Thermodynamic parameters for the thermal denaturation, folding reversibilities using consecutive upscan method (UU) and enzymatic activities of crystallised lysozyme samplesa.
1. Introduction
An important aspect in the preparation of proteins as pharmaceutical products is stabilisation of the native protein conformation (folded, three-dimensional, tertiary state), which is required for biological activity. Moreover, it is not enough for this conformation to be stable, but the protein must be able to find the state or folding pathway in a short time from a denatured, unfolded conformation [1]. Folding minimises exposure of non-polar groups and maximises exposure of polar groups to the solvent [2].
Lysozyme, a globular protein, molecular weight 14,300 Da, was chosen as a model protein; it consists of a single 129 amino acid chain divided into two domains cross linked by four disulfide bridges. The hydrophilic groups tend to concentrate on the surface and the hydrophobic groups in the core [3]. The goal of this study was to investigate the influences of crystallisation on folding reversibility of lysozyme in solution as assessed calorimetrically.
Many literature reports cited the value of High Sensitivity Differential Scanning Calorimetry (HSDSC) for determining thermodynamic parameters (transition temperature,
Differential scanning calorimetry has the ability to provide detailed information about both the physical and energetic properties of a substance [11]. The HSDSC technique was described in detail by Cooper and Johnson (1994) [12]. In summary, all equilibrium processes involving molecules are governed by free energy changes (G), made up of enthalpy (H) and entropy (S). The relationship between (H) and (S) is given by the Gibbs free energy equation:
where T is the temperature. Differential scanning calorimeters measure enthalpies, which provide the basis for determining the thermodynamic properties of a system. Both enthalpy and entropy are related to the heat capacity of the system. The enthalpy is the total energy (at constant pressure) required to heat the system from absolute zero to the required temperature:
where Cp (T) is the temperature-dependent heat capacity at constant pressure. The total entropy of the system can be expressed as:
Accordingly, differences in H and S can be expressed as:
and
Thermodynamic parameters depend on conditions, such as temperature, pressure, concentration and composition. Thus, it is necessary to correct experimental results to standard conditions (standard states) denoted by the superscript 0 (
where R is the gas constant and K is the equilibrium constant for the process and is related to (H0) and (S0) and to the absolute temperature by:
assuming that (H0) and (S0) do not vary significantly with temperature over the range of interest.
The HSDSC provides an insight into the thermal stability and instability (e.g. formation of soluble and insoluble aggregates) of solutions of different formulations. HSDSC was used to assess the thermal stability of lysozyme solutions after storage at stressed conditions [13]. Consecutive heating scans indicated the folding reversibility of thermal transitions [8, 14] and the validity of calorimetrically measured protein folding reversibility [15-17]. Creighton (1994) [18] reported mechanisms and thermodynamic factors controlling protein folding-and-unfolding.
In the present study, HSDSC investigated thermal changes; in particular protein refolding performance, of crystallised samples (in low and high protein concentrations) upon heat treatment. The thermal structural transition of lysozyme involves two thermodynamic states, native and denatured [19] as for other globular proteins [20]. However, Hirai et al. (1999) [21] indicated that folding-and-unfolding kinetics of proteins depend on the number of amino acid residues. Proteins with residues above ~100 do not follow simple two state kinetics in a folding-and-unfolding process as a single cooperative unit. Accordingly during HSDSC analysis of lysozyme, there might be formation of intermediates between native and denatured states. The thermodynamic stability of proteins not only requires that the transition temperature (
Consequently, formation of the native state is a global property of the protein as described [1]. This state is necessary for stability and activity; proteins are marginally stable and achieve stability only within narrow ranges of conditions of solvent and temperature. The free energy of stabilization of proteins under ordinary conditions is ~ 5-15 kcal mol-1 [1].
Proteins undergo various structural changes if physiological conditions alter. Accordingly, they may denature and the denatured protein tends to adsorb to surfaces and aggregate with other protein molecules. Katakam et al. (1995) [23] proposed that denaturation of recombinant human growth hormone involves unfolding of the molecule; the unfolded part adsorbs to surfaces and aggregates with neighbouring molecules. Shaking and exposure to an air/water interface, heating, lyophilisation or reconstitution of lyophilised protein may aggregate protein with subsequent loss of stability and activity.
The combination of HSDSC and enzymatic activity determined if refolding of denatured crystallised lysozyme after thermal denaturation in HSDSC arises from the nativeness, three-dimensional folded state, of the initial lysozyme structure. This means that enzymatic activity was employed to investigate if folding reversibility of the thermal transition reflects the renaturation of the unfolded protein to folded native structure.
2. Materials and methods
2.1. Materials
Chicken egg white lysozyme (purity 95%, 5% sodium chloride and sodium acetate), sodium chloride (99.5%), sodium phosphate (99.3%) and
2.2. Preparation of crystallized lysozyme
Lysozyme was crystallised using a published method [24]. Crystals formed were filtered, dried and kept in a freezer (-15°C) until tested.
2.3. High Sensitivity Differential Scanning Calorimetry (HSDSC)
Solution samples of crystallised lysozyme were analysed with a Microcal MCS differential scanning calorimeter (Microcal Inc., MA, USA). Degassed samples (5 and 20 mg product / 1 mL 0.1M sodium acetate buffer, pH 4.6) and reference (0.1M sodium acetate buffer, pH 4.6) were loaded into cells using a gas tight Hamilton 2.5 mL glass syringe. The folding reversibility of lysozyme denaturation was assessed by temperature cycling using two scan calorimetric methods. The upscan-upscan method (UU) employed two consecutive upscans from 20-90°C at 1°C/min. After the first upscan, the sample was immediately cooled in the calorimeter (downscan) to 20°C at 0.75°C /min (the fastest cooling rate allowed by the instrument) and the heating cycle was immediately repeated. Transition reversibility was measured as ratio (%) of enthalpy change of second upscan (H2) over that of first upscan (H1). The upscan-downscan method (UD) involved heating of protein solution from 20-90°C at 1°C/min immediately followed by downscan (cooling) from 90-20°C at a cooling rate of 0.75°C/min. Enthalpies were measured and downscan (H3) / upscan (H1) enthalpy ratios were calculated as a measure of folding reversibility. The calorimeter was temperature- and heat capacity-calibrated using sealed hydrocarbon standards of known melting points and electrical pulses of known power, respectively.
Experiments were performed under 2 bar nitrogen pressure. A base line was run before each measurement by loading the reference in both the sample and reference cells; this base line was subtracted from the protein thermal data and the excess heat capacity was normalized for lysozyme concentration. Data analysis and deconvolution employed ORIGIN DSC data analysis software. The
2.4. Enzymatic assay
Biological activities of thermally denatured crystallised lysozyme were determined after cooling (in HSDSC) to determine whether the renaturation is due to the nativeness of the protein structure i.e. to correlate the folding reversibility with biological activity. In this assay, a bacterial suspension was prepared by adding 20 mg of
All data were presented as mean of three determinations standard deviation. The Student’s
3. Results and discussion
3.1. High Sensitivity Differential Scanning Calorimetry (HSDSC)
Differential scanning microcalorimetry experiments can thermodynamically characterise the unfolding transition by determing heat capacities, enthalpies and melting temperatures of native and denatured protein [25]. HSDSC monitored thermal stability and folding reversibility of reconstituted lysozyme preparations. For samples, traces for thermal denaturation and folding reversibilities, using (UU) method, of reconstituted crystallised lysozyme are illustrated in Figure 1(a) and (b) for 5mg/mL and 20mg/mL protein concentrations, respectively. Thermodynamic parameters and enzymatic activities are in Table 1. Figure 2 shows an example for folding reversibility of unprocessed lysozyme using (UD) method. As is evident in Figures 1 and 2, HSDSC profiles of all samples showed a single endothermic peak (first upscan). Lysozyme crystals started to unfold at ~65°C with a mean
It is noticeable that rescan profiles, whether endothermic (second heating cycle, Figure 1a and b dotted lines) or exothermic (downscan upon cooling, Figure 2 lower trace) showed two peaks, a main one and a small peak or shoulder. Deconvolution of the data (using ORIGIN DSC data analysis software) revealed two transition regions characterised by
The other reasonable explanation is that lysozyme, when its folding process is analysed using circular dichroism, does not obey a single co-operative transition, but the process involves several parallel folding pathways. Each of the two domains stabilises with different kinetics [26]. In particular, the amides in the -helix are involved in the formation of stable helical structure and assembly of the hydrophobic core. Then a stable hydrogen bonded structure in the - domain forms. Accordingly, partially structured intermediates develop during the folding of lysozyme. This explanation is supported by Buck et al. (1993) [27] who reported that lysozyme consists of two structural domains that are stabilised by different pathways.
Lysozyme Sample | Tm1 (°C) | Tm2 (°C) | % folding reversibility (H2 /H1) | % enzymatic activity |
Crystallised 5 mg mL-1 20 mg mL-1 | 76.10.21 75.60.07 | 75.90.15 75.20.02 | 66.51.4 52.41.6 | 65.71.4 52.72.2 |
Lysozyme Sample | % folding reversibility (H3 /H1) | |
Crystallised 5 mg mL-1 20 mg mL-1 | 43.6 (1.6) 48.6 (3.8) |
A comparison of calorimetric enthalpy (Hcal) and the theoretical enthalpy (HVH, van’t Hoff enthalpy) changes, to judge the validity of a two-state mechanism for the unfolding of lysozyme, reveals the presence of intermediates [25]. It was reported that, in the unfolded state, proteins aggregate and react chemically with amino acid residues exposed to the solvent; this can lead to misfolding or irreversible denaturation [28]. Also, the formation of any irreversible component alters the shape of a HSDSC thermodynamic peak over the temperature range at which it forms [29].
For low protein concentration (5mg/mL, Figure 1a),
Table 1 shows no significant difference between
It was reported that determination of the mechanism and pathway of unfolding and refolding depends on the identification of the intermediates that may not be stable at the equilibrium [18]. Thus, detection and characterisation of kinetic folding intermediates is complex. This intricacy can arise from accumulation of intermediates or from subpopulations of the unfolded state refolding at different rates. Also, events in folding are obscure [1]. With respect to samples with high protein concentration (20mg/mL, Figure 1b),
Table 1 demonstrates that
On comparing low and high protein concentrations, thermal stabilities (
For folding reversibility calculated by (UD) method, Table 2 above summarises the results of folding reversibilities of crystallised lysozyme at low and high protein concentrations. From Table 2, it is apparent that the percentage folding reversibility calculated by (UU) method (Table 1) was significantly higher
3.2. Enzymatic assay
Lysozyme solutions upon cooling in the HSDSC after thermal denaturation were assayed for biological activity towards Micrococcus lysodeikticus. Based on the HSDSC results, all samples renatured to some extent after thermal stress. Thus, enzymatic assay should answer an important question. Is this renaturation or folding reversibility related to regain of the native structure of lysozyme (which is essential for biological activity), or does it result from misfolding, i.e. folding of the protein in a manner different from the original natured structure which subsequently leads to loss of activity?
Table 1 presents percentage enzymatic activities of preheated solutions, in HSDSC, of crystallised lysozyme (relative to an aqueous solution of a fresh sample). It is evident that the biological activity of lysozyme was maintained by crystals (5mg/mL and 20mg/mL). The results were consistent with data of folding reversibilities. This answers the question posed previously in that folding reversibility was related to the native structure of lysozyme that is required for its activity, as the greater the folding reversibility, the higher the enzymatic activity. The results illustrated that lysozyme crystals maintained structural integrity even after heating in the HSDSC. A review by Jen and Merkle (2001) [32] showed that hydrated protein within crystals is present in a folded, native form.
From the HSDSC and enzymatic activity results, the folding reversibility, calculated by consecutive upscans (UU, Tables 1), correlated with enzymatic activity of lysozyme, confirming that the upscan-downscan method (UD, Table 2) underestimates the magnitude of folding reversibility. However, proteins are diverse molecules and the presence of correlation between folding reversibility and biological activity of lysozyme, as demonstrated in this study, may not be applicable to other proteins.
4. Conclusions
The overall results suggested that reconstituted lysozyme crystals were able to refold after heating. The folding reversibility arises from the nativeness of the initial lysozyme structure as demonstrated by biological activity data. The results indicated that the upscan-downscan method underestimated the extent of folding reversibility. Consequently, it is preferable to calculate this reversibility, employing high sensitivity differential scanning calorimetry, by the consecutive heating upscan method.
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