Thermal stability of high concentration lysozyme across varying pH: A Fourier Transform Infrared study.

AIM: The current work is aimed at understanding the effect of pH on the thermal stability of hen egg white lysozyme (HEWL) at high concentration (200 mg/mL).
MATERIALS AND METHODS: Fourier Transform Infrared (FTIR) Spectroscopy with modified hardware and software to overcome some of the traditional challenges like water subtraction, sample evaporation, proper purging etc., are used in this study.
RESULTS: HEWL was subjected to thermal stress at pH 3.0-7.0 between 25°C and 95°C and monitored by FTIR spectroscopy. Calculated Tm values showed that the enzyme exhibited maximum thermal stability at pH 5.0. Second derivative plots constructed in the amide I region suggested that at pH 5.0 the enzyme possessed higher amount of α-helix and lower amount of aggregates, when compared to other pHs.
CONCLUSIONS: Considering the fact that HEWL has attractive applications in various industries and being processed under different experimental conditions including high temperatures, our work is able to reveal the reason behind the pH dependent thermal stability of HEWL at high concentration, when subjected to heat denaturation. In future, studies should aim at using various excipients that may help to increase the stability and activity of the enzyme at this high concentration.

Physical and chemical instabilities of proteins are considered a bottleneck for a successful drug development. Specifically, protein aggregation is considered as one of the most common issues that significantly affects the quality and efficacy of therapeutic candidates.[1] Protein aggregation is induced by several factors such as temperature, pH, ionic strength, vortexing, surface/interface adsorption and protein concentration. Of all these, protein concentration is of particular interest, since subcutaneous route of delivery demands a highly concentrated protein.[2] At such a high protein concentration (>0.1 g/L), aggregation due to self-association (macromolecular crowding) is expected, that affects the solubility, stability and activity of drugs.

Lysozyme, a globular protein of 14.4 kDa molecular weight is a bacteriolytic enzyme that is used as a natural food preservative.[3] The bacteriostatic and bactericidal properties of lysozyme have generated numerous applications in medicine and in the pharmaceutical industry.[4] Due to its antibacterial activity, lysozyme has been of a considerable interest within the food industry.[5] The enzyme finds its major applications as an antimicrobial in cheese, as an antibiotic in chicken feed,[6] and has been shown to inhibit the growth of HIV in vitro.[7] Hen Egg White is the main source of lysozyme in industries not only due to its rich lysozyme content, but also because the enzyme can be easily purified from the source in a cost effective way.[8] Due to the enzyme's attractive application as a food preservative, it is highly important to understand the relationship between the protein's stability and activity at different experimental conditions. A study on the heat treatment on human milk found that pasteurization at temperatures above 70°C resulted in the destruction of lysozyme with only 3% activity retained at 100°C.[9] Both pasteurization treatments at low temperature over longer periods of time (63°C for 30 min) and high temperatures over shorter time frames (74°C for 16 s) resulted in 11.5% and 24% activity loss, respectively, in lysozyme in cow's milk,[10] while that in human milk was 47% and 30% respectively.[11] Also, a recent study aimed at understanding the relationship between pH, thermal stability, and activity of lysozyme showed that the enzyme is most stable at pH 5.2 and its activity is significantly affected at higher temperatures and pH.[12] Enzymes are very sensitive to the experimental conditions such as pH and temperature that profoundly affect their activities. For industrial applications of enzymes, these two factors are very important to be considered in order to extract the maximum application of the enzymes.

While there are extensive reports already available related to the thermal stability of lysozyme using different biophysical methods such as Differential Scanning Calorimetry and spectroscopic methods like Fluorescence, Circular Dichroism (CD), Fourier Transform Infrared (FTIR) and UV-Vis, no study so far has been able to shed light on the thermal denaturation of lysozyme at high concentrations (>100 mg/mL) across varying pH. Given the fact that high concentration protein solutions are being increasingly used in industries, it is mandatory to understand the structural integrity of such concentrated proteins, when subjected to various stress.

FTIR spectroscopy is a very well-known spectroscopic method for characterizing protein aggregates at high concentrations.[1314] In FTIR, amide I band region between 1700/cm and 1600/cm is very useful to monitor protein's conformational changes.[15] However, generally speaking, conducting FTIR experiments using commercial instrument poses certain difficulties with respect to water subtraction, proper purging, cleaning of sample cells, evaporation of the sample and reproduction of data with minimum errors. Historically, to combat such challenges, researchers used deuterated samples to avoid the high absorbance of water in the amide I region that might otherwise mask protein's secondary structure signal during measurement. Even in one of the most recent FTIR based study of high concentration lysozyme, deuterated sample of the enzyme was used probably to overcome the problems with water subtraction.[13] Neither in the food processing industry nor in pharmaceutical applications of lysozyme is the enzyme used in deuterated solvents.[4] This fact highlights the importance of understanding the conformational changes of enzymes in water-based buffers. In addition, it is well known that amide – I frequencies are strongly affected by H-D exchange in the peptide linkages.[16] According to the literature, protein's secondary structure might be altered in deuterated buffers and water-based buffers provide more native environment.[15]

In this study we were able to overcome many of the challenges traditionally facing thermal investigations by modifying the hardware and software that was previously validated in a study on a high concentration monoclonal antibody.[14] In the current study, we followed the thermal denaturation of hen egg white lysozyme at a very high concentration across varying pH to understand the pH dependent thermal stability of the enzyme.

Materials and Methods

Hen egg white lysozyme was bought commercially (Sigma Aldrich, >95% pure). Enzyme at a concentration of 0.2 g/L was dialyzed against a buffer containing 25 mM each of borate, citrate and phosphate supplemented with 0.1 M NaCl (between pH 3.0 and pH 7.0). Protein samples were concentrated (Microcon centrifuge tubes, MWCO 3000 Da, Millipore) and filtered through 0.2 μm Millipore syringe filters. The pH of both the buffers and samples were confirmed by the pH meter (with deviations less than 0.1 pH units) and the concentration of the enzyme samples was measured by UV-spectroscopy at 280 nm and was found to vary between 0.195 g/L and 0.205 g/L. All samples were freshly prepared prior to thermal denaturation experiments.

For detailed description of the method (experimental details and data analysis), refer to our previously published work.[14]

Results and Discussion

Thermal stability of lysozyme across varying pH

Lysozyme at an enzyme concentration of 0.2 g/L was thermally stressed from 25°C to 95°C in a buffer cocktail containing 10 mM each of Boric, Citric and Phosphoric acid (Ionic strength provided by 0.1 M NaCl). Taking into account that the enzyme is most stable around pH 5.0 according to other studies,[1217] we tried 2 pH units above and below pH 5.0 for our studies. Thermal unfolding was monitored by FTIR spectroscopy. The change in the intensity at 1654/cm-1 corresponding to native α-helix was plotted against temperature [Figure 1a] in Origin, and the transition temperature was calculated by fitting the data to a Boltzmann Sigmoid equation. Figure 1b gives an overview of the effect of pH on the transition temperatures of lysozyme at 200 mg/mL. The measured intensity values (both from the absorbance and second derivative curves) at 1654/cm remain unchanged between 25°C and 50°C implying that temperature had minimum or null effect over the secondary structure of lysozyme. Hence, for the calculation of transition temperatures, only data points between 50°C and 95°C were taken into consideration [insert in Figure 1a]. From the Tm bar diagram [Figure 1b], lysozyme exhibits maximum thermal stability at pH 5.0, the observation being consistent with other studies.[1217] Such reports confer that the thermal instability (irreversibility and inactivation of the enzyme) is due to both physical and chemical degradation such as asparagine deamidation,[1819] disulfide scrambling[20] and aggregation.[21] Second derivative plots were constructed for all experimental conditions in an effort to understand the conformational changes in lysozyme induced by increasing temperatures that may be responsible for affecting the thermal stability [Figure 2].

Figure 1

Tm plot of lysozyme at 200 mg/mL (a) Plot of second derivative curve intensity of lysozyme at 1654/cm-1 against temperature (25-95°C) at pH 5.0. The inset is a similar plot where Tm is calculated from the sigmoid fitting between 50°C and 95°C at pH 5.0. (b) Bar diagram of Tm plot of lysozyme at varying pH calculated between 50°C and 95°C. For the transition temperature values [Table 2]

Figure 2

Second derivative plots of lysozyme at all pH (a-e). The curves correspond to 25°C (black), 65°C (red), 70°C (green), 75°C (blue), 80°C (cyan) and 90°C (pink). For the assignment of the amide I frequencies refer to Table 1. For convenient purpose, peak intensities at 1617/cm and 1654/cm are indicated

Table 2

Tm values of lysozyme at different pHs

Table 1

Secondary structure assignment of lysozyme in the amide I region

Analysis of the second derivative plots of lysozyme

The second derivative plots of lysozyme between pH 3.0 and 7.0 are shown in Figure 2a–e. Owing to the subtle structural changes of lysozyme below 50°C, limited temperature plots are shown here. At all pH, the secondary structure of lysozyme at 25°C is dominated by α-helix band at 1654/cm-1 with minor contributions from 1613 and 1686 (intermolecular anti-parallel β sheets due to aggregation), 1638 (intramolecular β sheets that are from the native structure) and 1673/cm-1 (turns) [see assignment according to the literature[22232425] in Table 1]. High protein concentrations most likely result in protein–protein interactions that could give rise to the formation of intermolecular β sheets. Hence it is not surprising to see aggregation peaks even at 25°C for lysozyme.

As the temperature was slowly increased we did not observe any noticeable change in the secondary structure until 65°C. At 65°C the peaks at 1613 and 1673/cm blue shifted to 1615 and 1675/cm respectively while the intensity of the peaks from α-helix (1654/cm) and β sheets (1638/cm) decreased. Decrease in the peak intensity at these frequencies clearly indicates the unfolding event of lysozyme due to higher temperatures (loss of native α-helix and β-sheets). In parallel, shift in the frequency from lower to higher values show that the aggregates and turns in the secondary structure of lysozyme are becoming less stable, with weak hydrogen bonding of amide groups. The transition temperatures calculated for lysozyme between pH 3.0 and pH 7.0 varies between 69°C and 71.9°C [Table 2]. At the transition zone at 70°C (close to the Tm value), the secondary structure of lysozyme is significantly perturbed. Though the enzyme retains most of the structural features displayed at 65°C, the intensity of the α-helix peak at 1654/cm has been affected noticeably as shown in Figure 3a. Specifically, there is a steep decrease in the α-helix content at pH 3.0 and pH 4.0 when compared to pH 5.0, 6.0 and 7.0 as the protein is heated above 65°C. While the native helical content of lysozyme steadily increases between pH 5.0 and 7.0, profound increase in aggregation (as evidenced by the intensity increase at peaks 1617 and 1690/cm) specifically at pH 6.0 and 7.0 is observed [Figure 3b aggregation plot]. Between pH 3.0 and pH 5.0, until 75°C, change in the aggregation pattern looks the same. When heated above 75°C, sample both at pH 3.0 and 4.0 shows increased aggregation than at pH 5.0. Further shift in the aggregation peak from 1615 to 1617/cm indicates that as the temperature increases, the protein aggregates become less solvent exposed.

Figure 3

Plot A is the a-helix plot of intensity values at 1654/cm from the second derivative plots at pH 3.0 (black), pH 4.0 (red), pH 5.0 (green), pH 6.0 (blue) and pH 7.0 (cyan). Plot B is the aggregation plot of intensity values at 1617/cm from the second derivative plots at pH 3.0 (black), pH 4.0 (red), pH 5.0 (green), pH 6.0 (blue) and pH 7.0 (cyan) against temperature

Interestingly, at 90°C, lysozyme undergoes the following conformational changes:

Significant decrease in the aggregation peaks (the intensity is almost the same as seen at 70°C). Previous research studies have shown this characteristic pattern of temperature induced disappearance of protein aggregates.[22]

Blue shift of the peak from 1654/cm to 1657/cm that indicates a change in the helical conformation induced by higher temperatures.

Slight increase in the peak intensity at 1675/cm (β turns) at all pH.

Partial unfolding of the protein after crossing the transition zone leads to aggregation as evident by the growth of the peak at 1617/cm. This common phenomenon is expected and been observed for highly concentrated proteins.[1421] However, what is unusual is the disappearance of such aggregates at further higher temperatures. According to the literature,[23] surface characteristics of the monomers such as its hydrophobicity, surface area and lower free energy determine the reversibility of protein aggregates. In parallel, it is also interesting to note that native α-helix of lysozyme often shows two spectral features (1654 and 1657/cm) that are associated with slightly different hydrogen bonding strengths of the carbonyl groups (lower and higher temperatures).[26] This also suggests that the native α-helix of lysozyme at high temperatures become less solvent exposed due to increased aggregation and the protein now can be visualized to have a compact tertiary structure without pronounced helical content.

Maximum thermal stability at pH 5.0 is due to minimum aggregation

We have studied the effect of pH on the thermal denaturation of lysozyme at 200 mg/mL by FTIR spectroscopy. Analysis of the second derivative plots show that when moving from pH 3.0 to 5.0, dominance of α-helix even at 70°C at pH 5.0 when compared to lower pH may play a crucial role in determining the thermal stability of the protein. When the pH is further increased from 5.0 to 7.0, increase in aggregation decreases the thermal stability at pH 6.0 and 7.0, leaving pH 5.0 as the most stable pH for lysozyme in terms of thermal stability. Hence, a competition between the existence of increased content of α-helix and aggregates determines the transition temperature of the enzyme across pH.

Prevention of aggregation is very important as these aggregates affect the solubility, stability, activity of the protein and ultimately pose immunogenic threats.[27] Studies have shown that use of excipients such as Sorbitol, Glycine, Sucrose, Trehalose, Hydroxyproline etc.,[2829] have significantly helped to reduce aggregation in lysozyme. Activity of an enzyme depends on the protein's stability when exposed to various experimental conditions like pH, temperature, buffers etc., Of all these, thermal inactivation was shown to be the primary cause for enzyme inactivation.[30] The benefits of lysozyme with intended uses in food products, cosmetics, and baby formulas has remained attractive and prompted a research group to produce cloned transgenic cattle that expressed recombinant human lysozyme in breast milk.[31] Heat treatments like pasteurization, a series of high temperature, short time exposures of the milk and milk powder (65-100°C) exposes the protein to thermal stress.[10] Hence, it is extremely important to maintain the enzyme's activity during such harsh treatments. In this study, we attempted to study the thermal stability of lysozyme at high concentrations across different pH. Using FTIR spectroscopy that is currently the best suited available technique to characterize the relationship between high concentration and secondary structural changes, we were able to show that lysozyme exhibits maximum thermal stability at pH 5.0 owing to reduced aggregation. On the other hand, its thermal stability is affected at other pH due to increased aggregation and reduced native helical content. Future efforts should be aimed to screen for excipients that may help to enhance both the native structure and decrease, if possible, the aggregation of lysozyme at high concentrations making the enzyme even more suitable for wide spread applications in industries.