COACERVATION OF AMYLOPHOSPHORIC ACID AND PROTEINS‘ P. KOETS uan’t Hoff Laboratory, University of Utrecht, Utrecht, Holland Received June 11, 1986
When hydrophilic colloids of opposite electric charge are mixed in solution, separation of a complex may be observed over a range of concentration of the components. In most cases this complex is of a liquid nature, separating out in the form of microscopic droplets. After some time these drops may unite to form a viscous liquid layer at the bottom of the container. This phenomenon has been observed before, but its underlying principles have been recognized lately by Bungenberg de Jong and Kruyt (3, l),who have given the name “coacervation” to this separation of a liquid phase from solutions of hydrophilic colloids (“complex coacervation” when two or more colloid components are involved). They assume that the difference in sign of the charge causes the micelles to approach, their double layers partly discharging each other. Owing to this approach the outer and less firmly bound water of hydration will be removed, but ultimately the approach will come to a halt, when the force of attraction, due to the contrast in charge, will be balanced by the resistance of the more rigidly bound water of hydration. They therefore consider the resulting droplet to be a conglomerate of positive and negative micelles which remain individually next to each other, each retaining part of its water of hydration (ucervus = swarm, heap). They showed that this phenomenon occurs generally in solutions of hydrophilic colloids, even when only one colloid is involved, the opposition in charge being located in this case on one and the same surface (either naturally as in the proteins or brought about artificially by adsorption of polyvalent ions of a charge opposite to that of the colloid). From measurements of viscosity it could be shown that partial dehydration takes place, the viscosity of the mixtures of the two colloid solutions being considerably lower than that calculated from additivity. On the other hand, these authors showed that every influence which diminishes the electric charge of the micelles and therefore lowers the attraction of the colloid particles, prevents the formation of the coacervate 1 Presented at the Thirteenth Colloid Symposium, held rtt St. Louis, Missouri, June 11-13, 1936. 1191
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P. KOETS
or disperses the complex again after it has formed. As two opposite potentials are involved, this influence is found for both cations and anions, the concentrations required being lower the higher the valency of the ion (double Schulze-Hardy rule). These coacervates may separate out in any form ranging from a fairly liquid fluid to a more or less plastic mass. The better the opposite charges are balanced, the higher the contrast in charge, and the greater will be the mutual dehydration. On the other hand, the effect of the attraction can be increased by the addition of dehydrating agents. It was found by these authors that the tendency of a hydrophilic colloid to enter into coacervation depends on its density of charge. Nucleic acid, having a high density of charge, combines readily with other biocolloids, and the resultant coacervate is highly resistant to electrolytes. No coacervates could, however, be obtained with the carbohydrate amylose as a component, a fact which may perhaps be significant in connection with the position of amylose as a reserve substance in nature. The negative electric charge of amylose is probably due, at least in part, to the dissociation of the hydroxyl groups in the surface of the micelle. ’This dissociation is a function of the hydrogen-ion concentration of the solution. The charge is small in comparison to that of other hydrophilic colloids as agar or gum arabic, where more strongly dissociated groups resulting from sulfuric acid esters or from the carboxyl group contribute to it. In alkaline niedium the dissociation of the hydroxyl groups of amylose is favored and, eventually, a completely developed double layer can be built up. In neutral or slightly acid solution, however, the negative charge of amylose is too small to bring about coacervation with positive proteins, the mutual attraction being too small to overcome the repelling force of the micelle hydration. The character of amylose can now be changed completely by introducing strongly negative groups into the surface of the micelle, for instance, by esterification with phosphoric acid. The resulting amylophosphoric acid shows an appreciable anodic migration and possesses a sufficiently high density of charge to be able to combine with positively charged proteins to droplets and floccula, which in their general behavior show the character of coacervates. We prepared amylophosphoric acid following the method described by Kerb (7, 21). Six grams of “amylum soluble Merck,” free from nitrogen, were dissolved in the usual way in 150 cc. of hot water. After cooling, the vessel was placed in ice and 24 g. of pure calcium carbonate was stirred in. In the course of two hours a solution of 5 g. of phosphorus oxychloride in 10 cc. of chloroform was gradually added under continuous vigorous stirring. An equal amount of water was then added and the precipitate centrifuged off after it had settled overnight. From the clear solution
COACERVATION OF AMYLOPHOSPHORIC ACID AND PROTEINS
1193
calcium amylophosphate was precipitated by addition of an equal volume of alcohol; the precipitate was washed with 50 per cent alcohol and redissolved in water. The solution was then electrodialyzed until free from calcium. In the mean the amylophosphoric acid so prepared contained 0.5 per cent of phosphorus pentoxide, calculated on the dry substance. When solutions of amylophosphoric acid and of a protein are mixed in different proportions at a pH below the isoelectric point of the protein, a pronounced turbidity, caused by the formation of microscopic droplets, may be observed in a distinct region of proportions of the two colloids. Gradually, the liquid droplets change to solid floccula and settle to the bottom of the vessel. Addition of alkali in small quantities causes the turbidity to disappear as the charge on the protein is reduced and ultimately reversed. Addition of acid has the same effect, as the charge of both colloids is diminished. In order to establish further the coacervate nature of the new phase, we measured the viscosities of the mixtures in comparison to those of the two components. When two solutions of hydrophilic colloids are mixed and no interaction of the micelles takes place, the viscosities of the mixtures can be calculated from those of the two components by the rule of additivity. I n the case of oppositely charged colloids a deviation from additivity is observed, which is greate? the more completely the opposite charges balance each other ( 2 , 4 ) . AMYLOPHOSPHORIC ACID-GELATIN
We used a 0.12 per cent solution of amylophosphoric acid and a 0.12 per cent solution of a pure gelatin (isoelectric point, pH = 5.0). The viscosities were measured in an Ostwald viscometer at 40°C. The required pH values of the solutions were obtained by adding the necessary amounts of sodium hydroxide or hydrochloric acid, and were controlled by means of a quinhydrone electrode and by color indicators. The results are collected in tdble 1 and represented in figure 1. The fourth column of the table gives the relative viscosities as found experimentally; the fifth, those calculated for additivity of the viscosities of the two unmixed solutions and their proportion in the mixture. The results are in complete accord with what may be expected in the case of a coacervate. The lower the pH, that is the farther the gelatin is removed from its isoelectric point (and therefore the larger its positive charge), the less is the total amount of gelatin necessary to bring about the minimum in the additivity curve. The optimum of attraction of the opposite charges of the micelles is found at a pH of approximately 3.5. Separation of coacervate droplets is found not only at the colloid proportions denoted by the minima, but also at some distance on either side of the minima. The droplets which separate at the conditions of the
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minimum are electrically neutral, and they show practically no cataphoretic movement. On either side of the minimum, however, the droplets show an outward electrophoretic charge of the sign of the colloid which predominates in the complex. At the proportion of exact balance, the TABLE 1 Viscosities of rntxtulm 01 a m y l a p h o s p h o r i c acid a n d gelatin sols PH
AXYLOPHOSPHORIC ACID SOL
PER CENT OF ADDITIVITY
OELATIN SOL
-1 cc.
2.6
95 80 .?I0 20
3 5
42
100 95 80 50 20
80 50 20
4.9
80 100
0 075 0 023 0 027 0 079 0 165 0 211
0 082 0.102 0.143 0.184
100 28 1 26 5 55 4 89 7 100
5 20 ,50 80 100
0 109 0 022 0 019 0 052 0 118 0 193
0 0 0 0
113 126 151 176
100 19 5 15 1 34 4 67 0 100
> 20
,io
100 80 50 30 20 100 80 50 30 20
100
0 111
100
90
4.6
cc.
100
10 20 50 80 100
0 064 0 041 0 023 0 043 0 078
0 108 0 105 0 094 0 085
100
0 113
20 50 70 80 100
0 064 0 028 0 028 0 035 0 067
0 0 0 0
104 090
081 076
0 099 0 066 0 059 0 061 0 065
100 61 5 31 2 34 6 46 1 100 100
0 115
20 50 70 80 100
59 3 39 0 244 50 6
0 105 0 090
0 080 0 075
94 73 73 81
3 3
7 3
100
micelles can approach each other to a maximum extent and the hydration is therefore at a minimum. When there is a preponderance of either of the two 'charges in the complex, the mutual repulsion of these excess charges causes the system to be less condensed and more hydrated.
COACERVATION OF AMYLOPHOSPHORIC
ACID AND PROTEINS
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AMYLOPHOSPHORIC ACID-LEUCOSIN
The viscosities of mixtures of a sol of amylophosphoric acid and of one of leucosin, prepared from wheat, were determined at, 3OoC. The results are tabulated in table 2 (figure 2). The phenomena are similar in all respects to those described for the system amylophosphoric acid-gelatin. AMYLOPHOSPHORIC ACID-POTATO
ALBUMIN
An albumin from potato was obtained by saturating the juice, squeezed out by means of a hydraulic press, wit,h ammonium sulfate. The precipitated impure proteins were shaken with cold water, and the filtered solution again saturated with ammonium sulfate. This was repeated four
times. The original deep-green color of the juice had then largely disappeared. The solution of the last precipitate in water was submitted to electrodialysis. Some globulin which separated out during this last process was removed by filtration. The viscosities of the sol mixtures were measured at 3OOC. The results are represented in table 3 (figure 3). The following general characteristics which these coacervates have in common may be mentioned. Immediately after mixing of the solutions, when the coacervate still exists in the form of liquid drops, it can be dissolved again completely by addition of either alkali, or acid, or neutral electrolytes. I n the case of peptization with neutral salts in small quantities, the coacervate can be made to reappear by addition of alcohol or
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P. KOETS
AYYLO-
PH
27
30
40
45
PHOSPHORIC ACID SOL
I
cc
1
100 95 80 50
I
100 95 80 50 20 100 80 50 20 100 80 50 20
LEUCOSIY SOL
I ~ ~ ) , , ,I , ,
PER CENT OF
( ' ~ ) o a , o d
ADDI+IVITY
cc
0 111 0 049
5 20 50 100
0 035 0 039 0 035
0 107 0 096 0 073
100 45 8 364 53 5 100
~
0 0 0 0 0 0
111 065 030 032 037 033
0 0 0 0
107 096 072 049
100 GO 7 31 3 44 5 75 5 100
112 049 034 033 027
0 095 0 070 0 044
100
0 0 0 0 0
100 51 6 48 5 74 9 100
20 50 80 100
0.114 0 076 0 044 0 on 0 020
0 095 0 067 0 038
100 801 65 7 76 1 100
5 20 50 80 100
20 50 80
FIQ.2
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TABLE 3 Viscosities of mixtures of ainylophosphoric acid and albumin sols AUYM-
PHOSPHORIC ACID SOL
ALBWMIN 8OL
cc.
cc.
2.6
100
90 80 50 25 3.5
100 95 75 50 25
4.4
100 80 50 25
PER CENT OF ADDITIVITY
10 20 50 75 100
0 0 0 0 0 0
115 071 055 040 034 028
5 25 50 75 100
0 0 0 0 0 0
120 085 037 023 021 026
20 50 75 100
0 0 0 0 0
122 069 031 024 018
100
0 0 0 0
107 098 072 050
66 3 56 1 55 5 68 0 100 100 73 9 38 6 31 5 42 8 100
0 115 0 096
0 073 0 049
100 68 4 44 3 54 5 100
0 101 0 070 0 044
IdC
n
c
rl,
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P. KOETS
another dehydrating agent. The droplets of these coacervates have, however, a pronounced tendency t o change to floccula on standing. They then become, at the same time, more resistant to dispersion; peptization may sometimes be obtained by potassium thiocyanate or alkali. AMYLOPECTIN
The observation that amylose can enter into coacervation with proteins only after its density of charge has been increased, for instance, by esterification with phosphoric acid, leads to some new speculations on the nature of amylopectin. It is to the presence of amylopectin that the high viscosity and the paste-forming qualities of a starch solution are ascribed. A solution of native starch in water is, however, not a true colloidal solution. Its viscosity does not follow Poiseuille’s law, indicating the presence of micelle conglomerates or gel fragments in the solution (12, 6). In the ultracentrifuge the amylopectin settles quickly and long before the amylose (8); even in the ordinary centrifuge, the amylopectin fraction can be separated from the bulk of the solution (22). We may consider a solution of native starch to be a solution of amylose in which amylopectin, in the form of gel fragments, is more or less finely suspended. Samec (18, 14,20, 17) showed that amylophosphoric acid is a component of the amylopectin and that, apart from silicic acid and fatty acids, it contains nitrogenous substances oftm to a considerable extent. I n potato starch the nitrogen content is small compared to that of phosphoric acid; from wheat, amylopectin fractions can be obtained in which the nitrogen content is equal to or even greater than the content of phosphoric acid. Samec assumed that protein and amylophosphoric acid are bound “saltlike” in amylopectin, but later he himself showed this view to be unsatisfactory (16). We suggest that it may be of advantage to consider amylopectin t o be of the nature of a coacervate as described in the first part of this paper, that is to say, that the binding of the components is not one of classical chemical mass stoichiometry, but rather a balancing of the opposite charges of the colloid micelles involved, these micelles largely retaining their individuality in the complex. It has been shown that the ultimate electrophoretic charge of the coacervate drops will depend on the proportions of two colloids present. I n potato starch the negative component largely predominates over the positive protein. I n accordance with the above theory one would expect the resulting coacervate to have a negative charge and to move cataphoretically t o the anode, as is found experimentally. Similarly Samec and Antonovic (19) found fractions of wheat amylopectin which differed in their cataphoretic behavior, those in which phosphoric acid is in excess moving to the
COACERVATION O F AMYLOPHOSPHORIC ACID AND PROTEINS
1199
anode, and those in which protein is in excess actually moving or at least having a tendency to move to the cathode. Samec’s original assumption that the high viscosity of starch solutions is solely due to the phosphoric acid content of the amylopectin has been the subject of controversy (13, 23, 6, 5). Indeed the viscosity of a solution of amylophosphoric acid, obtained from pure amylose, is not very much higher than that of a solution of amylose. The excessively high viscosities originally mentioned by Samec may be explained by the fact that the amylose which he esterified with phosphoric acid was obtained from amylopectin fractions from which the phosphoric acid had been removed previously by saponification. The nitrogenous substances probably remained in solution and were able to give rise to new coacervates as soon as amylophosphoric acid was formed again. These complexes, remaining dispersed in solution as gel fragments, could then cause a high viscosity. Indeed we found that the complexes of amylophosphoric acid and proteins described in this paper can be dispersed by bdling in a solution of pure amylose, from which they do not separate again on cooling. The viscosity of a solution so obtained shows an appreciable increase compared to that of an equally concentrated amylose solution. Samec distinguished amylopectin fractions according to the color produced with an iodine-potassium iodide solution, the color changing from blue to red the higher the nitrogen content of the fraction. Whereas a solution of amylophosphoric acid gives a blue color with this reagent, we found that the color obtained with the coacervates previously described turned from blue to violet and red, the more the protein content was increased. Many experiments have been recorded in which the properties of native starch have been changed by treatment with either dilute hydrochloric acid or potassium hydroxide. From our point of view, removal of protein from wheat amylopectin in larger proportion than phosphoric acid (15) must necessarily lead to a product resembling potato amylopectin (the coacervate, by removal of the positive component, changing to a complex in which the negative amylophosphoric acid micelles predominate), and ultimately to a product having the properties of a soluble starch (the system being incapable of coacervation owing to the absence of one of the components). Considering potato amylopectin to be a coacervate complex with a large negative surplus charge, one may expect it to be able to undergo renewed coacervation with positively charged protein, ultimately leading to a product resembling wheat amylopectin, as has indeed been found (16, 11). Many authors have described amylopectin as differing only from amylose by being more interlocked (more dense) and less hydrated (24, 9, 10, 5). These properties are inherent in coacervate complexes, the opposite charges
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P. KOETS
causing the micelles to approach more closely to each other and t o be less hydrated than in free colloidal solution. I n conclusion the author wishes to express his thanks to Prof. H. R. Kruyt, in whose laboratory this investigation was carried out, for his continual interest. REFERENCES (1) BUNGENBERG DE JONG AND COLLABORATORS: Biochem. Z. 1929-32. For sumDE J O N Q :Protoplasma 16, 110 (1932). mary see BUNGENBERG DE JONG AND DERKER:Biochem. Z. 212, 318 (1929). (2) BUNGENBERG (3) BUNGENBERG DE JONG AND KRUYT:Proc. Roy. Acad. Amsterdam 32,849 (1929); Kolloid-2. 60, 39 (1930). (4) BUNQENBERG DE JONG AND ONGSIANGWAN:Biochem. Z. 221, 166 (1930). ( 5 ) HIRST,PLANT,AND WILKINSON: J. Chem. Soc. 1932, 2375. (6) KARRERAND KRAUSZ: Helv. Chim. Acta 12, 1144 (1929). (7) KERB:Biochem. Z. 100, 3 (1919). (8) LAMM:Kolloid-Z. 69, 44 (1934). (9) LEPISCHKIN: Kolloid-2. 32, 42 (1923). (IO) NOWOPOKROWSKI: Kolloid-Z. 62, 302 (1930). AND DOBROWOLSKA: Biochem. Z. 246, 388 (1932); 248,16 (1932). (11) YON PRZYLECKI (12) ROTHLIN:Biochem. 2. 98, 34 (1919). (13) SAMEC: Kolloidchemie der Starke, pp. 26, 27. (14) SAMEC:Biochem. 2. 186, 337 (1927); 196, 72 (1928). (15) SAMEC:Kolloidchem. Beihefte 33, 95 (1931). (16) SAMEC:Kolloidchem. Beihefte 40, 449 (1934). (17) SahiEc: 2.ges. Getreide-Muhlenw. Backereiwesen 21, 111 (1934). (18) SAMEC:Trans. Faraday Soc. 31, 395 (1935). AND ANTONOVIC: Kolloidchem. Beihefte 23, 377 (1926). (19) SAMEC (20) SAMEC AND BLINC:Kolloidchem. Beihefte 30, 163 (1930). AND MAYER:Kolloidchem. Beihefte 16, 91 (1929). (21) SAMEC (22) SHERMAN AND BAKER:J. Am. Chem. S O C . 38, 18% (1916). (23) TAYLOR AND WALTON: J. Am. Chem. Soe. 61, 3431 (1929). (24) DE VRIES: Botan. Jahresber. 1, 122 ( 1 M ) .
