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(3) GREEN,H . : Ind. Eng. Chem. 17, 802 (1925). (4) HAUSER,E.A.: Latez. The Chemical Publishing Company, Xew York (1930). ( 5 ) HAWSER, E.A., AND SZE,M. C.: J. Phys. Chem. 46, 118 (1942). (6) KAO,J. Y. L.: Sc. D. Thesis, Massachusetts Institute of Technology, 1942. (7) MCHUGH,J.: Sc. B. Thesis, Massachusetts Institute of Technology, 1942. (8) SCHIDROWITZ, P.:British patent 193,451 (1921). P.:British patent 208,235 (1922). (9) SCHIDROWITZ, (10) SCHOLZ, P.: Ksutschuk 1937, 101. (11) SPENCE,D., AND YOUNG, J.: Kolloid-Z. 11, 28 (1912). D.F.:Trans. Inst. Rubber Ind. 6, 419 (193C-31). (12) TWISS,
SOME PHYSICAL PROPERTIES OF LIVING MATTER' CHARLES H. NORRIS Department of Biology, University of Colorado, Boulder, Colorado Received August 4 , 1948
One of the characteristics of living matter which distinguishes it from nonliving is its organization. It is probably no misstatement to say that all other properties of living matter, i. e., metabolism, growth, irritability, and reproduction, depend for their manifestations on the structure of that living matter. It must be understood, however, that when one speaks of structure, one must include the submicroscopic, as well as the gross and the microscopic. Following Purkinje, who in 1840 applied the name protoplasm to the basic substance of living organisms, the biologists of the nineteenth century considered protoplasm to be a single chemical substance, and we find Huxley in 1868 speaking of molecules of protoplasm. Since that time, however, biologists have come to recognize that living matter is composed of highly complex colloidal systems. Furthermore, many of the early microscopists felt that protoplasm was characteristically foamy, while others believed that it was fibrillar, and still others were sure that it was granular. Today, we recognize the fact that all were partially right, and yet none completely so, for there are many kinds of protoplasm, and an ameba is just as truly living as a human muscle cell. Investigations of the gross anatomy and the microscopic anatomy have proceeded regularly, so that a vast body of knowledge in these fields has been amassed, but the ultrastructure of living matter has been investigated for such a comparatively short time, and so many technical difficulties beset the problems here encountered, that very little is known about this important aspect of the organization of organisms. Inasmuch as direct methods of ascertaining the intimate structure of organisms are inadequate in many cases, it is necessary to make indirect investigations by way of study of certain properties which will suggest the possible types of organization which one may encounter in cells. Presented a t the Kineteenth Colloid Symposium, which was held a t the University of Colorado, Boulder, Colorado, June 18-20, 1942.
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To some extent, studies of the physical properties of living matter have been valuable in making such suggestions. RRsearphes on permeability, electrophoretic mobility, electrical impedance, double refraction, and x-ray diffraction have all been extremely important. In this discussion, however, it will be necessary to limit the considerations to three other types of physical property: tension of the surface membrane, viscosity, and elasticity. These three properties have been investigated to a considerable extent, and have been important in indicating certain possibilities of cell organization, especially when considered with evidence from other types of study. THE TENSION OF THE SURFACE MEMBRANE
Every living cell is separated from its environment by a definite, living membrane, i. e., an envelope or surface layer which prevents the mixing of the cell and its environment. This region, taken i n toto, determines many of the properties of the cell as a whole, for, in addition to the maintenance of the cell integrity, it is probably of importance in the transmission of the excitation impulse, it is responsible for the permeability of the cell, and it may in some cases be important in the maintenance of cell shape. In attempting to arrive a t some adequate picture of the structure of this layer, studies of the tension of this membrane, considered with other types of investigation, have been of some value. If we define the tension of the cell membrane as the force within the membrane due to intermolecular attraction, which tends to exert an inwardly directed pressure, we are not necessarily indicating that we are dealing with true interfacial tension. As a matter of fact, subsequent evidence will indicate that we are not doing so, but that we are dealing with a region of definite, but as yet inadequately measured, thickness. For this reason it is convenient to express the force in terms of force per unit length, or the same units as used in expressions of surface tension. It is possible to get approximate values for this membrane tension by using techniques which are valid for true surface or interfacial tension. The first successful determination of membrane tension of a living cell was made by Harvey (S), using the centrifuge-microscope developed by Harvey and Loomis (10). The techniwe depends on the principle that when a fluid drop is stretched from the spherical shape to a cylinder, it becomes unstable when the length is equal to times the diameter. The unfertilized egg of the sea-urchin, Arbacia punctulata, is peculiarly well adapted for such a study, for it possesses a semifluid surface membrane, and in the fluid contents one finds granules and globules of varying densities. Under the influence of centrifugal force the contents of this egg are stratified, and when placed in a medium of the same density as the average for the egg, the stratified contents pull the cell into a cylinder, which divides into light and heavy spherical halves when the conditions of instability are reached, as described above. Study of motion pictures of the fragmentation of such eggs, with suitable calculations, enabled Harvey to show that the membrane tension for this cell was about 0.2 dyne per centimeter. A modification of this technique, in which an included oil drop is pulled out of a cell, enabled Harvey and his coworkers to show that in many kinds of cell the tension
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in the membrane is of the same order of magnitude as that shown for the Arbacia egg (see reference 9 for details). Cole (3) developed a technique for the study of the membrane tension of spherical cells, in which he compressed the unfertilized Arbacia egg with a gold microbeam, and again found low values for the membrane tension. He further showed that he was dealing with a true elastic membrane, rather than with a simple interface, for he found that the tension value obtained increased with increases in degree of compression. Values ranged between an extrapolated value of 0.08 dyne per centimeter for zero compression to 0.13 dyne per centimeter for about one-third compression. Norris (17), using a technique which involved stretching of the cell under consideration, was able to show that the nucleated erythrocyte of the Japanese newt, Triturus pyrrhogaster, was possessed of a membrane where the tension was very low, and that this membrane was elastic. This type of cell is rather interesting, for here we find a cell with no readily detectable shape-supporting structure in the cell as a whole, yet the cell is of such a shape that some support must be present. Sorris concluded that the membrane or envelope is the principal shape-support. Harvey and Shapiro (12),in studies of the tension between the egg cytoplasm and the contained oil drop of mackerel eggs, found that this interfacial tension, as studied by the sessile-drop method, was low, i. e., 0.6 dyne per centimeter. Danielli and Harvey ( 5 ) were able to show that this sort of value could be obtained in vitro by having an interface between mackerel egg oil and sea water which contained some of the globulin fraction extracted from the mackerel eggs. However, when the interface mas that between the oil and plain sea water, such a value could not be attained. They concluded that in this case it was probable that the outermost layer of oil, next to the cytoplasm, formed a Langmuir film with the hydrophil portions out, and that to this layer was adsorbed a layer of the globulin. From this, and other data, Danielli (4) reasoned that the most probable sort of cell membrane which could be postulated for the cell would be one in which there would be two monomolecular flms of lipoid, with the long axes of the molecules arranged radially with respect to the cell surface, and the hydrophil groups oriented so that they point in and out. Then, adsorbed to the surface, would be placed protein molecules. Such a system would account for the general low tension of the membrane which has been observed for all cells investigated. Furthermore, the lipoid content in such a system would be in agreement with the extraction determinations of Gorter and Grendel (7) and of Dziemian (6) for the red blood cell. The thickness of such a film would be of the same order of magnitude as that determined for the red cell by Waugh and Schmitt (27) by the analytical leptoscope, i. e., in the neighborhood of 100 A. Such a picture would also account for the elasticity of the surface membrane, since Langmuir (15) has indicated the pronounced elasticity of protein monolayers. The differencesin rigidity of the cell membrane could be accounted for on the basis of differences in the kind of proteins present, since some thin layen of protein are very rigid while others appear semifluid. On the other hand, it seems difficult to reconcile this relatively simple picture
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of the cell membrane with the pronounced differences in permeability which are observed in erythrocytes of different species (14). Nor would it explain the striking constancy observed in the electrophoretic behavior of the erythrocyte of a given species when that erythrocyte is subjected to pronounced changes in the environment, as shown by Abramson, Gorin, and Ponder (I). These investigators are convinced that a uniform film of protein does not exist a t the cell surface. All in all, it can be said that as yet there is no really adequate description obtainable for the structure of the cell membrane. It may be that Danielli’s picture is correct, or the true situation may turn out to be quite different. It is possible that here we are dealing with an intricate, highly developed mosaic structure, quite different from the old mosaic picture in which patches of lipoid and protein were postulated, or yet again, it may be that the true picture will be one in which there are lipoprotein complexes, with very unstable bonds between the components. However, whether Danielli’s idea is right or wrong, it has stimulated tremendous interest, and will continue to stimulate research until it is either proved or disproved unequivocally. THE VISCOSITY RELATIONS OF CELL CONTENTS
I t would be better, it is true, if this section were to have the word Consistency substituted for the word viscosity. However, the latter term is so widely used in the literature that it will be used here as well, with the understanding that we may not always be dealing with true viscosity. As a matter of fact, it is hard to say whether we are ever dealing with true viscosity in biological systems. In most elementary textbooks one finds descriptions of protoplasm as varying from the consistency of water to that of solid gels. Measurements of the viscosity of cell contents do show that there is very pronounced variation in viscosity from region to region in the same cell, as well as variations between different kinds of cells. Two principal techniques have been used in attempts to evaluate the viscosity of cell contents. The rate of sedimentation of included particles in a centrifugal field, using Stokes’ law with Cunningham’s correction, has allowed many investigators to calculate the viscosity of cells, and the Einstein equation for Brownian movement has also been used. In both there is considerable experimental error possible, especially when one considers the difficulty of accurate measurements of the very small moving particles. By the use of such techniques, values have been obtained for various kinds of cells which range from 3 centipoises to over 8000 centipoises (see, e. g., Heilbrunn (13) and Harvey (9) for reviews of the literature). That there is variation in the consistency of portions of the same cell has been shown clearly by a number of investigators, and we find Harvey and Marsland (11) noting that granules move in a rather jerky manner when an ameba is centrifuged, thus dislodging the granules. This is a clear indication that there are present in the ameba structures of different consistency from the more fluid portion, perhaps forming a sort of meshwork. We are thus confronted with the possibility that figures for the average rate of sedimentation of particles mean nothing, for such particles would tend to fall more rapidly in the
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interstitial spaces and be caught for a short time in the mesh. Furthermore, in the determinations of Brownian movement, values which are too low would be attained in many cases if the moving particles were moving in the interstitial, more fluid material. Finally, it should be noted that when particles are centrifuged down in a cell, the internal structure may be disrupted by these particles. Of considerably more interest from the biological standpoint are the studies which have been carried out on the effects of various agents in changing the relative viscosity of living systems, and the effects of such changes on the activities of living systems. Among the most illuminating of these investigations are those which have been carried out by Marsland and his collaborators, in which cells have been subjected to high hydrostatic pressure, and where correlated studies have been made of the effects of the pressure on the relative viscosities and the rates of occurrence of certain types of cell movements. Marsland has recently (16) reviewed this interesting field, so a very brief summary of the general results will suffice. There is very good evidence that the relative viscosities of a number of protoplasmic systems are decreased markedly by high hydrostatic pressure and, further, that such protoplasmic movements as ameboid movement, animal-cell division, cyclic streaming in certain plant cells, and contraction of melanophores are inhibited in a way which correlates closely with the viscosity changes. From this he reasons that the maintenance of such activities depends on the maintenance of delicately poised sol e gel transformation systems. Seifrir has reached a similar conclusion with regard to the mechanism of protoplasmic streaming, in the slime mold especially, and recently (25) has concluded that anesthetics act on such processes by changing the physical state relations in protoplasm. Northen (19) has shown that the viscosity of Spirogira protoplasm is lowered by such anesthetics as ether, chloroform, and certain alcohols. However, magnesium ions seem to increase viscosity, and here again we are dealing with an anesthetic (see Korthen and Sorthen (20) ). Thus, if anesthetics do inhibit action by shifting the physical state relations, it must be concluded that a shift either way would result in anesthesia. If this is the case, how is one to explain the non-anesthetic effects of such agents as temperature, which do change viscosity to a considerable extent? (See Heilbrunn (13) for examples.) All in all, one may say that viscosity studies have been useful in proving the wide differences found in different kinds of protoplasm, as well as in showing that, in some ways, there is definite similarity in the protoplasm of such widely different organisms as Ameba and Elodea. THE ELASTICITY OF LIVING SYSTEMS
William Seifriz has done more than any other one individual to stress the importance of elasticity and contractility as fundamental general characteristics of living systems. In many of his publications (22, 23,24, 25) he has laid emphasis on these properties as important in indicating structure of living matter. He and his collaborators have repeatedly demonstrated the widespread occurrence of the elastic behavior of cytoplasm, nucleus, and cell surface. However,
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relatively little of an exact nature is known about the elastic properties of living systems. The first really accurate determinations of Young’s modulus for a relatively uniform material were carried out by Sichel (26), who found this value for isolated resting muscle fibers to be about 2.5 X 106 dynes per em*. Norris (18) demonstrated that this value for the slime mold channel wall was about 9.0 X lo4 dynes per cm.l In both cases it was found that uninjured material conformed to Hooke’s law within a considerable range of elongation. Furthermore, Norris was able to show that with increase in temperature there was a drop in the value of Young’s modulus, while a decrease in temperature increased its value. It was also demonstrated in this investigation that the elasticity was primarily in the gelled region, and not localized t o the cell membrane. However, Ramsey and Street (21)have evidence to show that the resting tension of muscle fibers is the result of membrane (sarcolemma) tension only. Their evidence depends on injury of the cells being studied, and does not agree with Sichel’s findings that with injury there is lack of conformity with Hooke’s law. The meaning of this pronounced elasticity is not as yet clear. It is, however, quite possible that in contractile protoplasm there are present long-chain protein molecules which are capable of being stretched or which may be folded more pronouncedly. Astbury (2) has demonstrated that contraction of muscle is the result, fundamentally, of superfolding processes in myosin molecules. Thus it may be that in the slime mold and other contractile systems we have similar situations, and that the differences in elasticity and contractility which can be observed are the results of variation in the kind and/or amount of such protein molecules present. In conclusion, it might be said that we are dealing with such complex situations in living systems that as yet no definite interpretations of structure of such systems can be made from the investigations which have been outlined here. That they are suggestive of possibilities there can be no doubt, but much time must elapse and much more work must be carried out before it will be possible to attain any real conception of the structure of protoplasm. REFERENCES (1) ABRAMSON, H. A , , GORIN,M. H., AND PONDER, E . : Cold Spring Harbor Symposia Quant. Biol. 8, 72 (1940). (2) ASTBURY, W.T.:Proc. Roy. Soc. (London) Bl29,307 (1940). (3) COLE,K . S.:J. Cellular Comp. Physiol. 1, 1 (1932). (4) DANIELLI, J. F.: J. Cellular Comp. Physiol. 7, 393 (1936). J . F.,AND HARVEY, E. N . : J. Cellular Comp. Physiol. S, 483 (1935). (5) DANIELLI, (6) DZIEMIAN, .4 J . : J. Cellular Cornp. Physiol. 14, 103 (1939). (7) GORTER, E.,AND GRENDEL, F.: J. Exptl. Med. 41, 439 (1925). (8) HARVEY, E.S . : Biol. Bull. 61, 273 (1931). (9) HARVEY, E.N.:J. Applied Phys. 9.68 (1938). (10) HARVEY, E. N., AND LOOMIS, A. L . : Science 76, 42 (1930). (11) HARVEY, E . N.,AND MARSLAND, D . A.: J. Cellular Comp. Physiol. Z, 75 (1932). E N.,AND SHAPIRO, H.: J. Cellular Comp. Physiol. S, 255 (1934). (12) HARVEY, (13) HEILBRUNN, L. V.:A n Outline of General Physiology. W. B. Saunders Company, Philadelphia (1937). (14) JACOBS, M . H . : J. Applied Phys. 9, 81 (1938).
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(15) LANGMUIR, I.: Cold Spring Harbor Symposia Quant. Biol. 6,171 (1938). (16) MARBLAND, D. A , : In The Structure of Protoplasm, edited by W. Seifriz. Iowa State College Press, Ames, Iowa (1942). (17) NORRIS,C. H.: J. Cellular Cornp. Physiol. 14, 117 (1939). (18) KORRIS,C. H.: J. Cellular Comp. Physiol. 16, 313 (1940). (19) KORTHEN,H . T.:Botan. Gaz. 100, 238 (1938). (20) NORTHEN, H. T., AND KORTHEN, R. T. Plant Physiol. 14,539 (1939). (21) RADISEY, R . W.,AND STREET,S. F: Biol. Symposium 3,9 (1941). (22) SEIFRIZ,W.: Am. Katuralist 80, 121 (1926). (23) SEIFRIZ, W.: Am. Naturalist 63, 410 (1929). (24) SEIFRIZ,W.:Protoplasm. McGraw-Hill Book Company, Inc., New York (1936). (25) SEIFRIZ,W : The Structure of Protoplasm. Iowa State College Press, Arnes, Iowa (1942). (26) SICHEL,F.J. M.: J. Cellular Cornp. Physiol. 6,21 (1934). (27) WAUGH,D.F.,AND SCHJIITT,F. 0.: Cold Spring Harbor Symposia Quant. Biol. 8, 233 (1940).
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NORM.4L VARIATIOK I S T H E CONCENTRATION OF FIBRINOGEN, ALBUMIN, AND GLOBULIN IN BLOOD PLASMA’ ROBERT hl. HILL
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VIRGIXIA TREVORROW
Department of Biochemistry and the Child Research Council, University of Colorado School of Medicine, Denver, Colorado Received August 4 , 194.9
Ten years ago, impressed by the lack of adequate data in the literature, we began a study of the concentrations of the albumins, globulins, and fibrinogen in the plasma of healthy individuals and the changes that these fractions undergo with age. The protein fractions were separated by a micro-modification of the “Howe technique,” Le., the fibrinogen and the globulin plus fibrinogen were salted out with appropriate concentrations of sodium sulfate. The albumin remains in solution after the latter precipitation. The nitrogen of the fractions, the total nitrogen, and the non-protein nitrogen were determined by a microadaptation of the Kjeldahl method. Details of our system of analysis have been published elsewhere (12). Division of the plasma proteins into these fractions-albumin, globulin, and fibrinogen-has proved to be very useful in physiological studies and perhaps even more so in the practice of medicine, but the validity of this classification has been frequently questioned during the past twenty years ( 6 ) . The fractions which we call “albumins,” “globulins,” and “fibrinogen” are said to be artifacts, having no real existence in native plasma. We are asked, therefore, to abandon the old nomenclature and adopt a new one (6, 13). We believe that the proPresented a t the Nineteenth Colloid Symposium, which was held at the University of Colorado, Boulder, Colorado, June 18-20, 1942.
