid line) and the sum of 1000 transitions (broken line) are shown.
Fig. 3. Plot of cp vs. T of human erythrocytes. (A) The original scan (solid line) and the rescan (broken line) after scanning to 100 C at 1 C/min are shown. (B) The corrected scan of cp(excess) vs. T after correction for the baseline curvature and Dcp and the theoretical fit to the Hb curve assuming two-state irreversible denaturation (broken line) are shown.
4. Differential scanning calorimetry profiles of cells Cells are a complex, interacting mixture of proteins, nucleic acids, and membrane lipids, each of which can undergo order–disorder, endothermic transitions
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Fig. 6. DSC scans of HeLa cells. (A) Original scan (solid line) obtained at a scan rate of 1 C/min with a fitted baseline (broken line). (B) Profile of cp(excess) vs. T obtained by subtracting the generated baseline from the original scan.
Fig. 4. Plot of cp vs. T of Bacillus stearothermophilus (ATCC 12016). (A) The original scan (a), the rescan after scanning to 100 C at a scan rate of 1 C/min (b), and a water–water baseline (c) are shown. (B) The scans corrected for baseline curvature for the original scan (a) and the rescan (see text for details) are shown. The features marked with the arrows and labeled A–D, L, and Tl are discussed in the text.
Fig. 7. DSC scans of Chinese hamster lung V79 cells. Shown are the profiles of cp(excess) vs. T obtained at a scan rate of 1 C/min for control cells (solid line) and cells exposed to 5% ethanol (dotted line) or 5% glycerol (dashed line).
Fig. 5. DSC scans of four species of Bacillus. Values of Tg, Tmax, and Tl are given in Table 2.
detectable by DSC, and numerous small molecules and ions. A DSC scan should be the sum of the transitions of all components. In addition, other processes such as aggregation and metabolism, both exothermic events, can be detected by calorimetry and must be considered in interpreting a DSC profile. The human erythrocyte
is a fairly simple cell which lacks a nucleus and consists primarily of hemoglobin. In addition, it has a low level of metabolism, and thus metabolic heat released during a DSC scan should not be a problem. These characteristics should lead to a less complex, more easily interpretable, DSC profile [23]. 4.1. Erythrocytes 4.1.1. Methodology Human erythrocytes were purified from freshly drawn blood by washing three times in a 10-fold excess solution of phosphate-buffered saline (PBS). The buffy
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coat and top of the pellet were removed after each centrifugation by aspiration to remove all white blood cells. Erythrocytes were used on the day of isolation. DSC scans were obtained with an adiabatic MicroCal-2 (MicroCal, LLC) scanning calorimeter (1.21 ml sample cells). The procedure used for higher sensitivity calorimeters such as the MicroCal VP-DSC or Nano DSC (Calorimetry Sciences) is identical. High quality scans of cells cannot be obtained using faster-scanning, non-adiabatic calorimeters. The sample and reference cells of the DSC were cooled to 0 C, and the degassed erythrocyte suspension in PBS (4–6 mg/ml protein) and the reference solution (PBS) were added. Degassing is necessary since bubble formation due to reduced air solubility at higher temperatures interferes with the scan. It is accomplished by exposing the cell suspension to a mild vacuum while gently stirring. The scan was started (1 C/min scan rate) when the sample and reference cells equilibrated at 0 C (approximately 40 min after addition of erythrocytes). The cells should settle to the bottom of the calorimeter cell during the equilibration period, but settling prior to scanning does not alter the shape of the profile. The scan was run to 100–102 C, the cells cooled to 0 C, and a second scan (the rescan) obtained which was used to correct for the non-linear, baseline curvature as previously described [20]. Fig. 3A shows the DSC scan and the rescan of human erythrocytes obtained using the procedure described above. The first step is to correct for intrinsic baseline curvature by subtracting the rescan. The second step is to correct for Dcp as was done for the DSC scan of citrate synthase as shown in Fig. 1. The resulting corrected scan of cp(excess) is given in Fig. 3B. This corrected scan is more appropriate for analysis. There are four resolvable endothermic peaks, labeled A, B, C, and Hb, each of which represents a single strong transition or a group of transitions with similar TmÕs. The evidence indicates that this profile is due almost exclusively to protein denaturation. The large endothermic peak with Tm = 72 C labeled Hb and the exotherm at 78 C are due to the denaturation of hemoglobin and its subsequent aggregation, respectively. This peak was identified by measuring the Tm of isolated hemoglobin, which was within 2 C of peak Hb in cells. In addition, hemoglobin is the major protein component of erythrocytes and this is by far the strongest transition. The hemoglobin peak can be theoretically fit using a two-state, irreversible model (Fig. 3B). Aggregation is exothermic, and the position of the exotherm is consistent with it being due to the aggregation of denatured hemoglobin. The first deviation of cp from the baseline occurs between 40 and 45 C, indicating that significant protein denaturation detectable by DSC first occurs in this temperature range. The peaks labeled A, B, and C were identified by comparing their TmÕs to those of membrane
components of isolated ghosts. The transitions in isolated erythrocyte membrane ghosts have been identified by a combination of DSC and a technique referred to as thermal gel analysis in which the loss of specific protein bands on a gel due to thermally induced aggregation are correlated with Tm on a DSC scan [24]. Peak A represents the denaturation of spectrin, peak B the denaturation of several other components of the membrane skeleton, and peak C the denaturation of the transmembrane portion of the band 3 protein. Thus, the membrane skeleton, including spectrin, is the most thermolabile major component of the erythrocyte. The erythrocyte is a particularly simple cellular system, but it illustrates the approach for using DSC to measure the stability of proteins in cells. Specific peaks are observed and correspond to the denaturation of individual or groups of proteins. Identification of individual peaks, if it can be done, allows a determination of which proteins are denatured at heat shockÔ temperatures. 4.2. Bacterial cells Different bacterial strains have widely different maximum growth temperatures (Tmax), defined as the maximum temperature at which continuous growth occurs [25]. DSC can be used to determine if Tmax is limited by protein denaturation at high temperatures. This can be tested using bacterial strains with a wide range of Tmax. Two thermophiles Bacillus stearothermophilus (ATCC 12016 and WAT), a mesophile Bacillus megaterium, and a psychrotroph Bacillus psychrophilus were used [25]. 4.2.1. Methodology Cells were grown in trypticase soy broth at their respective growth temperatures (Tg, see Table 2). Bacterial cells were harvested when the A650 of the culture medium reached 1.0. The procedure was to cool the cultures quickly to 4 C and employ centrifugation and two washes in phosphate buffer (10 mM, pH 7.0) containing chloramphenicol to block further protein synthesis during harvesting. The final bacterial suspension and reference solution were degassed under mild vacuum at 4 C for 5 min immediately before addition to the calorimeter Table 2 Maximum growth temperatures (Tmax) vs. onset of denaturation (Tl) for various species of Bacillus
Bacillus psychrophilus Bacillus megaterium Bacillus stearothermophilus (WAT) Bacillus stearothermophilus (ATCC 12016)
Tg (C)
Tmax (C)
Tl (C)
20 37 56
32.5 48 56
30 46 55
56
69
65
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cells. The calorimetry procedure was identical to that described above for erythrocytes. Shown in Fig. 4A is the original scan of B. stearothermophilus (ATCC), a rescan, and a water–water baseline illustrating the intrinsic curvature of the calorimeter. The scan has a series of endothermic peaks from 65 to 100 C and an obvious endotherm near 15–20 C. The rescan, which consists of all reversible transitions, contains two endothermic transitions at approximately 30 and 93 C. Both scan and rescan were corrected for intrinsic curvature, which is partially visible in the water–water baseline (curve c). Before the rescan could be subtracted from the scan, it was necessary to remove the two reversible peaks. This was done by removing the two peaks from the rescan by numerical methods and fitting the remaining curve with a fourth-order polynomial to capture the intrinsic curvature. The fourth-order fit was then subtracted from the scan and rescan to correct for intrinsic baseline curvature. The corrected curves are shown in Fig. 4B. The specific features are more clearly illustrated in these curves. There are four endothermic transitions above 65 C, three of which (labeled A–C) are irreversible and apparently due to protein denaturation. The onset temperature for denaturation is defined as the temperature at which these endothermic peaks begin (labeled Tl). The fourth (peak D) is reversible, as can be seen in the rescan, and has a Tm in the range expected for DNA. A well-defined endotherm (labeled L) in the rescan occurs near the temperature of the low temperature exotherm (15– 20 C) in the scan. These presumably are related, although the transition in cells is more complex in shape, and appear to be membrane lipid transitions. One last feature is a broad exotherm from 50 to 65 C with a minimum at 60 C marked with an arrow. This is due to the metabolic heat released in this temperature range. This metabolic exotherm makes an accurate determination of Tl difficult. Fig. 4B illustrates the main thermic transitions and events detectable by DSC in cp vs. temperature scans of cells. The shape of the profile is influenced by metabolism, lipid transitions, and DNA transitions in addition to protein denaturation. Corrected DSC scans for psychrotroph B. psychrophilus, mesophile B. megaterium, and other thermophile B. stearothermophilus (WAT) in addition to B. stearothermophilus (ATCC) are shown in Fig. 5. The ranges over which protein denaturation can be detected for each species differs considerably. The onset temperatures for denaturation Tl and the maximum growth temperature Tmax for each species are given in Table 2. There is a very good correlation between the onset of denaturation and the maximum growth temperature for these bacteria from 30 to 70 C. This illustrates how DSC determinations of protein denaturation can be correlated with growth.
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4.3. Mammalian cells Dividing eukaryotic cells are the most commonly used category of cells in studies of heat shock, and protein denaturation can be measured in them using DSC as described above for erythrocytes and bacteria. DSC profiles with common features have been obtained from mammalian Chinese Hamster Lung V79, CHO, 3T3, HeLa, A549, and L929 cells as described below. 4.3.1. Methodology Cells can be grown in suspension or attached. Cells grown in suspension require less manipulation, since they do not need to be removed from culture plates, than cells grown attached. For example, Chinese Hamster Lung V79 and HeLa cells, adapted to suspension culture, can be grown in suspension in minimal essential medium (MEM), with no added calcium, containing 10% fetal calf serum, 26 mM Hepes, and 10 mM sodium bicarbonate at pH 7.4 [26]. Cells are harvested, after reaching a density of 5 · 105 per ml, by mild centrifugation before resuspension in Hepes-buffered saline. DSC scans and rescans are obtained as described above for erythrocytes and bacteria. Depending on the size of the cell and the sensitivity of the calorimeter, suspensions of 1–10 · 107 cells/ml are required for high quality, noise-free scans. After degassing, the viability is checked using erythrosine B. The effect of ethanol and glycerol on the DSC profile was determined by adding these compounds after degassing. In general, most additional compounds can be added to the Hepes-buffered saline before resuspension and degassing of cells. However, volatile solvents should be added after degassing to ensure that vacuum treatment does not alter concentration. Shown in Fig. 6 is the original scan of HeLa cells at a concentration of 3 · 107 cells/ml in the calorimeter. The original scan, as for erythrocytes and bacteria, shows a large Dcp after going through the endothermic transitions from 40 to 100 C. This can be corrected as described above, and shown in Fig. 6A are the original scan and the generated baseline used to correct for Dcp. The corrected curve is shown in Fig. 6B. Scans of all tissue culture cells, both rodent and human, with a relatively high nuclear/cytoplasmic volume ratio are similar to this scan. There are five main peaks labeled A–E with transition temperatures of 50–55, 62– 64, 70–75, 85, and 96–98 C, respectively. Peaks C, D, and E are primarily nuclear transitions [6]. There is evidence that peaks D and E are due to the melting of relaxed and supercoiled DNA, respectively, and that peak C represents the denaturation of histones [27]. Peaks A and B are due primarily to the denaturation of cytoplasmic proteins, with a contribution from thermolabile nuclear proteins [6]. The proteins denaturing in the temperature region of 40–50 C should be the
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ones damaged during heat shock. The identity of the proteins denaturing at this temperature is largely unknown. DSC profiles are altered by conditions that also alter cellular sensitivity to heat shock. Shown in Fig. 7 are DSC scans of CHL V79 cells and cells in the presence of ethanol, which sensitizes to heat shock and is an Hsp inducer, and glycerol, which protects from heat shock and inhibits Hsp induction. Of particular interest is the low-temperature region of the profile near 40 C. It can be seen that ethanol shifts this region to lower temperatures, indicating that it lowers the Tm for these proteins, while glycerol shifts the region to higher temperatures, indicating it raises the Tm of these proteins. This is consistent with the effects of these compounds on cellular sensitivity to heat shock. 4.4. Quantification of protein denaturation The fractional protein denaturation on scanning to a given temperature is given by the area under the DSC profile of cp(excess) vs. temperature as described above for the protein citrate synthase (Fig. 1). The DSC profiles for V79 cells shown in Fig. 7 can be used to estimate the amount of denaturation for specific temperature exposures. Plots of fraction denaturation as a functional of temperature are given in Fig. 8. These plots were generated by first removing peaks D and E, which appear to be due to transitions in DNA not protein [27]. The resulting curves are integrated and normalized to yield fractional denaturation (fD) (Fig. 8). An important assumption made in this analysis is that the specific DHcal for each protein is the same. The value of DHcal for individual proteins decreases as the Tm decreases as a consequence of positive Dcp [6]. Thus, the curves probably somewhat underestimate the fractional denaturation occurring at lower temperatures. When survival of V79 cells is measured under the same heating conditions as for DSC (i.e., heating rate of 1 C/min), killing is first detectable after reaching
Fig. 8. Plots of estimated fractional protein denaturation (fD vs. T) for V79 cells (solid line) and for the same cells exposed to 5% ethanol (dotted line) or 5% glycerol (dashed line).
45 C with 100% killing occurring at about 49 C (results not shown). At 45 C and 49 C, fD is approximately 0.07 and 0.2. Thus, to a rough approximation killing occurs over the range of 7–20% denaturation. Ethanol increases the amount of protein denaturation while glycerol decreases it. For example, on scanning to 45 C, ethanol (5%) increases denaturation by approximately 3-fold while glycerol (5%) reduces it by 2-fold.
5. Summary DSC is a technique that is sensitive to protein denaturation in intact cells including erythrocytes, bacteria, yeast [28], mammalian cells, and tissue [29]. Irreversible endothermic transitions are detectable in the relevant heat shock range for the above cells. These endothermic transitions can be identified as protein denaturation based on irreversibility, the values of DHcal obtained, the expected large contribution of protein denaturation due to the cellular content of proteins, the lack of strong lipid transitions in the heat shock range, the relatively low enthalpy associated with changes in protein interactions (i.e., polymerization and depolymerization), and the association of transitions in erythrocytes with the denaturation of specific proteins [6]. The onset temperature of protein denaturation correlates well with the inhibition of growth and the onset of killing. The amount of protein denaturation occurring as a function of heating can be measured for specific heat treatments. A rough estimate for CHL V79 cells is that killing occurs in the range of 7–20% total denaturation.
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J.R. Lepock / Methods 35 (2005) 117–125 [16] H.H. Kampinga, J. Cell Sci. 104 (1993) 11–17. [17] J.R. Lepock, H.E. Frey, M.L. Heynen, G.A. Sennisterra, R.L. Warters, Cell Stress Chaperones 6 (2001) 136–147. [18] J.M. Sturtevant, Ann. Rev. Phys. Chem. 38 (1987) 463–488. [19] W. Hemminger, G. Hohne, translated by: Y. Goldman, Calorimetry-fundamentals and Practice, Verlag Chemie GmbH, Weinheim, (1984). [20] J.R. Lepock, K.P. Ritchie, M.C. Kolios, A.M. Rodahl, K.A. Heinz, J. Kruuv, Biochemistry 31 (1992) 12706–12712. [21] S.E. Zale, A.M. Klibanov, Biochemistry 25 (1986) 5432– 5444. [22] J.R. Lepock, H.E. Frey, A.M. Rodahl, J. Kruuv, J. Cell Physiol. 137 (1988) 14–24.
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[23] J.R. Lepock, H.E. Frey, H. Bayne, J. Markus, Biochim. Biophys. Acta 980 (1989) 191–201. [24] J.F. Brandts, L. Erickson, K. Lysko, A.T. Schwartz, R.D. Taverna, Biochemistry 16 (1977) 3450–3454. [25] J.R. Lepock, H.E. Frey, W.E. Inniss, Biochim. Biophys. Acta 1055 (1990) 19–26. [26] J.R. Lepock, H.E. Frey, M.P. Heynen, J. Nishio, B. Waters, K.P. Ritchie, J. Kruuv, J. Cell. Physiol. 142 (1990) 628–634. [27] N.A. Touchette, R.D. Cole, Biochemistry 31 (1992) 1842–1849. [28] K. Obuchi, H. Iwahashi, J.R. Lepock, Y. Komatsu, Yeast 16 (2000) 111–119. [29] K.P. Ritchie, B.M. Keller, K.M. Syed, J.R. Lepock, Int. J. Hyperthermia 10 (1993) 605–618.
Measurement of protein stability and protein denaturation in cells using differential scanning calorimetry James R. Lepock Department of Medical Biophysics and Ontario Cancer Institute, University of Toronto, 610 University Ave., Toronto, Ont., Canada M5G 2M9 Accepted 20 August 2004 Available online 19 December 2004
Abstract Many methods exist for measuring and studying protein denaturation in vitro. However, measuring protein denaturation in cells under conditions relevant to heat shock presents problems due to cellular complexity and high levels of light scattering that interfere with optical techniques. A general method for measuring protein denaturation in cells using high sensitivity differential scanning calorimetry (DSC) is given. Profiles of specific heat (cp vs. temperature) are obtained providing information about transitions in cellular components including the denaturation of proteins. The specific approaches employed with erythrocytes, bacteria, and mammalian cells are described, and an identification of several features of the DSC profiles is given. Protein denaturation on the level of roughly 7–20% occurs for commonly used heat shocks in mammalian cells. 2004 Elsevier Inc. All rights reserved.
1. Introduction Cells and organisms respond to supra-optimal temperatures, referred to as hyperthermia or heat shock, in several ways. Cytotoxicity results if the heat shock is severe enough [1]. Milder heat shocks produce sublethal damage that can result in sensitization to other stressors. Thermal radiosensitization is of particular interest because of its potential in cancer therapy [2]. Heat shock also results in the induction of heat shock proteins, which correlate well, in general, with thermotolerance [3]. Each of these responses appear related and in large part due to a single initiating event. For nucleated cells, especially mammalian cells, temperatures only 3–5 C above normal growth temperature are needed to induce a response to heat shock. There is considerable evidence that this initiating event, occurring after a small increase in temperature, is the denaturation of thermolabile cellular proteins. Some of the earliest evidence implicating protein dena-
E-mail address: [email protected]. 1046-2023/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2004.08.002
turation followed from the thermodynamics of cell killing. A high activation energy, usually in the range of 600–800 kJ/mol, is associated with hyperthermic cell killing [1]. In the simplest interpretation, the activation energy is just the temperature dependence of killing. However, the Arrhenius model predicts that the underlying mechanism of killing should have the same activation energy. Most enzymatic processes have activation energies in the range of 20–100 kJ/mol, while molecular transitions have much higher activation energies in the range of 400–800 kJ/mol. In addition, the thermal inactivation of enzymes also requires a high activation energy [4]. These observations are consistent with a temperature-induced transition such as protein denaturation being the initiating event during heat shock. The implication that the high Arrhenius activation energy of killing suggests that protein denaturation is the initial event in thermal damage was recognized independently by two groups as early as 1971 [1,5]. A number of chemical agents, referred to as sensitizers or protectors, respectively, either sensitize cells to heat shock or protect when present during heating. Table 1 is a partial listing of the known cellular sensitizers and
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Table 1 Hyperthermic sensitizers and protectors Sensitizers
Protectors
Alcohols (methanol to octanol) H+ (low pH) Solvents (e.g., DMSO) Sodium arsenite Sulfhydryl reagents (e.g., diamide) Amino acid analogs Local anesthetics Amphotericin B Alkylating agents
Polyhydroxyl alcohols (e.g., glycerol) D2O [Thermotolerance] [Cycloheximide]
protectors. In addition to their effects on cell survival during heat shock, nearly all the sensitizers have been shown to induce heat shock protein (Hsp) synthesis at normal growth temperatures, while the protectors inhibit Hsp synthesis, and reduce the level of thermotolerance when present during heating (for a review see [6]). The agents given in Table 1 have little in common except that most, if not all, of the sensitizers destabilize or perturb protein structure. The protectors glycerol and D2O are protein stabilizers. The other two protective conditions, acquired thermotolerance and the tolerance induced by cycloheximide, are associated with protein stabilization in cells [6]. That numerous chemical agents sensitize cells to heat shock and induce Hsp synthesis and thermotolerance suggests that common initiating signals are responsible for these responses. Similar observations led Hightower to suggest in 1981 that Hsp induction is due to damaged or denatured protein [7]. Since then it has been shown that injection of denatured protein into cells is sufficient to induce Hsp synthesis [8,9]. The correlation between Hsp synthesis and thermotolerance also suggests that protein denaturation is the initiating signal for both and also the initiating event for thermal damage resulting in death.
2. Assays for protein denaturation If protein denaturation is the initial event occurring during heat shock that is ultimately responsible for thermal damage, then it is important to directly measure protein denaturation in cells during heat shock. Protein denaturation refers to a conformational transition resulting in a partial or total unfolding, depending on denaturing conditions, of a protein from its native state to a more disordered conformation. This transition is usually of first-order, and for thermal denaturation usually it results in a molten globule-like structure [10]. Thermal denaturation is accompanied by enzymatic inactivation, but thermal inactivation can occur without the unfolding characteristic of denaturation. Thus, the
most direct and reliable way of detecting protein denaturation is to monitor a parameter that is sensitive to protein conformation. The basic concept is that if a parameter y is sensitive to a conformation so that its value differs for a protein in the native state (yN) compared to the denatured state (yD) then the fraction of denatured protein (fD) is given by fD ¼ ðy y N Þ=ðy D y N Þ; where y is the value of the parameter under particular denaturing conditions. This approach has been extensively used to monitor the denaturation of proteins in vitro using various spectroscopic techniques such as UV/ Vis absorbance, intrinsic (trp and tyr) fluorescence, extrinsic fluorescent probes, IR absorbance, circular dichroism (both near and far UV), Raman spectroscopy, and electron paramagnetic spectroscopy (EPR) using spin labels. For example, protein denaturation during heating in membranes isolated from normal and thermotolerant HeLa cells has been monitored using EPR spectroscopy [11]. It was found that proteins are more stable in membranes isolated from thermotolerant cells, suggesting that protein stabilization may play a role in thermotolerance. The optical spectroscopic techniques listed above are applicable to isolated proteins and membranes but are not suitable for studying protein denaturation in intact cells due to light scattering and the complexity of cellular systems. Two other techniques have been applied to cells: differential scanning calorimetry (DSC) and protein insolubilization. The principle underlying protein insolubilization is that irreversible protein denaturation nearly always results in aggregation. These aggregates are resistant to solubilization by mild, non-ionic detergents. Thus, a measure of protein denaturation is the difference between the amounts of detergent-insoluble protein in control compared to heated cells. The detergents most commonly used for this assay are Triton X-100 and NP-40. For unheated cells, these detergents extract all cytoplasmic proteins and the more soluble fraction of nuclear proteins [12]. Protein aggregation in the nucleus results in a reduction in protein extraction and an increase in the protein associated with the nucleus. This approach was first used to detect protein aggregation in nuclei of HeLa cells, measured as an increase in the nuclear protein/DNA ratio of isolated nuclei following heat shock [13]. This increase in insoluble protein is due to heat-induced protein aggregation resulting in a decrease in the amount of soluble nuclear protein extracted by the detergent treatment used during isolation of nuclei [12]. Several enzymes involved in DNA replication and repair and several nuclear oncoproteins and proto-oncoproteins have reduced extractability following heat shock [14]. The thermal inactivation of a number of reporter enzymes in mouse and Drosophila cells
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has been shown to be due to the insolubilization of these proteins [15]. Protein aggregation and insolubilization occurs not only in the nucleus. Detergent extraction using the plasma membrane permeabilizer digitonin has shown that aggregation also occurs in the cytoplasm, although cytoplasmic aggregates are more easily dispersed than nuclear aggregates [12]. Protein aggregation influences the response of a cell to heat shock since the state of thermotolerance is associated with either a reduced level of protein insolubilization, indicative of reduced thermal damage, or an increased rate of resolubilization during recovery, indicative of faster repair [16]. Protein insolubilization is only an indirect measure of protein denaturation as defined above. The problem is that the insoluble aggregates consist of both denatured and native protein. For example, the majority of the aggregated, insoluble protein in nuclei isolated from L929 and CHL V79 cells following heat shock is native [12,17]. Denatured protein in the cell appears to act as a nucleation site for further aggregation of native, non-denatured protein [6,17]. Thus, while protein insolubilization is indicative of protein denaturation, the amount of insoluble protein is not an accurate measure of the amount of protein actually denatured.
3. Protein denaturation by differential scanning calorimetry Differential scanning calorimetry (DSC) is a powerful technique for studying the thermodynamics of transitions in biological macromolecules [18]. The specific heat (cp) as a function of temperature can be determined for a protein solution or more complex biological structure. Differential scanning calorimeters ordinarily consist of two cells: one containing sample and the other the reference solvent. The heat flow into the cells is monitored as the temperature is increased at a constant rate. The heat absorbed by the sample due to temperature increase and any conformational transitions is monitored. From the differential heat flow, the specific heat can be determined. Fig. 1A shows the plot of cp vs. temperature for citrate synthase, which undergoes a reversible unfolding near 52 C represented by an endothermic transition. There is an intrinsic curvature in the baseline due to instrumental effects which can be corrected by subtracting the rescan. The intrinsic curvature is not visible in the rescan of Fig. 1A because of the limited temperature range shown (20 C), but it is visible in the rescans in Fig. 3A and 4B which cover a range of 100 C. Below the denaturation transition, the specific heat of the native state increases with temperature while that of the denatured state is usually independent of temperature (not enough of the scan is shown clearly demonstrate this). There is an increase in the cp upon denaturation (Dcp), which is shown by the higher value of cp of the scan above
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Fig. 1. DSC analysis of the denaturation of citrate synthase. (A) Uncorrected DSC scan of cp vs. T (solid line) and the rescan obtained after scanning to 100 C at a scan rate of 1 C/min (broken line). (B) Scan corrected for intrinsic baseline curvature by subtraction of rescan (solid line) and the generated baseline used to correct for Dcp (broken line). (C) Corrected plot of cp(excess) vs. T. (D) Plot of fractional denaturation (fD vs. T) obtained by integration of scan shown in (C).
60 C compared to the rescan value. This must be corrected for to determine the excess cp due to the transition itself. A baseline is generated under the assumption that Dcp at any point through the transition is given by the difference between cp for the denatured and native forms of the protein. The method of Hemminger and Hohne [19] was used to generate the baseline shown in Fig. 1B. Subtraction of the baseline gives the plot for cp(excess) due to the transition itself (Fig. 1C). The fractional denaturation (fD) upon heating to any temperature is given by the fractional area under the cp vs. T curve to that temperature (Fig. 1D). The transition temperature (Tm) is defined as the temperature at which one-half of the protein has denatured, which is approximately 52 C for citrate synthase. The fractional denaturation on heating to any temperature can be determined from fD vs. T curves such as that shown in Fig. 1D. For reversible denaturation, the thermodynamic parameters for protein unfolding such as the transition temperature Tm, DHVH (vanÕt Hoff enthalpy), DHcal (calorimetric enthalpy), and DS (entropy) can be determined from curves such as those shown in Figs. 1B and C. The simplest case of reversible denaturation follows the two-state model: N $ U; where N and U represent the native and unfolded denatured states, respectively. The presence of multiple domains in a protein leads to a more complex
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denaturation profile and is indicated by the presence of multiple transitions, often superimposed. However, very few proteins denature reversibly, especially in the extremely crowded interior of the cell. Irreversible denaturation alters the shape of the cp vs. temperature profile such that it is ordinarily impossible to extract the reversible thermodynamic parameters. Irreversible denaturation of a protein can be modeled by the three-state transition N $ U ! D; where D represents an irreversibly unfolded state due to a second, irreversible step following the initial reversible unfolding [20]. For proteins denaturing at lower temperatures (e.g., below 60 C) and neutral pH, irreversibility is usually due to aggregation [21]. The three-state model can be approximated by N ! D: This is the simplest model for an irreversible process and is identical to the model assumed for cell killing during heating (i.e., Cell(live) fi Cell(dead)) that was used to calculate the Arrhenius activation energy of cell killing [1]. The critical parameter in this model is the apparent rate constant for the transition N to D. The temperature dependence of this rate constant is the Arrhenius activation energy. The activation energy of denaturation can be determined from curves of cp vs. T for irreversible denaturation [6]. This allows a direct comparison between the thermodynamics of cell killing due to heat shock and protein denaturation as determined by DSC [22]. In addition to protein denaturation being largely irreversible in cells, the denaturation profile of whole cells would be expected to consist of multiple transitions since cells contain large numbers of proteins. This should lead to a very complicated profile of cp vs. T. In the simplest case, one would expect the profile to be the sum of the transitions of all major proteins in the cell, possibly altered by protein–protein interactions occurring in supramolecular structures and organelles. Expected DSC profiles for cells were modeled by summing various numbers of independent transitions with the Tm, peak amplitude (DHcal), and peak width (activation energy) of each transition chosen randomly as described in the legend for Fig. 2. The overall profile and individual transitions are shown in Fig. 2 for a DSC scan consisting of 20 independent transitions. These 20 transitions yield a profile consisting of three resolvable peaks, which is roughly similar to what is observed for the cell scans shown in Figs. 3–7. For a very large number of independent transitions (e.g., 1000), the transitions overlap to such an extent that a smooth profile results (Fig. 2). This is very different than what is observed for cells and suggests there are a limited number of independent denaturation transitions in complex cellular systems.
Fig. 2. Simulation of a multi-component DSC profile (cp vs. T) of a cellular system consisting of multiple protein denaturation transitions. Twenty transitions of random amplitude (DHcal) with Tm selected from a Poisson distribution with an average Tm of 55 C and a minimum of 40 C (small peaks). The sum of these 20 transitions (solid line) and the sum of 1000 transitions (broken line) are shown.
Fig. 3. Plot of cp vs. T of human erythrocytes. (A) The original scan (solid line) and the rescan (broken line) after scanning to 100 C at 1 C/min are shown. (B) The corrected scan of cp(excess) vs. T after correction for the baseline curvature and Dcp and the theoretical fit to the Hb curve assuming two-state irreversible denaturation (broken line) are shown.
4. Differential scanning calorimetry profiles of cells Cells are a complex, interacting mixture of proteins, nucleic acids, and membrane lipids, each of which can undergo order–disorder, endothermic transitions
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Fig. 6. DSC scans of HeLa cells. (A) Original scan (solid line) obtained at a scan rate of 1 C/min with a fitted baseline (broken line). (B) Profile of cp(excess) vs. T obtained by subtracting the generated baseline from the original scan.
Fig. 4. Plot of cp vs. T of Bacillus stearothermophilus (ATCC 12016). (A) The original scan (a), the rescan after scanning to 100 C at a scan rate of 1 C/min (b), and a water–water baseline (c) are shown. (B) The scans corrected for baseline curvature for the original scan (a) and the rescan (see text for details) are shown. The features marked with the arrows and labeled A–D, L, and Tl are discussed in the text.
Fig. 7. DSC scans of Chinese hamster lung V79 cells. Shown are the profiles of cp(excess) vs. T obtained at a scan rate of 1 C/min for control cells (solid line) and cells exposed to 5% ethanol (dotted line) or 5% glycerol (dashed line).
Fig. 5. DSC scans of four species of Bacillus. Values of Tg, Tmax, and Tl are given in Table 2.
detectable by DSC, and numerous small molecules and ions. A DSC scan should be the sum of the transitions of all components. In addition, other processes such as aggregation and metabolism, both exothermic events, can be detected by calorimetry and must be considered in interpreting a DSC profile. The human erythrocyte
is a fairly simple cell which lacks a nucleus and consists primarily of hemoglobin. In addition, it has a low level of metabolism, and thus metabolic heat released during a DSC scan should not be a problem. These characteristics should lead to a less complex, more easily interpretable, DSC profile [23]. 4.1. Erythrocytes 4.1.1. Methodology Human erythrocytes were purified from freshly drawn blood by washing three times in a 10-fold excess solution of phosphate-buffered saline (PBS). The buffy
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coat and top of the pellet were removed after each centrifugation by aspiration to remove all white blood cells. Erythrocytes were used on the day of isolation. DSC scans were obtained with an adiabatic MicroCal-2 (MicroCal, LLC) scanning calorimeter (1.21 ml sample cells). The procedure used for higher sensitivity calorimeters such as the MicroCal VP-DSC or Nano DSC (Calorimetry Sciences) is identical. High quality scans of cells cannot be obtained using faster-scanning, non-adiabatic calorimeters. The sample and reference cells of the DSC were cooled to 0 C, and the degassed erythrocyte suspension in PBS (4–6 mg/ml protein) and the reference solution (PBS) were added. Degassing is necessary since bubble formation due to reduced air solubility at higher temperatures interferes with the scan. It is accomplished by exposing the cell suspension to a mild vacuum while gently stirring. The scan was started (1 C/min scan rate) when the sample and reference cells equilibrated at 0 C (approximately 40 min after addition of erythrocytes). The cells should settle to the bottom of the calorimeter cell during the equilibration period, but settling prior to scanning does not alter the shape of the profile. The scan was run to 100–102 C, the cells cooled to 0 C, and a second scan (the rescan) obtained which was used to correct for the non-linear, baseline curvature as previously described [20]. Fig. 3A shows the DSC scan and the rescan of human erythrocytes obtained using the procedure described above. The first step is to correct for intrinsic baseline curvature by subtracting the rescan. The second step is to correct for Dcp as was done for the DSC scan of citrate synthase as shown in Fig. 1. The resulting corrected scan of cp(excess) is given in Fig. 3B. This corrected scan is more appropriate for analysis. There are four resolvable endothermic peaks, labeled A, B, C, and Hb, each of which represents a single strong transition or a group of transitions with similar TmÕs. The evidence indicates that this profile is due almost exclusively to protein denaturation. The large endothermic peak with Tm = 72 C labeled Hb and the exotherm at 78 C are due to the denaturation of hemoglobin and its subsequent aggregation, respectively. This peak was identified by measuring the Tm of isolated hemoglobin, which was within 2 C of peak Hb in cells. In addition, hemoglobin is the major protein component of erythrocytes and this is by far the strongest transition. The hemoglobin peak can be theoretically fit using a two-state, irreversible model (Fig. 3B). Aggregation is exothermic, and the position of the exotherm is consistent with it being due to the aggregation of denatured hemoglobin. The first deviation of cp from the baseline occurs between 40 and 45 C, indicating that significant protein denaturation detectable by DSC first occurs in this temperature range. The peaks labeled A, B, and C were identified by comparing their TmÕs to those of membrane
components of isolated ghosts. The transitions in isolated erythrocyte membrane ghosts have been identified by a combination of DSC and a technique referred to as thermal gel analysis in which the loss of specific protein bands on a gel due to thermally induced aggregation are correlated with Tm on a DSC scan [24]. Peak A represents the denaturation of spectrin, peak B the denaturation of several other components of the membrane skeleton, and peak C the denaturation of the transmembrane portion of the band 3 protein. Thus, the membrane skeleton, including spectrin, is the most thermolabile major component of the erythrocyte. The erythrocyte is a particularly simple cellular system, but it illustrates the approach for using DSC to measure the stability of proteins in cells. Specific peaks are observed and correspond to the denaturation of individual or groups of proteins. Identification of individual peaks, if it can be done, allows a determination of which proteins are denatured at heat shockÔ temperatures. 4.2. Bacterial cells Different bacterial strains have widely different maximum growth temperatures (Tmax), defined as the maximum temperature at which continuous growth occurs [25]. DSC can be used to determine if Tmax is limited by protein denaturation at high temperatures. This can be tested using bacterial strains with a wide range of Tmax. Two thermophiles Bacillus stearothermophilus (ATCC 12016 and WAT), a mesophile Bacillus megaterium, and a psychrotroph Bacillus psychrophilus were used [25]. 4.2.1. Methodology Cells were grown in trypticase soy broth at their respective growth temperatures (Tg, see Table 2). Bacterial cells were harvested when the A650 of the culture medium reached 1.0. The procedure was to cool the cultures quickly to 4 C and employ centrifugation and two washes in phosphate buffer (10 mM, pH 7.0) containing chloramphenicol to block further protein synthesis during harvesting. The final bacterial suspension and reference solution were degassed under mild vacuum at 4 C for 5 min immediately before addition to the calorimeter Table 2 Maximum growth temperatures (Tmax) vs. onset of denaturation (Tl) for various species of Bacillus
Bacillus psychrophilus Bacillus megaterium Bacillus stearothermophilus (WAT) Bacillus stearothermophilus (ATCC 12016)
Tg (C)
Tmax (C)
Tl (C)
20 37 56
32.5 48 56
30 46 55
56
69
65
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cells. The calorimetry procedure was identical to that described above for erythrocytes. Shown in Fig. 4A is the original scan of B. stearothermophilus (ATCC), a rescan, and a water–water baseline illustrating the intrinsic curvature of the calorimeter. The scan has a series of endothermic peaks from 65 to 100 C and an obvious endotherm near 15–20 C. The rescan, which consists of all reversible transitions, contains two endothermic transitions at approximately 30 and 93 C. Both scan and rescan were corrected for intrinsic curvature, which is partially visible in the water–water baseline (curve c). Before the rescan could be subtracted from the scan, it was necessary to remove the two reversible peaks. This was done by removing the two peaks from the rescan by numerical methods and fitting the remaining curve with a fourth-order polynomial to capture the intrinsic curvature. The fourth-order fit was then subtracted from the scan and rescan to correct for intrinsic baseline curvature. The corrected curves are shown in Fig. 4B. The specific features are more clearly illustrated in these curves. There are four endothermic transitions above 65 C, three of which (labeled A–C) are irreversible and apparently due to protein denaturation. The onset temperature for denaturation is defined as the temperature at which these endothermic peaks begin (labeled Tl). The fourth (peak D) is reversible, as can be seen in the rescan, and has a Tm in the range expected for DNA. A well-defined endotherm (labeled L) in the rescan occurs near the temperature of the low temperature exotherm (15– 20 C) in the scan. These presumably are related, although the transition in cells is more complex in shape, and appear to be membrane lipid transitions. One last feature is a broad exotherm from 50 to 65 C with a minimum at 60 C marked with an arrow. This is due to the metabolic heat released in this temperature range. This metabolic exotherm makes an accurate determination of Tl difficult. Fig. 4B illustrates the main thermic transitions and events detectable by DSC in cp vs. temperature scans of cells. The shape of the profile is influenced by metabolism, lipid transitions, and DNA transitions in addition to protein denaturation. Corrected DSC scans for psychrotroph B. psychrophilus, mesophile B. megaterium, and other thermophile B. stearothermophilus (WAT) in addition to B. stearothermophilus (ATCC) are shown in Fig. 5. The ranges over which protein denaturation can be detected for each species differs considerably. The onset temperatures for denaturation Tl and the maximum growth temperature Tmax for each species are given in Table 2. There is a very good correlation between the onset of denaturation and the maximum growth temperature for these bacteria from 30 to 70 C. This illustrates how DSC determinations of protein denaturation can be correlated with growth.
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4.3. Mammalian cells Dividing eukaryotic cells are the most commonly used category of cells in studies of heat shock, and protein denaturation can be measured in them using DSC as described above for erythrocytes and bacteria. DSC profiles with common features have been obtained from mammalian Chinese Hamster Lung V79, CHO, 3T3, HeLa, A549, and L929 cells as described below. 4.3.1. Methodology Cells can be grown in suspension or attached. Cells grown in suspension require less manipulation, since they do not need to be removed from culture plates, than cells grown attached. For example, Chinese Hamster Lung V79 and HeLa cells, adapted to suspension culture, can be grown in suspension in minimal essential medium (MEM), with no added calcium, containing 10% fetal calf serum, 26 mM Hepes, and 10 mM sodium bicarbonate at pH 7.4 [26]. Cells are harvested, after reaching a density of 5 · 105 per ml, by mild centrifugation before resuspension in Hepes-buffered saline. DSC scans and rescans are obtained as described above for erythrocytes and bacteria. Depending on the size of the cell and the sensitivity of the calorimeter, suspensions of 1–10 · 107 cells/ml are required for high quality, noise-free scans. After degassing, the viability is checked using erythrosine B. The effect of ethanol and glycerol on the DSC profile was determined by adding these compounds after degassing. In general, most additional compounds can be added to the Hepes-buffered saline before resuspension and degassing of cells. However, volatile solvents should be added after degassing to ensure that vacuum treatment does not alter concentration. Shown in Fig. 6 is the original scan of HeLa cells at a concentration of 3 · 107 cells/ml in the calorimeter. The original scan, as for erythrocytes and bacteria, shows a large Dcp after going through the endothermic transitions from 40 to 100 C. This can be corrected as described above, and shown in Fig. 6A are the original scan and the generated baseline used to correct for Dcp. The corrected curve is shown in Fig. 6B. Scans of all tissue culture cells, both rodent and human, with a relatively high nuclear/cytoplasmic volume ratio are similar to this scan. There are five main peaks labeled A–E with transition temperatures of 50–55, 62– 64, 70–75, 85, and 96–98 C, respectively. Peaks C, D, and E are primarily nuclear transitions [6]. There is evidence that peaks D and E are due to the melting of relaxed and supercoiled DNA, respectively, and that peak C represents the denaturation of histones [27]. Peaks A and B are due primarily to the denaturation of cytoplasmic proteins, with a contribution from thermolabile nuclear proteins [6]. The proteins denaturing in the temperature region of 40–50 C should be the
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ones damaged during heat shock. The identity of the proteins denaturing at this temperature is largely unknown. DSC profiles are altered by conditions that also alter cellular sensitivity to heat shock. Shown in Fig. 7 are DSC scans of CHL V79 cells and cells in the presence of ethanol, which sensitizes to heat shock and is an Hsp inducer, and glycerol, which protects from heat shock and inhibits Hsp induction. Of particular interest is the low-temperature region of the profile near 40 C. It can be seen that ethanol shifts this region to lower temperatures, indicating that it lowers the Tm for these proteins, while glycerol shifts the region to higher temperatures, indicating it raises the Tm of these proteins. This is consistent with the effects of these compounds on cellular sensitivity to heat shock. 4.4. Quantification of protein denaturation The fractional protein denaturation on scanning to a given temperature is given by the area under the DSC profile of cp(excess) vs. temperature as described above for the protein citrate synthase (Fig. 1). The DSC profiles for V79 cells shown in Fig. 7 can be used to estimate the amount of denaturation for specific temperature exposures. Plots of fraction denaturation as a functional of temperature are given in Fig. 8. These plots were generated by first removing peaks D and E, which appear to be due to transitions in DNA not protein [27]. The resulting curves are integrated and normalized to yield fractional denaturation (fD) (Fig. 8). An important assumption made in this analysis is that the specific DHcal for each protein is the same. The value of DHcal for individual proteins decreases as the Tm decreases as a consequence of positive Dcp [6]. Thus, the curves probably somewhat underestimate the fractional denaturation occurring at lower temperatures. When survival of V79 cells is measured under the same heating conditions as for DSC (i.e., heating rate of 1 C/min), killing is first detectable after reaching
Fig. 8. Plots of estimated fractional protein denaturation (fD vs. T) for V79 cells (solid line) and for the same cells exposed to 5% ethanol (dotted line) or 5% glycerol (dashed line).
45 C with 100% killing occurring at about 49 C (results not shown). At 45 C and 49 C, fD is approximately 0.07 and 0.2. Thus, to a rough approximation killing occurs over the range of 7–20% denaturation. Ethanol increases the amount of protein denaturation while glycerol decreases it. For example, on scanning to 45 C, ethanol (5%) increases denaturation by approximately 3-fold while glycerol (5%) reduces it by 2-fold.
5. Summary DSC is a technique that is sensitive to protein denaturation in intact cells including erythrocytes, bacteria, yeast [28], mammalian cells, and tissue [29]. Irreversible endothermic transitions are detectable in the relevant heat shock range for the above cells. These endothermic transitions can be identified as protein denaturation based on irreversibility, the values of DHcal obtained, the expected large contribution of protein denaturation due to the cellular content of proteins, the lack of strong lipid transitions in the heat shock range, the relatively low enthalpy associated with changes in protein interactions (i.e., polymerization and depolymerization), and the association of transitions in erythrocytes with the denaturation of specific proteins [6]. The onset temperature of protein denaturation correlates well with the inhibition of growth and the onset of killing. The amount of protein denaturation occurring as a function of heating can be measured for specific heat treatments. A rough estimate for CHL V79 cells is that killing occurs in the range of 7–20% total denaturation.
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