A Comparison between Gelatin and Trehalose Matrixes - American

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Biopreservation of Myoglobin in Crowded Environment: A Comparison between Gelatin and Trehalose Matrixes Enrico F. Semeraro,*,†,§ Sergio Giuffrida,*,†,∥ Grazia Cottone,*,†,‡ and Antonio Cupane*,† †

Dipartimento di Fisica e Chimica, Università di Palermo, Viale delle Scienze 17-18, I-90128 Palermo, Italy School of Physics, University College of Dublin, Dublin, Ireland

‡

ABSTRACT: Biopreservation by sugar and/or polymeric matrixes is a thoroughly studied research topic with wide technological relevance. Ternary amorphous systems containing both saccharides and proteins are extensively exploited to model the in vivo biopreservation process. With the aim of disentangling the effect of saccharides and polypeptidic crowders (such as gelatin) on the preservation of a model protein, we present here a combined differential scanning calorimetry and UV−vis spectrophotometry study on samples of myoglobin embedded in amorphous gelatin and trehalose + gelatin matrixes at different hydrations, and compare them with amorphous myoglobin-only and myoglobin-trehalose samples. The results point out the different effects of gelatin, which acts mainly as a crowding agent, and trehalose, which acts mainly by direct interaction. Gelatin is able to improve effectively the protein thermal stability at very low hydration; however, it has small effects at medium to high hydration. Consistently, gelatin appears to be more effective than trehalose against massive denaturation in the long time range, while the mixed trehalose + collagen matrix is most effective in preserving protein functionality, outdoing both gelatin-only and trehalose-only matrixes.

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INTRODUCTION The issue of stability and protection of biological structures has a large relevance because of its applications in food industry, pharmaceutics, and medicine. Many processes contribute to the degradation of biological matter, involving both chemical (oxidation/reduction, pH alteration, de/polymerization) and physical (aging, crystallization of amorphs, phase separation) phenomena. The study of the mechanisms regulating the degradation/protection processes has therefore been the subject of considerable efforts, which are of a particular relevance also because they might contribute to explain the processes of stabilization of biomolecules in vivo. In what follows we introduce and briefly discuss the issues of bioprotection by saccharides and by polymers, and the role of crowding. Biopreservation by Saccharides. Many amorphous saccharide matrixes have been proven to be very efficient in protecting biostructures against adverse conditions, such as drought or extreme temperatures.1 In particular, trehalose (α-Dglucopyranosyl-α-D-glucopyranoside) is generally considered one of the best bioprotecting agents.2 The origin of the bioprotective effectiveness of trehalose, and of the low molecular weight (low MW) saccharides in general, is not fully understood. It is accepted that a sugar should meet at least two important requirements to be a reliable stabilizer: it must remain in the glassy state during storage, since in the rubbery state the high molecular mobility would compromise the stabilization, and it must contain no (or a very limited number of) reducing groups. This condition favors sugars with © 2017 American Chemical Society

a high glass transition temperature (Tg), such as polysaccharides or some disaccharides, and virtually excludes all monosaccharides. Moreover, di- (or oligo-) saccharides appear to perform better than polysaccharides due to the limited flexibility of the latters’ molecular chains, which cause excessive steric hindrance and difficulties in an efficient vitrification at the surface of the embedded macromolecules.3,4 Several hypotheses have been proposed in the current literature on the mechanisms underlying bioprotection, in particular in the trehalose case. The high viscosity of a trehalose matrix would reduce the large-scale internal protein motions that lead to loss of structure and denaturation.5 A special role is attributed to the modulation of water concentration in the surroundings of the protected biostructure, either through the formation of direct hydrogen bonds (HBs) with the disaccharide, or through the entrapment of residual water at the interface by glass formation, thus preserving the “native” solvation.6−8 It must be pointed out that the above hypotheses are not mutually exclusive. The formation of a glassy state does not imply particular hydrogen bonding patterns; the efficiency of trehalose may be due to its ability to form glassy structures in a wide hydration range, along with its influence on the HB network. Conversely, the occurrence of chemical reactions in Received: July 23, 2017 Revised: August 21, 2017 Published: August 22, 2017 8731

DOI: 10.1021/acs.jpcb.7b07266 J. Phys. Chem. B 2017, 121, 8731−8741

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The Journal of Physical Chemistry B the glassy systems implies that the glassy state cannot be the only reason for preservation.9 On the other hand, sugars may substitute for water, thus stabilizing the protein secondary structure, but this phenomenon cannot, in general, be related directly to the protein degradation rate in the glass.10 More generally, conformational studies in full equilibrium conditions for proteins in anhydrous sugars are very difficult to perform due to the very slow dynamics of these glassy systems, where ergodicity cannot be attained on a practical time scale. It has also been reported that differences in the number of protein−sugar HBs do not influence enzymatic activities in the first stages of preservation, but their effects become apparent only during long-term storage,4 i.e., where glass dynamics is generally deemed dominant.9 From an atomistic point of view, trehalose molecules have been shown to reside preferentially close to polar residues (ASP and GLU),7,11 which are preferentially exposed to the solvent in native or nativelike, partially folded proteins: this would reduce the conformational entropy of the protein and slow down the unfolding dynamics. The same effects would be produced by the viscosity increase or when the system is below Tg and the proteins become “locked” in a distribution of nativelike conformational states, also in the absence of direct interactions. The same is observed with simple dehydrated protein powders.8 The two effects can be seen as “specific” and “aspecific” components of the same preservation mechanism.12,13 As concerns protein dynamics, a strong inhibition has been reported for carboxymyoglobin (MbCO) embedded in trehalose glassy matrixes,6,14−17 which resulted in a marked dependence on the traces of residual water.18,19 The inhibition of internal protein dynamics can be rationalized in terms of a tight anchorage of the protein surface to the matrix, which is at the basis of the coupling of protein and matrix dynamics. FTIR studies pointed out that, within a matrix allowing only smallscale motions, tighter protein−solvent coupling corresponds to better bioprotective properties. This is to be expected since structural alterations of biomolecules must come along with structural alterations to the surrounding matrix.18,20,21 Protein− matrix coupling has been further confirmed through differential scanning calorimetry (DSC), which showed the existence of a linear correlation between the protein denaturation temperature and the Tg of the whole protein−trehalose−water system.22−24 Even though the above results depict trehalose as an ideal biopreserver, from an applicative point of view the addition of a low MW sugar to food or chemicals may cause problems, due to some unfavorable characteristics of sugar chemistry (hygroscopicity, crystallization, sensitivity to athmospheric moisture). For this reason polymers are often added to saccharides in formulations for the preservation of biological matrixes.25 Biopreservation by Polymers. Polymers are another class of molecules, which were found to improve preservation in food and pharmaceuticals, besides their use to tailor the rheological properties of the system. In particular, long chain carbohydrates (such as dextrans or dextrins) are widely used as additives; they have been shown to be effective biopreservers because of their high Tg and of excluded volume effects. High molecular weight polymer molecules are strongly excluded from protein surfaces; hence, they stabilize the protein by crowding more than by direct interaction.26 Their high Tg

values result in strong correlations to the shelf life of food products, with the rate of degradation linearly dependent on the difference between storage temperature and Tg.27 From a molecular point of view, high molecular weight polymers tend to self-organize to form porous media above a given concentration (crossover polymer fraction). If the pores are large enough, proteins can associate, and the kinetics of both protein folding and aggregation may accelerate.28−30 If two or more proteins can aggregate, the final state might occupy a smaller volume fraction,31 so an aggregated state may be more favored than the native one. On the other hand, the enhanced solution density and viscosity in a crowded environment slow down molecular motions, which include degradation events, contributing to protein stabilization. For the same reason, too high a level of crowding could even inhibit both folding and aggregation, by reducing the related kinetic constants.29 Because of this possible structuring, the modeling of crowded systems often leads to misestimations.10,32 Small sugars and large polymers exemplify two different types of stabilization mechanisms: direct or mediated interaction with the biostructures and excluded volume (crowding) effects. Within the same series of substances, increasing the molecular weight causes a shift of the protective mechanism from direct interactions to crowding, as shown in the case of polyethyleneglicols and oligo-/polysaccharides, bringing about an increase of complexity in the matrix modeling.32,33 Oligo-/polysaccharides and disaccharides have been reported to contribute synergistically to bioprotection when used as mixtures, since the latter might “fill in” the porous matrix of the former, improving the interaction with the embedded biomolecules and providing a proper coating.34,35 In these cases, the low molecular weight component is supposed to be the main contributor to the effective stabilization, while the high molecular weight component would provide a high Tg, improving the physical stability of the whole matrix. The obvious downside is that the small disaccharide would act as a plasticizer for the polymer, whose Tg needs to be chosen to be high enough to compensate.3 However, as long as vitrification is achieved in the whole temperature range, a higher Tg does not increase stability.4 A similar concurrent effect has been reported with nonsaccharidic polymers, such as polyethylene glycols, and proteins (BSA, gelatin) mixed with disaccharides. Also in these cases the polymeric components appear to contribute with superior glass transition effects, while the sugars contribute with direct interaction.36 In mixtures of proteins and saccharides, either simple or polymeric, a synergistic effect on molecular packing density and apparent viscosity was observed: a network of weak electrostatic interactions and HBs forms, which increases viscosity with respect to a single component system, granting at the same time a large water retention.37 Moreover, no concurrent increase of the rate of carbohydrate crystallization occurs, granting a good environment for biomolecule stabilization.38 In the case of mixtures of protein and other polypeptides, i.e., a purely proteinaceous matrix with neither saccharides nor other polymers, the H-bond network is left substantially unaltered, except for a slowing down of its dynamics due to an increase of local viscosity.39 This has been shown to be roughly independent of either protein size or secondary structure; a certain dependence of the protein conformational fluctuations on the hydration shell dynamics is observed.40 8732

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water at 353 K, to guarantee its complete solubilization, and then cooled down to room temperature before mixing with the other components. With this procedure it was possible to obtain unstructured gelatin, without degradation. None of our samples showed signals of “structural” gelatin.44 Weight ratios used for the preparation of stock solutions are reported in Tables 1 and 2. Mb/Clg samples were prepared

The aim of this work is to link the biopreservation studies on the stability of saccharide coated proteins to the macromolecular crowding topic, trying to figure out the interplay between excluded volume effects and specific interactions and their different effects on a probe protein, myoglobin. We studied highly concentrated myoglobin samples embedded in solid amorphous matrixes composed by gelatin and gelatin + trehalose, and compared them with analogous samples of pure myoglobin and of myoglobin embedded in amorphous trehalose matrixes (data taken in part from Bellavia et al.23). Gelatin, a polypeptide with a quite simple and regular structure, was used here as a crowding agent. In gelatin matrixes the limited intermolecular association leaves substantial parts of the network in the flexible disordered form,41 where aspecific interaction with small polyhydroxy compounds, such as sugars, occurs. Therefore, mixed matrixes are stabilized against crystallization, a result that is suitable for preservation scopes.42 Moreover, high concentrations of saccharides and collagen (which is a more structured form of gelatin) are commonly found in the extracellular matrix of many organisms in the animal kingdom. The study of these two components yields therefore samples with a composition more similar to in vivo systems, with respect to analogous studies with, e.g., synthetic polymers. The role of water in tuning the physicochemical processes taking place in preserving matrixes is well-known. In particular, the peculiarity of trehalose with respect to other bioprotectants clearly emerges from measurements on samples at a very low hydration level, where both the protein and the sugar compete for the few residual water molecules.2,17 The effects of changes in hydration have been exploited in previous studies to identify the conditions for different preserving mechanisms, along with the structural and/or thermodynamics processes involved.6,15,21,23,24,43 Also in this work we deemed hydration a fundamental property, and therefore, we prepared our matrixes at different water contents, from high (95% w/w) to very low (13% w/w) hydration level. Both thermal properties and structural/functional stability of these samples were studied by differential scanning calorimetry and UV−vis spectroscopy, respectively. The differences and complementarity of the two techniques will be discussed below (see in Materials and Methods section), as DSC measurements are performed under energy flux, while spectroscopy probes the behavior of the system upon prolonged isothermal treatment and can also distinguish the fraction of protein still functional after prolonged thermal stress. It is noteworthy that functional preservation after long-term, roughly isothermal conditions is the main requirement for a protecting agent in practical applications.

Table 1. Weight Ratios Used in Stock Solutions for DSC Measurementsa sample type

Mb% w/w

Clg% w/w

Tre% w/w

Mb Mb/Clg Mb/Clg/Tre

4.34 ± 0.03 4.43 ± 0.03 9.11 ± 0.01

4.04 ± 0.03 3.87 ± 0.01

4.21 ± 0.01

a

Values refer to the mass of the pure components, corrected for the possible presence of hydration water.

Table 2. Weight Ratios of Stock Solutions for UV−Vis Spectroscopy Measurements sample type met-Mb met-Mb/Clg met-Mb/Tre met-Mb/Clg/Tre MbCO MbCO/Clg MbCO/Tre MbCO/Clg/Tre

Mb% w/w 1.99 0.88 0.98 1.00 1.89 1.02 0.99 1.00

± ± ± ± ± ± ± ±

0.05 0.04 0.01 0.02 0.03 0.01 0.02 0.02

Clg% w/w

Tre% w/w

0.88 ± 0.04 0.48 ± 0.03

0.98 ± 0.01 0.49 ± 0.03

0.95 ± 0.01 0.48 ± 0.03

1.00 ± 0.02 0.49 ± 0.03

with roughly the same mass of the two proteins (1:1). Mb/Clg/ Tre samples were prepared with approximately the same mass of gelatin and trehalose, and a double mass of Mb (2:1:1). These choices allowed an effective protein preservation and a good resolution of denaturation signals. The same protein/ matrix mass ratios have already been used in the study of the behavior and preservation of proteins embedded in saccharide matrixes,16,18,23,24,45,46 and will be used here to compare the present results with previous DSC measurements.23,24 With respect to the Mb-only sample, it must be pointed out that our procedure was meant to produce an amorphous myoglobin sample, and not a dehydrated powder. Real amorphous samples of single proteins are difficult to obtain, as dehydration leads generally to crystalline powders by simple water loss. In our case the presence of the buffer salt and the strong nitrogen flux utilized during dehydration prevented crystallization and allowed us to obtain reasonably good amorphous samples. Differential Scanning Calorimetry. Samples for DSC measurements were prepared by pouring aliquots of the stock solution, with multiple depositions if needed, in an aluminum pan with maximum capacity ∼20 μL. Stock solutions for DSC measurements were prepared at the highest possible concentration (see Table 1) suitable to obtain homogeneous and wellpreserved samples, to avoid long, time-wasting, drying procedures and to reduce the number of depositions. Each sample was then dried at room temperature in a desiccator with silica gel (or calcium chloride, for the samples at lowest hydration), connected to a continuous flow of dry nitrogen. During this drying procedure, the samples were monitored by weighing, in order to obtain samples with the required hydration. The pans were then hermetically sealed. With such

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MATERIALS AND METHODS Lyophilized ferric horse myoglobin (Mb) and lyophilized gelatin (type A, produced by acidic hydrolysis from pigskin collagen, Clg) were purchased from Sigma (Sigma-Aldrich, St. Louis, MO). Both proteins were used without further purification. Trehalose (Tre) from Hayashibara (Hayashibara Shoji inc., Okayama, Japan) was used after recrystallization from aqueous solutions. All the other reagents, of analytical grade, were from Sigma-Aldrich (Sigma or Fluka brand), and were used without purification. Stock solutions of Mb, Mb/Clg, and Mb/Clg/Tre were prepared by dissolving the solutes in a buffered water solution (20 mM TRIZMA buffer, pH 6.7). Gelatin was predissolved in 8733

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Figure 1. Soret (left panel) and Q (right panel) bands for met-Mb and MbCO samples after subtraction of background and scattering contributions, including tails from other Mb bands.

solutions were measured at 296 K in quartz cuvettes of 1 cm optical path, on a JASCO V-570 UV−vis−NIR spectrophotometer, equipped with a JASCO ETC-505T temperature controller. Before each measurement the samples were sealed, to avoid gas exchange with atmosphere, and were left 10 min to allow possible aggregates to sediment. With the aim to test any possible alteration Mb might undergo during the sedimentation and measurement steps, we tested the measurement procedure on a MbCO solution prepared at both the concentrations utilized. Data indicated that a sample in a closed cuvette does not undergo any significant change (oxidation, CO loss, precipitation) within 30 min. The whole spectra were analyzed by heuristic fits using pseudo-Voigt curves to fit Q and Soret bands and Gaussian curves for all the other bands in the spectrum. Tabulated band parameters were used as starting values.47 A background, which included the Rayleigh scattering contribution, was also included. All the bands were fit at the same time by minimizing the global residues. This was needed to evaluate at the best the scattering background, which is almost completely hidden under the bands. As for the MbCO samples, in these systems Mb undergoes two processes: oxidation to met-Mb as well as complete degradation and aggregation. The first process makes the MbCO bands disappear in favor of typical met-Mb bands; moreover, in most of the MbCO spectra, MbCO and met-Mb bands are superimposed. This makes the fitting procedure very difficult due to the huge set of fitting parameters to be considered. On the other hand, aggregation makes the bands of both species disappear, with a concomitant appearance of a scattering contribution. Our strategy to perform the data analysis was to fit MbCO spectra by including a curve representing the whole met-Mb spectrum multiplied by a factor representing the fraction of MbCO altered to met-Mb. This curve was obtained from the fitting of the corresponding met-Mb sample (identical thermal treatment times, to take into account the possible alterations of the band parameters). The multiplying factor was considered as a fitting parameter. Then, by minimizing the global residues, we obtained all the parameters of the MbCO bands, and the fractions of met-Mb and MbCO species. The procedure was applied to the analysis of both the Soret and Q bands, for each sample, at each thermal treatment time. In Figure 1 the shapes of the Soret and Q bands for MbCO and met-Mb, after subtraction of the scattering and background contributions, are shown together with the fits for a typical sample. The corrected areas for both species were expressed as a fraction of the total Mb content measured in the reference

a drying procedure we achieved hydration values from 95% w/ w to 13% w/w. DSC measurements were performed with a PerkinElmer Pyris Diamond Power Compensation calorimeter, equipped with a Cryofill device. Indium was used to calibrate the temperature and heat flow. The heat flow error was 0.05 mW. The temperature program consisted of two identical cycles performed as follows: cooling from 303 to 188−133 at 500 K min−1, holding 2 min, then warming to 363−403 at 10 K min−1. An empty sealed pan was used as a reference. The upper heating and lower cooling temperatures were chosen in order to avoid high pressure and allow the observation of both glass transition and myoglobin denaturation. To check the baseline stability and to match the aluminum mass contribution, a temperature cycle on a second empty pan was performed after each measurement. The specific heat variation was evaluated by subtracting the heat flow measured by using empty aluminum pans, normalized to the pan mass, and then dividing by the scan rate and the sample mass. After subtraction of a suitable cubic polynomial baseline, the myoglobin denaturation temperature (Tden) was determined from the position of the irreversible endothermic peak and the denaturation enthalpy from its area. The temperature value at the onset of the low temperature leap of specific heat was assigned to Tg. Absorption Spectroscopy. For UV−vis spectroscopy measurements, stock solutions were prepared with the same component ratios used for DSC (see Table 2). Samples were prepared with either ferric myoglobin (met-Mb) or MbCO. To obtain the latter, the solutions of met-Mb were pre-equilibrated with CO, and Fe(III) was reduced to Fe(II) by addition of sodium dithionite (∼5 mM). Afterward, samples were prepared by layering aliquots (385 μL) of solutions on ∼2 cm diameter round glass windows. Mb-only samples were prepared with smaller aliquots (150 μL) on ∼13 mm diameter CaF2 windows, because their low viscosity made a full coverage of the glass windows impossible. All samples were initially dried for ∼20 h in a silica gel desiccator at room temperature. Met-Mb samples were then dried under vacuum, while MbCO samples were dried in a low pressure CO atmosphere. All dried samples were then put into a vacuum oven at 353 ± 2 K. The heating procedure was stopped after the required time span (1−66 days). Then, just before the measurement, each sample was dissolved in water (previously saturated with either N2, in the case of met-Mb samples, or CO, in the case of MbCO samples) in order to obtain a solution with suitable concentration to get an optimal resolution of the Q band (∼0.3 μM), and then diluted to obtain a second solution with suitable concentration to get an optimal resolution of Soret bands (∼0.03 μM). These 8734

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The Journal of Physical Chemistry B stock solutions. A value at heating time t = 0 was included and refers to a sample desiccated and redissolved before heating. As can be observed in Figure 1, the Q band undergoes a larger modification than the Soret band upon CO loss and oxidation, as it is more sensitive; however, it is more affected by the background and scattering subtraction. Since degradation is expected to affect both the bands, the fraction of a given species was obtained by averaging the values of both bands. Throughout this analysis we assumed that MbCO and metMb are the only species present in the system, or that every other species (e.g., MbO2 or deoxy-Mb) was negligible. Actually in all our heuristic fittings the best fit required only the presence of the two species taken into account, and they were always able to correctly describe the data, making us confident of the procedure. Comparison between DSC and Spectroscopy Results. To best exploit the results of both techniques, we remind the reader about the differences between them. DSC measures the heat flow under a temperature gradient. The heat flow depends both on the thermodynamic heat capacity (multiplied by the applied heating rate) and on a kinetic response factor, which conveys information on all those slow relaxation processes, which do not reach equilibrium in the time scale of the experiment. The measured heat flow can therefore be expressed as Φq = Cp(dT/dt) + kf(T, t), where the first term stands for the thermodynamic (ideal) heat flow, while the second is related to processes whose temporal evolution is not fully accomplished in the experiment time window, and depend also on the specific instrumental setup. In jammed systems, like those studied here, many slow relaxation processes might be present, and kinetic contributions are a considerable fraction of heat flow, in particular in the very low hydration range, where the systems are far from ideality. In addition, these contributions are at the basis of the differences between upscans and downscans (hysteresis) usually noticed in DSC measurements. Note that in our case it is not possible to suppress the kinetic contributions using techniques such as, e.g., Stepscan DSC, in view of the very slow relaxation processes present in our glassy systems. Conversely, our spectroscopy measurements are performed on samples kept for many days at a fixed temperature, and therefore allowed to relax, and then dissolved just prior to the measurement. Even if we cannot exclude relaxation processes with time scales longer than our time spans, there is no question that these contributions should be smaller than in DSC, and further abated by dissolving. Therefore, in the very low hydration range kinetic contributions have different effects on DSC and spectroscopy data. In particular, spectroscopy results should be almost devoid of them and represent the equilibrium behavior to our best ability. They are more useful to draw information on the behavior of a real sample in a preserving matrix, with a usable shelf life. Conversely, DSC results may be used to draw information on, e.g., resistance to heat shocks. Moreover, since CO loss and iron oxidation are local processes at the level of the active site, spectroscopy results on MbCO/met-Mb conversion can be assumed as a measure of functional alteration; i.e., of the processes which are able to hamper the function of the protein, but do not imply massive degradation/alteration. This information has a peculiar relevance from an application point of view, as it allows us to evaluate alterations which happen in long time ranges before full denaturation; these

result in proteins that are unable to properly work, which retain nativelike structures anyway. If a preservation matrix must be used for active biomolecules (vaccines, enzymes), the evaluation of loss of functionality is more relevant than any evaluation of massive degradation or aggregation.

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RESULTS AND DISCUSSION DSC Results. From DSC thermograms, reported in Figure 2, we were able to obtain information about properties of both

Figure 2. Example of thermograms obtained for samples at high (water ∼83% w/w, line a), intermediate (water ∼44% w/w, line b), and low (water ∼16% w/w, line c) hydration. The dashed lines are second scans, after protein denaturation. Baselines are shifted to avoid superposition of the curves.

matrix (Tg) and protein (Tden and ΔHden, Mb denaturation enthalpy) at the same time. Concerning these data, in particular, ΔHden, we stress that they are to be considered apparent values, e.g., not measured under strict thermodynamic equilibrium conditions, in particular in the very low hydration range. In this work we mostly utilize DSC data to compare different systems under the same conditions. As long as we take into account these deviations from ideality, we expect that the conclusions derived from the comparison are convincing. Gelatin glass and melting transitions are not observable in unstructured gelatin systems since they are both very broad signals, with smooth alterations upon phase change. It has been reported that the exact positions of these transitions are hard to observe, because of the molecular polydispersity and the presence of various concurrent relaxation processes.42 Therefore, we will neglect gelatin transitions in the following discussion. It must be remarked that Tg values are obtained from an operational definition, making them very dependent on the method used and the system studied. In any case, since Tg is not an equilibrium quantity, it is very sensitive to system history and state. In particular Tg is not likely to have a single, precise value in complex and intrinsically heterogeneous systems, like the gelatin matrix. Indeed, in our systems some samples showed a clearly measurable glass transition, which yielded a quite scattered set of Tg values, while in others the signals were broad and noisy, in particular in the low hydration range. For this reason we reported only on the former data and abstained from doing statistical analyses. Information on measurement error can be obtained only indirectly from the dispersion of experimental data. Glass Transition Temperature. In Figure 3 the values of Tg as a function of water percentage with respect to the total 8735

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Table 3. Values of Tg′ and Hydration Range for the Transition between Inhomogeneous Ice/Glass Mixture and Homogeneous Glass ([C′g])a system Mb Mb/Clg Mb/Tre Mb/Clg/Tre

T′g (K) 224.1 239.6 212.0 238.5

± ± ± ±

0.9 0.7 1.0 0.5

[C′g] (% w/w water) [31−35] [29−44] [47−57] [25−50]

a

The high and low limits of these ranges are calculated, respectively, as the lowest value at which an inhomogeneous system and the highest value at which a homogeneous system are certainly obtained, each widened by the respective errors on the water/protein ratio.

Figure 3. Onset values of glass transition temperature as a function of hydration. Mb/Tre data are taken from Bellavia et al.23 The vertical bars mark the disappearance of ice melting peak for each system. Error bars are not present as no statistical analysis was done on Tg data (see text).

weight (used as a measure of the hydration level) are reported for the four different systems studied here. In all of them Tg has an almost constant value at high hydration. This is expected since the amount of water exceeds what the glassy system can withstand, and even though the temperature is lowered very fast, a fraction of solvent crystallizes instead of being incorporated in the glass. The higher the hydration is, the higher the fraction of excess water that crystallizes is. This is confirmed by the presence of an endothermic peak of ice melting (see, e.g., Figure 2, line a) in the thermograms. An inhomogeneous mixture of glass and ice is therefore present, and the Tg values, which refer only to the glassy fractions, remain roughly constant. These Tg values correspond to the glass transition temperatures of maximumfreeze-concentrated solution (T′g).9 Conversely, when the thermograms lack the endothermic peak of ice melting, the systems are expected to be homogeneous glasses. At these low hydrations, Mb/Tre system data have been reported to follow the Gordon−Taylor equation23 with a sharp departure from the T′g value. This difference between low hydration Tg and Tg′ is far less evident in all the other systems, and the Tg dependence on hydration is impossible to check in the region of homogeneous glass even in the mixed matrix, where an intermediate behavior might be expected.42,48 This could be caused by nonideal mixing or segregation of the different components of the matrix, a known cause for apparently erratic behavior of Tg in systems with few (or loose) hydrogen bonds.49 The general low quality of data and the absence of a clear Tg signal in some systems make it impossible to deepen the study of Tg. Also, the linear relation between Tg and Tden, reported in saccharide-containing systems,23 cannot be confirmed in collagen-containing samples. To characterize our samples we therefore report in Table 3 the Tg′ values, as obtained from the fitting of the temperature data in the inhomogeneous region, and a transition hydration range ([C′g]), corresponding to the range of the disappearance of the ice melting peak. The Tg′ values are in line with the known behavior of the amorphous systems, as those with higher MW components have higher T′g.9,35 It is worth noting that the T′g and [C′g] values of Mb/Clg and Mb/Clg/Tre systems are very close. This suggests that gelatin, rather than trehalose, dominates the glassy matrix properties, at least in the inhomogeneous region. Role of Collagen as Crowder. In Figure 4 the values of Tden and ΔHden at different hydrations are reported for Mb and Mb/Clg systems.

Figure 4. Denaturation temperature Tden (upper panel) and denaturation enthalpy ΔHden (lower panel) as a function of hydration: comparison between Mb and Mb/Clg. All the data points have both xand y-error bars, though they are often smaller than the point symbols. The vertical lines mark the disappearance of the ice melting peak; thus, the systems are homogeneous glasses to the left of the lines and heterogeneous mixtures to the right.

Two different behaviors can be clearly distinguished, at high and low hydration, respectively. In the high hydration range, in both systems Tden and ΔHden decrease with decreasing water content, down to about 30% w/w water. Then, ΔHden exhibits an apparent change of regime with an enthalpy steep decrease of about 110 kJ mol−1 Mb (see Figure 4, lower panel). At lower water content, the ΔHden trend inverts. In the same hydration intervals, the Tden trend also inverts, slowly increasing after a minimum; this happens in particular in the case of the Mb/Clg system. The behavior in the high hydration regime clearly indicates a progressive destabilization of the native structure that undergoes denaturation at a lower temperature and with reduced enthalpy change. The destabilizing effect of collagen appears therefore to be enthalpy driven. To rationalize this effect we use a model in which protein denaturation is followed by aggregation, according to the following scheme: N ⇋ D′ ⇀ A 8736

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The Journal of Physical Chemistry B Here, D′ is a fully or partially denatured state in equilibrium with the native state N; D′ may be seen as a metastable state needed in the aggregation process.30,50−52 Tden and ΔHden measured with calorimetry refer to the N ⇋ D′ process. In this model the excluded volume effects favor aggregation and shift the equilibrium constant toward D′. This becomes apparent as a reduction of both Tden and ΔHden. It is clear that this is possible as long as enough water remains in the system to allow the protein large-scale fluctuations, needed to expose hydrophobic residues and to access the D′ state. Reduction of water content in this hydration range could cause an increased probability of interaction among hydrophobic patchy areas on the protein surfaces, leading to denaturation and precipitation. Since collagen is also a protein, the same mechanism could be effective in gelatin-containing matrixes, leading to the observed destabilizing behavior, barely distinguishable from the Mb-only one (see Figure 4). By further lowering the water content, the crowded protein matrix becomes stiff due to the lack of free water; this inhibits large-scale diffusive-like motions and encumbers the protein high tier dynamics, thus stabilizing the native state. The low hydration behavior observed for the Mbonly samples confirms that crowded myoglobin can be stabilized against thermal denaturation, even in the absence of preserving cosolutes. It is remarkable that the stabilization observed at low hydration is larger for Mb/Clg than for Mb-only systems and can reach Tden values larger that 370 K, well above the Tden values observed at high hydration. One could suggest that, in the Mb/Clg system, collagen molecules could interpose among myoglobin molecules, leading to a crowded stiff matrix wherein protein denaturation is inhibited. Indeed, in a system where the accessible volume is already low, the Mb fraction able to denature and aggregate would be further reduced, causing the steep increase of Tden and ΔHden. This kind of behavior is common for diffusion-limited processes in crowded systems.30,50,51 It should also be noted that, notwithstanding the marked Tden increase, ΔHden values increase only moderately, not even recovering their high hydration values. This is indicative of entropy driven effects, typical of crowded systems. Role of Trehalose in Crowded Environment. In Figure 5 the values of Tden and ΔHden at different hydrations are reported for Mb/Tre and Mb/Clg/Tre systems. Tden and ΔHden data for Mb/Clg are also reported for the sake of comparison. The plot of Tden as a function of hydration for the Mb/Tre system (Figure 5, upper panel) is noticeably different from analogous plots for all the other systems. Tden increases continuously in the whole temperature range, with increasing slope at low hydration. No sign of protein destabilization is observed, even though the highest value in Mb/Tre does not reach the values of Mb/Clg at very low hydration. Also, ΔHden values (see Figure 5, lower panel) show a unique regime, with a continuous, mild, decrease in the whole range. No steep decrease is observed at low hydration as in other cases. Furthermore, at each water content, higher values than those for Mb/Clg and Mb/Clg/Tre systems are observed. The behavior of both Tden and ΔHden stems from the nature of the Mb/Tre system. At variance with the other cases, trehalose systems are homogeneous solution and homogeneous glasses before and after Tg′, respectively. From the molecular point of view, it has been demonstrated, by direct and indirect

Figure 5. Denaturation temperature Tden and denaturation enthalpy ΔHden as a function of hydration: comparison of Tden among Mb/Clg, Mb/Clg/Tre, and Mb/Tre systems (upper panel, the latter data are taken from Bellavia et al.23); comparison of ΔHden between Mb/Clg, Mb/Clg/Tre, and Mb/Tre systems (lower panel). The vertical lines mark in the disappearance of the ice melting peak; thus, the systems are homogeneous glasses to the left of the lines and heterogeneous mixtures to the right. In the upper panel also the fitting curve for the Mb/Clg/Tre system in the high hydration range is shown.

measurements, that in a trehalose−water matrix HB networks encompassing the sample are present, involving protein groups, sugar, and water molecules and in which the matrix rigidity can be uniformly tuned by lowering the water content.14,17 Here macromolecular crowding plays little effect, if at all, whereas the specific interaction between trehalose and the Mb hydrophilic surface is the main stabilizing mechanism. As has been observed, the decrease of water content promotes peculiar sugar−protein bonds stabilizing the protein.11 A direct matrix− protein interaction could in turn rationalize the behavior of the thermodynamics quantities, as already discussed by Bellavia et al.23 Indeed, by definition, the ratio ΔHden/Tden is the denaturation entropy (ΔSden). Since Tden increases while ΔHden decreases, it follows that ΔSden decreases more rapidly than ΔHden. This means that the stability increase in water/ trehalose matrixes at low hydration is an entropy driven process. Quoting from Bellavia et al. one could conceive that “...lowering the water content affects the protein−matrix interactions so that the reduction of the degeneracy of the unfolded state dominates the free energy differences between the folded and the unfolded state”.23 In the Mb/Clg/Tre system both Tden and ΔHden exhibit a pattern similar to what is observed in the Mb/Clg one, with values intermediate between those of Mb/Clg and Mb/Tre systems. It seems that the presence of trehalose attenuates, but does not cancel, the myoglobin destabilization at mid-to-high hydration. As reported in the literature this behavior might arise from the superimposition of contrasting effects of trehalose and protein concentration (crowding).53 Moreover, the separation in two distinct regimes and the sharp increase in Tden at very low hydration hold, although the increase is less steep. 8737

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The Journal of Physical Chemistry B It is tempting to try a description of the behavior of the Mb/ Clg/Tre system as a combination of the behaviors of Mb/Clg and Mb/Tre systems. With this aim, we did a heuristic fitting of the Tden versus water/protein ratio in terms of a weighted sum of polynomial curves for both the binary systems. We obtained the best fit of Tden for Mb/Clg/Tre in the high hydration range by using a linear combination with a weight for the Mb/Clg curve close to twice the weight for the Mb/Tre curve (see in Figure 5, upper panel, green solid line). However, at lower hydration the fitted curve never matches the data, regardless of the weight choice. This leads us to suppose that trehalose and collagen contributions to Mb stabilization/destabilization are simply additive at high water content,53 with the destabilizing effect of collagen being twice more effective than the stabilizing effect of trehalose; differently, the collagen/trehalose reciprocal interaction is more complex at low hydration. Role of Crystallizable Water in Protein Preservation. In this final section, we would speculate on the role of crystallizable water in our systems. As shown in Figures 4 and 5, in Mb-only, Mb/Clg, and Mb/Clg/Tre the inversion point in the ΔHden regime is located roughly in the same hydration range of the disappearance of the ice melting peak (see in Figures 4 and 5, the vertical lines in the 27−33% range). Since partial denaturation always requires a residual protein mobility (or low viscosity), it can happen only if an excess water is present. The disappearance of the ice melting peak corresponds to depletion of excess water, and hence to the formation of a stiffer matrix in which protein denaturation is prevented just by crowding. Therefore, the disappearance of free water appears to trigger the onset of preserving effects. Remarkably in the trehalose system the disappearance of crystallizable water, which happens at a rather high water content (purple vertical line at about 45% w/w), has no apparent effects. This is in line with the consensus molecular view of trehalose−water matrixes, in which lowering the water content smoothly affects the protein−matrix interaction.6,14,17 Indeed, when water is removed, trehalose hydroxyl groups replace it and directly bind to protein residues (“water replacement hypothesis”, see Introduction). Stabilization is accomplished here by specific, noncrowding, effects. Spectroscopy Results. Before discussing the spectroscopy results we remind the reader that when our dry samples aged at 353 K are dissolved in water, myoglobin molecules that have undergone the massive denaturation process aggregate and precipitate out of the solution. Undenatured myoglobin molecules that have undergone an oxidation process are found in solution as met-Mb, while the native ones are found as MbCO. The procedure described in the Materials and Methods allows us to quantify, as a function of the heating time, the total fraction of undenatured protein molecules (referred to as “recovery fraction”) and the fraction of oxidized protein molecules (referred to as “CO loss”). Results are reported in Figure 6 (upper panel, recovery fraction; lower panel, CO loss). We stress that recovery fractions represent the loss of folded Mb caused by denaturation and subsequent aggregation: they stand for the structural/spectroscopic counterpart of thermodynamics results obtained by DSC measurements. However, we remark that spectroscopic data are obtained after prolonged treatments, lasting many days, and therefore might include also the outcomes of phenomena on very long time scales. Conversely, “CO loss” represents the oxidation process of native Mb molecules and gives a measure of local alteration of the heme

Figure 6. Total Mb recovered fraction (upper panel) and fraction of CO loss by the MbCO species (lower panel). Lines are only visual guides.

pocket region (the functional group of Mb), which does not involve massive denaturation. Therefore, CO loss will be used here as a measure of “functional” degradation, i.e., degradation which impairs protein functionality leaving the overall tertiary structure roughly unaltered. In Table 4 values for “half-life” Table 4. Recovery Half-Life for Denaturation and CO Loss

a

system

[t1/2] (h)

Mb Mb/Clg Mb/Tre Mb/Clg/Trea MbCO MbCO/Clg MbCO/Tre MbCO/Clg/Tre

[130−380] [930−1500] [510−630] [1080−1350] [95−115] [330−380] [540−580] [770−940]

This result was calculated by extrapolation.

time ranges ([t1/2]) are reported for both the processes. They represent the time span of 353 K treatment after which the amount of a given species is halved. With respect to massive degradation, the data show that all the matrixes have some protecting effect. The Mb-only system is, as expected, the less effective: after about 300 h at 353 K only half the total amount has not denatured. This value is however considerably higher than that for a protein solution, where massive denaturation happens in a few hours, but is lower than in solid systems containing preserving agents. As for the Mb/Clg, Mb/Tre, and Mb/Clg/Tre matrixes, taking into account the error bars, they have roughly the same efficiency against denaturation up to about 350 h of heating, after which Mb/Tre starts to fail with respect to the gelatin-containing systems. The gelatin matrix appears to be considerably more protectant than trehalose: half-life values of about 1250 h are observed for both Mb/Clg and Mb/Clg/Tre. This is 8738

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The Journal of Physical Chemistry B compatible with DSC results in the very low hydration regime (see Figure 5) and confirms that at low hydration crowding effects prevail upon specific interactions. We turn now to functional degradation. Also, in this case, some protection against CO loss is evident in all the matrixes; actually, CO loss begins within minutes in MbCO dilute solutions at 353 K. In the Mb-only matrix the half-life of bound CO is on the order of a few days, showing the limited effect of simple self-crowding. Gelatin matrix appears to be more effective, with 50% of the CO present up to nearly about 350 h (2 weeks) of heating, with 20% possibly remaining also at very long times (>1000 h). Different from the massive denaturation, the trehalose matrix is able to preserve half the initial amount of CO for more than about 500 h, with this effect being even larger for the mixed trehalose/collagen matrix. In trehalose the preservation appears to be stronger in the initial and middle times. Comparing the results of massive denaturation and functional degradation it can be seen that functional degradation and massive denaturation occur in the same heating time range in the trehalose matrix: the trehalose preserving action appears to be particularly relevant at short times and against CO loss. On the other hand, myoglobin in gelatin is less able to withstand the CO loss. In this case, myoglobin is not directly stabilized and may easily undergo small scale conformational fluctuations, that cause deformation at the heme pocket site. Steric hindrance, caused by crowding, likely hinders only very large-scale fluctuations, but still allows the local structural fluctuations needed for CO loss, even at low hydrations. Therefore, massive denaturation will be prevented with respect to functional degradation, explaining the inversion in relative efficiency between sugar and gelatin. These results point out the role of trehalose in the preservation process, and confirm what was reported in previous works.11,18,45 In fact, functional degradation does not require complete denaturation, and occurs as a consequence of the motion of residues in the proximity of the heme pocket. Trehalose interacts, either directly or indirectly, with protein surface, and inhibits these motions.11,54 Conversely, gelatin matrixes do not exhibit such localized effects. In the mixed gelatin + trehalose matrix the two preserving agents likely act in synergy giving the best results against functional degradation. Here protein denaturation is prevented with the same efficiency of the gelatin matrix, but the presence of trehalose gives a better functional preservation efficiency. We suppose that in this system the fraction of inaccessible volume occupied by collagen acts to both lower Mb high tier dynamics and force trehalose molecules near the Mb surface, increasing the local concentration of trehalose and granting the best efficiency.

while trehalose decreases it; at low hydration (homogeneous samples) a correlation between Tg and protein denaturation temperature is present only for trehalose, but absent for gelatincontaining matrixes. Concerning the protein massive degradation, at medium to high hydration trehalose stabilizes myoglobin while gelatin destabilizes it. A possible explanation for this different behavior could be as follows: at these water contents, trehalose preserves protein’s native solvation by water entrapment, impairing protein−protein interactions leading to denaturation and precipitation. At variance, the hydrophobic nature of collagen surfaces would favor myoglobin−collagen hydrophobic patching, as long as enough water is present to allow protein and/or collagen dynamics. The effects of the two agents are additive, as shown by the behavior of the mixed matrixes. At low hydration, gelatin matrixes are very effective against massive degradation, leading to a very sharp rise of the Tden and to an increase of ΔHden. This has been suggested to arise from curbing the high tier motions leading to denaturation. Gelatin superiority over trehalose is remarkable, with the pure gelatin matrix performing even better than the mixed gelatin + trehalose matrix. The DSC results are fully confirmed by the spectroscopy results obtained in thermally equilibrated samples at low hydration where gelatin-only and gelatin−trehalose mixed matrixes perform similarly, both outdoing the trehalose-only matrix. The crowding-related effects observed in both Mb-only and Mb-gelatin samples appear to be analogous: a destabilization in the high hydration range and a stabilization at low hydration, with the transition between the two regimes being roughly marked by the disappearance of ice melting peaks. The ability of retaining bound CO molecules after thermal treatments is a measure of functional preservation, i.e., the preservation of the protein in native or native-like forms, which are still able to perform their original functions. Spectroscopic measurements performed at very low hydration indicate a preserving effectiveness higher in trehalose than in gelatin, in particular at low heating times, when a large fraction of the probe protein is still in nativelike form. This could be expected as trehalose mainly interacts with the protein surface, thus impairing local structural deformation, while gelatin is mainly effective where large-scale conformation fluctuations and/or volume variations occur. Conversely, at long heating times, when also massive denaturation is occurring, the mixed matrix appears to have the best performance, likely taking advantage of the combined effects of both the components. The reported results highlight the different behaviors of the two matrixes, and their possible synergy; this parallels what is reported in the literature about the combined effect of, e.g., glucose and hydroxyethylstarch on liposomes, in which the monosaccharide depresses the dry lipid melting temperature, with little inhibition of fusion, except at extremely high concentrations. Conversely, the polymer has no effect on the phase transition, but inhibits fusion.55 Such combinations of molecules are likely to be found also in nature, in anhydrobiotes. As an example, in the desiccation tolerant alga Nostoc, a glycan and some oligosaccharides apparently work in conjunction, as proteins and saccharides also do, being both required for stability. Also, the saccharide phase state in vivo was shown to be dependent on protein content.55 The results of this work contribute to identifying which stages of the biopreservation process are influenced by either

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CONCLUSIONS In this work we studied the preserving properties of solid amorphous systems under conditions of thermal stress. We compared a simple crowded system constituted by amorphous myoglobin with matrixes containing preserving agents, working either by crowding or by direct interaction. The results point out that specific and aspecific preservation effects have outcomes that are clearly distinguishable; hence, their use can be tailored to requirements. Our results can be summarized as follows. Concerning the glass transition properties, at medium to high hydration (inhomogeneous samples) gelatin increases T′g 8739

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The Journal of Physical Chemistry B

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the polymeric component or the saccharide one, and when the mixture gives the best benefit; these results may also be exploited to tailor the preserving agents to the real degradation process and to allow a good preservation of biological material.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: *E-mail: *E-mail: *E-mail:

[email protected]. sergio.giuff[email protected]. [email protected]. [email protected].

ORCID

Enrico F. Semeraro: 0000-0002-6096-1108 Sergio Giuffrida: 0000-0003-3704-7358 Present Addresses §

Laboratoire de Rhéologie et Procédés, CNRS, UMR 5520, F38041, Grenoble Cedex 9, France. ∥ Direzione Centrale Analisi Merceologica e Laboratori Chimici, Agenzia delle Dogane e dei Monopoli, via M. Carucci 71, I00143, Roma, Italy. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the financial support by MIUR (Grant PRIN 2008ZWHZJT “Struttura-Dinamica-Funzione di Biomolecole in Sistemi lontani dall’idealità termodinamica”) and the University of Palermo (FFR Program 2012/2013).

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