206
WALTER
H. SEEGERS
(38) SEEGERS, W. H., ASD SMITH,H. P.: Am. J. Physiol. 137,348 (1942). W.H., ASD MCGISTY,D. M.: J. Biol. Chem. 146,511 (1942). (39) SEEGERS, (40) SEEGERS, W.H., A N D SMITH,H. P.: Proc. SOC.Exptl. Biol. Med. 62, 159 (1943). (41) SEEGERS, W . H., LOOUIS,E. C . , A N D VAKDENBELT, J. hl.: Arch. Biochem. 6,85 (1945). W.H., SIEFT, hl. L.,A K D VANDENBELT, J. M . : Arch. Biochem. 7, 15 (1945).. (42) SEEGERS, (43) S T ~ B E X L ,. : Arch. ges. Physiol. (Pfliiger’s) 181, 285 (1920). (44) TOCANTINS, L. hI.: .4m. J. Physiol. 114, 709 (1936). (45) T O C A N T I N S , L. hI.: Medicine 17, 155 (1938). (46) WILSOK,S. J.: Arch. Internal X e d . 69, 647 (1942). (4i) WARNER, E.D., BRISKHOUS, K. M.,ASD SMITH,H. P.: Am. J . Physiol. 114,667 (1936).
HEJIOCYASIKS OF THE GASTROPODS1 SVEN BROHLLT The Institute o.f Physical C h e m i s t i y , Cniuersity of Cpsala, C p s a l a , Sweden Receiued August 8 , 1946
Svedberg and collaborators (9, 13, 16) have shown that the hemocyanins are giant molecules with molecular Tyeights from about half a million up t o about ten million. They have also found that the hemocyanins may dissociate into Jyell-defined submultiples upon a change in the pH, this dissociation often being reversible. Among the hemocyanins the largest molecular weights are observed for the Gastropods (9). The dissociation reactions of these hemocyanins have been investigated more in detail (3, 4). It was then found that for certain species (e.g., Helix pomatia) the dissociation depends not only upon the p H but also upon the nature and the amount of electrolytes or non-electrolytes present in the solution. For other species (e.g., Paludina vivipara) the dissociation is influenced only by the pH. Helix pomatia hemocyanin (abbreviation, H.P.h.) and some other hemocyanins of the Gastropods have been investigated by the author (3) and by Borgman and the author (4,5). Paludina oivipara hemocyanin (abbreviation, P.V.h.) has been studied by Ekvall and the author ( 7 , 6). I. MOLECULAR XYEIGHT AND MOLECULAR SHAPE
The molecular constants have been determined for the two species Helix pomatia and Paludina civipara.
A . Xolecular weight The sedimentation constant, s, the diffusion constant, D, and the partial specific volume, V , are given in table 1 for H.P.h. and P.V.h. and for their 1 Presented a t the Twentieth Sational Colloid Symposium, which was held a t Madison, Wisconsin, May 28-29,1946.
207
HEMOCYANIKS OF THE GASTROPODS
dissociation products. of Svedberg (12):
The molecular weight \vas calculated from the formula
The values are found in table 1. It has not been possible to isolate the first dissociation component of P.V.h. and to determine its molecular neight, since this component only appears in a very narrow pH region. It follows from table 1 that the dissociation products correspond to one-half and one-eighth of the original molecule. The molecular constants of the two species of hemocyanins are equal within the limits of experimental error. TABLE 1 Molecular constants of Helzx pomatza and P a l u d i n a t i t i p a r a hemocyanzns and t h e w dzssocaation products
IO-'cm.
Helix pomalia.. . .
. . . . . . . I 103.0 I 65.7 1 19.7
P a l u d i n a viaipara . . . .
i
. 102.5 64.5 21.8
!
*
I
1.07 0.738 1.41 0.738 1 . 7 7 0,738
8 91 x 10' 4 31 X loa 1 03 X lo6
1
1 45 1 40 1 79
1.09 0.738 0.738 1.79 0,738
8 70 X lo6
1
1 43
1.13 X lo6
1.72
' I
__ A.
A30 820 820
890 890 960
1090 790
__
the sedimentation constant at zero concentration, is expressed in Svedbergs (S). t Length of the molecules from j/fo $ Length of the molecules from stream double refraction (&)
0,
B. Molecular shape (1) Frictional ratio, f/fo
It is possible t o calculate the lengths, L , of the hemocyanins and their dissociation products from the frictional ratio, f/fo, by assuming that the molecules are unhydrated and that they behave hydrodynamically like oblong ellipsoids of revolution (16). The values of f/fo and the lengths, L , appear in tables 1 and 2 (L = L1 in table 1). The calculated minor axes, d, are given in table 2. (2) Stream double refraction Snellman and Bjornetfihl (11) have determined the lengths of H.P.h. and its dissociation products by stream double refraction, assuming that the molecules behave like cylinders. The values of L are given in tables 1 and 2. (L = Lf in table 2.) L and ilf being known, the minor axes, d , may be calculated (table 2). It may be concluded from the figures in table 1 that the wholes, halves, and eighths are equal in length. The dissociation down to eighths therefore probably occurs parallel to the longer axes (cf. Polson (10)).
n.
210
SVES BROHL‘LT
(5) Comparison between the different methods The lengths of the molecules calculated from the frictional ratio agree rather ne11 with those obtained by the stream double refraction (tables 1 and 2). Since a calculation from the frictional ratio, f/fo, is valid only for unhydrated molecules, it may be concluded that there is no considerable hydration of the hemocyanin components. I t should be of great importance to compare the molecular sizes obtained by indirect methods with those estimated dit ectly from electron micrographs. As already mentioned, reproducible micrographs have not yet been obtained. The disagreement betwen the length estimated from figure 1 and that obtained from stream double refraction is too large to permit the assumption that the particles observed are single molecules. Resides, if they ivere single molecules, their thickness xould be more than twice that observed by Trurnit and Berc:old (table 2 ) . Therefore the particles in figure 1 probably are some split products of the original hemocyanin. The length estimated from figure 2 is too uncertain to permit a comparison ivith other values. The ividth of the filaments in figure 2 is somewhat larger than that obtained by the first two methods (table 2). One must consider that some deformation may occur when the hemocyanin solution is evaporated on a film of cellulose nitrate before the esposure to the electron beams. The preparations of samples for electron micrographs and of monolayers are siniilar, and lye might expect a certain agreement in the results. It is also found (table 2) that the ividth calculated from measurements of rnon”1ayei-s agrees with that estimated from the filaments in figure 2. 11. DISSOCIATIOX Ah-D ASSOC1,ATIOK REACTIOKS
A . Helix pomatia hemocyanin (1) Dissociation upon change in p H
The H.P.h. may, upon a change in the pH, dissociate into halves, eighths, and lower components. Near the isoelectric point only whole molecules exist, provided the salt concentration is not too high (see figure 5 ) . If the solution is not too acid or too alkaline, the dissociation reaction is reversible (Svedberg and Heyroth (15) ; Eriksson-Quensel and Svedberg (9)). The sedimentation diagram of a hemocyanin solution a t pH 7 . 2 appears in figure 3. h certain amount of “intermediate compounds”-1-ith no definite boundary-is found between the peaks corresponding to whole and half molecules. We assume that these “intermediate compounds” are composed of swelling whole molecules. The assumed mechanism is shown schematically in figure 4. The original hemocyanin molecule where the halves are close t o each other is indicated in the figure by 1. K i t h successively increasing distance between the halves, molecules such as 2 and 3 may be obtained. The forces between the fragments are still great enough to keep them together. The solvent may t o some extent pass through molecules such as 2 and 3 during the sedimentation,
211
HEMOCYAXISS OF THE G.4STROPODS
and therefore a decrease in the sedimentation constant is to be expected until freely sedimenting half molecules (4 and 4’) occur. The mechanism described above gives a plausible explanation of the existence of “intermediate compounds.” The swelling of the molecule may be the first step in the dissociation process. By diffusion experiments we have tried to prove the assumption that the swelling of the molecule is a process preceding the dissociation. The diffusion constant of the “intermediate compounds” should be lower than that of the
-
t
JI
I
FIG.3. Sedimentation diagram by the refractmn method, shoving the “intermediate compounds”
2.
3.
4
4‘
... FIG.4. Swelling of the homocyanin molecule. 1, whole molecule; 2 and 3 “intermediate compounds”; 4 and 4’, half molecules.
whole molecules. A hemocyanin solution containing 49 per cent wholes, 22 per cent halves, and 29 per cent “intermediate compounds” gave a diffusion constant, D, equal t o 1.07. Accordingly, the diffusion experiment supports the assumption of the “intermediate compounds” being swelling molecules, as the diffusioii constant of wholes is 1 . O i and that of halves is 1.41. (2) Dissociation by electrolytes The H.P.11. is dissociated not only by a change in the pH but also by addition of electrolytes (3, 0 ) . The dissociation effect increases in general with the
212
SVEN BROHULT
amount and with the valence of the ions, both cation and anion being of importance. Certain ions, however, have special effects, particularly in high concentrations. In dilute salt solutions the dissociation caused by different types of salts is a s follo!vs, the valence combinations being arranged in decreasing order of effect :
>
1-4 Na4Fe(CN)s
>
1-3 XaFe(Cf\')s
2- 1 1-2 CaCh SanSOa
>
1-1
S'aCI
The valence rule does not apply to the 2-2 valent magnesium sulfate.
50
/
,p
-
0 I'
I
A+ ;,
I'
I
/x :;'
25I
k 7'
A
'
A *A
i
/
I
I
I
I
e
.
,
I
I
HEMOCTASIXS O F THE GASTROPODS
213
of the hemocyanin. Dissociation to eighths and lower components takes place when concentrated solutions of calcium chloride are used, while an association t o whole molecules occurs in concentrated solutions of sodium sulfate (3).
(3) Dissociation by non-electrolytes The H.P.h. is also dissociated by non-electrolytes such as sugars, glycerol, and urea (3). The effect is, honever, in general less pronounced than with electrolytes. (1) The hemocyanin of Helix pomatia is composed of two kinds of molecules When the H.P.h. is dissociated by addition of salts, e.g., sodium chloride (figure j),the dissociation increases v i t h the concentration of the electrolyte and suddenly ceases ivhen 75 per cent of the hemocyanin is dissociated. S o further dissociation occurs upon increasing the concentration of the electrolyte. The hemocyanin behaves as if it n-ere composed of two different kinds of molecules: one, A, whose concentration is 75 per cent and which is dissociated by electrolytes, and another, B, which is not dissociated. The existence of tn-o kinds of molecules, 9 and B, has been proved by the following experiments. ( a ) Separation by centrificyation: -1separation has been done with the preparative centrifuge of Beams. The hemocyanin solution used for the experiment was one where the dissociation had reached its maximum value, Le., 75 per cent. By repeated centrifugation it \vas possible to obtain two solutions, one containing only half molecules and one containing whole and half molecules in about equal proportions (figure 6). The dissociation properties of these solutions as a function of the concentration of electrolytes are given in figure 7, together with those of the original solution. This figure shows that we have obtained two solutions differently influenced by electrolytes : one tvhich can be completely dissociated into half molecules, and one iThere the dissociation is less than in the original solution. The first solution contains only molecules of kind A ; the second one has a larger amount of kind B than the original solution (cf. also 4). ( b ) Precipitation by ammonium sulfate: The hemocyanin ordinarily precipitates between 0.40 and 0.45 Am2S04.* The precipitation is so carried out that seven fractions are obtained. The deposits are redissolved and dialyzed against a buffer (1 31 sodium chloride f 0.08 A1 acetates), where the original hemocyanin is dissociated to 75 per cent. By sedimentation analysis it was found that the hemocyanin of the first fraction was dissociated only to 60 per cent and the hemocyanin of the last fraction was completely dissociated t o half molecules. This experiment shows that the properties bf the tn-o kinds of molecules, A and B, differ so widely that they-although equal in size-can be fractionated by ammonium sulfate. (c) M i z e d molecules: The two kinds of hemocyanin molecules, iz. and B, can both be dissociated into eighths by a change in the pH. The eighths originating 2 0.40.4rnd304 designates a solution that in a total of 100 ml. contains 40 ml. of 4 '1.I ammonium sulfate.
214
SVEK I3ROHULT
60
70 X
65
2
I
bo
65
70 x
1
i
I
t
J
FIG.6. Sedimentation diagrams showing the separation of the two kinds of hemocyanin molecules. A : original hemocyanin; whole molecules; buffer, 0.2 M sodium chloride 0.08 M acetates; pH, 5.3. B: original hemocyanin; maximum dissociation b y electrolytes; buffer, 1.00 .M sodium chloride 0.08 M acetates; pH, 5 . 2 . C : separated hemocyanin; same buffer as in B ; the solution contains only half molecules. D: solution of the redissolved centrifuge deposit; same buffer as in B. Concentration of the hemocyanin about 0.3 per cent
+
+
%
!OO -
75 -
50 -
I
05
io
i5 n
FIG.7. Dissociation into half niolecules as a function of the molarity, N , of sodium chloride. Curve I, original hemocyanin; curve 11, hemocyanin of kind A, completely dissociated into half molecules when the concentration of the salt is above 0.8 M ; curve 111, solution of the redissolved deposit containing the kinds.4 and B i n about equal proportions.
215
HEMOCYANINS OF THE G.ISTROPODS
from A and those from B should have different properties. When eighths are reassociated to wholes, we might expect mixed hemocyanin molecules. It was now found that reassociated hemocyanin molecules have other dissociation properties than the original hemocyanin. h reassociated hemocyanin was dissociated to 88 per cent by addition of electrolytes instead of 75 per cent for the original hemocyanin (cf. also 4 ) .
B. Paludina vivipara hemocyanin (1) Dissociation by change of pH The P.V.h. dissociates like H.P.h. into halves and eighths by a change in the pH (cf. 8 ) . The halves appear in a very narrow pH region (pH 7.2-7.8) and
% io0
75
50
15
0
0.1
0.5
1.0
1.5
FIG.8 . Paludina ziuipara hemocyanin. Percentage of different componmts as a function of the ionic strength, I . 0 , percentage of wholes; X , percentage of halves; percentage of eighths; ., percentage of association products. Solvent: sodium chloride phosphate buffer of pH 7.5.
+,
+
aliyays together with whole molecules or with wholes and eighths. Eighths occur in the pH region 7.i-10. Loner-not well-defined-components than eighths are observed on the alkaline side. Coagulation takes place on the acid side, starting a t pH 4.5.
(2) Effect of electrolytes Contrary to H.P.h. the P.T’.h. is not dissociated by electrolytes. The effect of salt is quite different: association occurs instead of dissociation. ( a ) Association in dilute salt solutions: The effect of the addition of sodium chloride appears in figure 8, where the hemocyanin solution investigated had a pH of i . 5 . At ionic strength below 0.05, wholes and halves have about the same concentration. Upon increasing the ionic strength, association to whole
216
S V E N BROHULT
molecules occurs. Between ionic strengths of 0.2 and 1.0 only whole molecules appear in the sedimentation diagram. ( b ) Association in concentrated salt solutions: New components of 120-130 S appear in concentrated salt solutions and probably correspond to double hemocyanin molecules. This association is always accompanied by a dissociation into halves and eighths (figure 8). The association phenomenon has been observed in the pH region 7.0-8.0. In some cases association products of 140-160 S have been found. 111. COMPARISON B E T W E E N HEMOCYANINS O F T H E GASTROPODS
Borgman and the author (4)have studied the influence of electrolytes also on some other hemocyanins of the Gastropods and found that this influence was very different for different species. The hemocyanins investigated may be classified in two groups (table 3) : one where dissociation occurs both on the addition of electrolytes and upon a change in the pH, and one where dissociation takes place only upon a change in the pH. TABLE 3 Dissociation brought about by change in the p H and by electrolytes ELECIPOLYTES
SPECIES
i H e l i z pomatia . . , . . . . . . . . . . . . H e l i z hortensis.. . . . . . . . . . . . . . . Heliz arbustorum.. . . , . . . . . . . . . P a l u d i n a civipara Littorina liltorea Buccinum undatum
Dissociation to 75 per cent Dissociation to 30 per cent Dissociation to 100 per cent I
Dissociation ' .Vo dissociation Dissociation X o dissociation Dissociation 1 .Vo dissociation
Svedberg and collaborators (9, 14)have found that the sedimentation constant and the dissociation upon change in pH shoT many regularities indicative of biological kinship. Considering the influence of electrolytes, the maximum dissociation differs widely from one species t o another (table 3) and this difference may also be biologically interesting. SUMMARY
1. The hemocyanins of Helix pomatza and Paludzna z'ivzpara have served as prototypes in this investigation. 2. The molecular constants of these hemocyanins are given in table 1 and are equal xithin the limits of experimental error. The first and the second dissociation products correspond to halves and eighths of the original molecule. 3. The wholes, halves, and eighths are equal in length. The dissociation therefore probably occurs parallel to the longer axis. 4. Lengths and widths obtained from the frictional ratio, from stream double refraction, from electron micrographs, and from measurements of monolayers are collected in table 2. A fairly good agreement is found between the different values.
HEMOCYANINS OF THE GASTROPODS
217
5 . The first step in the dissociation process probably is a swelling of the hemocyanin molecule. 6. The hemocyanin of Helix pomatia is dissociated not only by a change in the pH but also by the addition of electrolytes. The dissociation effect increases with the valence of the ions, both cation and anion being of importance. 7 . The hemocyanin of Heliz pomatia is composed of two kinds of molecules: one, A, whose concentration is 75 per cent and n-hich is dissociated by electrolytes, and another, B, which is dissociated only by a change in the pH. A has been isolated by centrifugation and by precipitation with ammonium sulfate. B has been enriched from 25 to 50 per cent. 8. The Paludina vivipara hemocyanin is dissociated by a change in the pH. The addition of electrolytes causes association instead of dissociation. The author wishes to express his sincere gratitude to the Head of the Institute, Professor The Svedberg, for his kind interest in this work and for the facilities which were placed a t his disposal. The expenses of this investigation were defrayed by grants from the Rockefeller Institute and the Wobel Fund of Chemistry. REFERENCES (1) (2) (3) (4) (5)
(6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (161 (17)
BLODGETT, K. B . : J. Am. Chem. SOC.67, 1007 (1935). BLODGETT, K . B., AKD LANGMCIR, I.: Phys. Rev. 61, 964 (1937). BROHCLT, S.: S o v a Acta Reg. SOC.Sci. Upsaliensis [4] 12, KO.4 (1940). BROHCLT, S., ASD BORGMAN, K.: In TheSvedberg 1884-19&, pp. 429-37. Almquist and Wiksells, Upsala (1944). BROHTLT,S., AND BORGMAN, K . : Unpublished work. BROHULT, S., ALNDCLAESSON, S.: Nature 144, 111 (1939). BROHCLT, S., AND EKWALL, P . : Unpublished work. EKWALL, P.: Finska Kemistsamfundets Medd. 61, 67 (1942). ERIKSSON-QUENSEL, I.-B., ASD SVEDBERG, T . : Biol. Bull. 71, 498 (1936). POLSOLN, -4.: Kolloid-Z. 88, 51 (1939). SKELLYAK, O., A K D B J ~ R N S T ~1.: H LKolloid-Beihefte , 62, 403 (1941). SVEDBERG, T . : Zsigmondy Festschrift (Erg. Bd. zu Kolloid-Z. 36) 53 (1925). SVEDBERG, T . : Ind. Eng. Chem., Anal. Ed. 10, 113 (1938); Kolloid-Z.86, 119 (1938). SVEDBERG, T . , ASD HEDESIUS,.4.: Biol. Bull. 66, 191 (1934). SVEDBERG, T., ASD HEYROTH, F. F.: J . Am. Chem. SOC.61,550 (1929). SVEDBERG. T.. ASD PEDERSES. K. 0 . :The Cllracenlrifuae. Oxford Universitv Press. S e w York (1940). TRVRSIT,H J., . ~ K DBERGOLD, G . : Kolloid-Z. 100, 177 (1942). .
I
