Some Protein Properties of Plant Protoplasm.*

SOME PROTEIN PROPERTIES OF PLANT PROTOPLASM.*

By W. H. PEARSALL AND JAMES EWING.

CONTENTS. not PA01 i. Precipitation • 349 5. Heat Coagulation 352 2. Chemical Combination • 349 General Relation of Tissues to 3. Changes in Volume . • 350 Hydrogen-Ion Concentration. 352 4. Viscosity .... • 351 References 356 IN attempting to explain the reactions and behaviour of living organisms, the experimental biologist is often tempted to fall back on the causal consideration of the observed phenomena in terms of the single living cell. While it is true that our physiological knowledge of this unit is almost negligible, there is at any rate a widely accepted belief that its properties may often be expressed in terms of two of its most important chemically active substances, the various proteins and their derivatives or the fatty bodies. So far as proteins are concerned, this assumption is justified by the classical analyses of Reinke and Rodewald,1' which show that in the plasmodium of Fuligo varians, about 60 per cent, of the dry weight of protoplasm is composed of protein. It is therefore legitimate to consider the hypothesis that certain properties of normal plant cells appear to be most simply expressed in terms of their protein reactions. Recent work on the proteins, particularly that of Loeb,6 has shown that hydrogen - ion concentration is the most important factor affecting their physical and chemical behaviour. At a particular hydrogen-ion concentration, the iso-electric point, most of the protein properties are at a minimum, the particles are electrically uncharged, and chemically least active. At this point, the protein is most easily precipitated or coagulated by heat. Both its volume and osmotic pressure are least, and it will absorb least water from the surrounding medium. Further, its viscosity is also at a minimum. If then the hydrogen-ion concentration is altered, the protein absorbs water, swells, and the viscosity of the solution increases. * Received October 1st, 1924. 347 W. H. Pearsall and James Ewing The particles become electrically charged and the solution is increasingly ionised as one passes further from the iso-electric point. On the alkaline side of the iso-electric point, the protein bears a negative charge and behaves as an anion. On the acid side of this point, the charges are positive and the protein acts as a cation. The chemical behaviour of a given protein largely depends, therefore, on the hydrogen-ion concentration of the medium. Now, if we are to define plant protoplasm in terms of its protein properties, we require to know the normal hydrogen- ion concentration of the tissue and that also of the iso-electric points of the proteins present. The normal reaction of the cell sap in a wide range of parenchymatous tissues is between /H 5.5 and 6.5, expressing the hydrogen-ion concentration in S0rensen's logarithmic notation. There are, of course, many exceptions to this general statement, especially in acid fruits ; but it covers the majority of cases, and particularly those tissues to which subsequent reference is made. The pW values at which representative vegetable proteins are iso-electric are given in the following table (Pearsall and Ewing9):—

/H Values ofIso-electric Points. Beans and Vicilin 3-4 Tomato—Globulin . . . 40 Peas—Legumin 4-4-4-6 Hemp—Edestin* ... 5-6 „ Legumelin • 4-2 Yeast—Globulin * ... 46 Wheat—Glutenin 4-4-4-S „ Albumin* ... 4-6 „ Gliadin 3-5-S-S „ cells .... 3-1-3-3 „ Leucosin * 4-5 Nitella extract * ... 4-6-4-7 Potato—Tuberin* 4-4 Bacterium colt . . . 3-2 Carrot—Globulin • 4-1-4-4 B. typhosui .... 3-5 It will be seen from this table that there is a general tendency for the proteins of the higher plants to be iso- electric about pH 4.0 to 4.5. This is particularly the case of those proteins asterisked, which are obtained from active tissues and which do not exist as reserve foods only. Further, since the tissues normally possess /H values higher than the iso- electric points indicated, these proteins must occur in nature on the alkaline side of their iso-electric points. There is one very striking fact which confirms this view. Almost all the vegetable proteins are or can be prepared by methods which involve the precipitation of the protein by the addition of small 348 Protein Properties of Plant Protoplasm quantities of a weak acid (see Osborne8). Precipitation is then more rapid because the protein is brought nearer to the iso-electric point by this treatment. If the protoplasm of the tissues mentioned in the above table possesses to any marked degree the properties of the proteins extracted from these tissues, then it should be possible to show that changes in the protoplasm occur when the pW value of the external medium is altered to the iso-electric point of the active proteins or below it The changes which we might expect to observe are five in number. There should be (i) precipitation, (2) alteration in the combining powers with acids and bases, changes (3) in volume and (4) in viscosity, while (5) coagulation by heat should be more easily produced. (1) Precipitation.—MeierT has shown by dark ground illumina- tion that the protoplasm of pea roots contains particles which move to the anode in an electrical field and which hence are negatively charged. By a similar method using root hairs of wheat, Addoms 1 found that the effect of solutions of/H 4.1 or less is to cause aggregation and precipitation of the particles existing in the protoplasm. The behaviour of these particles is then exactly what would be expected if they were protein, and the physiologically active protein in wheat, leucosin, would precipitate in the same way under such treatment. (2) Chemical Combination.—Typical uniform tissues like potato tuber and bean cotyledons (or embryos) stain more strongly with basic dyes under normal conditions, between /H 5 to 7. On the other hand, at /H 3 to 4, these tissues stain more readily with acid dyes, and basic dyes can be largely removed by washing with acidulated water. Similar results have been obtained for potato by Robbins." Since the observed effects are chiefly protoplasmic, it appears that the protoplasm combines more readily with bases under normal conditions, but more readily with acids below pH 4 to 5. It behaves, therefore, as do the extracted proteins. A similar conclusion holds for yeast, and this may be justified by treating similar samples of yeast with solutions of various hydrogen-ion concentrations for twelve hours. These are then treated with dilute silver nitrate solution (o. 1 M) in the dark for an hour, and repeatedly washed in the dark with ice-cold water. On 349 W. H. Pearsall and James Ewing exposing the samples to the light, those in solution above pH 5 develop a pronounced silver stain, while no stain is visible in those originally treated with solutions below pH 4.5. The protoplasm combines with silver (i.e. behaves as an anion) above/H 5. It may be noticed that this is an interesting case because yeast cells themselves are iso-electric at pH 3.1 to 3.3 (by precipitation and cataphoresis). Their extracted proteins are iso-electric about/H 4.6, and this point appears to be the one about which the combining power of the protoplasm centres. The properties of the protoplasm appear, therefore, to be closely aJlied to those of the main constituent proteins. (3) Changes in Volume.—Marked changes in volume may be observed by treating living tissues with solutions of different pW value, but reliable data are more easily obtained by measuring the absorption of water by weight. The following experiment was carried out with potato tuber cut into small round discs, as described by Stiles and J0rgensen.ls Roughly equal weights of these discs, dried on filter paper, were placed in each of a number of different solutions of very dilute hydrochloric acid or sodium hydroxide at various pH values. The quantity of solution taken, in each case, was very large, and the solutions were changed every two hours, so that no significant change in/H value occurred. The discs of tissue were dried on filter paper and weighed at intervals. The results of three experiments are given below, for an exposure of ten hours in each case. Between 25 and 30 gms. of potato were taken for each sample and the percentage increase in weight calculated. Absorption of Water by Potato at Different /H Values. fiU. I. 1I. III. /H. I. II. III. 7-i 147 ... 4-3 ... 13-8 6-3 ... 12-2 I2-I 4-1 ... 137 6-1 ... 12-4 4-0 142 6-o 13-9 .. ... 3-9 ... 12-5 5-9 ... I 33 ... 37 127 124 .. 57 136 ...... 3-6 ... 12-8 5-4 ... .. n-5 3-5 ... 11-8 4-9 ... .. 131 3-35 11-8 47 139 146 3-1 102 9-7 .. 4-5 ... 12-4 12-4 2-9 ... 79 .. 4-4 137 ...... 2-8 5-6 ... .. Figure* are percentage increase in weight. 35O Protein Properties of Plant Protoplasm If these results are expressed graphically, it will be seen that a marked depression in the swelling curve occurs aboutpW 4.5, which is approximately the iso-electric point of the chief protein, tuberin, obtained from potato. Hence the properties of the tissue resemble those of its chief protein in that water absorption is at a minimum at this point. It is obvious that water absorption by a plant tissue is a complex process, involving other factors besides the imbibition of water by a single colloidal substance. There are hence other minima on the swelling curve, notably aboutpH 5.4, 6.2, and belowpH 3.3. Into the causes of these depressions we need not now inquire since they do not affect the present argument. Similar swelling curves can be obtained using buffer solutions, although the exact position of the various depressions varies slightly with different buffer mixtures. Other plant tissues show similar swelling curves with a series of depressions. In the case of beans, whose proteins are included in the table, there is, as in potato, a depression between pH 4.3 and 4.7, and another one in the vicinity of/H 3.0. These depressions occur, therefore, approximately at the iso-electric points of the chief proteins. It may be noted that actual measurements of volume give results in agreement with those obtained by measuring the absorption of water, although accurate results are much more difficult to obtain. (4) Viscosity.—Changes in the viscosity of plant protoplasm in contact with acid substances have been studied by Jacobs, who used Spirogyra along with various animals. Jacobs * found that short exposure to carbon dioxide caused a decrease in the viscosity of Spirogyra protoplasm, while further exposure caused a subsequent increase in viscosity. The behaviour of the protoplasm is thus analogous to that of a protein solution which, originally on the alkaline side of its iso-electric point, is first made iso-electric by weak acid, and then on further increasing the hydrogen-ion concentration, passes over on to the acid side of its iso-electric point. It has not yet proved possible to obtain proteins from Spirogyra which give sharp iso-electric points. Proteins are only obtained in small quantity in crude extracts and these are iso-electric between pW 3.7 and 5.4. The normal sap reaction of the species of Spirogyra examined W. H. Pearsall and James Ewing by us varies between pH 6.2 and 7.0, so that the proteins in the cell would be on the alkaline side of the iso-electric point. (5) Heat Coagulation. — Spirogyra was also used by Lepeschkin,* who showed that the protoplasm was coagulated more easily by heat in the presence of dilute solutions of weak acids than it was either in water or in weak alkalies, which made coagulation more difficult. A solution of an albumin is found to coagulate on heating most readily at its iso-electric point, and it would behave like Spirogyra protoplasm if it were on the alkaline side of this point. In the case of potato (and of beetroot) heat coagulation is produced most rapidly by increasing the hydrogen-ion concentration of the external solution to /H 4.4, the iso-electric point of the chief protein present. It will be seen from the above examples that plant protoplasm shows properties which are also those of the proteins extracted from the same tissue. Not only so, but the hydrogen- ion concentration required to produce the observed effects is the same for both the protoplasm and the proteins. It would appear to be legitimate to assume that the proteins may behave in protoplasm as they do in vitro, and hence that the properties of the protoplasm are to some extent those of the included proteins. We have confined the evidence above to cases where the proteins have been extracted and their properties determined. General Relation of Tissues to Hydrogen-Ion Concentration.— Besides the exhibition of properties which may perhaps be directly referred to as protein reactions, plant tissues also show some general properties which may also be related to the presence of proteins in the protoplasm. The permeability of the protoplasm to dissolved substances would obviously be affected by the changes in the colloidal state of the proteins present. Coagulation of the protoplasm by heat, for instance, produces almost complete permeability. In the same way, the visible aggregation of the protoplasmic particles observed by Addoms,1 appears to be associated with increased permeability. It will be evident, therefore, that if protoplasm is treated with a solution of such hydrogen-ion concentration that the proteins become iso-electric, then a change in permeability ought to 35* Protein Properties of Plant Protoplasm result, since the protoplasm will be precipitated in part, it will lose water and its viscosity will decrease. A useful method of observing such changes is to follow the rate of diffusion of chlorine ions from tissue at various pW values. In solutions containing hydrochloric acid, with or without dilute sodium chloride, the rate of outward diffusion of chlorine from potato remains approximately constant between pH 7.2 and pW 4.4; but it is greatly increased below the latter point, which is approximately the iso-electric point of potato globulin. A similar result is obtained for carrot, rapid outward diffusion taking place below pH 4.25, the approximate iso-electric point of carrot globulin (Pearsall and Ewing10). The colour diffuses out of beetroot rapidly below pH 4.6, which is also the point below which heat coagulation is most rapid. In the case of Nitella we have a fairly full picture of the changes in ion movement produced by changing pH values. Here also, chlorine diffuses out rapidly below pH 4.6 (Hoagland and Davis1), and this is found to be the iso-electric point of suspensions of Nitella protoplasm (Pearsall and Ewing). Hoagland and Davis, loc. cit., also find that absorption of nitrate decreases as the/H value is raised, and Irwin8 observed that a basic dye enters more rapidly as the external pW value is increased, but that it diffuses outward most rapidly as this pH value falls. (These results for nitrate and the basic dye refer only to pW. values above 5.4.) It seems from these statements that as the hydrogen-ion concentration is reduced anions enter less easily, cations more easily. These results would be most simply interpreted by assuming that the protoplasm is more strongly charged negatively as its proteins get further from their iso-electric points. Hence there would be increased "attraction" for cations, and decreased attraction for anions. Some tropisms belong to the second type of general protoplasmic reaction which may be examined along with the protein properties. An example of such a reaction has been given by Spruit1* for Chlamydomaaas. The cilia of this organism adhere to glass only in slightly acid solutions. This property is lacking in neutral or alkaline media. Since glass in aqueous solutions is electro-negative, it is suggested that the 353 W. H. Pearsall and James Ewing material of which the cilia are made are iso-electric slightly on the acid side of neutrality, and positively charged in media of higher hydrogen-ion concentration. In alkaline media, the cilia are presumably charged negatively and hence not attracted by glass. The last general application of the protein properties to protoplasm refers to the theory of plant growth (Pearsall and Priestley11). Most metabolic processes in living cells appear to be of the reversible type, hydrolyses in one direction and condensations in the other, as expressed by the general equation :— A + B^=C + HSO, where A and B are relatively simple substances and C the complex product of the reaction. The concentrations in which these substances exist at equilibrium are then expressed by the mass law as— (cone. CXConc. H,O) „ . . —? Xw £r^ = K (a constant). (cone. A)(conc. B). v ' It is evident from this equation that the concentration of C can be most simply increased by reducing the concentration of water. In a word, starting from a given point, synthesis of C would be favoured by low concentrations of water, hydrolysis of C would result from increased concentrations of water. There is nothing to suggest that the metabolic reactions involved in cell-growth differ from those familiar in normal differentiated cells, and it is fruitful to suppose that the dividing cell differs from the non-growing cell only in the fact that its metabolism is oriented in the direction of synthesis by some such mechanism as that indicated above. Structurally, the typical dividing cells in plants present one very significant feature. They normally contain a dense mass of protoplasm, in which vacuoles are quite or almost absent. The non- dividing cells, on the other hand, contain large vacuoles and a relatively small proportion of protoplasm. They are obviously full of water, in contrast to the dividing cells, which appear to contain minimal quantities of water. This characteristic appears to be of great significance when considered in con- junction with the facts observed during the origin of dividing cells from normal vacuolated tissues. Such a process takes place during cambium formation at the cut surface of a potato. 354 Protein Properties of Plant Protoplasm Owing to the liberation of fatty acids at the cut surface, the hydrogen-ion concentration may rise to pH 3.5, while that of the normal cells inside is usually betweenpH 5.4 and 6.0. In this case, the cambium arises across a gradient of hydrogen- ion concentration, and apparently at a pH value of approximately pH 4 to 5. When a cambium arises around the vascular tissues of a flowering plant, a somewhat similar gradient seems to exist. The sap in the xylem is usually acid, values between pW 4 and 4.5 being common, while, as pointed out by Sachs, the reaction of the phloem is more alkaline, the pH values varying about pH 7.5. The inner cambium develops between these two tissues, again across a gradient of hydrogen-ion concentration. It is suggested (Pearsall and Priestley") that the cambium arises at or near the iso-electric point of the protoplasm or of important constituent proteins in the protoplasm. Since proteins possess minimum affinity for water at this point, water is withdrawn on each side by cells of greater osmotic activity, so that synthesis is favoured by the low concentration of water in the iso-electric cells. The direct determination of the hydrogen-ion concentration of dividing cells is impossible, as they are nearly impermeable. In the case of potato cambia, however, the hydrogen-ion concentrations on either side of them suggest that their reaction is approximately pH 4 to 5, and as shown in the table this is sufficiently near to the iso-electric point of the potato globulin (pH 4.4) to agree with theory. It would obviously suffice if the reaction of the dividing cells occurred at any of the points of minimal swelling which have already been given for potato. A simpler and critical case is that of yeast. The yeast proteins are iso-. electric at pW 4.6. According to Mr F. A. Mason of Leeds, the growth-optimum for brewer's yeast occurs between pH 4.6 and 5. This definitely supports the suggestion that cell-growth proceeds most rapidly at or near the iso-electric point of important constituent proteins. It is obviously probable that the question is complicated by the necessity for the cells to maintain suitable exchange of ions with the external medium. It may be suggested as of interest to animal physiologists that the classical experiments on the artificial fertilisation of sea-urchin eggs might repay investigation from this point of view. The 355 W. H. Pearsall and James Ewing most successful treatment involves the addition of weak acid to the eggs, which are subsequently treated with hypertonic sea water. The processes involved appear to resemble to a certain extent those outlined in the above hypothesis, viz., the protoplasm is brought to a point of minimum swelling by acid and then partly dehydrated by the strong salt solution. This comparison of the properties of plant protoplasm with those of the proteins might well be extended over a much wider field than has been covered in the preceding paragraphs. We have, however, attempted to confine the evidence to those cases in which the behaviour of the extracted proteins is known. The result of the comparison would seem to justify the working hypothesis that the properties of the protoplasm can be foretold, to a certain extent, if those of the more important protein constituents are known. References. 1 Addoms, Ruth M. (1923), "The Effect of the Hydrogen-ion on the Protoplasm of the Root Hairs of Wheat," Amtr. Journ. Bot, 10, 211-20, pL 23. 1 Hoagland, D. R., and Davis, A. R, (1923), "The Composition of the Cell Sap of the Plant in Relation to the Absorption of Ions," Journ. Gen. Physiol., 0,629-46, fig. I. 3 Irwin, Marian (1932), "The Permeability of Living Cells to Dyes as affected by Hydrogen-ion Concentration," Journ. Gen. Pkysiol., 6, 223-24. * Jacobs, M. H. (1922), "The Effects of Carbon Dioxide on the Consistency of Protoplasm," Biol. Bull, 42, 14-3a 4 Lepeschkin, W. W. (1910), "Zur Kenntnis der Plasmamembran,"Ber. deut bot Ges. 28, 91-103, 383-93 ; and 29, 181-90. ' Loeb, Jacques (1922), Prottins and the Theory of Colloidal Behaviour, New York, pp. 292. 7 Meier, Henry F. A. (1921), "Effect of Direct Current on Cells of Root Tip of Canada Field Pea," Bot. Gaseite, 72, 113-38, figs. 3, pis. 2. 8 Osborne, T. B. (1924), The VtgttabU Proteins, London : Longmans, Green, pp. 154. 8 Pearsall, W. H., and Ewing, James (1924), "The Iso-Electric Points of Some Plant Proteins," Biochenu Jourtu, 18, 329-39. 10 Pearsall, W. H., and Ewing, James (1924), "The Diffusion of Ions from Living Plant Tissues in Relation to Protein Iso-Electric Points," New Phytol (in the press). 11 Pearsall, W. H., and Priestley, J. H. (1923), " Meristematic Tissues and Protein Iso- Electric Points," New Phytol, 22, 185-91. 18 Reinlce, J., and Rodewald, H. (ib8ij, "Studien iiber das Protoplasma," Unters. a. d. bot. Lab. d. Univ. Gottingen, 2, 1-75. a Robbins, W. j. (1923), "An Iso-Electric Point for Plant Tissues and its Significance," Amer. Journ. Bot., 10, 412-39, figs. 6. 14 Spruit, C. (1920). "The Influence of Electrolytes on the Tactical Movements of Chlamydomonas variabilis," Recueil Trav. Bot. Neerland, 17, 129-204, figs. 7. 16 Stiles, Walter, and J0rgensen, Ingvar (1917X "The Swelling of Plant Tissue in Water and its Relation to Temperature and Various Dissolved Substances," Ann. of Bot, 81, 4«5-34i figs. 9. 356