THE RELATIONS BETWEEN YOLK AND WHITE IN THE HEN'S EGG

Transcription

1 293 THE RELATIONS BETWEEN YOLK AND WHITE IN THE HEN'S EGG II. OSMOTIC EQUILIBRATION. BY MICHAEL SMITH AND JAMES SHEPHERD. (From the Low Temperature Research Station, Cambridge.) (Received 8th May, 1931.) (With Seven Text-figures.) CONTENTS. PAGE I. Osmotic equilibration in intact eggs protected from evaporation (a) Methods 293 (b) The uptake of water by the yolk (c) The equilibration of freezing-points of white and yolk (d) The mechanism of equilibration 296 (e) The effect of temperature 299 II. Osmotic equilibration with rapid evaporation (a) Methods 300 (6) Results 300 III. Osmotic relations of separated yolks (a) Recovery of hypertony by diluted yolks when replaced in egg-white 302 (b) E f f e c t o f p e r i o d o f i m m e r s i o n i n e g g - w h i t e ( c ) E f f e c t o f p e r i o d o f i m m e r s i o n i n w a t e r (d) Effects of heterogeneity of the egg-white (e) Osmotic relations of yolks in various aqueous solutions (/) T h e uptake of water from aqueous solutions IV. S u m m a r y References I. OSMOTIC EQUILIBRATION IN INTACT EGGS PROTECTED FROM EVAPORATION. (a) Methods. THE difference in freezing-point between white and yolk of the hen's egg is not permanently maintained, but is smaller in eggs which have been stored for a long period. For instance Straub (7) found a difference of only o-o2-o-o5 C. in eggs which had been preserved in lime, as contrasted with the normal difference of O-IO-O-I6 C. At the same time there is during storage, as Greenleetz) showed, an increase in the percentage water content of the yolk and a decrease in that of the white, which

2 294 MICHAEL SMITH and JAMES SHEPHERD latter, under normal storage conditions, is partly accounted for by the evaporation of water through the shell. Evaporation was not a controlled variable in Greenlee's experiments, but it must be expected to influence the uptake of water by the yolk, for as the white becomes more concentrated the passage of water yolkwards should tend to be checked and ultimately to be reversed in direction. Experiments by the present writers have shown that this reversal does in fact occur when evaporation is rapid. Evaporation can be eliminated altogether by storing the eggs in saturated air, but under these conditions moulds develop rapidly on the shell, particularly at higher temperatures. A humidity of 80 per cent, will prevent any visible development of moulds after prolonged storage, and the rate of evaporation at this humidity can be further reduced by partially blocking the pores of the shell with a mixture of paraffin and paraffin wax of suitable consistency. By adopting this procedure, evaporation has been reduced to about gm. per day per egg at o C, and to gm. per day at the other extreme temperature of 25 0 C. The disadvantage that the thin wax coating might modify the normal behaviour of the egg by hindering the diffusion of carbon dioxide outwards, was regarded as less serious than the possible effects of the penetration inwards of any sterilising solution which could alternatively be applied to the shell. The 400 eggs used in the following experiment were obtained from White Leghorn hens at the Papworth Poultry Farm, and were laid within a perio"d of 18 hours prior to the commencement of the observations. 100 eggs were stored at each of four temperatures, o, io, 18 0 and 15 0 C. Samples of six eggs were removed periodically for measurement of the weight of the yolk and of the freezing points of yolk and white. (b) The uptake of water by the yolk. The gain or loss of water by the yolk may be measured with sufficient accuracy by its gross change in weight, for the amounts of solid substances transferred during storage across the vitelline membrane are relatively inconsiderable, as may be shown by dry-weight determinations. It does not follow, of course, that the passage of ions across the membrane is an unimportant factor in the attainment of osmotic equilibrium, since the exchange necessary for this purpose would be too small to affect the weighings: the passage of as little as 0-22 millimol (13 mg.) of sodium chloride from yolk to white would suffice to equalise the two freezing-points. Since the same yolk cannot be weighed twice, a sampling method becomes necessary. The actual weights of yolks from different eggs vary considerably, but the ratio of the initial weight of the yolk to the initial weight of the whole egg is reasonably constant for individual eggs of a well-selected batch. It is thus possible to compute from the initial weights of whole eggs the initial weights of their yolks, and to compare these with the actual weights measured after a period of storage. Sample eggs from the batch gave the following figures for the initial weight of the yolk as a percentage of the initial weight of the whole egg : 28-9, 27-4, 28-4, 26-1, 28-0, 28-4; mean 27-8.

3 Relations between Yolk and White in the Hen's Egg 295 (Of the remaining 72-2 per cent, the white accounted for approximately 62-2 and the shell for io-o per cent.) The initial water contents of white and yolk were as follows: White 88-5, 90-8, 877, 87-8, 86-8, 88-4; mean 83-3 per cent. Yolk 467, 47-6, 46-5, 46-4, 46-6, 47-0; mean 46-8 per cent. The six eggs of a sample were broken open separately, the yolks were wiped free from adhering traces of egg-white, and were then transferred to Petri dishes and weighed Days Fig. 1. Gain in weight of yolks in eggs stored at o C, io C. and 25 C. (Evaporation prevented.) The sampling method is likely to give rather irregular results, since although the final weight can be obtained with considerable accuracy, a small difference in the mean percentage weight of the yolk in individual samples will produce a relatively large error in the estimate of the original weight. But the general trend of the results, which are plotted in Fig. 1, is clear. The uptake of water by the yolk is fairly rapid for a few days about 0-5 gm. in 7 days at io C. for example. After about 15 days, however, the weights become steady, the yolks now having taken up approximately 0-4 gm. at o C, o-8 gm. at io C. and 1-3 gm. at 25 0 C. (c) The equilibration of freezing-points of white and yolk. When the six yolks of a sample had been weighed, they were mixed together; the six whites were also mixed, and the freezing-points of the two fluids were measured. These observations were continued for a period of about 80 days, at each of the four temperatures, 0, io, 18 0 and 25 0 C. Table I summarises the results.

4 296 MICHAEL SMITH and JAMES SHEPHERD Table Days Yolk o C. White Difference Yolk io C. White Difference 0 3 S o o' O O-II o-ioo O-557 O O O O O At each temperature the freezing-point depression (A) of the yolks steadily decreased, and that of the whites steadily increased, during the storage of the eggs. The changes were most rapid initially, and became slower and slower the more nearly osmotic equilibrium was approached. After 75 days at the highest temperature (25 0 C.) the two freezing-points had become equal; at o C. the indications were that many months would elapse before this point was reached. The data for o C. and for 25 0 C. are shown graphically in Fig. 2. (d) The mechanism of equilibration. The exchange of water between yolk and white is not in itself sufficient to explain the freezing-point figures. The changes in freezing-points corresponding to the uptake of water shown for each temperature in Fig. 1, may be calculated by making use of the following data: Mean weight of egg Mean weight of white Mean weight of yolk Total water in white initially Total water in yolk initially Free water in white Free water in yolk Salts in white (as millimols NaCl) ~/T x 2^'^ Salts in yolk (as millimols NaCl) -z- x gm % x 50-0 = 31-1 gm. 278 % x 500 = 13-9 gm % x 311 =27-4gm. 468 % x 13-9 = 65 gm. 97 % x 274 = 266 gm. 85 % * 6-5 = 5-5 gm. = 3'5I i-ogm. The results are shown by the dotted curves in Fig. 2, and it will be seen that there are very marked discrepancies from the observed freezing-point values, shown by the full curves. The separated vitelline membrane is permeable to ions, as will be shown in a later paper of this series, but if a hypothetical exchange of ions is superposed on the observed exchange of water it is still impossible to reproduce the observed curves. Since the amount of free water in the white is nearly five times as great

5 I. Relations between Yolk and White in the Hen's Egg 297 Days / Yolk i8 C. White Difference Yolk 25 0 C White Difference IS o-6io '573 O O-5SO O O O' O O O O O-IO O-545 O O O-49S O O-O2I O-OI5 OOO \ V k A 4\ i \ to OJ O, -o -t-» 'o - bd C" 0) «,~ Calculated from water uptake at 0 \x X e^ A y^~~^-. v \ Calculated from water -^~_^ "* - v uptakeatjy ~ / * v ^^ 1 /?. T^ ^^"^ /vvcalculated from water^--^ V7.. Yolk White "^ Yolk ^ - ^^White 0-46 ^"^ Calculated from water loss at 0 _J c Fig. 2. Equilibr&tion of freezing points of yolk and white, x x = At o C

6 298 MICHAEL SMITH and JAMES SHEPHERD as the amount in the yolk, a simple transfer of water or ions, or both, in either direction, should produce nearly five times as great a change in the freezing-point of the yolk as it does in that of the white. Actually the ratio of the rate of decrease in the freezing-point depression of the yolk to the rate of increase in the freezingpoint depression of the white was always much less than five (Fig. 3) except in the first few days; in fact after a month at o C, and after about 45 days at io C, the white was actually changing more rapidly than the yolk. It is impossible therefore to fit the observed results by assuming that x gm. of water pass from white to yolk, and y gm. of salts from yolk to white. 6 <A t - o B \ Expected constant value 4 '1 A X? \. ^ «N. / 18 C a ^SV-.. o c 10 C. ~ ^ o * Days Fig. 3. Ratio between rate of change of freezing-point of the yolk and that of the white. The direction of the discrepancies may be seen from Fig. 2. The freezing-point depression of the white invariably increased more than it should have done in view of the amount of water lost. The yolk was less regular in its deviation. In the first 5 days at all temperatures the observed change in freezing-point agreed well with the observed uptake of water; later on at 25 0 C. (and perhaps also at io C.) the change in freezing-point was less than calculated from the uptake of water; and finally at all temperatures the uptake of water practically ceased while the freezingpoint depression continued to decrease. The explanation of these deviations may be found to lie either in the formation of fresh osmotically active molecules or ions on one side of the membrane, or by the adsorption on one side of the membrane of substances which have passed through from the other, or possibly by changes in the ratio of bound to free water in white or yolk. A 0

7 Relations between Yolk and White in the Hen's Egg 299 (e) The effect of temperature. The rate of a process of equilibration by simple diffusion ought to be proportional at any time to the osmotic gradient. In Fig. 4 the rate of equilibration (in C. change of freezing-point difference per 10 days) is plotted against the actual freezing-point difference (yolk-white). The curves are not linear: the rate of change per unit freezing-point difference is much greater in the initial stages when the freezing-point difference is large, than later on when it is small. 25 C Fig. 4. Rate of freezing-point equilibration as a function of freezing-point difference and temperature. The influence of temperature on the rate of equilibration can be expressed by comparing the rates corresponding to the same value of the freezing-point difference. Taking the rate at o C. in each case as unity, the following relative figures are obtained from Fig. 4. Freezing-point diff. At 0 C. At 10 C. At 18 C. At 25 C. i-6o i 40 I-2O i-oo o-8o M M M M M S

8 300 MICHAEL SMITH and JAMES SHEPHERD The temperature coefficient appears to be a function of the freezing-point difference, in the sense that the rate of equilibration is less considerably affected by temperature when the difference is great (that is to say when the eggs are fresh) than it is subsequently. This variability is not surprising if, as is indicated by the evidence already given, the process of equilibration is a complex one involving different factors at different stages. If the freezing-point difference between yolk and white is maintained by a "Lebenswirkung," the slow equilibration could perhaps be regarded as marking the gradual death of the stored egg. But it is not clear why the egg should die progressively more and more rapidly as the temperature is raised from o to 25 0 C. Moreover the viability of the embryo in fertile eggs shows quite a different relationship to temperature, since according to Moran(s) there is an optimum at 8 to io C, and a slow decline of viability on either side of this point. On the other hand, the shape of the curves and the general relation to temperature strongly suggest that the egg, commencing its existence in a state of disequilibrium between yolk and white, slowly attains an equilibrium by diffusion. The problem then is to account for the extreme slowness of the process. The transfer of about i-6 gm. of water from white to yolk or of about 0-22 x io~ 3 mol. of salt (expressed as NaCl) from yolk to white, would suffice to equalise the freezingpoints; diffusion can occur across a membrane with a superficial area of about 30 sq. cm. which is apparently permeable both to water and to salts; yet equilibrium is only reached at 25 0 C. after about 70 days, while at o C. the indications are that many months would be necessary. II. OSMOTIC EQUILIBRATION WITH RAPID EVAPORATION. (a) Methods. If the yolk is actively maintaining its hypertony to the surrounding medium, it is perhaps to be expected that when the osmotic concentration of the medium is increased, that of the yolk will rise with it. The white can easily be concentrated in an intact egg by evaporation. Under these conditions, will the yolk still maintain its hypertony, or, at least, will the normal tendency to dilution be checked? In order to obtain evidence on this point a second batch of eggs was stored at io C. over concentrated sulphuric acid, and evaporation was promoted by removing about 15 per cent, of the shell around the air space. The rate of loss of water under these conditions was 1-65 gm. per day, that is to say between 5 and 6 per cent, per day of the total water content of the white. Samples of six eggs were withdrawn from time to time, yolks and whites were separated, and the freezing-point of the mixed yolks and of the mixed whites was determined as before. (b) Results. The results are shown in Fig. 5, together with those obtained at the same temperature when evaporation was negligibly small. The difference in the initial freezing-points of the yolk in the two cases is due to a seasonal variation. With

9 Relations between Yolk and White in the Hen's Egg 301 rapid evaporation the freezing-points became equal in about 100 hours, as against 150 days (estimated) when evaporation was prevented. Thereafter the white had a greater freezing-point depression than the yolk, the difference now presumably representing the gradient of osmotic pressure which is to be expected inside an evaporating system possessing a considerable internal resistance to water movement. The particularly noteworthy result is that the curve of freezing-point changes in the yolk runs parallel to the corresponding curves for the yolks of eggs which are not evaporating. The course of changes in the freezing-point of the yolk was not appreciably affected by the rapid concentration of the white Yolk no evaporation.0 Whiterapid evaporation Yolk rapid evaporation 0-50 White no evaporation Hours Fig. 5. Equilibration of freezing points when evaporation is rapid. There is again a discrepancy between changes in freezing-point and changes in water content in the white. The rate of evaporation is known, and its calculated effect on the freezing-point of the white is shown by the curve XY in Fig. 5. The actual curve at first rises more steeply than the calculated one, but subsequently becomes much flatter. Meanwhile the yolk is presumably taking up some water from, or losing some ions to the white, but this exchange does not appreciably help to bring the calculated curve into agreement with the observed one in the earlier stages, while in the later stage the small correction it involves is in the wrong direction. A study of possible freezing-point changes in egg-white when kept, or of the relation between freezing-point and water content, may provide the explanation of these results. The main point of interest for the present problem is that the experi-

10 302 MICHAEL SMITH and JAMES SHEPHERD ment fails to provide any evidence in favour of the maintenance of hypertony in the yolk by a "Lebenswirkung." III. OSMOTIC RELATIONS OF SEPARATED YOLKS. (a) Recovery of hypertony by diluted yolks when replaced in egg-white. From a thermodynamical point of view the most impressive of Straub's experiments was that in which an egg-yolk, diluted by immersion in water to a freezingpoint depression smaller than that for egg-white, recovered its hypertony in 24 hours on being transferred back from water to egg-white. At first sight it seems to follow conclusively that we cannot be dealing with a system which is drifting, with extreme slowness, towards an osmotic equilibrium; for when the point of equilibrium is overstepped, the system will still return to its original state on replacing the original conditions. As the result of further observations a simpler explanation can now be suggested which does not presuppose the expenditure of energy to maintain a steady state. When yolks are immersed in water, or for that matter in diluted egg-white, or hypotonic glycerol solutions, or perhaps in any solution from which water is rapidly taken up by the yolk, a heterogeneity appears. Individual yolks differ somewhat, but a characteristic feature is the appearance of blisters or cushions of a clear solution just inside the membrane; sometimes this is accompanied by a change in the appearance of the peripheral part of the yolk which becomes white and pasty. If the yolk is rolled over the cushions of clear fluid tend to rise to the top of it. By means of a fine pipette it is possible to extract some of the clear solution, though hardly without a certain admixture of material from the yolk proper, but even so its freezing-point depression is found to be much smaller than that of the yolk taken as a whole. Presumably then the water which passes through the vitelline membrane is not immediately incorporated in the yolk, perhaps because there is a considerable resistance to water movement in the yolk emulsion. These observations suggest that Straub's result could perhaps be accounted for as follows: after immersion in water, although the yolk as a whole is hypotonic to egg-white, a central core remains hypertonic; on returning the yolk to egg-white the dilute superficial layer will tend to become isotonic with the white, by the passage of water outwards or of dissolved substances inwards; and the over-all effect of an isotonic superficial layer and a core still hypertonic, will be that the yolk contents as a whole will be found hypertonic to the white, although less so than initially. This is in fact the result found. (b) Effect of period of immersion in egg-white. Support for such an explanation is afforded by the behaviour of yolks which after dilution with water are kept for varying periods in egg-white. Attention was first called to the importance of this last factor by an unsuccessful attempt to repeat Straub's original observation. In order to reduce the experimental errors ten yolks were mixed for each freezing-point determination, the temperature was kept at io C, a litre of egg-white was employed, and, to give ample time for the recovery of hyper-

11 Relations between Yolk and White in the Hen's Egg 303 tony, a period of 72 hours was allowed in the egg-white. The initial mean freezingpoint of the yolks was C.; after 24 hours' immersion in 1 litre of water it was C.; after 72 hours in egg-white the mean freezing-point of the yolks was C. and that of the whites C-> tnat * s t0 sav > almost complete isotony had been attained by leaving the yolks in the egg-white for a longer period. The results of a further series of observations are shown in Table II. Table II. Freezing-points of diluted yolks after different periods in egg-white. Freezing-point of fresh yolks After 18 hr. in water Replaced in egg-white at After 24 hr After 48 hr After 72 hr C. C. Diff. C. Freezing-point water Freezing-point white >) >j The diluted yolk first of all becomes hypertonic to the white (in the first 24 hours), and then with further immersion tends to become isotonic. This result is to be expected if the water taken up, which initially forms a superficial dilute layer, gradually mixes uniformly in the yolk, and as a matter of fact it is generally observed that the superficial blisters or cushions of clear fluid disappear slowly when the yolk is replaced in egg-white, until after about 3 days the yolk is macroscopically homogeneous once more, except for a slight coagulation. The re-concentration of the diluted yolk is effected by an uptake (or production) of dissolved substances rather than by a loss of water, as may be shown by following the changes in weight. In water the yolk shows a considerable gain in weight; but so far from returning to its original weight on being replaced in egg white, there is generally a further gain. The figures in a typical case are given below : Original weight of yolk After 24 hr. in water Gain in water After 30 hr. in egg-white After 54 hr. in egg-white After 74 hr. in egg-white Further gain in white 144 gm. 194 gm. 2-0 gm. 193 gm gm. 202 gm. 08 gm. (c) Effect of period of immersion in water. The period of preliminary immersion in water also affects the result obtained, as is shown by Table III. In Fig. 6 the experimental points are plotted, and the dotted curves represent the general scheme into which the results seem most reasonably to fit. In its bearing on the problem of the osmotic relations of white and yolk the most important result is that the effects of dilution are more complicated than Straub considered, that his experiment is not really crucial in showing that work must be done at the membrane, and that an alternative explanation on the basis of a temporary heterogeneity of the diluted yolk is supported by visual observation and is in line with all the recorded data. If, on the other hand, the hypertony of the yolk is imagined to be maintained by a "Lebenswirkung," these results would have to be accounted

12 304 MICHAEL SMITH and JAMES SHEPHERD for by supposing that the yolk dies after about 72 hours in water or after about 24 hours in water followed by 72 hours in egg-white; and, although this could not perhaps be definitely disproved, it is not in line with the general behaviour of separated yolks. Yolks placed straight away in egg-white certainly maintain their hypertony for much longer periods than these, and, as will be shown in a later paper of this series, the same thing happens when yolk, white and vitelline membrane are all removed and then re-constituted as a diffusion-system; so that if hypertony were maintained by a " Lebenswirkung" it would have to be assumed that the system was a fairly viable one. 0-6r x Egg-white Yolks in water Yolks restored to egg-white 0-2 Hours Fig. 6. Recovery of hypertony by diluted yolks Table III. Freezing-points of yolks placed in egg-white after different periods in water. c. gp Freezing-point of fresh yolks hr. in water: yolk hr. in white hr. in water: yolk hr. in white hr. in water: yolk hr. in white hr. in water: yolk hr. in white hr. in white Freezing-point of fresh white Into white at Final freezing-point of white Into white at Final freezing-point of white Into white at Final freezing-point of white Into white at Freezing-point of white Freezing-point of white Final freezing-point of water C. 3 C. O O Q' O-4S O Diff. C

13 Relations between Yolk and White in the Hen's Egg 305 (d) Effects of heterogeneity of the egg-white. The white of an egg is a complicated physical system, and it is not to be expected that separated yolks immersed in mixed egg-white will necessarily behave in the same way as the yolks of intact eggs. In fresh eggs two zones of white are distinguishable at a glance an outer layer of clear more or less watery fluid (thin white), and an inner layer of viscous jelly-like substance (thick white). In addition a third zone is distinguishable immediately adjacent to the vitelline membrane. Usually in fresh eggs the amounts of thin and of thick white are approximately equal, but the proportion of thick white slowly diminishes if eggs are preserved at a fairly high temperature (25 0 C. for several weeks), and perhaps, even more slowly, at lower temperatures. The enormous difference in viscosity is not accompanied by any considerable difference in total water content, for Romanoff has found the values P er cent, and P er cent, for the outer and middle layers respectively, and P er cent, for the small layer immediately next the yolk. In fresh eggs the present writers have found a difference of only about C. in the freezing-points of thin and thick white (the latter being consistently the higher) but the relative values obtained for stored eggs depend on the rate of evaporation from the egg. This is illustrated by the following figures: (i) Eggs stored at 25 C. and 100 per cent, humidity, after treatment with HgCl 2 ; no evaporation: Thin white Thick white Initial freezing-points After 72 hr (ii) Eggs rapidly evaporating at 10 C. over concentrated H 2 SO 4 with 15 per cent, of shell removed; mean rate of loss of water 2-2 gm. per day: Thin white Thick white Initial freezing-points After 72 hr In case (i) the only water movement is a slow one from thick white yolkwards. In case (ii) the thin white is rapidly concentrated by evaporation. An osmotic pressure gradient of considerable magnitude appears to be necessary to supply water from thick to thin white. This evidence of a resistance to the movement of water between thick and thin egg-white, suggested that the rates of osmotic equilibration of yolks in thin and in thick white respectively might be considerably different. This is in fact the case: (i) Fresh eggs at io C. Yolk Thin white Thick white Initial freezing-points After 72 hr. in thick white After 72 hr. in thin white Rate of equilibration ( C. per day) per unit mean freezing-point difference: For yolks in thick white 008 For yolks in thin white 015 (ii) Eggs stored 2 months at io c C. Thin white Thick white Yolk - 050S Initial freezing-points After 96 hr. in thick white 0511 After 96 hr. in thin white Rate of equilibration ( C. per day) per unit mean freezing-point difference: For yolks in thick white 027 For yolks in thin white 047

14 306 MICHAEL SMITH and JAMES SHEPHERD In each case equilibration proceeds only about half as rapidly in thick white as in thin. This result helps to explain why equilibration is much more rapid in separated yolks immersed in egg-white than in intact eggs. The differential resistance to water movement in thin and thick white is not sufficiently great to show itself in an effect on the rate of evaporation from exposed surfaces of the two liquids in an atmosphere of still air, which in each case is found to be practically identical with that from a free water surface of the same form and area. But this result only means that the resistance in the liquid is small compared with that interposed by the stationary film of air over the surface, which is very great. The rate of uptake of water by dried gelatin from thick and from thin white was also investigated. No significant difference was found, but here again it is not unlikely that some other factor, and not the actual resistance to water movement in the liquid, was limiting the rate of the process. (e) Osmotic relations of yolks in various aqueous solutions. The complexity of egg-white suggests a study of the behaviour of egg-yolks in simple aqueous solutions of electrolytes and non-electrolytes. Glucose, glycerol and Ringer solutions (the latter made up according to the formula, NaCl 24, KC1 o-6, CaCl 2 o-6, NaHCO 3 0-4, and diluted to the required freezing-point) have been employed. Yolks immersed in these solutions were weighed periodically, and finally removed in order to determine their freezing-points. Two sets of solutions were employed one of approximately the same freezing-point ( 0-44 C.) as egg-white, the other hypertonic to the yolk ( 0-72 C.) to about the same extent as the white is hypertonic. Observations were made at two temperatures, o and 15 0 C. The results, as far as equilibration of freezing point is concerned, are summarised in Table IV, where As = freezing-point depression of solution which, owing to the relatively large volume, is sensibly constant during the experiment. A^ = original freezing-point depression of yolk. Ay 2 = final freezing-point depression of yolk. E = rate of equilibration ( C. per day). r) = rate of equilibration per C. freezing-point difference between solution and (mean) yolk. Equilibration is about twice as rapid at 15 0 C. as at o C, and this agrees approximately with the temperature coefficient for intact eggs mentioned in an earlier section. It is less rapid in hypotonic solutions, when the yolk is gaining water, than in hypertonic solutions, when as will be shown later, the yolk is not losing water but is gaining dissolved substances. The rate seems to be of the same order whether a non-electrolyte (glycerol, glucose) or an electrolyte (Ringer) is employed and the dialysate of egg-white, reconcentrated to a freezing-point depression of C. gives a figure which is again similar, though perhaps significantly smaller.

15 Relations between Yolk and White in the Hen's Egg 307 Table IV. Rates of freezing-point equilibration between yolks and aqueous solutions. Solutions at 15 0 C. Time 48 hr. (a) Hypotonic Glucose Glycerol Ringer (6) Hypertonic Glucose Glycerol Ringer Solutions at 0 C. Time 72 hr. (c) Hypotonic Glucose Glycerol Ringer (d) Hypertonic Glucose Glycerol Ringer White dialysate As A Vl * 41 hours No result O No result * E No result No result O-02I V O-27 > O-22 O-2O7 No result o-391 n.? A 0-29/ > O-I2j OI No result oi ^017 o-i7j 012 (/) The uptake of water from aqueous solutions. Since the amounts of dissolved substances transferred one way or the other in the process of equilibration are small, a combination of weighings and freezing-point determinations will, as before, serve to show whether equilibrium is being approached primarily by the movement of water or by changes in the amounts of dissolved substances on opposite sides of the membrane. Table V gives the changes in weight of the yolks whose freezing-point changes have been set out in Table IV. Table V. Increase of weight of yolks in aqueous solutions. (a) Hypotonic solutions: Glucose (0449) Glycerol (0-431) Ringer (0-444) White dialysate (0425) (b) Hypertonic solutions: Glucose (0727) Glycerol (0-717) Ringer (0-726) White dialysate (0-730) 24 hr. (gm.) o-oo o At 15 0 C. 48 hr. (gm.) o-88* i-4it 052 o hr. (gm.) o-oo I-2I No result o-33 At 0 C. 72 hr. (gm.) o-oo 1-19 No result o-6i o-59 o-54 * Probably punctured. f Punctured. Although there appears to be a rather considerable variation in the behaviour of individual yolks, one result stands out very clearly in none of the jeb-vmiii 21

16 308 MICHAEL SMITH and JAMES SHEPHERD solutions was there a decrease in weight: that is to say, water never passed out of the yolk. In hypertonic solutions osmotic equilibrium was presumably approached by an increase of osmotically active substances in solution. White dialysate seems to behave like the other solutions in this respect. The behaviour in hypotonic glucose is perhaps anomalous in that there is often no change of weight, and this point needs further investigation.,*144 hours 120 hours > Freezing-point depression of solution Fig. 7. Uptake of water by egg yolks from Ringer solutions of different concentrations. A more detailed picture of the uptake of water by yolks from Ringer solutions of different concentrations at o C. is given in Fig. 7. The solutions used had freezing-points of o-i 1, 0-22, 0-44, 0-58, 0-73, 0-96 and 1-20 C. respectively, and a parallel set of observations was made with distilled water. The yolks initially had a mean freezing-point (based on controlled determinations) of 0-58 C. The yolks were weighed after 24, 48, 72, 120 and 144 hours and a curve has been drawn through each of these five sets of points. From the most dilute solutions there is a rapid uptake of water; in the case of pure water the uptake is followed by a partial loss, due perhaps to the slow passage of dissolved substances out from the yolk. From moderately hypertonic solutions (0-73, 0-96, 1-20 C.) there is also an uptake of water which appears to be directly proportional to the concentration of the solution and to the time of immersion. Finally, there is a broad zone of concentration on either side of the point of isotony, where the uptake

17 Relations between Yolk and White in the Hen's Egg 309 of water is small (although quite definite) and almost independent of the concentration. Osborne and Kincaid (6) have observed the uptake of water by yolks from concentrated salt solutions (of much greater strength than those employed above) and have attributed it to a solution of the vitellin resulting in the development of a high osmotic pressure inside the yolk membrane. This does not fit the present case. The freezing-point depressions of the yolks when eventually removed from the solutions are given in Table VI, where as before A.? = freezing-point depression of solution. Ayi = original freezing-point depression of yolk. Aj 2 = final freezing-point depression of yolk (after 6 days). E = rate of equilibration ( C. per day). Table VI. Freezing-point changes of yolks in Ringer solutions of various concentrations. As C. O-II A Vl 0 /-i A* C o-s4 o- S 8» o-8 3» E o-i35 * After 2 days only instead of 6. All the yolks tend towards equality of freezing-point with the solutions in which they are immersed, and the more rapidly the greater the osmotic pressure difference between yolk and solution. The yolks in hypertonic solutions are therefore taking up their water against the gradient of osmotic pressure as measured by the freezingpoint depression. Equilibrium is much more rapidly approached in these solutions than in intact eggs. The following figures for the rate of equilibration ( C. per day) per unit mean freezing-point difference, may be placed together for comparison. Table VII. Rates of osmotic equilibration under different conditions. Intact eggs Yolks in thick white thin white,, white dialysate,, hypotonic solutions,, hypertonic solutions At 15 0 C O-22 O-34 At o C o-io 017 A bridge between the extreme values is provided by the behaviour of yolks in thick white, thin white, and white dialysate. This suggests that a resistance to the movement of water in the intact egg-white is partly responsible for the slowness of equilibration. 21-3

18 310 MICHAEL SMITH and JAMES SHEPHERD The yolk also has a physical structure which may considerably impede the passage of water through it, and this is shown not only by the behaviour of diluted yolks already commented upon, but also by the fact that evaporation from an exposed surface of egg-yolk soon results in a marked difference of water-content between the superficial layer, which eventually forms a solid crust, and the underlying fluid portion. But the supposition of an immense resistance to equilibration centred in the yolk (or for that matter in the membrane) is difficult to reconcile with the fact that equilibration is relatively rapid when yolks are placed in these aqueous solutions, unless it is assumed that the removal of the yolk from the egg or contact of the membrane with the aqueous solution, themselves induce profound changes in the system. IV. SUMMARY. 1. The freezing-points of white and yolk in the hen's egg gradually approach equality when the egg is kept for long periods; and the rate of the process of equilibration is rapid at first but becomes very slow as equality is more closely approached. 2. Between o and 25 0 C. the rate of equilibration has a temperature coefficient (Q 10) of from 1-5 to 2. At 25 0 C. equality of freezing-points is reached after about 70 days. 3. Equilibration is achieved partly by the passage of water across the vitelline membrane from white to yolk, but partly also by more complicated changes of osmotic concentration occurring more or less independently in white and yolk. 4. The recovery of hypertony by a yolk, previously diluted by immersion in water, when it is replaced in egg-white can be explained on the basis of a temporary heterogeneity of the diluted yolk, and this explanation is supported by experimental evidence. 5. The rate of equilibration is much greater when the separated yolk is placed in mixed egg-white than in the intact egg, but since it is also greater in thin white than in thick white, and greater again in the white dialysate, the structure and viscosity of the white are probably important factors. 6. There is evidence of an appreciable resistance to water-movement both in egg-white and in egg-yolk. 7. In hypotonic or hypertonic aqueous solutions of glucose or glycerol, or in Ringer's solution, the rate of equilibration is greater than in egg-white and many times greater than in the intact egg. Water is taken up by the yolk both from hypotonic and hypertonic solutions of Ringer, within the range A = o-io to 1-20 C, at a rate which increases the further the solution is removed from the point of isotony. 8. Evidence that the apparent disequilibrium in intact eggs is not a steady state maintained by a "Lebenswirkung," is afforded by: (i) the form of the equilibration curves, which strongly suggest the slow attainment of an equilibrium by diffusion, rather than a steady state terminated by death;

19 Relations between Yolk and White in the Hen's Egg 311 (ii) the temperature relations of equilibration, which are consistent with the former assumption, but which do not agree at all with the effect of temperature on the viability of fertile eggs; (iii) the absence of any tendency of the yolk to maintain its hypertony when the white is concentrated by rapid evaporation; (iv) the alternative explanation for the recovery of hypertony by diluted yolks, which was the most crucial evidence for the existence of a steady state maintained by the expenditure of energy. REFERENCES. (1) BIALASCEWICZ, K. (1929). Protoplasma, 6, i. (2) GREENLEE, A. R. (1912). Journ. Amer. Chem. Soc. 34, 539. (3) HILL, A. V. (1930). Proc. Roy. Soc. B, 106, 477. (4) (1930). Proc. Faraday Soc. (Symposium). (5) MORAN, T. (1925). Proc. Roy. Soc. B, 98, 436. (6) OSBORNE, W. A. and KINCAID, H. E. (1914). Biochem. Journ. 8, 28. (7) STRAUB, J. (1929). Rec. Trav. Chitn. Pays-Bas, 48, 49.