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Conformational changes of gelatine molecules during melting of gels
CONFORMATIONAL CHANGES OF GELATINE MOLECULES DURING MELTING OF GELS* V. 1~. IZMAILOVA, V. A. PCHELIN and SAMIR ABU ALI Lomonosov State University, Moscow (Received 30 December 1964) OUR previous work [1] investigated the effect of concentration and pH on the melting temperature of gelatine gels. It was shown that the melting temperature of gels increased with their concentration. The deviations from an isoelectrical state towards either higher or lower pH values resulted in the decrease of the melting point. I t was assumed that three types of bonds take part in gelatine gel formation. The groups carrying a charge are important because they determine the rate of gel formation. Hydrogen bonds are responsible in 1 to 20% of the gels, but there are in addition also hydrophobic bonds, which are responsible in 30 to 55% of the gels. It was also shown that the more concentrated gels melted at above helix-to-coil transition temperature, while the less concentrated gels melted below that temperature. There is at present a large number of works dealing with the optical rotation study of conformational states of the gelatine molecules in solutions [2-16]. One of the papers [16] reported the transition due to a change in temperature and the effect of electrolyte and nonelectrolyte additions on the helix-to-coil conformation in a 0.1% gelatine solution. The mean helix-to-coil transition temperature was the measure of the stability of the helical conformation. However, these authors did not study the gels. The aim of the present work was to study the helix-to-coil transition in the solations and gels of gelatine, and to clarify the effect of gel-formation on it. Conformational changes of the gelatine molecule were studied by heating gels having melting points below the temperature of the total helix-to-coil transition. For example, a 2% gel started to decompose at 25°C, while the helix to statistical coil transition took place at 30--35°C; a 5% gel melted at 31°C and the helix-to-coil transition took place at the same temperature. The effect of the gel p H on the rate of the helix-to-coil transition was also studied. EXPERIMENTAL
The conformation of polypeptide chains in a solution was determined by measuring the specific optical rotation in a Hilger polarimeter with a 0.01° reading accuracy at ~----5850-
5750/~. The experiments were made with the "Foto" brand of gelatine, which was purified and made in the isoelectrical state by the Loeb method. The gelatine solutions were prepared * Vysokomol. soyed. 7: No. 11, 1985--1988, 1965.
2178
Conformational changes of gelatine molecules
2179
as follows: a weighed amount of gelatine was placed in a volumetric flask, one haft of the required water was added, and the flask then kept 12 hours at 5°C to allow for swelling. The flask containing the swollen gelatine was immersed in water heated to 60°C, and then shaken until the gelatine was fully dissolved. The solution was cooled to room temperature, water was added to the mark and the contents heated again to 60°C. The investigated solution was poured into a thermostatically controlled cell, 0,5 dm long, having an aperture in the centre through which the end of a thermocouple was lowered. The specific optical rotation during the melting of gels was measured at a temperature gradient of I°C in 10 minutes. The solutions of various concentrations (0.5, 2 a n d 5 0 ) a n d p H ' s (from 2 to 11) were first investigated during natural cooling from 40 to 20°C; the specific optical r o t a t i o n increased during cooling, as we have shown earlier [17]. The gel solutions were t h e n cooled in a refrigerator to 5°C. Special tests showed t h a t the changes of specific optical r o t a t i o n were practically complete after 4 d a y s storage at this temperature. This was due to the t e r m i n a t i o n of the conformational changes of the gelatine molecule a n d a m a x i m u m transition to a helix. The d e t e r m i n a t i o n was followed b y slowly heating, the gelatine-containing cell, a n d the specific optical rotation was determined for all concentrations a n d pHvalues as a function of temperature. A typical curve of a gelatine gel is shown in Fig. 1. I t could be seen t h a t the specific optical rotation decreased with t e m p e r a t u r e increase. This was connected with the disappearance of helical shapes and their gradual change to r a n d o m coils. The curve m a y be divided into three sectors; t h a t of a constant m a x i m u m specific rotation, approximately up to 15°C; t h a t of the rapid change of specific rotation; a n d finally, the constant, b u t m i n i m u m specific optical rotation, above 30°C.
-~]o '
3oo
250
200
10
20
3O
#077C
FIG. 1. The effect of temperature on the specific optical rotation ofa 5~o gelatine gel.
2180
V. N. IZM~SOV.~ et d.
The effect of gel structure on the helix-to-coil transition was examined in experiments with 5 and 2 ~ gels, and 0 . 5 ~ gelatine solutions were used for comparison. Figure 2 shows the curves of the temperature coefficients of specific optical rotation of the solution and the gels in isoelectrical state as a function of temperature. The temperature coefficient of specific optical rotation is zero at 36°C and above. The gelatine molecules existed only in the state of statistical coils at these temperatures. The rate of helix formation increased on cooling to 20°C. This is connected with the greatest probability of the formation of hydrogen bonds, which at these temperatures strengthen the helices [18]. The temperature coefficient of specific optical rotation had a maximum and constant value at 17 to 20°C, and the value then decreased. The mobility of molecules and its segments apparently decreased on lowering the temperature, thus impeding the formation of the helical shape, and also, the great majority of gelatine molecules had then completed the transition from a statistical coil to a helix. Figure 2 sthows also that the maximum temperature coefficients of specific optical rotation of both, the gels and gelatine solutions, were the same. This indicates that the conformational from helix-to-statistical coil did not depend on the molecules being present as a solution or being part of the gel structure. 4o
~t Aa f~t 15 o
o2 "3
10 ° °
~
\
°, •
,,
5-
^
f
r
FIG. 2
,A A~'%..^
FIG. 3
FIG. 2. The effect of temperature t on the J~/ztt value: /--for 0.5% solution; 2--2% gel; 3--5% gel in an isoelectric state. FIG. 3. The effect of temperature t on the z~/zt~ value for a 5% gel at various pH: 1--pH 5.2; 2--pH 10.3~ 3--pH 2.
Conformational changes of gelatine molecules
2181
The effect of the charge of gelatine molecules on the temperature coefficient of specific optical rotation of gels was investigated in 5 ~ gels at an isoelectrica] state and acid or alkaline p H (Fig. 3). On heating the gel, the conformation changes of the gelatine molecules progressively increased, and the coefficient then remained constant for various pH values at certain temperatures. This happened at 18 to 20°C at pH 5.2, 14 to 18°C at pH 10, and 13 to 16°C at p H 2. Any further increase of temperature caused the transition from helix to chaotic coil to increase. The earliest rapid transition was observed at pH 2 (curve 3), then at pH 10 (curve 2), and the last at p H 5.2, this corresponding to the isoelectrical state. The general appearance of curves of acid and alkaline gelatine gels was the same. However, the temperatures of ,the complete helix-to-coil transition differed substantially when the temperature coefficient of specific optical rotation equalled to zero. I t was 36°C at pH 5.2, 29°C at pH 10.3, 25°C at pH 2, i.e. the complete transition occurred in the acid and in the alkaline range 7 to ll°C below t h a t of the isoelectrical state. This m a y be explained by a lower stability of helical shapes in alkaline and acid media owing to the charge on the gelatine molecules having the same sign. CONCLUSIONS
(1) Maximum temperature coefficients of specific optical rotation of gels and gelatine solutions are the same. This proves t h a t the conformation transition of gelatine molecules from helices to statistical coils does not depend on the molecules being in a solution or taking part in the formation of the gel structure. (2) The complete transition from a helix to a statistical coil takes place on heating in the case of acid and alkaline conditions at temperatures 7 to ll°C lower t h a n the temperature of the same transition in an isoleectric state. This m a y be explained by a lower stability of the helix conformations in alkaline and acid media which is the result of all molecules having the same charge. Translated by K. A. ALLEI~ REFERENCES
1. V. N. IZMAILOVA, V. A. PCHELIN and SAMIR ABU ALI, Vysokemol. soyed. 6: 2197, 1964 2. E. 0. KREMER and J. R. FANSLOW, J. Phys. Chem. 29: 1169, 1925 3. D. C. CARPENTER and F. E. LOVELACE, J. Amer. Chem. Soc. 67: 2342, 2337, 1935 4. C. ROBINSON and M. J. BLOTT, Nature 168: 325, 1951 5. C. COHEN, Nature 175: 129, 1955
6. 7. 8. 9.
C. COHEN, J. Biophys. and Biochem. Cytol. 1: 203, 1955 A. ELLIOT, Recent advances in gelation and glue research, 1957 A. R. DOWl~C and A. ELLIOT, Prec. Roy. Soc. 242: 325, 1957 I. T. YANG and P. DOTY, J. Amer. Chem. See. 79: 761, 1957 10. W. F. HARRINGTON, Nature 14: 997, 1958 11. A. VIES and J. COHEN, Nature 186: 84, 1960 12. P. Y. FLOIX and E. S. WEAVER, J. Amer. Chem. Soc. 82: 4518, 1960
2182
O. V. S M m N O V A eta/.
13. 14. 15. 16. 17.
W. F. HARRINGTON and P. HIPPEL, Arch. Biochem. and Biophys. 92: 1O0, 1961 P. H. HIPPEL and KWOK-I-WONG, J. Biochem. 4: 664, 1962 A. COURTS, J. Biochem. 88: 124, 1962 P. H. HIPPEL and KVgOK-I.WONG, J. Biochem. 2: 1387, 1399, 1963 V. A. PCHELIN, V. N. IZMAILOVA and V. P. MERZLOV, Vysokomol. soyed. 5: 1429, 1963; Doldady AN SSSR 150: 1307, 1963 18. V. A. PCHEL1N, N. V. GRIGOR'EVA and V. N. IZMAILOVA, Doklady AN SSSR 151: 134, 1963
SYNTHESIS AND STUDY OF POLYCARBONATES PREPARED BY INTERFACIAL POLYCONDENSATION OF DI-(4-HYI)ROXYPHE~NYL)-P~NYLMETHANE (DHPM) * O. V. SMIRNOVA, 0 . G. FORTUNATOV, 1~. M. GARBAR a n d G. S. KOLESNIKOV D. I. Mendeleyev Chemico-technological Institute, Moscow
{Received 31 December 1964) T H E p o l y c a r b o n a t e s belong to t h e plastics h a v i n g g o o d p h y s i c o - m e c h a n i c a l properties. So far, p o l y c a r b o n a t e s were b a s e d on 2,2-di(4-hydroxyphenyl)p r o p a n e ( d i a n ) a n d its d e r i v a t i v e s , a n d these h a v e b e e n described in some detail. T h e i n f o r m a t i o n a b o u t a p o l y c a r b o n a t e b a s e d o n cU-(4-hydroxyphenyl)phenylm e t h a n e ( D H P M ) is limited. I t was p r o d u c e d b y Schnell b y t h e direct p h o s g e n a t i o n m e t h o d a n d its m e l t i n g p o i n t was 210-220°C [1, 2]. W e were i n t e r e s t e d in a p r e p a r a t i o n o f this p o l y c a r b o n a t e b y interfacial p o l y c o n d e n s a t i o n , which p e r m i t s t h e p r o d u c t i o n o f a high p o l y m e r in r e l a t i v e l y simple a p p a r a t u s , a n d in t h e s t u d y of t h e r e a c t i o n m e c h a n i s m .
EXPERIMENTAL The polycondensation was carried out at 20°C in a condensation tube fitted with a stirrer and a drop funnel. The temperature was thermostatically controlled. To start an experiment, the tube was charged with an alkaline diphenol solution, stirred at the start for 20 min to bring the solution up to temperature, and a phosgene solution in CC14was added through the drop funnel. The ratio of the aqueous to the organic phase was 1 : 1 (by volume). Stirring was continued for 40 minutes at 2,800 r.p.m, and a 1% HC1 solution was added to neutralize the alkali. The solvent was stripped from the produced polycarbonate by steam distillation, the polymer was dried and reprecipitated with methanol from its methylene chloride solution. The reprecipitated polycarbonate was vacuum-dried at 40-50°C to constant weight. The yield of the polymer and its specific viscosity in methylene chloride was determined on 5 g/1. concentrations in each experiment. * Vysokomol. soyed. 7: No. 11, 1989-1992, 1965.
The conformation of polypeptide chains in a solution was determined by measuring the specific optical rotation in a Hilger polarimeter with a 0.01° reading accuracy at ~----5850-
5750/~. The experiments were made with the "Foto" brand of gelatine, which was purified and made in the isoelectrical state by the Loeb method. The gelatine solutions were prepared * Vysokomol. soyed. 7: No. 11, 1985--1988, 1965.
2178
Conformational changes of gelatine molecules
2179
as follows: a weighed amount of gelatine was placed in a volumetric flask, one haft of the required water was added, and the flask then kept 12 hours at 5°C to allow for swelling. The flask containing the swollen gelatine was immersed in water heated to 60°C, and then shaken until the gelatine was fully dissolved. The solution was cooled to room temperature, water was added to the mark and the contents heated again to 60°C. The investigated solution was poured into a thermostatically controlled cell, 0,5 dm long, having an aperture in the centre through which the end of a thermocouple was lowered. The specific optical rotation during the melting of gels was measured at a temperature gradient of I°C in 10 minutes. The solutions of various concentrations (0.5, 2 a n d 5 0 ) a n d p H ' s (from 2 to 11) were first investigated during natural cooling from 40 to 20°C; the specific optical r o t a t i o n increased during cooling, as we have shown earlier [17]. The gel solutions were t h e n cooled in a refrigerator to 5°C. Special tests showed t h a t the changes of specific optical r o t a t i o n were practically complete after 4 d a y s storage at this temperature. This was due to the t e r m i n a t i o n of the conformational changes of the gelatine molecule a n d a m a x i m u m transition to a helix. The d e t e r m i n a t i o n was followed b y slowly heating, the gelatine-containing cell, a n d the specific optical rotation was determined for all concentrations a n d pHvalues as a function of temperature. A typical curve of a gelatine gel is shown in Fig. 1. I t could be seen t h a t the specific optical rotation decreased with t e m p e r a t u r e increase. This was connected with the disappearance of helical shapes and their gradual change to r a n d o m coils. The curve m a y be divided into three sectors; t h a t of a constant m a x i m u m specific rotation, approximately up to 15°C; t h a t of the rapid change of specific rotation; a n d finally, the constant, b u t m i n i m u m specific optical rotation, above 30°C.
-~]o '
3oo
250
200
10
20
3O
#077C
FIG. 1. The effect of temperature on the specific optical rotation ofa 5~o gelatine gel.
2180
V. N. IZM~SOV.~ et d.
The effect of gel structure on the helix-to-coil transition was examined in experiments with 5 and 2 ~ gels, and 0 . 5 ~ gelatine solutions were used for comparison. Figure 2 shows the curves of the temperature coefficients of specific optical rotation of the solution and the gels in isoelectrical state as a function of temperature. The temperature coefficient of specific optical rotation is zero at 36°C and above. The gelatine molecules existed only in the state of statistical coils at these temperatures. The rate of helix formation increased on cooling to 20°C. This is connected with the greatest probability of the formation of hydrogen bonds, which at these temperatures strengthen the helices [18]. The temperature coefficient of specific optical rotation had a maximum and constant value at 17 to 20°C, and the value then decreased. The mobility of molecules and its segments apparently decreased on lowering the temperature, thus impeding the formation of the helical shape, and also, the great majority of gelatine molecules had then completed the transition from a statistical coil to a helix. Figure 2 sthows also that the maximum temperature coefficients of specific optical rotation of both, the gels and gelatine solutions, were the same. This indicates that the conformational from helix-to-statistical coil did not depend on the molecules being present as a solution or being part of the gel structure. 4o
~t Aa f~t 15 o
o2 "3
10 ° °
~
\
°, •
,,
5-
^
f
r
FIG. 2
,A A~'%..^
FIG. 3
FIG. 2. The effect of temperature t on the J~/ztt value: /--for 0.5% solution; 2--2% gel; 3--5% gel in an isoelectric state. FIG. 3. The effect of temperature t on the z~/zt~ value for a 5% gel at various pH: 1--pH 5.2; 2--pH 10.3~ 3--pH 2.
Conformational changes of gelatine molecules
2181
The effect of the charge of gelatine molecules on the temperature coefficient of specific optical rotation of gels was investigated in 5 ~ gels at an isoelectrica] state and acid or alkaline p H (Fig. 3). On heating the gel, the conformation changes of the gelatine molecules progressively increased, and the coefficient then remained constant for various pH values at certain temperatures. This happened at 18 to 20°C at pH 5.2, 14 to 18°C at pH 10, and 13 to 16°C at p H 2. Any further increase of temperature caused the transition from helix to chaotic coil to increase. The earliest rapid transition was observed at pH 2 (curve 3), then at pH 10 (curve 2), and the last at p H 5.2, this corresponding to the isoelectrical state. The general appearance of curves of acid and alkaline gelatine gels was the same. However, the temperatures of ,the complete helix-to-coil transition differed substantially when the temperature coefficient of specific optical rotation equalled to zero. I t was 36°C at pH 5.2, 29°C at pH 10.3, 25°C at pH 2, i.e. the complete transition occurred in the acid and in the alkaline range 7 to ll°C below t h a t of the isoelectrical state. This m a y be explained by a lower stability of helical shapes in alkaline and acid media owing to the charge on the gelatine molecules having the same sign. CONCLUSIONS
(1) Maximum temperature coefficients of specific optical rotation of gels and gelatine solutions are the same. This proves t h a t the conformation transition of gelatine molecules from helices to statistical coils does not depend on the molecules being in a solution or taking part in the formation of the gel structure. (2) The complete transition from a helix to a statistical coil takes place on heating in the case of acid and alkaline conditions at temperatures 7 to ll°C lower t h a n the temperature of the same transition in an isoleectric state. This m a y be explained by a lower stability of the helix conformations in alkaline and acid media which is the result of all molecules having the same charge. Translated by K. A. ALLEI~ REFERENCES
1. V. N. IZMAILOVA, V. A. PCHELIN and SAMIR ABU ALI, Vysokemol. soyed. 6: 2197, 1964 2. E. 0. KREMER and J. R. FANSLOW, J. Phys. Chem. 29: 1169, 1925 3. D. C. CARPENTER and F. E. LOVELACE, J. Amer. Chem. Soc. 67: 2342, 2337, 1935 4. C. ROBINSON and M. J. BLOTT, Nature 168: 325, 1951 5. C. COHEN, Nature 175: 129, 1955
6. 7. 8. 9.
C. COHEN, J. Biophys. and Biochem. Cytol. 1: 203, 1955 A. ELLIOT, Recent advances in gelation and glue research, 1957 A. R. DOWl~C and A. ELLIOT, Prec. Roy. Soc. 242: 325, 1957 I. T. YANG and P. DOTY, J. Amer. Chem. See. 79: 761, 1957 10. W. F. HARRINGTON, Nature 14: 997, 1958 11. A. VIES and J. COHEN, Nature 186: 84, 1960 12. P. Y. FLOIX and E. S. WEAVER, J. Amer. Chem. Soc. 82: 4518, 1960
2182
O. V. S M m N O V A eta/.
13. 14. 15. 16. 17.
W. F. HARRINGTON and P. HIPPEL, Arch. Biochem. and Biophys. 92: 1O0, 1961 P. H. HIPPEL and KWOK-I-WONG, J. Biochem. 4: 664, 1962 A. COURTS, J. Biochem. 88: 124, 1962 P. H. HIPPEL and KVgOK-I.WONG, J. Biochem. 2: 1387, 1399, 1963 V. A. PCHELIN, V. N. IZMAILOVA and V. P. MERZLOV, Vysokomol. soyed. 5: 1429, 1963; Doldady AN SSSR 150: 1307, 1963 18. V. A. PCHEL1N, N. V. GRIGOR'EVA and V. N. IZMAILOVA, Doklady AN SSSR 151: 134, 1963
SYNTHESIS AND STUDY OF POLYCARBONATES PREPARED BY INTERFACIAL POLYCONDENSATION OF DI-(4-HYI)ROXYPHE~NYL)-P~NYLMETHANE (DHPM) * O. V. SMIRNOVA, 0 . G. FORTUNATOV, 1~. M. GARBAR a n d G. S. KOLESNIKOV D. I. Mendeleyev Chemico-technological Institute, Moscow
{Received 31 December 1964) T H E p o l y c a r b o n a t e s belong to t h e plastics h a v i n g g o o d p h y s i c o - m e c h a n i c a l properties. So far, p o l y c a r b o n a t e s were b a s e d on 2,2-di(4-hydroxyphenyl)p r o p a n e ( d i a n ) a n d its d e r i v a t i v e s , a n d these h a v e b e e n described in some detail. T h e i n f o r m a t i o n a b o u t a p o l y c a r b o n a t e b a s e d o n cU-(4-hydroxyphenyl)phenylm e t h a n e ( D H P M ) is limited. I t was p r o d u c e d b y Schnell b y t h e direct p h o s g e n a t i o n m e t h o d a n d its m e l t i n g p o i n t was 210-220°C [1, 2]. W e were i n t e r e s t e d in a p r e p a r a t i o n o f this p o l y c a r b o n a t e b y interfacial p o l y c o n d e n s a t i o n , which p e r m i t s t h e p r o d u c t i o n o f a high p o l y m e r in r e l a t i v e l y simple a p p a r a t u s , a n d in t h e s t u d y of t h e r e a c t i o n m e c h a n i s m .
EXPERIMENTAL The polycondensation was carried out at 20°C in a condensation tube fitted with a stirrer and a drop funnel. The temperature was thermostatically controlled. To start an experiment, the tube was charged with an alkaline diphenol solution, stirred at the start for 20 min to bring the solution up to temperature, and a phosgene solution in CC14was added through the drop funnel. The ratio of the aqueous to the organic phase was 1 : 1 (by volume). Stirring was continued for 40 minutes at 2,800 r.p.m, and a 1% HC1 solution was added to neutralize the alkali. The solvent was stripped from the produced polycarbonate by steam distillation, the polymer was dried and reprecipitated with methanol from its methylene chloride solution. The reprecipitated polycarbonate was vacuum-dried at 40-50°C to constant weight. The yield of the polymer and its specific viscosity in methylene chloride was determined on 5 g/1. concentrations in each experiment. * Vysokomol. soyed. 7: No. 11, 1989-1992, 1965.