Thermally induced globular protein gels: peculiarities of formation, melting, and restoration of gels of different structure

Food Hydrocolloids Vol.6 no.1 pp.97-114, 1992

Thermally induced globular protein gels: peculiarities of formation, melting, and restoration of gels of different structure LV.Sochava and T.V.Belopolskaya Research Institute of Physics, Leningrad State University, Petrodvorets, 198904 Leningrad, USSR Abstract. The results of the calorimetric study of the globular protein heat-set gels performed by the authors during recent years have been generalized in the present paper. The conformational variations have been studied which took place after the denaturation in globular protein concentrated solutions with continuous heating in the temperature range 20-140°C. The effects of pH, the ionic strength of the solution and the protein concentration on the formation of heat-set gels and their melting have been studied in parallel with visual observations of the solution states under the same conditions. Four different types of globular protein gels have been discovered (e.g. lysozyme), and the ranges of their existence have been determined. The results obtained have shown that two types of gel can be formed within the pH range 1.7-9.0 at fixed ionic strength and protein concentration: clear meltable gel (type I) and a turbid one which cannot melt (type II). The heating thermograms of the type I gel demonstrate besides denaturation heat absorption an additional high temperature maximum, which is the calorimetric manifestation of cooperative transformation proceeding at the melting of this gel. The incipient stage of the melting gel formation is described at critical values of pH, ionic strength and protein concentration. The adjustment of protein aggregation in solution with the help of pH, ionic strength and concentration has been shown to broaden considerably the pH range in which thermoreversible gel is formed and to provide such changes in the structure of the turbid gel which make it meltable (type III). This results in the appearance of a high temperature maximum in the thermograms of such a gel. The melting of such a gel induces cooperative stratification of the system into the collapsed gel (type IV) and water.

Introduction

It is well known that as the temperature of concentrated solutions of globular

proteins increases, along with the destruction of the native molecular structure, i.e. denaturation, network formation of heat-set gels can proceed. Both processes are connected with sharp changes of structure. In the first case it is the structure of the individual protein molecule that is varied; in the second it is the structure of the solution as a whole. Gels of globular proteins are extremely weak solid substances. They have not been adequately investigated, not least because of lack of data until recently. Gels of denaturated proteins are widely encountered in the food industry as structurants in both natural and processed foodstuffs and their study is clearly of topical interest. Studies in the last decade have concentrated on mechanical and rheological characteristics of heat-set gels of globular proteins (1,2) and structural investigations by, in the main, electron microscopy, X-ray analysis, infrared spectroscopy and circular dichroism (3-6). This paper is concerned with the application of differential scanning calorimetry (DSC) , which we have shown can provide new valuable information on protein thermodynamics in aggregation conditions. The results of detailed systematic calorimetric studies of globular protein denaturation in concentrated solutions have been summarised in the authors' 97

I.V.Sochava and T.V.Belopolskaya

review (7). In the study of irreversible thermal denaturation of some globular proteins (lysozyme, myoglobin, bovine serum albumin and human serum albumin) the heating thermograms reveal, besides the maximum of denaturation heat absorption, an additional endothermic maximum at higher temperatures (8-10). This observation has stimulated a series of systematic investigations on the effect of the aggregational interactions between protein molecules during denaturation and the dependence of the higher temperature endotherm on solution conditions (11-13). This additional endotherm has been shown to be connected with the melting of the heat-set gel of globular proteins, suggesting an additional phase transition not previously observed for these systems. The present paper reviews the studies in this field published during the last five years, and includes some new results as well. The combined consideration of denaturation, gelation and gel melting as interconnected processes is unconventional, and in our opinion has proved to be fruitful. Materials and methods The study was performed using a Setaram differential scanning calorimeter, DSC-III, with an on-line computer IN-50 (France). A sample of the globular protein lysozyme (supplied by Sigma) was studied. Lysozyme belongs to the class of so-called small globular proteins with a single-stage denaturation transition, demonstrating only a single heat absorption peak in dilute solutions (14). The determination of the denaturation thermal effect (aQd) and the maximum heat absorption temperature (Td ) have been described in detail in (7) for both reversible and irreversible denaturation processes. Heat-set gels can be formed in concentrated solutions of lysozyme after denaturation. The parameters of the protein environment affecting the gelation process include protein concentration, pH and ionic strength of the solution as well as the temperature of the process. The protein concentration in the studies performed varied between 1.5 and 25%. Lysozyme solutions with a given pH were prepared by traditional dialysis of aqueous solutions of the sample with respect to a suitable buffer solution. Throughout the pH range 1.7-9.0 a glycine buffer was used. The pH region of 4-8, where this buffer loses its properties, was specially controlled by simultaneous pH measurements at the protein denaturation temperature. The role of the buffer in this region is reduced solely to setting the required pH value and to maintaining the ionic strength of the solution identical for the whole pH range. The effect of ionic strength on lysozyme gelation was studied with the addition of different concentrations of NaCI (0-0.5 mol/dm") to the protein solution in glycine buffer (0.25-1.0 rnol/dm') at pH 2.0 and 4.2 as well as to its aqueous solution (pH 6.4). The gelation temperature in scanning calorimetry, in contrast to the other physical methods mentioned, is a variable parameter; therefore, the gelation process is non-isothermal. As the ampoules with the solutions being studied are hermetically plugged and can withstand high pressure, the temperature range of calorimetric studies has no methodical limitations from above. The limitation depends only on the thermal destruction of the protein molecules themselves. 98

Globular protein gels

The duration of the solution at any given temperature above Td also affects the gelation process . This parameter can be varied in calorimetric experiments by changing the heating rate of the solution. We have studied the lysozyme solutions in the temperature range 20-140°C at the heating rates Vh = 5 and lSC/min. Along with DSC measurements, visual observations of the state of all the solutions were made in special transparent glass ampoules under identical heating conditions. If necessary , the heating could be stopped , and the ampoule withdrawn and later opened at room temperature . This enabled us to estimate qualitatively the gel elasticity, homogeneity and ability to retain water.

Results and discussion Dependence of lysozyme gel formation and melting on the pH value of the solution

DSC studies of lysozyme solutions were performed at temperatures from 20 to 140°C in the wide pH range of 1.7-9.0 at different ionic solution strengths and protein concentrations. In this section we consider the results for lysozyme solutions in 1M glycine buffer with protein concentration c = 10% , only one environmental parameter (pH) being varied (Figures 1-3). All the thermograms present the temperature dependences of lysozyme heat capacity, normalized to the protein mass in the solution and the temperature, given for the individual experiment most characteristic of the conditions considered . The lysozyme denaturation has proved to be irreversible in the whole pH range for all the solutions studied. This means that the maximum , corresponding to protein denaturation , is missing on repeated heating in the plots of protein heat capacity versus temperature. One should consider as the most important result the appearance of an additional endothermic high-temperature maximum (HTM) , besides the one of denaturation heat absorption, in the heating curves of lysozyme solutions with pH 1.7-2.7. At pH >2.7 only one peak appears on thermograms , this being related to protein denaturation . The results obtained are given in Table I and Figure 1, where the most characteristic thermograms are given . The complementary visual observations of the solutions during their heating, performed at temperatures up to 140°C in the whole pH range, have shown that as a result of aggregation after protein denaturation three different states arise, depending on the pH value . In the narrow region of pH 1.7-2.7, just after denaturation a clear, elastic, weak gel is formed, which does not repel water under mechanical action (type I gel). At pH 2.8-5.0, after denaturation a weak, turbid, viscuous solution occurs . And finally, at pH >5.0, gel arises again (type II) , though quite different from type I gel in its appearance and mechanical properties. As pH increases up to 9.0, type II gel is smoothly changed from being quite weak and turbid, easily transforming into the flow state when shaken , into a white , non-transparent, more rigid and elastic gel. Under mechanical action and heating this gel repels water quite easily . 99

I. V.Sochava and T. V.Belopolskaya

a.

Z 1 fO

30

5D

70

90

lID

T,(°c)

Fig. 1. Temperature dependence of heat capacity of lysozyme solution in glycine buffer. (a) pH = 1.7; mol/drrr' = 1.0; c = 10%; (b) pH = 5.3; mol/dm ' = 1.0; c = 10%. 1 and 2 denote the succession of heating; V he a t = SO/min. Table I. Effect of pH of the solution on the temperature and heat of conformational transitions in lysozyme during its denaturation and gel melting! pH 1.7 2.0 2.4 2.7 3.4 3.8 5.3 6.5 8.0 IVheat;ng

AQHTM (J/g) 75.1 76.3 76.0 75.0

= 5°C/min; c = 10%;

48.6 51.2 55.6 62.1 70.8 78.0 80.0 78.0 75.0

8.8 9.6 8.8 5.5

mol/drn"

= 1.0

13.9 15.1 16.4 26.5 31.5 34.9 34.4 33.6 31.7

(buffer).

The comparison of DSC results with visual observations shows that the curves with HTM refer to the solutions in which type I gel is formed during heating. HTM is absent in the thermograms of lysozyme solutions in which either type II gel or no gel at all is formed after the denaturation. What does HTM mean in thermograms of solutions with pH 1.7-2.7, which form type I gel? The answer to this question was obtained during visual observations of the state of these solutions in glass ampoules under the same heating conditions as in the DSC measurements. We observed that, when heating native solution in aggregation conditions immediately after denaturation, the clear heat-set gel is formed. Increasing the temperature further causes the sample to become liquid again; this corresponds to the gel melting. The temperatures of gel formation and melting were shown to coincide with those of denaturation and HTM respectively. It was thus proved that heat-set gel is formed during the 100

Globular protein gels

Op

/

I

I

I

, ...\

\

\

\

/

... /

JO

40

60

/

70

80

Fig. 2. Heat capacity of lysozyme solution versus temperature during the cycle of heating (1)cooling(2)-heating(3). pH = 2.0; mol/dm ' = 1.0; c = 14%; v = IS/min. , Table II. Effect of pH of the solution on the temperature and heat of conformational transitions in lysozyme during its denaturation and gel melting and restoration! pH 1.7 2.0 2.4 2.7 IVheahng

t;

(0C) 47.6 49.0 53.6 60.5

BQd

THT M

(J/g)

eC)

14.3 14.7 16.4 22.3

74.2 74.0 75.0 73.0

~QHTM

TrestoL

~Qrestor.

(J/g)

eC)

(J/g)

9.7 9.6 9.2 9.6

62.2 62.0 62.3 62.0

7.6 8.4 9.2 8.4

= 1.SOC/min; c = 10%; mol/drn' = 1.0 (buffer).

denaturation process as soon as it reaches its temperature range, and the HTM observed is closely related to its melting. Note that similar thermal behaviour of lysozyme solutions (pH = 2.0, c = 15%) has been observed in IR absorption spectra within the amide I-band with scanning over temperature (4). On the thermograms of the second heating (see, for example, Figures 1 and 2) the HTM is always achieved; the denaturation process is either missing or is quite negligible. The existence of the HTM in the curves of repeated heating shows that gel formed under our conditions at the first heating is thermally reversible. The value of the HTM thermal effect, LlQHTM, has been shown to depend only slightly on scanning rate during the first heating. This means that the gelation rate is comparable with the heating rates used. Measurements at the heating rates of 5 and 1.5°C/min have shown that at Vheat = 1.5°C/min the time is sufficient to complete the process. Thermodynamic HTM parameters, which can be obtained by slow heating, characterize the thermostability of the gel, being the new form of protein molecular arrangement arising at the transition from the native protein state into the denaturated one (see Table II).

101

I. V.Sochava and T. V.Belopolskaya

The thermoreversible character of the gel formed reveals itself not only in the presence of an HTM on repeated heating. The cooling of the melted lysozyme gel in these conditions has been shown to lead to gel restoration even at temperatures above Td ; in this case an exothermic effect is observed in thermograms which is close in value to the heat of gel melting during the following heating. Figure 2 presents the lysozyme solution thermograms for pH 2.0, demonstrating the successive cycle of heating-cooling-heating undertaken at a scanning rate v = 1.5°C/min. Similar results have been obtained for other pH values and the corresponding thermodynamic data are given in Table II. It should be stressed that overall equilibrium gel structure, controlling its thermostability, is formed at T ~ Td • This refers to both processes of gelation under the first heating of the native solution and of gel restoration from the melt. Further cooling down to room temperature introduces no essential changes into the heat-set gel structure. This distinguishes the gels of globular proteins from the ones of fibrillar proteins, in which both gelation and the gel structure variation exist well below the denaturation temperature. It has been shown experimentally in this work that thermostability and, hence, gel structure depend noticeably on the temperature to which the melt is heated and on its exposure at this temperature. The gel's thermostability decreases with the increase of both factors, as follows from the data of Figure 3. The important consequence of these data (the results are similar for other solutions with pH

-----

30

50

70

91)

110

Fig. 3. Gel melting temperature and its thermal effect as a function of the melt prehistory. pH = 2.0; mol/dm" = 1.0; c = 10%; v = SO/min. (a) The dependence on maximum heating temperature. 1 = the first heating up to 100°C; 2, 3, 4 and 5 = after heatings up to 100, 110, 125 and 135°C respectively. (b) The dependence on the exposure time at high temperature. 1 = the first heating up to 100°C; 2 = after heating up to 100°C, t = 10 min; 3, 4 = after heatings up to 120°C, t = 30 and 90 min respectively. (c) The repeated heatings of identical heating-cooling regimes.

102

Globular protein gels

1.7-2.7) is that one can obtain thermoreversible gels with different thermal stabilities and ordering degrees while varying the maximum temperature (Figure 3a) or the time of melt exposure at high temperatures (Figure 3b). Note that the repeated heating in the same heating-cooling regime does not influence practically the thermograms obtained (Figure 3c) and, hence, the gel properties. It appeared unexpected and interesting that by quenching (sharp cooling) the melt immediately after gel melting one could avoid gel restoration down to T roo m and obtain again the renaturated protein solution. The thermograms of the subsequent heating of such a solution are close to the initial ones (Figure 4) and again contain the denaturation maximum. Figure 4(a and b) demonstrates also that protein renaturation can take place during the quenching of melted gels with a different thermal prehistory. In such a way the conditions have been established under which the protein, after irreversible denaturation (due to gelation), acquires the possibility of renaturation. The above data characterizing the relationship between stability and quality of protein structures formed with their thermal prehistory can, in our opinion, be useful when creating gels with specific properties for their practical application. As shown in Table I, the thermal effect of the high-temperature maximum ~QHTM decreases as pH increases; the HTM vanishes at pH >2.7. It has been observed that TH T M does not depend on pH in the pH range 1.7-2.7, while Td increases with increasing pH, as should be expected. The constancy of T HTM and the increase of Td lead to the overlapping of both maxima, and their temperatures coincide at around pH 3.4. This means that the thermostability of part of the gel at first, and then all of the gel, becomes equal to that of the globule. At T HTM = Td this gel cannot be formed after denaturation; therefore the ~QHTM in the thermograms decreases at first while pH increases, and then turns to zero. (As Td decreases with the decrease of Vheating in the irreversible denaturation process, the coincidence of Td and T HTM occurs at different pH

Cp a.

30

50

70

90

Fig. 4. The effect of cooling rate of lysozyme gel melt on its renaturation ability. pH = 2.0; moll drrr' = 1.0; c = 10%; v = 5° min. 1,2 and 3 denote the succession of solution heating. (a) 2 = after conventional cooling in the device; 3 = after quenching. (b) 2 = after quenching; 3 = after cooling in the device.

103

I. V.Sochava and T. V.Belopolskaya

values for heating rates of 5 and 1.5°C/min.) The aggregation bonds arising in these conditions seem to have short life spans and the denaturation process should have acquired its practically reversible character. This is in good agreement with the visual observations, which have shown the reappearance of the solution after denaturation at pH >3.5. However, protein aggregation increases with increasing pH, which can be judged from the growth of denaturation irreversibility with repeated heating of the sample as well as from the turbidity of the solution after denaturation. Remember that lysozyme isoelectric point occurs at pH 11, i.e. intermolecular protein interaction should really grow in the direction of pH from 1.7 to 9.0. In this connection the absence of an HTM at those pH values where type II gel is formed could seem incomprehensible at first sight. The experiments performed have shown that type II gel does not melt while being heated up to 140°C. On the contrary, as the temperature increases this gel gradually repels the water and becomes more and more opaque and elastic. Hence, the absence of any HTM in the heating thermograms type II gels can only be the consequence of their thermoirreversibility; at least, up to the temperatures of protein destruction onset. The formation of gels of this type as well as of aggregates in solution can be seen calorimetrically in heating thermograms through recording the thermal effect of molecular aggregation. This thermal effect can be determined as a difference between the denaturation thermal effects of reversible and irreversible processes (e.g. for pH <5). Thus, the DSC studies as well as visual observations of the changes of rheological properties of the samples have shown that two possible types of heatset gels can be formed in the pH range 1.7-9.0. They are the meltable, clear gel and the turbid (non-transparent) gel which cannot be melted. The high temperature maximum is the calorimetric manifestation of cooperative conformational transition, proceeding in the type I gel at its melting. A similar classification of heat-set gels of globular proteins, but based on structural investigations, has been presented in (4-6). It has been shown there that the homogeneous clear gel, arising far from the isoelectric point, has a regular linear structure, being formed by successive attachment of disc-shaped globules, partially unfolded after the denaturation. The turbid non-transparent gel has a heavily branched and cross-linked structure, based also on bonded, partially unfolded protein molecules. For clear gel uniform protein density distribution is characteristic in the network structure over large distances; for the non-transparent one the densified regions are randomly distributed in the network. The results of the present research provide additional confirmation of these concepts. On the one hand, the existence of a rather narrow HTM in the thermograms of clear gels, which shows the cooperative conformation transition, is in favour of the ordered but not random arrangement of protein molecules throughout the bulk during the formation of heat-set gels far from the isoelectric point. On the other hand, the absence of any HTM in the thermograms of turbid gels highlights the lack of transition, which in turn indicates the absence of a long-range order in their structure. 104

Globular protein gels

Effects of the ionic strength of the solution and protein concentration on formation and melting of lysozyme gels of different types In this section we deal with the effects of the ionic strength of the solution and protein concentration on the behaviour of lysozyme solutions during their heating in the above-mentioned pH range. To begin with we will concentrate on the pH region where, as we have shown, regular thermoreversible gel can be formed. For this purpose we have studied solutions with different lysozyme concentrations (c = 1.5-25%) at glycine buffer molarities of 0.5 and 1.0 moll drrr', and with the addition of different NaCI quantities in some cases. Clear homogeneous gel

Consider the incipient gelation. We have found that lysozyme solution denaturation during scanning over temperature can be either reversible (Figure 5a) or irreversible (Figure 5c), depending on the protein concentration, buffer molarity, composition and pH. In the reversible process the thermal effect of denaturation .lQd is equal to the denaturation enthalpy and is related to the conformational variations, proceeding at the level of individual molecules. In an irreversible process the thermal effect is provided by the structural variations in molecules in the course of their interactions. The transition from a reversible to an irreversible process appears to proceed sharply in a narrow range of each parameter variation when the other parameters are kept unchanged. A critical

j

~~I ~ ~

a 2

i

b 2-I

,_---'-_~-'--_L-..- L------J'-----'---~~

30

50

70

90

T, roC)

Fig. 5. Temperature dependence of lysozyme solution heat capacity, one of the parameters (pH, mol/drrr', c%) passing through its critical value. (a) Reversible, (b) partially reversible, (c) irreversible denaturation. 1 and 2 denote the succession of heating.

105

I. V.Sochava and T. V.Belopolskaya

value exists for each parameter at which denaturation is reversible at first heating and only partially reversible with the appearance of the HTM at the second heating (Figures 5b and 6c). The latter case is related to the incipient gelation, and the corresponding magnitudes of protein concentration, molarity and pH of the solution represent their critical values. For completely irreversible denaturation the HTM appears in the heating curves even at the first heating. As follows from Figure 5, the decrease in the thermal effect of denaturation aQd' the appearance of the HTM and the growth of aQHTM are observed at the transition from reversible to irreversible denaturation. The decrease of aQd is caused by an increase in aggregational molecular interaction. Aggregation in this case is realized in the form of the regular heat-set gel. As the experiment has shown, irreversible lysozyme denaturation, for example at pH 2.0, can proceed at the following magnitudes of parameters: 0.5 mol/drrr', c ~1O% and 1.0 moll dm", c ~3%. Here, C = 10% and c = 3% are the critical concentrations of gelation under these particular conditions. The heat of the reversible gel melting QHTM is the thermodynamic parameter of gel-melt transition, which characterizes the depth of gelation and depends on the parameter set: pH, mol/dnr' and c%. There exists a definite set of these parameters for which the heat of gel melting reaches its maximum, indicating the optimum gelation conditions. In our experiments a Q~!f~ = (10.9 ± 1.3) J/g. The plots of the heat of gel melting in glycine buffer versus buffer molarity and protein concentration at constant pH (pH 2) are given in Table III. The decrease of both mol/dm" and c results in the decrease of thermal effect. The character of aQHTM (mol/din", c%) dependence does not contradict the concept of the binding energy changes and protein-protein interaction probability which are possible in the conditions used. However, in the dependence LlQHTM (rnol/drrr', c%) the most important factors are the possibility of overlapping of the melting and denaturation temperature regions occurring at decrease solution ionic

Table III. Plot of temperature and heat of conformational transitions in lysozyme during its denaturation and melting of type I gel versus ionic strength of the solution and protein concentration 1

THTM

c

Glycine buffer (mol/drrr')

NaCl (mol/drrr')

(%)

(0C)

(Jig)

Td ("C)

(Jig)

0.5 0.5

0 0.1

10.0 4

pH 2.0 68.6 70.5

5.9 7.1

53.6 55.0

21.0 22.1

0.5 0.5

0.25 0.5

4 4

79.5 90.0

9.2 6.7

54.0 49.0

16.3 16.8

1.0 1.0 1.0 1.0 1.0

0 0 0 0 0

71.5 74.0 76.3 81.0

6.3 9.2 9.6 10.9

54.0 53.0 51.9 51.2 51.0

23.5 19.3 15.5 15.1 15.1

1 Vheating

106

= 5°C/min.

aQHTM

1.5

3.5 6.0 10.0 16.0

aQd

Globular protein gels

strength and protein concentration. As has been shown above, the variation of pH leads also to overlapping of the maxima of denaturation and gel melting. While pH affects only the denaturation temperature (Td ) , the ionic strength and c% influence mainly the temperature of gel melting (THTM)' Therefore overlapping can occur with increased pH as well as with decreased ionic strength and protein concentration. The maximum gel melting temperature (THTM) is the second thermodynamic characteristic of this transition. As has already been mentioned, the experiment showed that the melting temperature of the regular linear gel is practically independent of pH. On the other hand, the experiments with solutions of variable ionic strength at constant pH and c% demonstrated the essential increase of T HTM with ionic strength (Table III). This can be seen in particular if one compares the variation of gel thermostabilities at different values of buffer molarity. From the data presented it also follows that the increase of the solution ionic strength due to NaCI addition increases the thermostability of clear gel. (One should note here that Td , unlike TH T M , decreases only slightly). Figure 6 shows the thermograms of successive heating of lysozyme solutions with different NaCI contents. A concentration of 0.1 mol/dm? NaCl, was shown to be the threshold value for the conditions chosen, as ge,l arises only on repeated heating (Figure 6c). At the same time 0.5 mol/drrr' NaCI changes the gelation condition drastically. In these conditions thermoreversible gel with maximum thermostability possible is formed at the first heating (Figure 6a). A further increase ofthe salt content (c >5%) induces turbidity, i.e. protein precipitation. The data presented are indicative of a substantially greater effect of NaCl on the gelation process than that of glycine buffer molarity. In all cases the high temperature maximum persists on the thermograms for the repeated heating, and gel which occurs at T < THTM always melts at

Cp

b

c

Fig. 6. Effect of NaCI molarity on the thermodynamic parameters of type I lysozyme gel melting. pH = 2.0; jlycine buffer, 0.5 mol/dm", v = SO/min. 1,2 and 3 denote the succession of heating. (a) 0.5 mol/dm NaCI; c = 8%; (b) 0.25 mol/drrr' NaCl; c = 5% (the third heating was performed after 21 days of the sample storage at room temperature); (c) 0.1 rnol/dm? NaCI; c = 3%.

107

I. V.Sochava and T.V.Belopolskaya

T> T HTM. From the identical phase states of the systems after gel melting for all magnitudes of ionic strength we assume that the increase of gel thermostability with ionic strength should be of energetic rather than entropic origin. Hence, for our experimental conditions, the observed increase of THTM should correspond to an increase in the thermal effect of -0.4 J/g, which is within experiment error. However, the magnitudes of .lQHTM given in Table III indicate considerably greater variations. In our opinion the reasons for these essential .lQHTM changes are quite different. At 0.5 mol/dnr' buffer they are related to the closeness of T HTM to T d ; on the other hand, for 0.5 mol/dm' NaCI they are related to the possible appearance of protein aggregates not participating in gel melting. We shall discuss this point later. The increase of the solution concentration from 3 to 16% at constant pH and ionic strength leads also to T HTM growth (Table III). Unlike the ionic strength, the increase in THTM is mainly entropic. The increase of protein concentration, while enhancing the probability of protein-protein bonding, does not change the energies of these bonds. Moreover, the greater the concentration, the smaller the conformational variation of entropy which takes place at gel melting. The fact that HTM exists in the heating curves of lysozyme solution, with NaCI added, and persists on repeated scanning, indicates that in this case the type I gel is also formed after denaturation. However, one should note that the visual observation of such a solution both at room temperature and during heating can reveal the features of its inhomogeneity. Thus at room temperature the 8% lysozyme solution in 0.5 mol/dm' glycine buffer with 0.5 mol/drrr' NaCl (pH 2) is highly turbid, i.e. the part of the protein in it is in the precipitated state. As one approaches the denaturation temperature (50-60°C) the solution becomes clearer, i.e. the process of protein dissolution takes place. After denaturation the gel is formed. The gel, though whitish, is essentially clearer than the initial solution. After HTM at T = 117°C one can see that the gel has melted and turned slightly turbid, with low viscosity. Upon cooling the gel is restored, acquiring again the whitish shade. All the data obtained show that aggregates can originate in the regular thermoreversible type I gel in the presence of NaCl which are the precursors of non-melting type II gel. This fact seems to explain the relatively low heat of gel melting in the presence of 0.5 moll dm' NaCl in spite of the high ionic strength in these experiments (Table III). Turbid inhomogeneous gel As already noted, the fast growth of globule thermostability with pH is a hindrance to clear gel formation from lysozyme solution in glycine buffer at pH >2.7. Because of this, the processes of denaturation, gelation and gel melting should occur in the same temperature range. The addition of 0.5 mol/dnr' NaCI to glycine buffer increases the T HTM of type I gel up to 90°C, which exceeds the maximum lysozyme denaturation temperature (Tcr ax =80°C) by lO°C. In this regard one could hope that the addition of NaCl will broaden the range of this gel existence up to the higher pH values. We have chosen pH 4.2, which is within the transition pH region at which, in glycine buffer, the type I gel is no longer 108

Globular protein gels

formed and the type II gel is not yet formed. At this pH value heating thermograms were obtained for solutions with NaCI molarities of 0.25, 0.35 and 0.5 mol/dm", and a protein concentration of c = 15% (Figure 7a). As can be seen from this figure, for 0.25 and 0.35 mol/dm? NaCI as well as for NaCl absence, HTM is not observed. Attention should be drawn only to the decrease in the denaturation thermal effect, which shows the growth of aggregational interaction with molarity, which proceeds further towards 0.5 mol/drrr'. On the lysozyme solution thermograms for 0.5 mol/drrr' NaCl, at the first heating HTM

Gp a

----J/\'-

8

1 2

-9-A-~--!

50

90

110

•

50

70

Fig. 7. Temperature dependence of heat capacity of lysozyme solutions in glycine buffer at pH = 4.2, NaCl being added. (a) Variable protein concentration and 0.5 mol/dm' NaCl; 1, 2 and 3 are for c = 15.0,6.3 and 3.6% respectively. (b) Variable NaCl molarity and c = 15.0%.4,5 and 6 are for mol/drrr' = 0.25, 0.35 and 0.5. The successive heatings are given by dashed lines. Table IV. Effect of NaCI molarity and protein concentration on temperature and heat of conformational transition in lysozyme at its denaturation and melting of type III gel'

THT M

Glycine buffer (rnol/drrr')

NaCI (mol/dm ')

c (%)

.iQHTM

t;

.iQd

(0C)

(Jig)

CC)

(Jig)

1

2

3

4

5

6

7

0.25 0.25 0.25 0.25 0.25

0.50 0.50 0.50 0.35 0.25

3.6 6.3 15.0 15.0 15.0

pH 4.2 92.5 92.5 91.2

5.9 7.1 9.2

73.0 72.0 72.2 74.2 75.5

23.9 23.9 24.8 29.8 35.7

0.25 0.25 0.25 0.25 0.0 0.25 0.25 0.25 0.25 0.25

0.50 0.45 0.39 0.25 0.0 0.50 0.50 0.50 0.50 0.50

15.0 15.0 15.0 15.0 15.0 30.0 15.0 10.0 5.0 4.0

pH 6.4 90.6 90.8 90.0

7.1 7.5 2.5

91.0 90.6 91.0 90.0 90.5

7.1 7.1 7.1 3.8 1.8

71.0 71.0 73.0 73.0 72.7 71.5 70.7 70.5 70.7 72.5

24.4 25.6 27.3 30.7 33.9 21.7 24.2 25.5 28.8 26.0

1 Vheating

= 5°C/min.

109

I. V.Sochava and T. V.Belopolskaya

was observed besides the denaturation maximum (Figure 7, Table IV). Thus, the addition of salt really results in HTM appearance at thermograms of lysozyme solutions within the pH range for which the HTM was missing in the case of pure glycine buffer. The peculiarity of this maximum is that its temperature position, shape and heat absorption value undergo strong changes upon repeated heating, i.e. the process providing this maximum is not completely reversible. To increase the reversibility of this transition the concentrations of solutions studied were decreased to c = 6.3% and 3.6% (Figure 7b). In fact, it appears that the lowering of the concentration to 3.6% gives total reproducibility of HTM upon multiple heating, i.e. for the solutions with pH 4.2 we obtained a similar calorimetric HTM behaviour to that for clear, homogeneous, meltable gel obtained at pH 1.7-2.7. The direct observation of this solution in glass ampoules has shown that an initially turbid solution of native protein at 55°C, i.e. before denaturation, becomes completely transparent. After denaturation at 85°C, a white, non-transparent weak gel is formed, which turns into a practically transparent solution after passing the HTM temperature region at 110°C. When the sample is cooled the gel is restored and becomes turbid. Thus, this HTM is also related to gel melting at pH 2. Also, the HTM thermodynamic characteristics (Table IV) are similar to the melting characteristics of the gel formed at pH 2 with the addition of 0.5 mol/dnr' NaCI (Table III). This allows us to think that, at pH 4.2, we are dealing also with the melting of type I regular gel. A quite different picture was obtained from visual studies of a concentrated solution (15%) of lysozyme. White non-transparent gel formed in this case after denaturation does not transfer into a liquid state above an HTM of 110°C, but turns to a more dense and rubber-like gel, repelling simultaneously a considerable quantity of water and becoming clearer. After the HTM, instead of the melt a densified gel was found that could be connected with the irreversible character of the HTM on the thermogram for high protein concentrations. Thus, the increase in concentration under the conditions chosen results in a change of aggregate state of the sample only at temperatures above T HTM; here, the HTM is always present in the thermograms at the first heating of the solution (Table IV). We think that the possible existence of a reversible and irreversible HTM at similar pH and mol/dm ' of the solution under only slight protein concentration variations enables us to consider the HTM in both cases as manifestations of the same process: namely, melting of regular gel. This is valid also for the case when, after HTM, the melt takes place as well as when dense, rubber-like gel is formed. It is known from the literature that the addition of NaCI promotes formation of the branched turbid gel. This is also confirmed by our experiments. Besides, the data presented here show that NaCI also promotes more intense formation of the regular melting gel, broadening essentially the pH range in which it is formed. We also considered non-buffered lysozyme aqueous solutions with different NaCl contents (pH 6.4, c = 15%). It was found that, in spite of high pH values, gel is not formed during the heating of lysozyme aqueous solutions. Instead,

110

Globular protein gels

after denaturation, a turbid solution of aggregates is formed. However, the addition of only 0.25 mol/dm' NaCl to the solution leads to the formation of a white gel after denaturation (82°C). Simultaneously, on the walls of the glass ampoule minor water drops appear. On increasing further the temperature the external appearance of the gel is not changed, but its elasticity grows and the quantity of water at the walls increases. Gel extracted from the ampoule at 105°C retains its shape but is easily separated into pieces under mechanical action repelling its water. The thermograms of the system, both for gel and aggregates formation, do not reveal the HTM. This indicates that it is a type II gel. The increase of NaCI content in the initial solution up to 0.5 mol/dnr' results in a gradual change in calorimetric behaviour which is manifested by the appearance of an HTM and the increase of its thermal effect up to Q = 7.1 Jig, T HTM being constant and equal to 90°C (Figure 8). Similar behaviour has been observed while studying the effect of protein concentration on heating thermograms (Figure 8b, Table IV). At the curves of the second heating both denaturation and HTM maxima are missing in all cases, which proves the complete irreversibility of the processes related to them. For all the gels with different NaCl molarity at a protein concentration of 15% and with variable protein concentration cOlo at 0.5 mol/drrr' NaCl, the white colour, low elasticity and beginning stratification are peculiar to the HTM: Above the HTM a sharp increase in gel elasticity and density occurs in all cases. The quantity of water repelled sharply increases while passing through the temperature region of the HTM; the system protein-water-salt becomes stratified. Note that, under the conditions chosen, as well as at pH 4.2, above the HTM the gel becomes noticeably clearer than below it.

Cp a

70

90

Fig. 8. Temperature dependence of heat capacity of aqueous lysozyme solutions at pH = 6.4; NaCI added. (a) Variable NaCI molarity and c = 15%; 1,2,3,4 and 5 are for mol/drrr' = 0, 0.25, 0.39, 0.45 and 0.5 respectively . (b) Variable protein concentration and 0.5 mol/drn" NaCl; 6, 7,8,9 and 10 are for c = 4.5, 10.0, 15.0 and 30.0%. The successive heating is denoted by dashed lines.

111

I. V.Sochava and T. V.Belopolskaya

Comparison of calorimetric and visual results, obtained for two pH values (4.2 and 6.4; c = 3.6%), shows that the difference in the behaviour of the studied systems is only evident above the HTM. Thus, the HTM at pH 6.4 as well as that at pH 4.2 should be related to the melting of the regular part of gel formed under these conditions. For pH 4.2 we have succeeded in choosing such mol/drrr' and c% at which melting has resulted in the transfer of the system into a liquid state. Changing these parameters so as to enhance the interaction, increasing pH in particular, results in the collapse of the gel network following immediately the destruction of part of the regular structure at gel melting. Here, above the HTM, instead of the liquid state we obtain a gel of high density (type IV). Thus, as a result of the type III gel melting, the cooperative stratification of the system into collapsed gel and water occurs. So, for lysozyme water solutions with pH 6.4, depending on NaCl content, turbid gels of two types can be formed: non-melting gel (mol/dm? <0.25), earlier called type II gel, and melting gel (mol/drrr' ~0.25), the mixed-type gel (type III). The melting gel which we observed at pH 4.2 for high protein concentrations should be referred to as a mixed-type gel. Concluding the consideration of the NaCI effect on gelation, we should like to stress once again that NaCI not only intensifies the process but broadens the pH range of the possible formation of regular melting gel. At the same time the branching/cross-linking of the turbid gel is enhanced. All these phenomena taken together enable us to explain the formation of mixed-type gel at high pH which, though retaining all the exterior features of the turbid gel, acquires the ability to melt. We have also revealed that NaCI addition to the lysozyme solution is not the only factor that can raise the HTM. An even stronger effect has been observed

.... 'CT".>

3

2 I

2 T 151

-e

151 ill

1S II)

lSI

~

151 N

....

....... 151

151 ill

....

T,COC) Fig. 9. Heat capacity of lysozyme solution in phos~hate buffer versus temperature. (a) pH = 7.0; mol/dm' = 0.25; c = 12%; (b) pH = 7.9; mol/dm = 0.25; c = 12%; 1 = the first heating up to T = 90°C; 2 and 3 = repeated heatings up to T = 140°C.

112

Globular protein gels

while studying the lysozyme solution in phosphate buffer (Figure 9), where T H"fM appears to be 108°C at Td = 71°C. Taking into account the rather low buffer molarity for the protein concentrations considered , such a high value of THTM should be related to the specific character of its ionic composition. The HTM is missing on repeated heating in phosphate buffer as is the case for saltwater solutions; hence, gel destruction is irreversible on heating. In the experiment described attention is drawn to a considerable difference between the temperature regions of gel formation and melting. This situation resembles the cold polymer crystallization from the glassy state in the course of which highly defective crystals are formed that are capable of refining their structure under further heating (15,16) . Thus, in the process of gelation under the conditions considered protein behaves as a polymer.

Conclusion By varying the parameters of the protein environment (the temperature included) for lysozyme, we have succeeded in revealing four different types of globular protein gel, both calorimetrically and visually, and in determining the regions of their existence. In their external appearance the gels noticeably differ from each other, with the exception of turbid gels of types II and III. The clear linear gel (type I) and turbid mixed gel (type III) are calorimetrically manifested as a cooperative heat absorption (HTM) due to the destruction of their regular structure. Collapsed gel (type IV), formed after melting type III gel, is responsible for the irreversibility of this process on repeated heating. The turbid non-melting gel is the only one which is not explicitly manifested calorimetrically. The analysis of the whole set of data obtained enables us to conclude that pH is the most important parameter controlling the gelation process. The pH specifies the path of the protein molecular aggregation , which can be either regular (directed) or irregular (branched) in the course of gelation. The ionic strength of the solution enables us to control its thermostability, i.e. the quality of the structure within the type predetermined. Controlling the protein aggregation in the solution by pH , ionic strength and concentration, one can essentially broaden the pH range in which the thermoreversible gel is formed and thus provide such changes in the structure of the turbid branched gel which make it meltable . Finally, changing the temperature of transparent gel melt and its exposure at this temperature, one can influence noticeably the structure and stability of the gel being restored on cooling.

Acknowledgements The authors express their deep gratitude to S.Yu.Kazitzina for participation in the experiments and to Dr G .I.Tsereteli for interest in this study and thoughtprovoking discussions. 113

I. V.Sochava and T. V.Belopolskaya

References 1. Richardson ,R .K. and Rose Murphy,S.B. (1981) Brit. Polymer. J. , 13, 11-1 6. 2. Bikbov,T.M. , Grinberg.VYa., Schmandke ,H. , Chaika ,T.S., Vaintraub,I.A. and Toistogusov,V.B . (1981) Colloid Polymer Sci., Z59, 536-547. 3. Clark ,A .H . and Tuffnell,C.D. (1980) Int. J. Peptide Protein Res., 16, 339-359. 4. Clark,A.H., Saunderson ,D .H.B. and Suggett ,A. (1981) Int. J. Peptide Protein Res., 17, 353364. 5. Clark ,A.H. , Judge,F.J., Richardson,J.B . and Suggett.A. (1981) Int. J. Peptide Protein Res., 17, 380-392. 6. Van Kleef ,F.S.M. (1986) Biopolymers, 25, 31-60. 7. Sochava.lV ., Belopolskaya ,T.V. and Smimova ,O .I. (1985) Bioph. Chemistry , ZZ, 323-336 . 8. Sochava,I.V., Kazitzina ,S.Yu. and Belopolskaya ,T.V. (1985) Proc. Symp. on phys.-chem. properties of biopolymers in solutions and cells, Pushchino , USSR, 62-63. 9. Belopolskaya ,T.V., Kazitzina,S .Yu. and Sochava ,I.V . (1985) Proc. of VI Symp. on conform. changes of biopolymers in solutions , Tb ilisi, USSR , 19. 10. Belopolskaya ,T.V ., Kazitzina,S.Yu. and Sochava ,I.V . (1989) Biofizika , 34, 520-521. 11. Belopolskaya,T.V. , Sochava ,T.V. and Kazitzina,S.Yu. (1990) Biofizika , 35, 751-755. 12. Sochava.l .V .; Belopolsk aya,T .V. and Kazitzina,S .Yu . (1990) Biofizika , 35, 756-761. 13. Belopolskaya,T.V. and Sochava,I.V . (1990) lIth IUPAC Conference on Chemical Thermodynamics, Como, Italy, Abstract, 27-28. 14. Chechinashvili,N.N., Privalov,P.L. and Tiktopulo,E.I. (1973) FEBS Lett., 30, 57-60. 15. Wunderlich ,B. (1970) Ber. Bunsen Phys. Chem. , 74, 768-770. 16. Sochava,I.V . and Smirnova,O .I. (1973) Fiz. Tverd. Tela. , IS, 3003-3005 .

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