THE SOLUBILIZING EFFECT OF GELATIN IN RELATIONTO THE CONFORMATION OF THE MOLECULES IN AQUEOUS SOLUTION* G. P. YAMPOL'SKAYA, V. :N. IZMAILOVAand V. A. PCHEI,I~ M. V. Lomonosov State University, Moscow (Received 5 November 1969)
IT IS now well known that proteins, the structure of which in aqueous solution is largely controlled by hydrophobic interaction, can become bonded to non-polar substances, especially hydrocarbons [1-6]. On the basis of the combined results of study of the protein-hydrocarbon interaction, it may be suggested that the bonding sites are non-polar regions of the three-dimensional structure of the proteins. It is therefore obvious that the ability to bond with hydrocarbons is determined by the nature of the spatial arrangement of the protein molecules. One oi the first indications that the solubilizing effect of protein molecules is dependent on their conformation was the result of work showing that for globular proteins this effect decreases with increase in the viscosity of the solution, and with increase in the specific optical rotation at p H values far from the isoelectric point [1, 2]. In a study of the interaction of gelatin with saturated and aromatic hydrocarbons it was found that solubilization is greatest at p H 4.9, i.e. at the isoelectric point of gelatin. Also the maximal bonding of hydrocarbons at the isoelectric p H values is much more clearly expressed in the case of gelatin than for globular proteins [7-9]. It is well known that under certain conditions gelatin can take up a highly ordered collagen-like structure similar to the poly-L-proline II helix. The reversible conformational transition of gelatin under heat treatment has been studied in a number of papers [10-13]. In aqueous solution gelatin can exist in various conformational states (a helix at low temperature and a coil above 35°). This makes gelatin an extremely interesting object of study for finding the correlation between the solubilizing effect of a protein and the conformation of its molecules. It was of value to study the changes in the conformation of the protein molecules and in the solubiliza~ion of benzene in aqueous solutions of gelatin with change in temperatures, because change in the solvent, addition of urea or other methods of altering the conformation of the protein can cause substantial changes in the true solubility of benzene in water and interfere with assessment of the adsorption of the hydrocarbon b y the protein. * Vysokomol. soyed. A12: No. 9, 1923-1927, 1970. 2177
G . P . YAMPOL'SKAYAet al.
The gelatin used in this work was the "Photo" grade, purified by the method of Leb  a n d the benzene was purified b y the special method described in reference . The solubility, of benzene in the gelatin solutions was measured b y the refractometric method [15, 16]. The refractive index of the solution was measured in an IRF-23 refractometer. The solubility of the hydrocarbon was eMculated from the additivity rule for specific refraction. nS--1 1 _ n ~ - - I 1
The quantities with the subscript i refer to the components of t h e mixture, P being the weight of the component and d the density. The aqueous solution of the protein is regarded as a single component, hence the solution of b~nzene in the protein solution is regarded as a " b i n a r y mixture", for which the following expression is valid
p ~-1 Pn~
dp -- n ~
- - - P a + s -'--PY" da n~,+ 2 4y
P u t t i n g P~ (1/dp) = V and (n ~ - 1)/(n 2 + 2) = / / w e obtain
Vp//p = V.//a + V~//~ where the subscripts a, p and y refer respectively to the gelatin solution, the gelatin solution saturated with the hydrocarbon and the pure hydrocarbon. Neglecting a n y deviation from additivity of the volumes, we assume that the volume of the solution is equal to the volume of the components, i.e. V p = V a + V ~ , , then V y ( H y - - I I p ) = V a ( I I p - - I I a ) and the value of Vy is given b y • //p--H,
The solubility of tho hydrocarbon, S, ws normally calculated in grammes per 100 ml of protein solution S = fIp -- ll*' dy 100.
//~-/Tp Special experiments showed that at 7° gelatin solutions become saturated with benzene in 4 hr and at 40 ° in 30 rain. The equilibrium value of the refractive index of the solutions was used for calculation of the solubility at all temperatures (7, 20, 40 and 55°). The solubility of benzene in water at these temperatures was measured as a control.
The solubilities of benzene at various concentrations of gelatin at the above temperatures are given in Table 1. At 7 and 20 ° gel formation occurs when the gelatin concentration is above 1 g/100 ml, therefore at the lower temperatures the solubilization of benzene was studied at concentrations not greater than 1.5 g/100 ml. A t the lower temperatures the solubility of benzene in gelatin solutions differs little from its solubility in water at the same temperatures. The solubility increases considerably in gelatin solutions at 40 and 55 ° . Figure 1 shows the dependence of the solubilization of benzene by gelatin in a gelatin solution ( c = l g/100 ml) and the dependence of the specific optical rotation of the gelatin molecule on temperatuTe. It is seen ~hat when t h e g e l a t i n has the collagen helix conformation (this conformation corresponds to a high specific optical rotation) the solubflizing effect of the gelatin molecules is low.
Solubilizing effect of gelatin
At 7° the solubility of benzene in the gelatin solution is practically the same as its solubility in water, the figure being 0.16 g/100 ml. As the temperature is raised the helices melt, this being shown by a fall in the specific optical rotation TABLE 1.
I1~ S O L U T I O N S OF
T E M P E R A T U R E S AND CONCENTRATIONS OF G E L A T I N , p H
Concentration of gelatin, g/100 ml Temperature, °C
Solubility of benzene, g/100 ml 7 20 40 55
0"16 0"17 0"21 0"23
0"20 0"20 0'23
0.20 0.25 , O.24 1 i -i i
0.17 0.27 0.30 0.60
(curve 2, Fig. 1). At the same time increase in the bonding between benzene and the gelatin molecules is seen. Gelatin molecules have the greatest solubilizing effect with respect to benzene when they have the conformation of random coils. In a gelatin solution at a concentration~ of 1 g/100 ml the solubility of benzene is about 0.30 g/100 ml at 40 ° and 0.60 g/100 ml at 55 °. Melting of the helices and formation of random coils causes increase in the solubility of benzene in gelatin solutions. This fact is very important in consideration of the mechanism of increase in the solubility of hydrocarbons in protein solutions. Some authors consider t h a t the protein and hydrocarbon molecules do not come into direct contact, but only through a layer of water, the structure of which is altered substantially by the non-polar groups on the surface of the molecule, and which arc in contact with the water, creating conditions for additional solution of the hydrocarbon in water [17, 19]. Others assume t h a t there is direct contact between the protein and hydrocarbon molecules [1-6]. Papers showing the incorrectness of the first hypothesis have already been published [10-22]. The results of the present work also support the second mechanism, since in [act the area of contact of the non-polar side groups of the gelatin with water is greater at low temperatures (and therefore the effect on the adjacent layer of water should be greater) than after melting of the ordeced structures and the occurrence of hydrophobic interaction, which leads to marked increase of the solubility of benzene in the aqueous solution. I t is therefore natural to assume t h a t at low temperatures the gelatin molecules do not form sites capable of bonding with hydrocarbons, but when the solutions are heated above 40 ° such sites arise. On the basis of this mechanism it is possible from the results obtained on the solubility of benzene in water and in aqueous solutions of gelatin, to calculate the distribution constant, K, of benzene between the protein and water, and t~
G. P. YA~IPOL'SKAYAet al.
d e t e r m i n e t h e change in the free e n e r g y o f b o n d i n g o f t h e h y d r o c a r b o n to t h e protein. T h e v a r i a t i o n in free e n e r g y was calculated f r o m t h e f o r m u l a A . F - = - - . R T In cl
c2 where c 1 is t h e c o n c e n t r a t i o n o f b e n z e n e in gelatin, a n d c 2 t h e c o n c e n t r a t i o n o f b e n z e n e in w a t e r (mole/1.). F i g u r e 2 shows t h e d e p e n d e n c e o f t h e l o g a r i t h m of t h e c o n s t a n t o [ d i s t r i b u t i o n of b e n z e n e b e t w e e n gelatin a n d w a t e r on reciprocal t e m p e r a t u r e • T h e slope of this line is equal to t h e change in e n t h a l p y on i n t e r a c t i o n o f b e n z e n e w i t h
. oI 5O
FIG. 1. Dependence of the adsorption (P) of benzene on gelatin (g/g of gelatin) (1) and the specific optical rotation of gelatin (2) on temperature. FIG. 2. Dependence of the logarithm of the distribution constant of benzene between gelatin and water on reciprocal temperature. gelatin. T h e v a l u e o f A H is a p p r o x i m a t e l y 4.5 k c a l / m o l e a n d t h e change in e n t r o p y is positive a n d equal to 20 kcal/mole/deg. T h e t h e r m o d y n a m i c c h a r a c t e r istics o f t h e process o f bonding o f benzene b y gelatin molecules are p r e s e n t e d TABLE 2. THERMODYNAMICCHARACTERISTICSOF THE PROCESS OF BONDING OF BENZENE TO GELATIN Temper ature, oK
47 48 204
--1.1 --2.5 --2.5 --3.2
4-5 4-5 4.5 4.5
AS, cal/mole. •deg 20 24 22 23
N* 14 70 90 360
* ~V is the number of moles of benzene bound to 1 mole of gelatin. Molecular weight of gelat i n - 70,000.
Solubilizing effect of gelatin
in Table 2. The values of these characteristics (small negative zlF, small positive AH and positive AS) correspond to transfer of a non-polar substance from a polar to a non-polar medium (the theory of hydrophobic interaction of Nemethy and Scheraga [23-25]) and do not correspond to the values characteristic of solution of a hydrocarbon in a structurized layer of water (this process involves a negative change in entropy of 16 kcal/mole). This study of the thermodynamic characteristics of the process of adsorption of benzene by gelatin suggests t h a t the higher solubility of benzene in aqueous gelatin solutions is due to penetration of the hydrocarbon into the less polar (in comparison with the aqueous environment) regions, formed by bonding between the propyl and hYdroxypropyl side groups of the amino-acid residues of the gelatin. CONCLUSIONS
(1) The solubility of benzene in gelatin solutions at 7, 20, 40 and 55 ° has been studied. I t is shown t h a t the solubility increases sharply at temperatures above 35 °, and t h a t this chan~e in the solubilizing effect of gelatin is associated with change in the conformation of the gelatin molecules. Gelatin molecules in the random-coil state have a greater solubilizing effect than when they are in the ordered state oi the collagen helix. (2) The thermodynamic parameters of the adsorption of benzene by gelatin have been calculated. It is shown t h a t the positive change in entropy, of 20 ca]/ /mole. deg, plays an important part. The change in the thermodynamic parameters corresponds to transfer of the hydrocarbon from the water to less polar regions, formed obviously by the bonding between non-polar side groups of the gelatin. Translated by E. O. PHILLIPS REFERENCES 1. V. A. PCHELIN, V. N. I Z I ~ L O Y A and L. E. BOBROVA, ?ysokomol. soyed. 3: 847,
1961 2. V. A. PCHELIN, V. N. IZMAILOVAand L. V. MITYUKItINA, Dokl. Akad. Nauk SSSI~ 149: 888, 1963 3. V. A. PCHELIN, V. N. IZMAILOVA and G. P. YAMPOL'SKAYA, Dokl. Akad. Nauk SSSR 148: 850, 1962; Vysokomol. soyed. 4: 938, 1962 4. V. N. I Z M A I L O V A , G. P. Y A M P O L ' S K A Y A , A. V. VOLYNSKAYAand V. A. PCHELIN, Dokl. Akad. Nauk SSSR 169: 143, 1966 5. D. WETLAUFER and R. LOVRIEN, J. Biol. Chem. 239: 596, 1964 6. A. WISHNIA and T. PINDER, Biochemistry 3: 1377, 1964; 5: 1534, 1966 7. V. A. PCHELIN, V. N. IZMAILOVA and K. T. OCHUROVA, Dokl. Akad. Nauk SSSP~ 123: 505, 1958 8. V. A. PCHELIN, V. N. IZMAILOVA and N. I. SERAYA, Vysokomol. soyed. 1: 1617, 1959 9. G. P. Y A M P O L ' S K A Y A , V. N. I Z M A I L O V A , V. A. PCHELIN and A. V. VOLYNSKAYA, Vysokomol. soyed. 7: 1956, 1965 10. C. COHEN, ~qature 175: 129, 1955; J. Biophys. and Bioehem. Cytol. 1: 203, 1955
V. A. K U z ' ~ A et al.
11. W. J. HARRINGTON, Nature 14: 997, 1958; W. J. HARRINGTON and P. HIPPEL, Arch. Biochem. and Biophys. 92: 100, 1961 12. P. HIPPEL and KWOK-J-W0NG, J. Biochem. 4: 664, 1962; 5: 1387, 1399, 1963 13. V. A. PCHELIN, V. N. IZMAILOVA and V. P. MERZLOV, Vysokomol. soyed. 5: 1429, 1963; Dokl. Akad. Nauk SSSR 150: 1307, 1963 14. Zh. LEB, Belki i teorii kolloidnykh yavlenii (Proteins and Theories of Colloidal Phenomena), p. 52, Gizlegprom, 1933 15. A. I. YURZHENKO, Zh. obshch, khim. 16: 1171, 1946 16. L. Ye. PEREGUDOVA and S. S. VOYUTSKII, Kolloid zh. 10: 309, 1948 17. I. M. KLOTZ, Brookhaven Symposium on Biology 13: 25, 1965 18. L M. KLOTZ, Federation Proc. 24: 24, 1965 19. M. R. V. SAHIUM, Life Science 5: 961, 1966 20. W. H. BISHOP and F. M. RICHARDS, J. Mol. Biol. 38: 315, 1968 21. R. G. SHORENSHTEIN and T. E. WAGNER, J. Amer. Chem. Soc. 22: 6199, 1968 22. R. FRANKE, Biochem. et Biophys. Aeta 160: 378, 1967 23. J. NEMETHY and H. SCHERAGA, J. Chem. Phys. 36: 3382, 1962 24. J. NEMETHY and H. SCHERAGA, J. Chem. Phys. 36: 3401, 1962 25. J. NEMETHY and H. SCHERAGA, J. Phys. Chem. 65: 1071, 1961; 66: 1773, 1962
THE CRYSTALLINE STRUCTURE AND UNIAXIAL DEFORMATION OF SPHERULITES IN THIN FILMS OF A LINEAR POLYURETHANE* V. A. KUZ'MI~A, Y r . V. PASECHNIK, L. P. GUL'KO, YU. S. LIPATOV a n d L. N. LISTROVAYA Institute of the Chemistry of Maeromolecular Compounds, Ukr. S.S.R. Academy of Sciences
(Received 5 M a y 1969) IT HAS been shown in a n u m b e r of papers t h a t t h e physicochemical a n d mechanical properties o f crystallizable p o l y m e r s are d e p e n d e n t on t h e m o r p h o l o g y as well as t h e size of t h e spherulites [1, 9, 15]. B y v a r y i n g t h e conditions o f crystallization ( t e m p e r a t u r e , time, concentration, phase state o f artificial s t r u c t u r e iorming agents, etc.) it is possible to affect substantially the spherulitic s t r u c t u r e and properties o f a polymeric article (film or fibre) . W e h a v e previously described the effect of the conditions of isothermal crystallization on the spherulitic s t r u c t u r e of thin films of a linear p o l y u r e t h a n e (LPU) from t r i e t h y l e n e glycol and h e x a m e t h y l e n e d i i s o e y a n a t e . This p o l y u r e t h a n e has been studied b y X - r a y analysis in a n u m b e r of p a p e r s [2, 16], b u t the t y p e of its u n i t cell and the unit-cell p a r a m e t e r s are n o t known. O£ all linear p o l y u r e t h a n e s the u n i t cell (triclinic) a n d its p a r a m e t e r s h a v e been d e t e r m i n e d o n l y for a p o l y u r e t h a n e f r o m 1,4b u t a n e d i o l a n d h e x a m e t h y l e n e d i - i s o e y a n a t e (Perlon I) [21-24]. * Vysokomol. soyed. A12: No. 9, 1928-1933, 1970.