- Email: [email protected]
Study of some globular proteins in aqueous organic mixed solvents by dispersion of optical rotation
Study of some globular proteins in aqueous organic mixed solvents
2925
9. 10. 11. 12.
J. H. FREEMAN and G. W. LEWIS, J. Amer. Chem. Soc. 76: 2050, 1954 A. A. VANSHEIDT a n d N. N. KUZNETSOVA, Zh. prikl, khimii 30: 1850, 1957 T. V. VETOSHIKINA, Zh. prikl, khimii 39: 2125, 1966 I. M. KOL'GOF, R. BELCHER, V. STENGER and Dzh. 1KATSUYAMA, O b " e m n y i analiz (Volumetric Analysis). Goskhimizdat, I I I , p. 643, 1961 13. Dzh. UOKER, Formal'degid (Formaldehyde). Goskhimizdat, p. 431, 1957
STUDY OF SOME GLOBULAR PROTEINS IN AQUEOUS ORGANIC MIXED SOLVENTS BY DISPERSION OF OPTICAL ROTATION* V. 13. MERZLOV, V. 1V[. GUREVICH, N . V. GmSHI~CA,A. B. Z E z I ~ a n d N. F . :BAKEYEV M. V. Lomonosov State University, Moscow
(Received 28 November 1968)
MAwr papers have now been published concerning the behaviour of proteins in mixed aqueous organic solvents [1-3]. On adding to an aqueous protein solution an organic solvent incapable of effectively competing for hydrogen bonds with a peptide group in the protein macromolecule (such as dimethylformamide, dioxane (DO), ethylene glycol, ethylene chlorohydrin (ECH), etc.), the proportion of helical conformations increases. With fairly high concentrations of the organic solvent the content (%) of amino-acid residues from a protein having an a-spiral conformation may considerably exceed the helical nature of the native molecule, which is accompanied by a marked increase in stability of the helical conformation [2]. The presence of an organic solvent in the aqueous protein solution weakens the hydrophobic reaction of non-polar side chains of amino-acid residues, thus considerably stabilizing the conformation of the protein globule [3]. A study of the behaviour of proteins in aqueous organic mixtures is directly related to the problem regarding the nature of the forces which stabilize the native form of protein. An attempt is made in this paper to examine the effect of the tertiary structure of egg albumen and human globin on the conformational stability of the a-spiral. EXPERIMENTAL Typical globular p r o t e i n s - - e g g albumen (EA) and h u m a n globin--were investigated. I t is well known t h a t E A contains one S -- S bond which closes a small segment of the polypeptide chain and cannot therefore greatly influence the globular stabilization of EA. The globin molecule does not contain S - - S bond at all. The stabilization of the globular form of these proteins is basically determined b y intraglobular hydrophobic reactions. E A is isolated b y well known methods, followed b y triple recrystallization from s a t u r a t e d amino* Vysokomol. soyed A l l : No. 11, 2571-2576, 1969.
V. P. MERZLOV et al.
2926
nium sulphate solution. Human globin was obtained by the Rossi-Fanelli method [4] in the albumen fractionating laboratory of TsOLIPK.* Organic solvents, ECH and DO, were purified by conventional methods [5]. Albumen solutions were studied by dispersion of optical rotation (DOR). The measurements were made in a Jasco ORD/UV-5 speetropolarimeter in the wave length range from, 550 to 300 rap, in thermostatically controlled cells 0.1, 0.2 and 1.0 dm in length. Information concerning DOR was analysed using the Mofflt~Youag equation, assuming that 20 =212 and b0 = - 630 mp for the a-spiral conformation. RESULTS AND DISCUSSION F i g u r e 1 shows t h e d e p e n d e n c e of p a r a m e t e r s a 0 and b0 in E A solutions in a p h o s p h a t e buffer ( p H = 9 . 4 , p----0.1) on solvent composition. A q u e o u s dioxane was used as solvent. I t can be seen t h a t in solutions containing less t h a n 1 2 % t .
ao,bo
0
20 D O , vol. %
FIG. 1
40
200
j
i
20
i
I
~
60 ECH ,
I
100 vol.%
FIG. 2
FIG. 1. Variation of parameters of the Mofflt-Young equation in aqueous dioxane solutions of EA according to solvent composition; c--0.09-0.12 g/100 ml solution; pH =9.4 (aqueous solution, phosphate buffer); p ~0"1; 1--ao, 2--bo. FIG. 2. Variation of parameters in the Moffit-Young equation in aqueous ethylene chlorohydrin solutions of native and denatured human globin, according to solvent composition; c =0-07-0.3 g/100 ml solution. Native human globin, pH =6.3-6.5 (aqueous solution, phosphate buffer); 1--ao, 2--bo. Denatured globin, pH=4.0-5.0 (aqueous solution); 3--ao, 4--bo.
DO no marked changes occur in parameters a0 and b0. This points to the absence of changes in the conformation of EA molecules. On further increasing the DO content in the solution a reduction is observed in the extent of the spiral structure * The authors are grateful to G. Ya. Rozenberg and M. M. Rudashevskaya for kin41y providing the native globin preparations. ~f When the question is to determine the percentage solvent composition, a volumetric percentage is always implied.
Study of some globular proteins in aqueous organic mixed solvents
2927
of the albumen. The addition of even larger amounts of DO results in a rapid increase in parameter b0 (the ]b01value is considered), which is typical of the formation of a dextro-a-spiral. With a DO content of 40% the b0 ~alue is higher than for albumen in an aqueous solution, i.e. the extent to which the amount of spiral structure is higher than for native albumen. This corresponds to the behaviour of EA in water and ECH mixtures containing large amounts of ECH [2, 6]. We carried out similar experiments with human globin solutions mixed with water and ECH. Results of measuring ]:)OR of these solutions are shown in Fig. 2. It can be seen that in an aqueous solution ( p H = 6 . 3 , #----0.1, phosphate buffer) this albumen is helical to the extent of 45%, which is in agreement with values previously obtained [7, 8]. Figure 2 indicates that the addition o£ small amounts of organic solvent to aqueous solution of this albumen markedly reduces the helical nature, as confirmed b y the variations of the parameters of the ~ o f f i t Young equation. The value of bo varies from --300 in an aqueous solution to --150 with an ECH content of 1 0 ~ and a 0 varies from --115 to --300. In proportion to the further increase in the ECH concentration in the solution a uniform change was observed in the dispersion parameters which in pure ECH were: b0=--410, a o = 150. These values correspond to a 75% spiral formation and agree with results previously obtained [9]. A reduction in spiral formation of native globin on adding small amounts of ECH is obviously due to rupture of the native structure and not the effect of ECI-I on the rotational ability of albumen as such, since for aqueous solutions of denatured g!obin (fraction insoluble at a of 6-7), containing small amounts of ECH the opposite pattern is observed. The addition of ECH to an aqueous solution of denatured globin causes an immediate change in the parameters
pit
bo
_3oof
a
-200 ~
~
-f00 25
5O
b
_80o~
75
3
O.
-400 i 100 T, °C
-200
25 5O 75T,'~ I
I
I
ao(b)
F JIG. 3. Dependence of parameters b0 (a) and on temperature in EA solutions of different compositions (water-DO): c=0.09-0.12 g/100 ml solution; /--water, pH=9"4, Z=0-1; 2--3.8; 3--12.4; 4--18.0 (a) and 40.0 (b); 5--36.0~ DO. of DOR, which corresponds to an increase in spiral formation of albumen (Fig. 2). Starting from a concentration of 20% a further increase in ECH content in solution up to 100% does not suggest any distinction in the behaviour of native
V. P. Mv.RZLOVet al.
2928
and denatured albumens. The ao and b0 values in this range of E C H concentrations practically coincide in both cases. Figure 1 shows that the addition to the EA solution of 12% DO does not cause marked changes in parameters a 0 and b0, compared with the aqueous solution. It may, however, be assumed that the organic solvent penetrates the protein globule even with low DO concentrations in the solution and influences the stability of the ~-spiral, since the secondary structure is to a certain extent stabilized b y the presence of a compact globular tertiary structure [3]. The thermal stability of the secondary EA structure was therefore studied in dioxane-water mixtures of various compositions. 80
65 o~
5
15 25 DO, col. %
35
FIG. 4. Dependence of melting point Tmeltof EA of helical structure on DO con~nt in aqueous 'albumen solutions. In an aqueous EA solution melting of the a-spiral is described by an S-shaped curve, which is indicated b y the dependence of parameters a 0 and b o on temperature and this occurs in the temperature range of 70-85 °. In solutions containing less than 25% DO the nature of the dependence of D O R parameters on temperature remains unchanged; however, as the DO concentration increases from 0 to 25~/o the range of transition increases and is displaced in the direction of lower temperatures. Figure 3 illustrates typical curves of the dependence of the parameters of D O R on temperature for some water-dioxane protein solutions. Figure 4 illustrates the dependence of the Tmelt value (which in diagrams showing the dependence of a o and b 0 on temperature corresponds to the temperature of halftransition) on the DO content in solution. The Figure shows that Tmelt for EA decreases with increase of the DO concentration in the solution, reaching a minimum when the DO concentration corresponds to the minimum degree of spiral formation in protein. With higher DO concentrations Tmelt increases. We could not measure Tmelt with a DO content higher than 40% since parameter bo in these cases remained practically unchanged on the altering temperature, which is obviously due to the well-known increase in stability of the a-spiral in non-polar solvents [2].
Study of some globular proteins in aqueous organic mixed solvents
2929
Results indicate (Figs. 1 and 2) that as with EA, for human globin the addition of certain amounts of organic solvent causes rupture of a large part of the spiral sections, which exist in native molecules of these albumens. However, if we consider the reaction of g-spiral sections not included in the tertiary or quaternary structure with organic solvents of low polarity, the degree of spiral formation should increase in their presence, or at least remain unchanged [10]. In fact, the a-spiral is stabilized by intramolecular hydrogen bonds, the energy of which is comparable with the energy of hydrogen bonds between the peptide group and water. The addition to this system of an organic solvent of low polarity, incapable of competing for the hydrogen bonds with a peptide group, increases (starting from a certain concentration of this solvent) the degree of spiral formation, since the reaction equilibrium of hydrogen bond formation within a single chain and between the peptide groups and solvent is displaced in the direction of intramolecular reactions. Similar considerations [3, 11] do not explain the breakdown effect of organic solvents in low concentrations on the secondary structure of EA and of native human globin. However, information obtained by the authors when investigating denatured globin (of a low degree of spiral formation) proves that a variation in spiral formation on adding ECH to the aqueous solution of this albumen takes place in accordance with the views described. A variation in parameters a 0 and b 0 in this case points to an increase in spiral formation in albumen on adding small amounts of ECH to the solution (Fig. 2). Thus, the results suggest that the compact globular structure of a native albumen molecule in aqueous solution stabilizes the spiral conformation of certain sections of the polypoptide chain. This assumption is satisfactorily confirmed by results of investigating the heat stability of the spiral conformation of EA in water-dioxane solutions. Information concerning the melting point of the secondary EA structure in the range of low DO concentrations is of particular interest, when judging from results of DOR no marked structural changes occur in protein. These results are shown in Figs. 1 and 4. As shown by Fig. 4, the stability of the a-spiral noticeably decreases on adding 2~o organic solvent and is minimum with a DO concentration corresponding to minimum spiral formation in EA. This is obviously due" to changes in the tertiary structure, since parameter b o, which characterizes the secondary structure, remains unchanged in this range of DO concentrations (Fig. 1), whereas the organic solvent should in principle increase the conformational stability of the ~-spiral. This m a y mean that the organic solvent, when contained in a small quantity in an aqueous albumen solution, is not distributed evenly in the volume but mainly penetrates the hydrophobic nucleus of the globule weakening intra-globular interactions. This assumption is quite reasonable, for the increased solubility in water of sparingly soluble hydrocarbons with detergents (above the critical concentration of micelleformation) and albumens is well known. It is assumed that the solubility of hydrocarbons increases in this case due to their penetration to the hydrophobic regions
2930
V. P. MERZLOV et al.
of the micelles. The addition to aqueous solutions oi detergents of an organic solvent, miscible with water, causes micelle rupture at a certain concentration [12]. A similar pattern was apparently observed in our case. Then, if the spiral conformation of certain parts of the polypeptide chain of albumen is stabilized not only by intramolecular hydrogen bonds, but also by hydrophobic reactions inside the globule, the gradual weakening of these reactions will inevitably reduce the stability of the ~-spiral, i.e. reduce the melting point Tmelt of the secondary albumen structure, as shown by Fig. 4. CONCLUSIONS (1) B y t h e dispersion o f optical r o t a t i o n , c o n f o r m a t i o n conversions in w a t e r organic m i x t u r e s o f globular a l b u m e n s were s t u d i e d using t h e e x a m p l e o f egg a l b u m e n (EA) a n d h u m a n globin. (2) I t is p o i n t e d o u t t h a t t h e a d d i t i o n o f c o m p a r a t i v e l y small a m o u n t s o f d i o x a n e (DO) to a q u e o u s E A solutions does n o t al~er t h e a l b u m e n c o n f o r m a t i o n . F u r t h e r increase in organic solvent c o n c e n t r a t i o n first lowers a n d t h e n increases t h e spiral f o r m a t i o n of a l b u m e n . Similar results were o b t a i n e d w h e n i n v e s t i g a t i n g solutions of n a t i v e h u m a n globin m i x e d w i t h w a t e r a n d e t h y l e n e c h l o r o h y d r i n
(ECH). (3) B y t h e e x a m p l e of E A it was f o u n d t h a t w i t h small a m o u n t s of D O in a n a q u e o u s a l b u m e n solution, w h i c h do n o t cause c o n f o r m a t i o n changes, t h e stab i l i t y of t h e spiral c o n f o r m a t i o n decreases. (4) T h e results indicate t h a t t h e helical c o n f o r m a t i o n of n a t i v e globular a l b u m e n s is stabilized b y t h e t e r t i a r y s t r u c t u r e . Tran~laSed by E. S~.~tERv. REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
11. 12.
S. J. SINGER, Advances Protein Chem. 17: 1, 1962 C. E. BRESIER, V. P. KUSHNER and S. Ya. FRENKEL', Biokhimiya 24: 685, 1959 O. B. PTITSYN, Usp. sovr. biologii 63: 3, 1967 A. ROSSI-FANELLI and E. ANTONIONI, Bioehim. et. biophys, acta 30: 608, 1958. A. VAISBERGER, E. PROSKAUER, Dzh. RIDDIK and E. TUPS, Organieheskie rastvoriteli (Organic Solvents). Tzd. inostr, lit., 1958 V. P. KUSHNER and S. Ya. FRENKEL', Biokhimiya 27: 1111, 1962 T. ASAKURA, S. MINAKAMI, Y. YONEYAMA and H. YOSHIKAWA, J. Biochem. 56: 594, 1964 Z. HRKAL and Z. VODRAZKA, Biochim. et biophys, acta 133: 527, 1967 P. URNES and P. DOTY, Advances Protein Chem. 16: 401, 1961 C. TANFORD, P. K. DE and V. G. TAGGART, J. Amer. Chem. Soe. 82: 6028, 1960; C. TANFORD, C. E. BUCKLY, P. K. DE and E. P. LIVELY, J. Biol. Chem. 237: 1168, 1962 T. M. BIRSHTEIN and O. B. PTITSYN, Konformatsiya makromolekul (l~Iacromolecular Conformation). Izd. "Nauka", 1964 K. SHINODA, T. NAKAGAVA, B. TAMAM'IJSI and T. ISEMURA, Kolloidnye poverkhnostnoaktivnye veshchestva (Colloidal Surface-Active Substances). Izd. "Mir", 1966
2925
9. 10. 11. 12.
J. H. FREEMAN and G. W. LEWIS, J. Amer. Chem. Soc. 76: 2050, 1954 A. A. VANSHEIDT a n d N. N. KUZNETSOVA, Zh. prikl, khimii 30: 1850, 1957 T. V. VETOSHIKINA, Zh. prikl, khimii 39: 2125, 1966 I. M. KOL'GOF, R. BELCHER, V. STENGER and Dzh. 1KATSUYAMA, O b " e m n y i analiz (Volumetric Analysis). Goskhimizdat, I I I , p. 643, 1961 13. Dzh. UOKER, Formal'degid (Formaldehyde). Goskhimizdat, p. 431, 1957
STUDY OF SOME GLOBULAR PROTEINS IN AQUEOUS ORGANIC MIXED SOLVENTS BY DISPERSION OF OPTICAL ROTATION* V. 13. MERZLOV, V. 1V[. GUREVICH, N . V. GmSHI~CA,A. B. Z E z I ~ a n d N. F . :BAKEYEV M. V. Lomonosov State University, Moscow
(Received 28 November 1968)
MAwr papers have now been published concerning the behaviour of proteins in mixed aqueous organic solvents [1-3]. On adding to an aqueous protein solution an organic solvent incapable of effectively competing for hydrogen bonds with a peptide group in the protein macromolecule (such as dimethylformamide, dioxane (DO), ethylene glycol, ethylene chlorohydrin (ECH), etc.), the proportion of helical conformations increases. With fairly high concentrations of the organic solvent the content (%) of amino-acid residues from a protein having an a-spiral conformation may considerably exceed the helical nature of the native molecule, which is accompanied by a marked increase in stability of the helical conformation [2]. The presence of an organic solvent in the aqueous protein solution weakens the hydrophobic reaction of non-polar side chains of amino-acid residues, thus considerably stabilizing the conformation of the protein globule [3]. A study of the behaviour of proteins in aqueous organic mixtures is directly related to the problem regarding the nature of the forces which stabilize the native form of protein. An attempt is made in this paper to examine the effect of the tertiary structure of egg albumen and human globin on the conformational stability of the a-spiral. EXPERIMENTAL Typical globular p r o t e i n s - - e g g albumen (EA) and h u m a n globin--were investigated. I t is well known t h a t E A contains one S -- S bond which closes a small segment of the polypeptide chain and cannot therefore greatly influence the globular stabilization of EA. The globin molecule does not contain S - - S bond at all. The stabilization of the globular form of these proteins is basically determined b y intraglobular hydrophobic reactions. E A is isolated b y well known methods, followed b y triple recrystallization from s a t u r a t e d amino* Vysokomol. soyed A l l : No. 11, 2571-2576, 1969.
V. P. MERZLOV et al.
2926
nium sulphate solution. Human globin was obtained by the Rossi-Fanelli method [4] in the albumen fractionating laboratory of TsOLIPK.* Organic solvents, ECH and DO, were purified by conventional methods [5]. Albumen solutions were studied by dispersion of optical rotation (DOR). The measurements were made in a Jasco ORD/UV-5 speetropolarimeter in the wave length range from, 550 to 300 rap, in thermostatically controlled cells 0.1, 0.2 and 1.0 dm in length. Information concerning DOR was analysed using the Mofflt~Youag equation, assuming that 20 =212 and b0 = - 630 mp for the a-spiral conformation. RESULTS AND DISCUSSION F i g u r e 1 shows t h e d e p e n d e n c e of p a r a m e t e r s a 0 and b0 in E A solutions in a p h o s p h a t e buffer ( p H = 9 . 4 , p----0.1) on solvent composition. A q u e o u s dioxane was used as solvent. I t can be seen t h a t in solutions containing less t h a n 1 2 % t .
ao,bo
0
20 D O , vol. %
FIG. 1
40
200
j
i
20
i
I
~
60 ECH ,
I
100 vol.%
FIG. 2
FIG. 1. Variation of parameters of the Mofflt-Young equation in aqueous dioxane solutions of EA according to solvent composition; c--0.09-0.12 g/100 ml solution; pH =9.4 (aqueous solution, phosphate buffer); p ~0"1; 1--ao, 2--bo. FIG. 2. Variation of parameters in the Moffit-Young equation in aqueous ethylene chlorohydrin solutions of native and denatured human globin, according to solvent composition; c =0-07-0.3 g/100 ml solution. Native human globin, pH =6.3-6.5 (aqueous solution, phosphate buffer); 1--ao, 2--bo. Denatured globin, pH=4.0-5.0 (aqueous solution); 3--ao, 4--bo.
DO no marked changes occur in parameters a0 and b0. This points to the absence of changes in the conformation of EA molecules. On further increasing the DO content in the solution a reduction is observed in the extent of the spiral structure * The authors are grateful to G. Ya. Rozenberg and M. M. Rudashevskaya for kin41y providing the native globin preparations. ~f When the question is to determine the percentage solvent composition, a volumetric percentage is always implied.
Study of some globular proteins in aqueous organic mixed solvents
2927
of the albumen. The addition of even larger amounts of DO results in a rapid increase in parameter b0 (the ]b01value is considered), which is typical of the formation of a dextro-a-spiral. With a DO content of 40% the b0 ~alue is higher than for albumen in an aqueous solution, i.e. the extent to which the amount of spiral structure is higher than for native albumen. This corresponds to the behaviour of EA in water and ECH mixtures containing large amounts of ECH [2, 6]. We carried out similar experiments with human globin solutions mixed with water and ECH. Results of measuring ]:)OR of these solutions are shown in Fig. 2. It can be seen that in an aqueous solution ( p H = 6 . 3 , #----0.1, phosphate buffer) this albumen is helical to the extent of 45%, which is in agreement with values previously obtained [7, 8]. Figure 2 indicates that the addition o£ small amounts of organic solvent to aqueous solution of this albumen markedly reduces the helical nature, as confirmed b y the variations of the parameters of the ~ o f f i t Young equation. The value of bo varies from --300 in an aqueous solution to --150 with an ECH content of 1 0 ~ and a 0 varies from --115 to --300. In proportion to the further increase in the ECH concentration in the solution a uniform change was observed in the dispersion parameters which in pure ECH were: b0=--410, a o = 150. These values correspond to a 75% spiral formation and agree with results previously obtained [9]. A reduction in spiral formation of native globin on adding small amounts of ECH is obviously due to rupture of the native structure and not the effect of ECI-I on the rotational ability of albumen as such, since for aqueous solutions of denatured g!obin (fraction insoluble at a of 6-7), containing small amounts of ECH the opposite pattern is observed. The addition of ECH to an aqueous solution of denatured globin causes an immediate change in the parameters
pit
bo
_3oof
a
-200 ~
~
-f00 25
5O
b
_80o~
75
3
O.
-400 i 100 T, °C
-200
25 5O 75T,'~ I
I
I
ao(b)
F JIG. 3. Dependence of parameters b0 (a) and on temperature in EA solutions of different compositions (water-DO): c=0.09-0.12 g/100 ml solution; /--water, pH=9"4, Z=0-1; 2--3.8; 3--12.4; 4--18.0 (a) and 40.0 (b); 5--36.0~ DO. of DOR, which corresponds to an increase in spiral formation of albumen (Fig. 2). Starting from a concentration of 20% a further increase in ECH content in solution up to 100% does not suggest any distinction in the behaviour of native
V. P. Mv.RZLOVet al.
2928
and denatured albumens. The ao and b0 values in this range of E C H concentrations practically coincide in both cases. Figure 1 shows that the addition to the EA solution of 12% DO does not cause marked changes in parameters a 0 and b0, compared with the aqueous solution. It may, however, be assumed that the organic solvent penetrates the protein globule even with low DO concentrations in the solution and influences the stability of the ~-spiral, since the secondary structure is to a certain extent stabilized b y the presence of a compact globular tertiary structure [3]. The thermal stability of the secondary EA structure was therefore studied in dioxane-water mixtures of various compositions. 80
65 o~
5
15 25 DO, col. %
35
FIG. 4. Dependence of melting point Tmeltof EA of helical structure on DO con~nt in aqueous 'albumen solutions. In an aqueous EA solution melting of the a-spiral is described by an S-shaped curve, which is indicated b y the dependence of parameters a 0 and b o on temperature and this occurs in the temperature range of 70-85 °. In solutions containing less than 25% DO the nature of the dependence of D O R parameters on temperature remains unchanged; however, as the DO concentration increases from 0 to 25~/o the range of transition increases and is displaced in the direction of lower temperatures. Figure 3 illustrates typical curves of the dependence of the parameters of D O R on temperature for some water-dioxane protein solutions. Figure 4 illustrates the dependence of the Tmelt value (which in diagrams showing the dependence of a o and b 0 on temperature corresponds to the temperature of halftransition) on the DO content in solution. The Figure shows that Tmelt for EA decreases with increase of the DO concentration in the solution, reaching a minimum when the DO concentration corresponds to the minimum degree of spiral formation in protein. With higher DO concentrations Tmelt increases. We could not measure Tmelt with a DO content higher than 40% since parameter bo in these cases remained practically unchanged on the altering temperature, which is obviously due to the well-known increase in stability of the a-spiral in non-polar solvents [2].
Study of some globular proteins in aqueous organic mixed solvents
2929
Results indicate (Figs. 1 and 2) that as with EA, for human globin the addition of certain amounts of organic solvent causes rupture of a large part of the spiral sections, which exist in native molecules of these albumens. However, if we consider the reaction of g-spiral sections not included in the tertiary or quaternary structure with organic solvents of low polarity, the degree of spiral formation should increase in their presence, or at least remain unchanged [10]. In fact, the a-spiral is stabilized by intramolecular hydrogen bonds, the energy of which is comparable with the energy of hydrogen bonds between the peptide group and water. The addition to this system of an organic solvent of low polarity, incapable of competing for the hydrogen bonds with a peptide group, increases (starting from a certain concentration of this solvent) the degree of spiral formation, since the reaction equilibrium of hydrogen bond formation within a single chain and between the peptide groups and solvent is displaced in the direction of intramolecular reactions. Similar considerations [3, 11] do not explain the breakdown effect of organic solvents in low concentrations on the secondary structure of EA and of native human globin. However, information obtained by the authors when investigating denatured globin (of a low degree of spiral formation) proves that a variation in spiral formation on adding ECH to the aqueous solution of this albumen takes place in accordance with the views described. A variation in parameters a 0 and b 0 in this case points to an increase in spiral formation in albumen on adding small amounts of ECH to the solution (Fig. 2). Thus, the results suggest that the compact globular structure of a native albumen molecule in aqueous solution stabilizes the spiral conformation of certain sections of the polypoptide chain. This assumption is satisfactorily confirmed by results of investigating the heat stability of the spiral conformation of EA in water-dioxane solutions. Information concerning the melting point of the secondary EA structure in the range of low DO concentrations is of particular interest, when judging from results of DOR no marked structural changes occur in protein. These results are shown in Figs. 1 and 4. As shown by Fig. 4, the stability of the a-spiral noticeably decreases on adding 2~o organic solvent and is minimum with a DO concentration corresponding to minimum spiral formation in EA. This is obviously due" to changes in the tertiary structure, since parameter b o, which characterizes the secondary structure, remains unchanged in this range of DO concentrations (Fig. 1), whereas the organic solvent should in principle increase the conformational stability of the ~-spiral. This m a y mean that the organic solvent, when contained in a small quantity in an aqueous albumen solution, is not distributed evenly in the volume but mainly penetrates the hydrophobic nucleus of the globule weakening intra-globular interactions. This assumption is quite reasonable, for the increased solubility in water of sparingly soluble hydrocarbons with detergents (above the critical concentration of micelleformation) and albumens is well known. It is assumed that the solubility of hydrocarbons increases in this case due to their penetration to the hydrophobic regions
2930
V. P. MERZLOV et al.
of the micelles. The addition to aqueous solutions oi detergents of an organic solvent, miscible with water, causes micelle rupture at a certain concentration [12]. A similar pattern was apparently observed in our case. Then, if the spiral conformation of certain parts of the polypeptide chain of albumen is stabilized not only by intramolecular hydrogen bonds, but also by hydrophobic reactions inside the globule, the gradual weakening of these reactions will inevitably reduce the stability of the ~-spiral, i.e. reduce the melting point Tmelt of the secondary albumen structure, as shown by Fig. 4. CONCLUSIONS (1) B y t h e dispersion o f optical r o t a t i o n , c o n f o r m a t i o n conversions in w a t e r organic m i x t u r e s o f globular a l b u m e n s were s t u d i e d using t h e e x a m p l e o f egg a l b u m e n (EA) a n d h u m a n globin. (2) I t is p o i n t e d o u t t h a t t h e a d d i t i o n o f c o m p a r a t i v e l y small a m o u n t s o f d i o x a n e (DO) to a q u e o u s E A solutions does n o t al~er t h e a l b u m e n c o n f o r m a t i o n . F u r t h e r increase in organic solvent c o n c e n t r a t i o n first lowers a n d t h e n increases t h e spiral f o r m a t i o n of a l b u m e n . Similar results were o b t a i n e d w h e n i n v e s t i g a t i n g solutions of n a t i v e h u m a n globin m i x e d w i t h w a t e r a n d e t h y l e n e c h l o r o h y d r i n
(ECH). (3) B y t h e e x a m p l e of E A it was f o u n d t h a t w i t h small a m o u n t s of D O in a n a q u e o u s a l b u m e n solution, w h i c h do n o t cause c o n f o r m a t i o n changes, t h e stab i l i t y of t h e spiral c o n f o r m a t i o n decreases. (4) T h e results indicate t h a t t h e helical c o n f o r m a t i o n of n a t i v e globular a l b u m e n s is stabilized b y t h e t e r t i a r y s t r u c t u r e . Tran~laSed by E. S~.~tERv. REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
11. 12.
S. J. SINGER, Advances Protein Chem. 17: 1, 1962 C. E. BRESIER, V. P. KUSHNER and S. Ya. FRENKEL', Biokhimiya 24: 685, 1959 O. B. PTITSYN, Usp. sovr. biologii 63: 3, 1967 A. ROSSI-FANELLI and E. ANTONIONI, Bioehim. et. biophys, acta 30: 608, 1958. A. VAISBERGER, E. PROSKAUER, Dzh. RIDDIK and E. TUPS, Organieheskie rastvoriteli (Organic Solvents). Tzd. inostr, lit., 1958 V. P. KUSHNER and S. Ya. FRENKEL', Biokhimiya 27: 1111, 1962 T. ASAKURA, S. MINAKAMI, Y. YONEYAMA and H. YOSHIKAWA, J. Biochem. 56: 594, 1964 Z. HRKAL and Z. VODRAZKA, Biochim. et biophys, acta 133: 527, 1967 P. URNES and P. DOTY, Advances Protein Chem. 16: 401, 1961 C. TANFORD, P. K. DE and V. G. TAGGART, J. Amer. Chem. Soe. 82: 6028, 1960; C. TANFORD, C. E. BUCKLY, P. K. DE and E. P. LIVELY, J. Biol. Chem. 237: 1168, 1962 T. M. BIRSHTEIN and O. B. PTITSYN, Konformatsiya makromolekul (l~Iacromolecular Conformation). Izd. "Nauka", 1964 K. SHINODA, T. NAKAGAVA, B. TAMAM'IJSI and T. ISEMURA, Kolloidnye poverkhnostnoaktivnye veshchestva (Colloidal Surface-Active Substances). Izd. "Mir", 1966