7208
Langmuir 2002, 18, 7208-7217
All Gelatin Networks: 1. Biodiversity and Physical Chemistry† Christine Joly-Duhamel, Dominique Hellio, and Madeleine Djabourov* Laboratoire de Physique et Me´ canique des Milieux He´ te´ roge` nes, Ecole de Physique et Chimie, UMR ESPCI-CNRS 7636, 10 Rue Vauquelin, 75231 Paris Cedex 5, France Received February 15, 2002. In Final Form: May 16, 2002 Gelatin gels are well-known for their ability to form nonpermanent, physical gels at room temperature and for their numerous applications in photographic and food industries. The difficulties in understanding the processes of gelation and the properties of the physical gels is partly due to the nature of the molecules that exhibit this state: these are often biopolymers. Concerning gelatin, a regain of interest appeared recently, in relation with the development of new types of moleculessmammalian gelatins were almost exclusively considered so far in the literature. In this series of two papers we investigated gelatin samples from various origins, mammalian and fish gelatins, with the aim of comparing their properties to the reference samples generally used in photographic and food applications. The samples were characterized by their imino acid composition, which indeed varies according to the biodiversity of the species from which they are extracted. The physical and chemical aspects are mainly reported in this paper, while the companion paper deals with the rheological properties. The influence of the thermal treatments, of the gelatin concentration, of the molecular weight, and of the solvent (aqueous and mixed solvents) was put in evidence and finally blending of samples was achieved. Optical rotation measurements were mainly performed; they allow us to fully characterize the development of the triple helices, which depends on the parameters mentioned before. A systematic comparison of the temperatures of helix formation and melting was undertaken. The results are discussed in the context of the models of helix-coil transitions often used for proteins and polypeptides and sometimes for polysaccharides.
Formation of physical networks in protein and polysaccharide solutions has been intensively investigated over the last years1-3 because of their many important applications or simply because they are difficult to understand. Some of these biopolymers undergo a specific conformational transition from coil to helix during which the physical network is formed. The relation between the molecular characteristics and the rheology of the networks is one of the important goals of these studies, compared to the more conventional rubberlike, chemical networks. The formation of extended helical structures characterizes the networks. The coexistence of ordered structures, which are often described as rigid rods, and of flexible coils is the starting point of theoretical models for the elasticity of these networks. The adequacy of these models to describe the real networks is difficult to assess. The physical networks obtained by cooling the solutions are disordered and their properties vary considerably from batch to batch, which makes comparisons between measurements obtained by different techniques or different authors very delicate. The variability of chemical composition of the biopolymer, the molecular weight, the polydispersity, the modifications of the environment such as pH, ions, additives such as sugars, polyols, etc., the thermal history, or the passage of time itself have been shown to substantially influence the mechanical and thermal properties of these physical gels. It is thus difficult to identify the relevant parameters, which should be kept in mind in † This article is part of the special issue of Langmuir devoted to the emerging field of self-assembled fibrillar networks. * To whom correspondence should be addressed: e-mail
[email protected].
(1) Faraday Discussion 101, Gels, 1995. (2) te Nijenhuis, K. Adv. Polym. Sci. 1997, 130. (3) Wiley Polymer Networks Group Review Series; Stepto, R. T. F., Ed.; John Wiley & Sons Ltd.: Chichester, U.K., 1998 and 1999; Vols. 1 and 2.
order to model their elasticity on the basis of a stressstrain relationship, at a molecular level. The question that still remains is how to find the right parameters to enter in such models? On one hand, many parameters have been identified experimentally; on the other hand, the models make use of very few of them. Also, one does not necessarily know how to measure some particular parameters that are introduced in the models. This is the paradoxical situation of physical gels! In this series of two papers we consider gelatin gels. When a gelatin solution is cooled below room temperature, the protein coils start to form triple helices and progressively a 3D network is formed. The triple helices are reminiscent of the native structure of collagen. The residues can only partially recover their native conformation; a certain proportion still remains in the random coil conformation, even when the samples are annealed for hours or days. The gel is definitely not an equilibrium state. When temperature is raised back above approximately 30 °C, the reverse transition, helix to coil, takes place and the gel becomes liquid. Many parameters, as those mentioned before, have a dramatic influence on the thermal and mechanical properties of the gels. The aim of this investigation is to take into account a large number of these parameters, which govern gel formation, and derive the common features that can serve as a basis for modeling the physical gels. We addressed these questions in the context of the emergence of new types of gelatins such as those extracted from fish skins of various species aimed to supplant the traditional ones. Fish gelatins are certainly different from mammalian gelatins. Fishes being, in general, nonhomoeothermic animals (they do not have a constant body temperature, like mammals), their body temperature depends on the temperature of the water where they live (rivers, seas, etc.). Therefore, fish collagens exhibit a large biodiversity in relation with their environment that the mammalians do not have, especially in their
10.1021/la020189n CCC: $22.00 © 2002 American Chemical Society Published on Web 07/10/2002
Gelatin Networks: Biodiversity and Physical Chemistry
imino acid composition. Accordingly, there is a wide range of gelation temperatures for these gelatins, different from the traditional ones.4-7 In the first paper of the series we compare the thermal properties of gelatin gels of various sources under various treatments: either cooling, annealing, or heating conditions. All gelatin samples were fully characterized at a molecular scale. Structural properties, basically the helical content, were determined for gelatins from different sources (mammalian and fish), various molecular weights, and isoelectric points. The fish gelatins came from fish skins either from cold seas (cod) or warm seas (tuna, megrim). They were compared to lime-processed bovine ossein, considered as a reference type for photography, and to pig skin gelatin, which is mostly used in food applications. We investigated these samples mainly in aqueous solutions, at their natural pHs. Their properties in the presence of additives (mixed solvents) were also briefly examined. We also investigated blends of gelatins. The structural data are discussed in the context of the well-known models for helix-coil transitions. In the second paper we focus on the rheological properties (shear moduli) in the linear regime, for gels formed in exactly the same conditions as for the structural investigation. The correlation between the two types of properties was systematically sought and allowed to define a unique master curve for all gelatins, for all conditions of gel formation, without any adjustable parameter. This paper contains three sections: materials and methods, results of optical rotation measurements, and a theoretical discussion based on the Zimm-Bragg model of helix formation. I. Materials and Methods The reference to collagen being necessary for understanding the difference between the various gelatin samples, we first recall some properties of these collagens. In particular, the melting temperatures of the collagens are important for the comparison between species. Collagens. Several solutions of soluble collagens were examined: calf skin (provided by SKW), sole from Coletica (Neptigene 2), and tuna and cod that we prepared ourselves with the following protocol: the fish skins were washed twice in demineralized water during 30 min, then in ammonium acetate (2 L, 0.5 M), and then again in demineralized water. The extraction of collagen was made in acetic acid in a cold store during 2 days. The skins first swelled and after 2 days in acetic acid were dislocated and collagen was dissolved. The solutions were filtered and centrifuged during 1 h at 12000g in order to eliminate impurities from skins. For further purification, the collagen was precipitated by adding salt to a final concentration of 0.8 M NaCl. The suspension was again centrifuged. The solid phase (collagen) was collected, washed, and once more dissolved in acetic acid in a cold store and centrifuged. Finally, the solution was cleaned by dialysis in order to eliminate the remaining salt in acetic acid. The concentration of collagen in solution was determined by solvent elimination (amount of dry material). The imino acid composition of mammalian and fish gelatins and the corresponding melting temperatures of the native collagens are presented in Table 1. Gelatin Samples. A large variety of gelatin samples was investigated. Two samples were from mammalians, provided by SKW (France): lime-processed demineralized bovine ossein extractions with two different molecular weights A1 and A2, acidic extractions from pig skins, also with two molecular weights, (4) Leuenberger, B. H. Food Hydrocolloids 1991, 5, 353-361. (5) Gilsenan, P. M.; Ross-Murphy, S.-B. In Wiley Polymer Networks Group Review Series; Stepto, R. T. F., Ed.; John Wiley & Sons Ltd.: Chichester, U.K., 1999; Vol. 2, Chapt. 28. (6) Gilsenan, P. M.; Ross-Murphy, S. B. Food Hydrocolloids 2000, 14, 191-195. (7) Gilsenan, P. M.; Ross-Murphy, S. B. J. Rheol. 2000, 44, 871-883.
Langmuir, Vol. 18, No. 19, 2002 7209 Table 1. Imino Acid Composition of Gelatins and Melting Temperatures of Corresponding Collagens imino acid (g/100 g of protein) bovine gelatin pig skin gelatin tuna sole or megrim cod a
Pro
Hypa
total
Tm(collagen), °C
13.03 13.99 11.61 12.86 10.29
12.23 11.15 10.49 9.06 6.72
25.26 25.14 22.1 21.92 17.01
36b 36b 29 28 15
Hydroxyproline. b Calf skin collagen.
B1 and B2. Various gelatin acidic extractions from fish skins were also studied: cod gelatin provided by Icetec (Reykjavik, Iceland), megrim from Instituto del Frio (Madrid, Spain), and tuna from SKW (Boulogne, France). The molecular characteristics and the isoelectric points of these gelatins are summarized in Table 2 and were provided by the laboratories mentioned above. The A1, B1, tuna, and cod samples may be considered as highquality extractions, containing high proportions of single, linear coils (called R-chains, Mw ≈ 125 000 g/mol): 48% in A1, 25% in B1, 31.5% in tuna, and 35% in cod. The megrim sample contained 35% β-chains (double the molecular weight of the R-chains) and only 22% R-chains, meaning that the extraction did not fully separate the single chains; they were still partially cross-linked. For each sample, the molecular weight distribution also includes some smaller molecular weights (limited to 25 000 g/mol) and occasionally larger ones (cross-linked coils). We also had at our disposal a very degraded gelatin (hydrolyzed sample) of type A from SKW, with a very low molecular weight, which is a nongelling sample (Mw ) 11 200 g/mol and an index of polydispersity I ) 1.6). Its molecular weight distribution is entirely located below the previous ones. This sample was interesting to investigate for illustrating the strong molecular weight effects in helix formation. The isoelectric points pI of gelatins depend on the extraction process: they are around 5 for basic extractions and around 8 or 9 for acidic extractions. Basic extractions increase the number of COO- groups by hydrolysis of lateral groups of Asp and Glu. The solvent for gelatin solutions was mainly demineralized water. We also used mixed solvents of water and glycerol (Rectapur 98%, Prolabo) or sorbitol [D-(-)-sorbitol, M ) 182.17 g/mol, Prolabo) from Merck Eurolab. The pH of the solutions was close to 5.7 for basic extractions and 5.1 for acidic extractions. Protocol of Gelatin Dissolution. The required quantities of gelatins were mixed with water and allowed to swell overnight at 4 °C. The A and B samples (the mammalian gelatins) were then dissolved at 45 °C during 30 min and the fish gelatins at 35 °C, by use of a magnetic stirrer. The tuna and cod solutions were filtered (0.45 µm disposable filters, Millipore). The crucial aspects in the protocol of dissolution are the control of temperature and time during which the solution is kept at high temperature, because of the risk of hydrolysis, which modifies the molecular weight distributions. It was also noticed that the solutions which gelled do not recover completely after heating their initial stage of dispersion, because some local associations of the chains (especially by hydrophobic groups or entanglements) still persist. Higher temperatures or longer periods of heating induce substantial degradation of the chains and must be avoided. Because of these restrictions, each solution was used only once. Methods. Optical rotation was measured on a Perkin-Elmer 341 polarimeter, equipped with a PC computer, with a software specially developed to collect simultaneously the optical rotation angle and the temperature of the sample versus time. The glass cell has an optical path of 1 cm and is jacketed to allow temperature to be controlled from an external circulating bath. The wavelength could be varied as the emission lines of Hg and Na lamps. Temperature was regulated by a Julabo FS18 bath that was programmed to execute various steps of cooling, annealing, or heating. Cooling ramps of 0.5 ˚C/min and heating ramps of 0.05 ˚C/min were mainly applied. The conformational change from coil to helix is accompanied by an important change in the optical rotation of the solution. In the case of collagen triple helices, it is possible to derive the amount of helices or the
7210
Langmuir, Vol. 18, No. 19, 2002
Joly-Duhamel et al.
Table 2. Molecular Characteristics of the Gelatin Samples gelatin Mw (g/mol) Mn I ) Mw/Mn pI
A1
A2
B1
B2
megrim
tuna
cod
145 700 86 300 1.68 5
102 200 52 400 1.95 5
168 500 88 370 1.91 8.7
74 600 39 540 1.89 7.6
226 300 136 325 1.66 ∼8.9
122 600 74 780 1.64 ∼8.9
120 500 62 435 1.93 ∼8.9
Table 3. Specific Optical Rotation of Gelatin Solutions in the Coil Conformation gelatin temperature (°C) coil [R]436nm
A1
A2
B1
B2
megrim
tuna
cod
35 -290 ( 2
35 -278 ( 1
35 -285 ( 1
35 -264 ( 1
30 -263 ( 2
30 -261 ( 1
15 -245 ( 1
fraction of amino acids in the helical conformation from the specific optical rotation of the solution. The procedure is explained in ref 8. The helix amount χ
χ)
number of residues in helical conformation total number of residues
is derived from
χ)
[R]λexp - [R]λcoil [R]λcollagen - [R]λcoil
(1)
where λ is the wavelength (most of the experiments were performed at λ ) 436 nm), [R]λexp ) R/cl is the specific optical rotation of the protein in solution, c is the concentration (grams per cubic centimeter), l is the optical path (decimeters), R is the optical rotation angle (degrees) measured experimentally, [R]λcollagen is the specific optical rotation of native soluble collagen (χ ) 1), which contains only triple helices, and [R]λcoil is the specific optical rotation of the coils (χ ) 0). An average value of 100 g/mol per amino acid was taken. At high temperature, the chains have a random coil conformation. The specific optical rotations for the different gelatins in coil conformation were derived directly from the measurement in solutions at high temperature. The exact concentration of gelatin was derived by weighing after drying the solutions (the granules of gelatin contain usually around 10% humidity). The specific optical rotations of the different gelatins in the coil state are given Table 3. The specific optical rotation in the coil conformation is independent of concentration below c ) 10% g/cm3. However, it varies slightly with temperature and solvent (for mixed solvents). We determined experimentally a drift of [R] (with the units given before) by ∆[R]/∆T ) 0.8/°C for sample A, 0.7/°C for tuna, or 0.5/°C for cod. This small variation may have at least two origins: changes of the index of refraction of the solvent or changes of local conformation of the coils. Thus, we kept the values of [R]coil436nm at the ultimate temperature before it starts to vary strongly at the beginning of the coil to helix transition. We were also able to measure the specific optical rotation of native collagens, calf skin, and sole skin at 436 nm. We found the following values: [R]helix436nm ) -800 deg cm3 g-1 dm-1 for calf skin collagen and [R]helix436nm ) -810 deg cm3 g-1 dm-1 for sole collagen. The value for calf skin is in agreement with the literature.8-9 We could not estimate precisely the values for all collagens, because all samples were not sufficiently pure; thus we decided to use a unique value for the specific optical rotation for all collagens, which is the value measured for the sole collagen and close to that of calf skin collagen (100% of residues in helix conformation). Viscosity measurements were performed in dilute and semidilute gelatin solutions at 40 °C. An Ubbelohde viscometer was (8) Djabourov, M.; Leblond, J.; Papon, P. J. Phys. France 1988, 49, 319-332; 49, 333-343. (9) von Hippel, P. H.; Wong, K.-Y. Biochemistry 1963, 2, 1399-1413.
Figure 1. Melting curves of soluble collagens of various origins in acidic solutions, derived from optical rotation measurements (dR/dT) at a heating rate of 0.05 °C/min. used for the determination of the intrinsic viscosity (c < 0.02 g/cm3) and a stress-controlled rheometer, AR 1000 from TA Instruments, with a concentric cylinder with double gap cup for larger concentrations (from 0.02 to 0.08 g/cm3).
II. Results: Optical Rotation Measurements Melting Curves for Collagens. The first important step for understanding the gelation of the different gelatin samples is the melting or the helix-coil transition of soluble collagens. The temperature of this transition is well characterized when the extraction of collagen preserved its native structure (helices). Optical rotation can be used to derive the melting temperature Tm and to establish the dependence of the helical content with temperature. In the experiments that we performed on fish collagens, the temperature was raised at a fixed rate of 0.05 °C/min. The optical rotation angle, R recorded during the temperature ramp, is a sigmoid going from R of the 100% helix to R of the 100% coil (χ varies between 1 and 0). The derivative of this curve, dR/dT or dχ/dT, exhibits a peak giving the average melting temperature. The width of the curve indicates the spread of temperatures. The results for our collagens are shown in Figure 1. The calf skin preparation was certainly a good one: there is a large peak and the width is only a few degrees. It exhibits the highest melting temperature. All the fish collagens melted at lower temperatures compared to calf skin, the cod having the largest deviation. The cod collagen extract was not as good as the other preparations, as it appears from the shape of the melting curve, where the total amount of soluble collagen was lower than expected and the width of the curve larger than in the other collagens.
Gelatin Networks: Biodiversity and Physical Chemistry
Figure 2. Helix formation for gelatins of various origins versus temperature at a cooling rate of 0.5 °C/min. (concentration 4.5% g/cm3).
The correlation between the imino acid content of the collagens, given Table 1 and their melting temperatures, is clear: the larger this content, the higher the melting temperature in acidic solutions. Gustavson10 first noticed the correlation between the denaturation temperatures of collagens and the environmental temperatures of the fishes. Later on, the relationship between the denaturation temperatures and the imino acid content (Pro + Hyp, hydroxyproline) has been established for vertebrate and invertebrate species11 in relation with the mechanism of stabilization of the triple helices by the pyrrolidine residues. More recent studies,12 however, stress the specific role of Hyp for collagens of vertebrates, because, in this case, the location of Hyp in the sequence of the protein is different from that in invertebrates. Renaturation and Melting of Triple Helices. The following gelatin solutions have been investigated: (a) aqueous solutions, single-component gelatins, (b) mixed solvents, single-component gelatins, and (c) aqueous solutions, blends of gelatins. We report first on the single component gelatins. (a) Aqueous Solutions, Single-Component Gelatins. Helix formation was investigated for gelatin solutions at a fixed concentration of 4.5% g/cm3; temperature was decreased linearly in time. Gelatins of various sources are compared in Figure 2, where helix amount is plotted versus temperature. The coil to helix transition starts at different temperatures; one can notice that it varies in the same order as the melting temperatures of collagen. However, this type of curve depends on the rate of cooling and concentration. Thus, from these experiments one cannot define precisely a temperature at which helices start to form but a range of temperatures obviously below the melting temperatures of the native collagens in aqueous solutions. The rate of helix formation follows the same trend for all gelatins: the lower the temperature, the larger the helix amount formed during the cooling ramp. A long annealing time provides additional amounts of helices, which never reach 100% within the “normal time scale” of observation. We could notice an increase, up to χ ) 0.55 for cod gelatin or 0.65 for A1 (see, for instance, Figure 4) (10) Gustavson, K. H. In The chemistry and reactivity of collagen; Academic Press: New York, 1956. (11) Harrington, W. F.; Rao, N. V. In Conformation of biopolymers; Ramachandran, G. N., Ed.; Academic Press: New York, 1967; Vol. 2., pp 513-531. (12) Privalov, P. L. Adv. Protein Chem. 1982, 35, 1-104.
Langmuir, Vol. 18, No. 19, 2002 7211
Figure 3. Helix formation versus time for A1, A2 and B1, B2 samples and for the hydrolyzed gelatin. The thermal history (cooling and annealing) is shown on the same graph.
Figure 4. Helix amounts at different temperatures after annealing for 15 h for the gelatins of various sources and molecular weights. The concentration is 4.5% g/cm3. The lines are guides for the eye.
The renaturation appears to be very sensitive to the molecular weight. This is illustrated in Figure 3 for A1, A2, B1, and B2 for the same concentration (4.5% g/cm3). The thermal history is also displayed, the final temperature being 20 °C. The kinetics of helix formation is shown. A1 and B1 reach a helix amount of about 0.5, while A2 and B2 reach only 0.15 or 0.25 after 20 000 s (5.5 h for the cooling ramp and annealing). The hydrolyzed sample (nongelling gelatin) is also shown on the same graph. The helical renaturation is much more restricted in the latter case (χ ≈ 0.03). We summarize in Figure 4 the results for the helix amounts, after annealing for 15 h at various final temperatures indicated in the plot. All gelling samples are represented with the exception of the very low molecular weight sample. The progressive shift of the characteristic temperatures of helix formation, in relation with the molecular composition, is clearly put in evidence. The helical content is lower for the low molecular weight gelatins, for the same temperature and annealing time than for the high molecular weight. Finally we examine the effect of gelatin concentration in Figure 5: helix amounts versus time are shown at four different concentrations (from 2% to 8% g/cm3), with the same thermal history. The curves corresponding to the highest concentrations were fairly noisy. This problem appeared several times at low temperatures for reasons
7212
Langmuir, Vol. 18, No. 19, 2002
Joly-Duhamel et al.
Figure 5. Helix renaturation for A1 at various concentrations and same cooling ramp.
that can either be the scattering of light, by some inhomogeneities of the gel, or a stress birefringence that accompanies the slight contraction of the gel, when helices are formed. This is the limitation of this technique, when used for higher concentrations and low temperatures. In Figure 5 one sees that the rate of helix formation during the cooling ramp was sensitive to the concentration, in the first stages: the larger the concentration, the larger the helix amounts. However, when the three samples were kept at constant temperature (10 °C) and allowed to mature, the amount of helices was very close, the more concentrated solution exhibiting even a slightly smaller amount. We proceed now with the melting or “unwinding” of the helices. The melting can be followed by optical rotation when temperature is slowly increased, after the solutions were kept for annealing. As for collagens, the derivative dχ/dT exhibits a peak that indicates the melting temperature range, and from the width of the peak one can deduce the sharpness of the transition. It is observed that the thermal stability of the helices is strongly related to the renaturation conditions: the position of the peaks and the width of the melting curves are related to the annealing temperature. This is clearly illustrated in Figure 6 for A1 and A2 (top panel) and for fish gelatins, cod and tuna (middle panel). The concentrations for these solutions was 4.5% g/cm3, and the annealing temperatures were different, as indicated by arrows. This is the temperature at which the heating ramp started. The hysteresis between the formation and maturation temperature (initial temperature) is thus observed and an average melting temperature appears for each sample. After maturation, the lowest temperatures provide the largest amounts of helices and the largest distributions of melting temperatures. Under identical conditions, the low molecular weight samples form less stable helices. Finally, the melting curves for three concentrations of A1 are shown in Figure 6 (bottom panel): there is no systematic shift of the peak position, but the shape of the melting curve and thus the distribution of the melting temperatures appears slightly different. The analysis of the melting curves is presented in the Discussion section. (b) Mixed Solvents, Single-Component Gelatins. The solvent composition also influences the formation and melting of helices. We used, as an example, mixtures of water and glycerol. Glycerol is miscible with water in all proportions and is used for making capsules for pharmaceutical applications. The densities of the mixed solvents were measured in order to determine the concentrations of gelatin solutions in grams per cubic
Figure 6. (a, top panel) Melting curves dχ/dT for A1 and A2 after cooling at different temperatures indicated by the arrows and annealing for 15 h. The melting temperatures vary according to the initial temperature and depend on the molecular weight. (b, middle panel) Melting curves dχ/dT for fish gelatins (cod and tuna) cooled and annealed at different temperatures indicated by the arrows. (c, bottom panel) Effect of concentration on the melting curves dχ/dT for A1.
centimeter. Densities, measured at 22 °C with increasing weight fractions of glycerol, vary between 1 g/cm3 for pure water and 1.25 g/cm3 for pure glycerol. To derive the helical content in these mixed solvents, we checked the validity of the Drude equation, which is known for gelatins in aqueous solutions.8 This equation was valid for the mixed solvents containing 30 and 50 wt % glycerol + water. Also, we noticed that the specific optical rotation of the coil conformation changes with the composition of the mixed
Gelatin Networks: Biodiversity and Physical Chemistry
Langmuir, Vol. 18, No. 19, 2002 7213
Figure 7. Helix formation in mixed solvents water + glycerol for A1 during cooling. The curves obtained with the mixed solvents are noisy and were not smoothed. There is no systematic correlation between noisy measurements and the use of mixed solvents as the problem appears even in pure water and it is not systematic.
solvent. We found for A1 in water [R]coil436nm ) -290 ( 2 coil deg cm3 g-1 dm-1, whereas with 30% glycerol [R]436nm ) coil 3 -1 -1 - 275 ( 1 deg cm g dm or 50% glycerol [R]436nm ) -252 ( 3 deg cm3 g-1 dm-1 measured at T ) 35 °C for the same wavelength. With these modifications, the helix amounts were derived, as in pure water. The helix amounts versus temperature, during cooling, are shown in Figure 7 for A1 at a concentration of 4.5% g/cm3. There is clearly a shift of the beginning of helix formation, toward higher temperatures, which is progressively enhanced by the addition of glycerol (up to 50 wt %, for larger amounts of glycerol; gelatin A1 was no more soluble). The same tendency was observed with sorbitol and glucose syrup at 30 wt % (data not shown). The melting of helices in these mixed solvents also exhibits a marked shift toward higher temperatures. According to the literature,13 the equivalent shift is observed in melting of soluble collagen with the mixed solvents (polyols). (c) Aqueous Solutions, Blends of Gelatins. We consider now the blending in aqueous solutions of two samples of gelatins, from different sources, with the aim of determining if two gelatins are compatible and can form helices containing chains of both species. To this end we designed the following experiments: we mixed A1 and cod gelatins, which lie at the two extremities of the melting temperatures of collagens. We considered single solutions of each species at 4% concentration and a blend at the total concentration of 8% g/cm3 containing equal proportions of the two gelatins. Solutions were cooled to 0.8 °C, annealed for 5 h, and finally heated again. In Figure 8 (top and middle panels), we see both steps, formation and melting of the helices. The formation step (top panel) clearly shows that the building of the helices was achieved in two waves: A1 starts first, during the cooling ramp, when the helix amount is superposed on the pure A1 data, followed by the cod sample. However, compared to the aqueous solutions of the cod alone, the gelation of cod started earliersat a higher temperaturesaround 15 °C instead of 4 °C. Eventually, after 5 h the total concentration of helices (cχ) in grams of helices per cubic centimeter was the same as calculated by the addition of the two populations (Figure 8, top panel). Melting is also achieved (13) Gekko, K.; Koga, S. J. Biochem. (Tokyo) 1983, 94, 199-205.
Figure 8. (a, top panel) Helix formation in a blend of cod and A1 gelatins at a total concentration of 8% g/cm3 and equal proportions of each species. (b, middle panel) Helix melting in the same blend after annealing of 5 h. (c, bottom panel) Helix melting of a blend of tuna and A1 gelatins with a total concentration of 9% g/cm3 and equal proportions of each species.
in two steps, each species separately: the derivative of dR/dT is seen in Figure 8 (middle panel) and also the curve calculated as the sum of the melting of A1 and cod in single solutions, which shows that the melting peak of A1 in the blend is almost identical to the single component, a small fraction of mixed helices melt in the intermediate range of temperatures, between the melting peaks of cod and A1 as single components. The melting peak of the cod component is broadened and apparently shifted toward higher temperatures. This peak may represent helices that are mixtures of cod and A1 and/or pure cod helices. It is therefore not possible to define “one average melting
7214
Langmuir, Vol. 18, No. 19, 2002
Joly-Duhamel et al.
the process toward higher temperatures. An equivalent shift appears on the native collagen when dissolved in such solvents. (iv) Blends of gelatins with extreme amino acid compositions show little synergy. Melting of the blends clearly reflects the presence of the two initial sets of populations. Formation of mixed triple helices is, however, favored in blends of gelatins with close compositions. III. Discussion
Figure 9. Melting temperatures for blends of tuna and A1 gelatins (total concentration of 9% g/cm3) and various proportions of tuna.
temperature”, the blend clearly containing the two populations and a small fraction of mixed helices. The total spread of the melting temperatures is equivalent to the addition of the two individual melting curves, with the differences given before. Blends of tuna and A1 were also investigated: they represent the closest melting temperatures of the collagens. The melting curve of a binary mixture of c ) 9% g/cm3 of equal proportions of the two constituents is shown in Figure 8 (bottom panel). There is a broad melting curve, but compared to the curve calculated as the sum of the two individual components, one can define in this case an average melting temperature at half-height of the χ versus T curves. For various proportions of the constituents the melting temperatures vary as shown in Figure 9. The total width of the melting peak varies between 6 and 3 °C and is much smaller than in Figure 8 (middle), for instance. A similar investigation based on rheological measurements was recently published by Gilsenan and Ross-Murphy.6 Their melting temperatures are different from ours because they are not derived in the same way. We also briefly investigated the effect of pH: as these gelatins (tuna and A1) have different isoelectric points, we suspected coacervation effects to play a role in this synergy. By changing the pH from the natural one (5.7 in demineralized water) to 3 or to 9, we noticed an effect on the kinetics of helix formation but not on the overall behavior under melting conditions. Thus, we conclude that the formation of mixed triple helices is enhanced between the species that have close imino acid compositions, independently of the pH. In conclusion of the experiments on helix formation and melting, the following features were established: (i) The temperature range for helix renaturation depends on the imino acid composition of the gelatin and is directly related to the collagen melting temperatures. (ii) The amount of helices for a given imino acid composition depends on temperature, but no equilibrium exists for the renaturation of helices. The helix amount versus temperature shows hysteresis between formation and melting and a distinct and marked dependence on molecular weight. The dependence of the helix amount on concentration (2 < c < 8 g/cm3) is not important after annealing, when the kinetic effects are damped. (iii) Mixed solvents (water + glycerol, or sorbitol, or sucrose,14 etc.) enhance the formation of helices by shifting (14) Nishinari, K.; Watase, M.; Kohyama, K.; Nishinari, N.; Oakenfull, D.; Koide, S.; Ogino, K. Polym. J. 1992, 24, 871-877.
The discussion is focused on the thermal properties of the helices. The well-known theory for coil-helix transitions proposed by Zimm and Bragg15 is based on a statistical mechanical model in which the conformations of the chains depend on two parameters, s and σ. The parameter s is an equilibrium constant for the addition of one residue to a sequence of residues in helical conformation, this residue being initially in the coil conformation adjacent to the helical portion. It characterizes the propagation step. The parameter σ is a factor that characterizes the difficulties of starting a new sequence (σ , 1) the nucleation step. σs is the equilibrium constant for the initiation of a new sequence by making the first hydrogen bond. The nucleation step is much less favorable than the propagation step. For very long chains, the Zimm-Bragg model assumes that helical and random coil sequences coexist in the same molecule in the temperature interval of the melting transition. s has a critical value at 1, in the neighborhood of which the transition from random coil to helical conformation occurs. The sharpness of the transition depends on both s and the chain length. However, above a certain value of the chain length the transition becomes independent of chain length. It is also assumed in this model that s depends on temperature and σ does not. The model describes an equilibrium transition and thus fully reversible. The relation of s to the temperature is in the form of a van’t Hoff relation:
d(ln s) ∆H ) dT RT
(2)
where T is the absolute temperature, R is the gas constant, and ∆H is the enthalpy change for adding one helical unit to the preexisting helical section. Thus s is related to the energy of hydrogen bonding through the Boltzmann factor and σ to the entropy of formation of the first turn of the helix, which is relatively independent of the temperature. Integration of eq 2 between the limits Tm and T allows us to relate the theory and the data:
ln s )
(
)
∆H T - Tm R TTm
(3)
Tm is the temperature of the midpoint of the melting transition for the very long chains. One can derive the fraction of residues in the helical form as a function of s, σ, and N, the number of residues (monomers) of the chain. From the Zimm-Bragg theory, Flory16 showed that the breadth ∆T for the transition from helix to random coil for very long chains is given by
∆T )
2RTm2σ1/2 ∆H
(4)
(15) Zimm, B. H.; Bragg, J. K. J. Chem. Phys. 1959, 31, 526-535. (16) Flory, P. J. Polym. Sci. 1961, 49, 105-128.
Gelatin Networks: Biodiversity and Physical Chemistry
Langmuir, Vol. 18, No. 19, 2002 7215
The melting temperature Tm is evaluated at half-height of the transition and the breadth ∆T between 3/4 and 1/4 of its amplitude. At the midpoint of the transition, the average number of residues in one sequence of triple helix is given by σ -1/2. In other words, σ -1/2 is a measure of the size of the cooperative unit. Melting curves of collagens have been analyzed in the framework of these statistical mechanical theories.11 From the breadth of the transition, the parameter σ can be derived. For different species of collagens, the enthalpy change ∆H of the transformation of one helical residue to random coil was taken from experimental data ∆H ≈ 1.2 kcal/mol ) 5 kJ/mol in acidic solutions. In the case of calf skin collagen with our data Tm ) 273 + 36 ) 309 K, ∆T ) 2 °C, and thus σ ) 0.4 × 10-4 or σ -1/2 ≈ 158 residues in the cooperative melting unit. The number of residues in the cooperative unit per chain is one-third of the total. The significance of the size of the cooperative unit in collagens was discussed by Privalov12 in the context of biological aspects. The author raises the question whether native collagen consists of a discrete or a continuous structure. The distribution of the imino acids in the sequence of various collagens suggests the existence of a repeat unit, as a reminiscence of a primordial gene. The pyrrolidine content of collagens modifies their melting temperatures. The imino acid content should mainly affect the entropy of the melting transition: the presence of pyrrolidine rings in the protein chain decreases the configurational entropy of the random coil, which was confirmed by the calculations by Josse and Harrington.17 On the basis of the assumption that the pyrrolidine rings have a conformational change that is zero at the coilhelix transition, the melting temperature is then related to the pyrrolidine content by
Tm )
∆H (1 - p)∆S
(5)
where p is the percentage of imino acids. Thus increasing the percentage of imino acids increases the melting temperature.18 In this model, the enthalpic term is constant, while in a more refined analysis,11-12 it was also assumed that the enthalpic term per residue may vary with the pyrrolidine content, decreasing with this content. One finds qualitatively a good agreement with the first assumption, but a more quantitative estimation of Tm is obtained with the more refined analysis. It is also known19 that the melting temperatures and the enthalpies of denaturation of collagen vary with solvent composition and pH and therefore there are no absolute values for these parameters. In agreement with Privalov,12 a more elaborate theory that takes into account the interaction of the macromolecules with the solvent should be necessary. The influence of the imino acid content on the cooperative unit is also difficult to corroborate: Harrington and Rao11 suppose that the cooperative unit diminishes when the total pyrrolidine content increases. The free energy of stabilization of the helical sequence will be increased by insertion of pyrrolidine residues and thus a smaller number of hydrogen-bonded residues can stabilize the sequence. Their experimental results do not show a clear correlation. Our results on tuna and sole do not allow us to either confirm or dispute this statement. The cooperative unit σ -1/2 varies as Tm2/∆T. For a lower imino acid content (17) Josse, J.; Harrington, W. F. J. Mol. Biol. 1964, 9, 269-287. (18) Harrington, W. F. J. Mol. Biol. 1964, 9, 613-617. (19) Harrington, W. F.; Rao, N. V. Biochemistry 1970, 9, 37143724.
Figure 10. (a, top panel) Melting temperatures versus annealing temperatures for A1 and A2. The extrapolation toward the melting temperature of mammalian collagens is shown. (b, bottom panel) Melting temperatures versus annealing temperatures for megrim, tuna, and cod.
Tm is decreased, the difference being 21 °C between the extremes, cod and calf skin (relative shift of 7%). The change of the breadth of the melting ∆T from one collagen to another is difficult to evaluate with great accuracy due to the significant effects of both the extraction conditions and the rate of heating known to modify the shape of the melting curves. Only fluctuations of the cooperative units are observed, which cannot be connected unambiguously to the imino acid content. While the Zimm-Bragg treatment could by applied for denaturation of native collagens (with the assumption of a continuous helical structure), the model used in the context of the gelatin gels gives inconsistent results. Two aspects were considered in this investigation: the influence of the gel formation temperature and of the molecular weight. We examine thus the helix-coil transition of the refolded triple helices. Influence of the Gelation Temperature. The melting temperatures of refolded triple helices were derived experimentally, as for collagen, as the temperature corresponding to the middle of the curve of R(T) or χ(T). Figure 10 summarizes these measurements: the melting temperatures of the gels are plotted versus annealing temperatures (gelation temperatures) in Figure 10 (top panel) for A1 and A2 and in Figure 10 (bottom panel) for the three fish gelatins, cod, megrim, and tuna. The melting temperatures were derived at a fixed concentration of 4.5%
7216
Langmuir, Vol. 18, No. 19, 2002
g/cm3. At different concentrations of the A1 sample (Figure 6, bottom panel), we observe a variation of melting temperature of the gels of 0.4 °C (between 26 and 26.4 °C) when concentration varied between 2 and 8% g/cm3 for gels cooled at 10 °C. Thus, we assume that the melting temperature is mainly related to the gelation temperature. The extrapolated melting temperatures, at the highest gelation temperatures, tend toward the native collagen of each species, i.e., the perfect sequence of triple helices. The comparison between the native collagen and the refolded triple helices, however, exhibits important limitations: renaturation is essentially a nonequilibrium, nonreversible process. The derivatives dR/dt or dχ/dt are nonsymmetrical peaks as shown in Figure 6, in particular for gels containing a large amount of helices (low gelation temperature). Using this model, Harrington and Rao11 have calculated the sizes of the cooperative units σ -1/2 from the melting of refolded helices, by normalizing to 1 the helix amounts observed at each cooling temperature and measuring the melting temperatures and the breadth for each thermal history (∆H is taken as a constant). These calculations, when applied to our own gels, show unexpected results in which the apparent cooperative lengths σ -1/2 strongly depend on the gelation temperature (for instance, σ -1/2 decreases with gelation temperature between 50 and 37 residues for A1). Both the melting temperatures and the cooperative units are much smaller for refolded helices than for native collagen and thus are not only a function of the imino acid and solvent composition, as stated in equilibrium transitions theories (Tm and σ should be independent of temperature). The apparent strong decrease in the cooperative unit size is due both to variations of the midpoint of the transition and of the breadth of the melting curve. A small proportion of very stable triple helices that have the stability of the collagen itself appears occasionally [see Figure 6 (top)]. An interesting feature appears also in Figure 10 related to the extrapolation of the melting temperatures versus gelation temperature. An interpretation of this effect is based on the nucleation theories in supercooled states. These well-known models of crystallization of liquids or polymers in solution rely on the presence of an interfacial tension between the crystal and the liquid. The nucleation theories state that the minimum number of molecules required for a stable nucleus to form in the supercooled state strongly depends on the supercooling, defined as ∆Tsuper ) |T - Tm|. The critical nucleus size is inversely proportional to ∆Tsuper. If this concept is applied to the incipient formation of the triple helices, one may expect that the lower the temperature of gelation, the shorter the nucleated sequences. At T ) Tm the nucleus is infinitely large and its melting temperature is that of the infinite crystal. The particular feature in gelatin gels is that the melting of the helices, even after long periods of annealing, at fixed temperatures, still keeps the memory of the cooling temperature: one can suppose that helices, while growing, “freeze in” the defects such as loops or mismatching of amino acid sequences (memory effect). A parallel between the critical size of the nucleus and the cooperative unit size is made in ref 19. Influence of the Molecular Weight. The effect of the molecular weight on helix renaturation and melting is even more pronounced than the effects mentioned above and has important practical consequences. The melting curves χ(T) for the various molecular weights for the A type samples are shown in Figure 11. The temperature range of this plot is chosen between 10 and 40 °C. The number of residues per chain N are calculated from Mw values. In the Zimm-Bragg model the influence of the
Joly-Duhamel et al.
Figure 11. Effect of the molecular weight on the melting of helices in the gels: Comparison with collagen.
length of the coils has been theoretically determined: two limits are generally considered, the infinitely long chains and the short chains. The difference in helix-coil transitions of long and short chains is that the latter tend to follow a “one sequence approximation” (called the “zipper model” for nucleic acids), whereas long chains have a tendency to alternate random and helical sequences, as stated before. The cutoff between the two regimes is the limit of N of the order of the cooperative unit length, σ -1/2; actually, the limit should be Νlimit ) 2σ -1/2. Applying this criterion to collagen, one finds Nlimit ≈ 300 (≈100 units per chain). This means that the hydrolyzed sample, which is the “nongelling sample”, N ) 120, should be considered as the limit of short chains. It is expected from these models that the helix-coil transition becomes substantially independent of the chain length above ∼Nlimit, as found experimentally by Zimm et al.20 for polypeptides with various molecular weights. The transition is particularly broad for the short chains and much steeper for the longest ones. The overall tendency observed for gelatin and collagen melting, Figure 11, is in agreement with this framework. However, one notices a large influence of the molecular weight on the melting curves even for gelatins of high molecular weights, which is not justified in the model. We also observed that the shorter chains exhibit a more reversible transition during cooling and slow melting (weak hysteresis). The shorter chains may indeed approach the “one sequence model” suggested by the Zimm-Bragg model. This is also in agreement with the nongelling property of these gelatins. Thus one may conclude that nonreversibility should be associated with branching or network formation. In conclusion, we stress the limitations of the equilibrium theories to describe the helix-coil transitions in gelatins and collagens. The fact that the renaturation of the helices creates junctions between the individual chains leads to a completely different situation from the classical models of the reversible transition. When the formation of a network is taken into account, one can explain, at least qualitatively, some of these differences that create a nonequilibrium situation for the helix-coil transition,8 with important hysteresis effects. It is interesting to compare these results to the model calculations proposed by Viebke et al.21 on the double helix formation of (20) Zimm, B. H.; Doty, P.; Iso, K. Proc. Natl. Acad. Sci. U.S.A. 1959, 45, 1601-1607. (21) Viebke, C.; Picullel, L.; Nilsson, S. Macromolecules 1994, 27, 4160-4166.
Gelatin Networks: Biodiversity and Physical Chemistry
κ-carrageenans. Experiments were performed with samples covering a large range of molecular weights. The authors arrived at the conclusion that branching on the helical level is not required for the formation of the gels (3D networks) of κ-carrageenans, which then are essentially built by the side-by-side aggregation of helices, at the superhelical level. The equilibrium models for coil to helix transitions that the authors propose, based on the ZimmBragg model, seem to satisfactorily reproduce their experimental data and allow them to derive the cooperativity parameter of the transition for this polysaccharide. Our conclusions and theirs thus complement each other. The comparison is, however, limited at this stage because gelation of carrageenans is related to a different mechanism than gelatin: side-by-side aggregation of the triple helices is not observed in gelatins. The triple helical sequences are stable in aqueous solutions. The fibers of triple helices, similar to native collagen ones, do not reform in gels. Mixed solvents have a marked shift on the melting temperature of the helices for single-component gelatin solutions. Our experiments concerned tuna, cod, and bovine gelatins. The increased thermal stability of collagen was known already,13 by thermodynamic measurements (calf skin collagen). The interpretation of this effect is mainly based on the influence of sugars and polyols on the structure of water. These compounds have a water structure-making character.13,22,23 This effect is also known for globular proteins. In the presence of these compounds a preferential hydration of the protein takes place such that the hydrophobic groups exclude the polyhydric compounds from their immediate vicinity. The hydrophobic interactions are strengthened and the protein selfassociation is enhanced. To get a better insight into these interactions, we performed intrinsic viscosity measurements of the gelatins in the presence of the mixed solvents such as water + glycerol. We found the following effects: for tuna gelatin the intrinsic viscosity is [η]) 53.8 cm3/g in pure water and 50.9 cm3/g with 30% glycerol, thus showing that the hydrodynamic volume is reduced in the presence of glycerol. The Huggins interaction coefficient kH is increased from 0.21 to 0.28, indicating a stronger attraction between the protein coils in the presence of glycerol. For A1, the effect was less pronounced and cannot be quantified. At higher gelatin concentrations, above 0.02 g/cm3, the viscosity η of semidilute solutions is strongly increased as shown in Figure 12 for tuna, and a similar effect is seen for A1 for the same concentration range. The enhancement of viscosity is noticed when either ηsolution, or the ratio ηsolution/ηsolvent, or the difference (ηsolution - ηsolvent) is plotted versus concentration. Such an effect can be ascribed to the formation of efficient entanglements between the protein chains in the presence of glycerol. These interactions may involve the hydrophobic groups, as observed in “associating polymers”, which are hydrophobically modified water-soluble polymers. However, gelatin solutions remain Newtonian, in contrast with the aqueous solutions of associating polymers. Recently, Wulansari et al.24 discussed the Newtonian behavior of (22) Arakawa, T.; Timasheff, S. N. Biochemistry 1982, 21, 65366544. (23) Gekko, K.; Timasheff, S. N. Biochemistry 1981, 20, 4667-4676. (24) Wulansari, R.; Mitchell, J. R.; Blanshard, J. M. V.; Paterson, J. L. Food Hydrocolloids 1998, 12, 245-249.
Langmuir, Vol. 18, No. 19, 2002 7217
Figure 12. Viscosity versus concentration for A1 in pure water and in a mixed solvent of water and glycerol.
gelatin solutions (in a phosphate buffer, at pH 7) in comparison with polysaccharide solutions, which in general are non-Newtonian. The authors raised the question of the entanglement mechanism of the protein coils. Our results also stress the role of the entanglements in the semidilute solutions and also the specific role played by the solvent. Conclusion In this paper we have extensively examined the helix formation of gelatin solutions of many sources, molecular weights, and isoelectric points and we have modified the thermodynamic conditions for gelation and melting by varying the temperature, changing the solvent (pure water, mixed solvents), and blending different gelatins. This investigation provides a clear comparison between the molecules themselves (imino acid compositions, molecular weights) and puts into evidence the influence of the environment on the helix formation. In view of such diversity of behavior, the search for the common features between all gelatin gels becomes imperative, and this is the topic of the next paper. Analysis of our experimental results in the context of the well-known Zimm-Bragg theory shows that a more elaborate theory that takes into account the building of the network and interaction of the macromolecules with the solvent should be necessary. Acknowledgment. This work was performed in the context of European Contract FAIR CT 97-3055. We thank all our partners for numerous and fruitful exchanges during the project. We are indebted in particular to Dr. M. Gudmundsson, Dr. P. Montero, and Dr. G. Takerkart for their expertise in gelatin preparation and characterization. We also thank Dr. M.-M. Giraud-Guille and Dr. L. Besseau for their precious help in the preparation of soluble collagen solutions and Dr. A. Ajdari for many stimulating discussions. We thank the reviewer for the thorough examination of our manuscript and for very interesting comments and suggestions that we took into account in the final version. LA020189N
