Measuring Osmotic Pressure of Hcemoglobin. 627rspa.royalsocietypublishing.org/content/royprsa/108/748/627.full.pdf · Measuring Osmotic Pressure of Hcemoglobin. 627 radiation emitted

Measuring Osmotic Pressure o f Hcemoglobin. 627

radiation emitted by the burning gases during the explosion period. The experimental procedure consisted in comparing pressure-time curves taken during the explosion of identical mixtures, taken first when the walls of the vessel were polished and then when they were blackened.

The inflammable gases experimented with consisted of hydrogen and air, carbon-monoxide and air and methane and air. An increased rate of combustion as indicated by the pressure-time curves was found in all cases when the superimposed radiation (first type) or increased radiation (second type) was of a kind which could be absorbed by the combustible gases.

While the experiments seem to prove conclusively that the absorption of radiation by the reacting gases promotes combustion, they also tend to focus attention upon intra-molecular energy (rotational and vibrational) of the reacting molecules as being the factor (or one of the factors) concerned in combustion.

A Critical Study o f the Direct Method o f Measuring the Osmotic Pressure o f

By G. S. A d a ir , Fellow of King’s College, Cambridge.

(Communicated by Sir William Hardy, Sec.R.S.—Received April 1, 1924.)

(From the Low Temperature Research Station, Cambridge.)

1. Introduction.A solution of haemoglobin and salts may be considered as a colloidal system.

Since the work of Thomas Graham, it has been recognized that such systems are unstable, for they are liable to undergo gradual changes in their state of aggregation. When the chief colloid is a protein, there may be in addition actual chemical breakdown. When, the chemical instability is associated with the lag normal to the colloidal state the application of the direct osmometric method becomes peculiarly difficult, because, if the experimenter elects to wait for the system to obtain equilibrium he is likely to encounter chemical breakdown, and if he accepts the first approximately steady readings and relies upon extrapolation to give the absolute values, his conclusions can never have the finality which would alone justify the use of so difficult a method.

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628 G. S. Adair. Direct Method o f

These disadvantages can be partly met by using osmometers designed to reach equilibrium rapidly (see Section 4), but they can be wholly overcome only by discovering conditions which ensure chemical stability. Of these the most important is a low temperature, and therefore all the critical measurements described in this paper and the papers which follow were made at 0° C.

Studies on the effect of solutes on haemoglobin revealed another condition. It was found that the stability was greatest, and apparently perfect at 0° C., in the presence of certain mixtures of salts. I t was remarkable that these salt solutions were similar to those which obtain in the blood. When the conditions for the chemical stability of haemoglobin were fulfilled, it was possible to give many days or weeks for the osmometer to reach a true osmotic equilibrium.

The most sensitive test for the stability of the protein is the osmotic pressure measurement itself, and the most conclusive proof of stability is given by the criteria for osmotic equilibrium described in Section 6. These criteria were supplemented by specific chemical tests to detect various types of breakdown.

The formation of methaemoglobin was noted in Experiment 135 and measured by van Slykes’ method. I t was found that 8 per cent, of the oxyhaemoglobin had changed in the course of 7 weeks. In order to estimate the effect of this change, the osmotic pressure of pure methaemoglobin was measured, and proved to be the same as for oxyhaemoglobin.

The outer liquids were tested for diffusible protein breakdown products by the usual methods, including the biuret reaction and the nitroprusside reaction, with negative results. These tests would not show non-diffusible breakdown products, but as my figure for the molecular weight is four times as large as the accepted value, the latter is more likely to have been subject to this source of error.

Denaturation and subsequent flocculation can be detected by the formation of a deposit in the osmometers, as well as by the accompanying change in osmotic pressure. These changes were most noticeable in dilute solutions at room temperature, but no measurable precipitation occurred in the experiments described in this paper.

Sources of error other than changes in the protein are imperfections in the membrane, changes in the outer liquid, and defects in the registering apparatus. The most important defect in the membrane is permeability by colloids as well as crystalloids. The former is a common type of error, sometimes overlooked because the diffusion velocity of colloids is small, and hours or days may elapse before appreciable quantities appear in the outer liquid. Membranes



629

permeable to colloids gave abnormally small pressures. More rarely the membrane proves to be impermeable by crystalloids and then gives excessive pressures. Certain types of membrane adsorb the protein and so diminish its concentration; others are elastic and allow volume changes. The avoidance of these errors depends upon the technique of membrane manufacture.

Changes in the outer liquid may prevent the system from obtaining osmotic equilibrium. This difficulty is at its greatest when it is desired to measure the osmotic pressure against pure water. The magnitude of the error is surprising. In one experiment, traces of carbon dioxide in the air, due to the respiration of a small sample of fruit and vegetables placed in the same cold store, doubled the osmometer readings within 48 hours. Changes in the outer liquid may be reduced or avoided by the use of vessels coated with hard paraffin and protected by soda-lime tubes, or by employing buffered solutions.

The most common defect in the registering apparatus is the sticking of the meniscus in the manometer tube, owing to capillary forces. This may give a false impression that equilibrium has been reached. In addition to these sources of error, which are essentially defects in technique, there is the possibility of mistaking a period of slow change for the true end point. For example, in the early stages of diffusion, before the salts have reached their equilibrium distribution, there may be temporary diffusion pressures, much larger than the true osmotic pressure (ref. Reid, 1904).

When the outer liquid is distilled water and the inner contains a protein salt, the rate of hydrolysis may be slow and an approximately constant pressure may be obtained for a time, although the osmometer is not really in equilibrium with distilled water.

In this work three main criteria have been relied upon to discriminate between true and false osmotic pressures, namely :—

(1) Steadiness of the pressures. Osmotic pressures were recorded daily for long periods, and all experiments were rejected if the pressure failed to maintain a constant level. This test eliminates errors due to changes in the protein or permeability of the membrane.

(2) Capacity for repeating the measurement. The osmometer was reset at a new level and allowed to come again into equilibrium. The new equilibrium should agree with the old. This test eliminates temporary diffusion pressures and effects due simply to inertia.

(3) Agreement of the results obtained when the technique was varied.

Measuring Osmotic Pressure o f Haemoglobin.




2. Methods for Preparing Hcemoglobin.

As a general rule the method of preparation influences the properties of a protein, therefore a variety of methods must be tested. The first and simplest was to lake the red corpuscles with distilled water, without removing the stromata. The second was to prepare crystals by the addition of twice the volume of saturated ammonium sulphate to a diluted haemoglobin solution. The third was to add ether to lake the corpuscles and then add salt to shrink the swollen stromata.

After the ether and salt treatment, the stroma protein was removed by centrifuging, and crystalloidal impurities were removed by dialysis in closed collodion sacs, as described by Adair, Barcroft and Bock (1921). Descriptions of other methods are given by Heidelberger and by Ferry.

There is a risk that complete removal of salts may cause irreversible changes, therefore, in many experiments, dialysis was conducted with a standard salt solution for outer liquid.

3. Manufacture of Membranes.A membrane for osmometer work should fulfil the following conditions ;

•firstly, it should be rigid and of sufficient strength to stand two atmospheres pressure unsupported. Secondly, it should be quite permeable by water and salts, but it should not allow any haemoglobin to pass through in the course of a fortnight’s test. Early experiments were made with Sorensen’s process, but about 90 per cent, of the membranes were penetrated by haemoglobin, therefore the procedure was modified. After a few years’ practice, the proportion of failures was below 10 per cent.

The membranes are now prepared by pouring four or five even coats of collodion over the outside of a glass mould, rotated in front of a gas fire or other source of radiant heat. The time intervals between the coats and the final drying time have to be judged by preliminary trials for each type of collodion.* After drying the membrane is immersed for a few hours in water, the upper end trimmed off, and removed from the mould by water pressure. A small hole in the end of the tube allows the water to creep in between the collodion and the glass.

Membranes can be mounted by softening the upper end in a mixture of ether

* In one type of collodion, made with 3-5 grams gun-cotton, 75 c.c. ether and 25 c.c. alcohol, the time intervals were 2-5 minutes, and the final time 20 minutes. The speed of rotation was 15 revolutions per minute.




and alcohol, drawing this end over a rubber cork or a glass tube, and allowing this part to dry in air, while the lower part of the membrane remained immersed in water. For very high pressures these mounts were strengthened by rubber rings and wire.

4. Construction of Osmometers *At ordinary temperatures, haemoglobin is rather unstable and the growth

of bacteria may affect the results, therefore much time was spent in devising models for rapid equilibration.

In model “ A,” adapted only for lowr pressures, a stirring system was used with a weight inside the collodion sac attached to a waxed thread which ran up the manometer tube. In “ B ” a very small membrane (2 c.c.) was used, in conjunction with a counter pressure apparatus for maintaining constant volume, as described by Sorensen (1917). Four models were used in the long duration experiments, conducted at 0° C. The proportions of types C and D are shown in the figure, drawn to scale by my friend Mr. Pique. Type C was used for pressures over 200 mm. of mercury (see overleaf).

A simple modification, CN was used for moderate pressures. In this type the manometer was made of capillary tubing throughout, and the pressure adjuster and the bend with paraffin omitted. Type D was used for pressures below 25 mm. of mercury. A smaller model DN was also used with a 2 c.c. collodion sac, 6 mm. in diameter, mounted on glass capillary tubing.

In the mercury manometers, the capillarity corrections were combined with the zero and density corrections in the following formula :—

p = (L - L0) + (B0 — B) — D /13-6 (B0 - B).

B and L are the mercury levels in limbs B and L in fig. 1, at osmotic equilibrium. L0 and B0 are measured levels at zero osmotic pressure, determined after the membrane and cork are removed. For the measurement of L0 and B0, the jar was slightly raised to bring the outer liquid to its original level. D is the density of the haemoglobin solution.

In the straight manometers, type D and DN, the following formula was used :—

p = [(Ri — R 2) — (R 3 — R 4)] X D/13-6.R x is the scale reading at the meniscus at osmotic equilibrium. R 2 is the scale reading at the level of the outer liquid. R3 is the meniscus reading when the tube is dipped in a tube of the inner liquid. R 4 is scale reading at the level of the inner liquid.

Although the capillarity corrections are small, they are rather uncertain on account of the surface film of protein ; they were measured with a rising and a falling meniscus ; as a rule, they took several days to reach equilibrium in manometer tubes below 1 mm. diameter.

* Useful descriptions of osmometers are given by Starling, Reid, Bayliss and Sorensen, and Morse.




Osmometer, Type D (left), with straight column of solution for measuring pressure. Scale in centimetres. Osmometer, Type C (right), for high pressures with mercury manometer : (1) A rigid collodion tube, 20 c.c. capacity, in jar holding 500 c.c. of outer liquid. (2) Rubber connection. The glass tube from (2) to (3) is filled with liquid paraffin. Beyond (3) there is water. (4) Rubber connection with clip (not shown here). (5) Glass T tube, with water down to level B. (6) Glass rod in rubber tube, for adjusting the pressure. (7) Glass tube, open to air, or fitted with a soda-lime tube. W = level of outer liquid ; B, level of mercury in manometer limb, 6 mm. in diameter ; L, level of mercury in manometer limb, 0 • 7 mm. in diameter. Above the mercury at L there is a drop of lactic acid.

5. Measurements of Concentrations.All measurements of concentrations were made at the end of the experiment.

Proteins and water were determined by drying a known weight of solution at 110° and ashing at a low red heat. The outer liquids were dried and ashed,



under the same conditions, to estimate the corrections due to the loss of water from phosphates, etc.

The dipping refractometer was used for most experiments, especially with small volumes of liquids, and factors for converting refractions to protein concentrations were determined empirically (Stoddard and Adair, 1923). Chlorides were determined by the method of Austin and van Slyke (1920). Phosphates were determined by the method of Bell and Doisy, as modified by Briggs (1922). Hydrogen-ion concentrations were determined colorimetrically in the outer liquids, and hydrogen-ion concentrations in the inner liquid were calculated with the aid of membrane potential determinations (ref. Bayliss, 1911; Loeb, 1922), as the hydrogen electrode gives unreliable readings in the presence of oxy-hsemoglobin.

6. Experimental Proof of Equilibrium.It has been stated that three criteria were to be employed in proving the

existence of osmotic equilibrium :—(i) The pressures must be permanent.(ii) The pressures must be reversible.

(iii) The pressures must be reproducible.In the experiments recorded below, haemoglobin was dissolved in the fol

lowing : potassium chloride, 7*456 grams; di-sodium phosphate crystals,22 • 69 grams ; potassium hydrogen phosphate, 0 • 454 grams ; with water to make up 1 litre. The temperature was -j-0*6° C. Under these conditions the haemoglobin appeared to be perfectly stable, and no protein breakdown products could be detected in the outer liquids. The different criteria are given in order below.

7. Criterion 1.—Permanence of Pressures.A set of six experiments was devised to test the permanence of the osmometer

readings. One was a failure (No. 202), for traces of haemoglobin appeared in the outer liquid, and one showed an unaccountable variation of 8 per cent., but the other four experiments appeared capable of maintaining a constant pressure indefinitely. A minute fall in pressure was observed, as the osmometers were left unattended throughout the summer vacation, but this change was accounted for by the small observed change in the hydrogen-ion concentration of the outer liquid.

In the first three weeks the range of variation is about 1 per cent., which is within the limits of experimental error.

2 x


VOL. CVIII.— A.




Table I.—Criterion I. Relation of Osmotic Pressure and Time.—Haemoglobin of sheep in standard phosphate at 0-6° C. Pressures in millimetres of mercury. Star (*) indicates a leak. Concentrations in grams per 100 c.c. solution.

Concentrations. 29-3 25-4 19-3 19-6 12-6 28-3

Time in Days. No. 200 No. 201 No. 202 No. 203 No. 204 No. 2052 277-5 191-5 117-7 116-5 50-7 2395 275-5 187-0 120-3 115-6 51-4 239

24 278-5 188-1 121-3 115-4 51-6 24231 274-2 184-0 120-1 110-8 48-5 _51 271-2 181-2 *109-1 106-9 48-7 _67 264-6 178-9 *104-2 — —

8. Criterion II. Reversibility of Pressures.In Experiment 200, when the osmometer was set, the initial reading of

limb L was 418 mm., but in two days it sank to 330-3 mm. It was then reset, with the initial level 273 mm., and it rose in two days to 328-5 mm., very near the first equilibrium value. I t was again reset at 363 mm., and in a day it fell to 330-5 mm., and remained within 1 mm. of this figure for weeks.

Table II.—Criterion II. Reversibility of Pressures.—No. 200, No. 204, experiment numbers as in I. B, L, actual scale reading on osmometers —compare fig. 1. Zero determination. Experiment 200 (Type C): B0 = 217, L0 = 63. Experiment 204 (Type CN): B0 = 3-2, L0 — 1 -2.

Time. Experiment 200. Experiment 204.

Days. Limb B. Limb L. Limb B. Limb L.

0 202 418-01 206 332-5 - 23-5 27-02 206 330-3 - 23-5 27-3

— Reset Reset Reset Reset3 208 273-0 - 13-0 16-53-4 206 323-0 - 23-0 27-04 206 328-5 - 23-0 27-85 206 328-5 - 23-9 27-6

— Reset Reset Reset Reset6 204 363-0 - 37-0 41-07 206 330-5 - 24-2 28-38 206 330-0 - 24-2 27-2

21 206 331-5 - 24-3 28-022 206 331-5 - 23-8 27-9



Table I I a .—Criterion II. Reversibility of Pressures.— Experiment 211 (Type DN) : Haemoglobin, 3*77 per cent. Heights in centimetres of solution (density, 1-0235). Corrections: zero, — 0-2; capillarity rising, 0-9; falling, 0-95. Col. 2 gives levels with the initial level too low. Col. 4 gives levels with the pressure reset at 17-4. Both tend towards 15-5 cm.

Measuring Osmotic Pressure o f Hemoglobin. 635

Time. Level. Time. Level.

0 11 40 6 17-401 12-30 7 16-802 13-65 8 16-603 15-00 9 16-405 15-28 12 15-726 15-35 13 15-70

These figures prove that the steady reading was a true equilibrium, because the osmometer always returned towards the same pressure when arbitrary variations were made in the levels.

Other examples are given in Tables II and IIa. It should be observed that there are small variations in the final pressures, in the region of 2 per cent. These are due to the capillarity corrections not being the same for a rising and a falling meniscus, and to the fact that the resetting and the subsequent osmosis must cause slight changes in concentration.

9. Criterion III. Reproducibility of Pressures.The errors due to bad membranes and the lack of salt equilibrium are not

unlikely to be reproduced if the experiments are made by a uniform method. In order to avoid the risk of constant errors in this work the experiments were made by a variety of methods. Membranes were prepared from different samples of collodion, and variations were made in the initial salt distribution and the initial hydrostatic pressure, therefore the agreement of the results is good evidence that the final osmometer readings were true osmotic pressures. Strict agreement can only be expected if the final conditions were exactly the same in each experiment, so that the observed variation is actually larger than the real range of experimental error.

The three final experiments in Table III are of theoretical significance. In Experiment 184 the corpuscles were laked with distilled water, and the hsemoglobin was kept free from contact with ether, or ammonium sulphate, or other reagents which might alter its constitution. In Experiment 211 the hsemoglobin was prepared by the ether and salt method, and in Experiment 222 it was prepared by three recrystallizations from 2/3 saturated ammonium

2 x 2




sulphate. Considering the extreme difficulty of the direct method of measurement, the results given in Table III are a satisfactory proof that haemoglobin will give a constant osmotic pressure.

Table I I I .—Criterion III. Reproducibility of Pressures.

Experiment No. 7r* CHbf Species.

51 2-5Per cent.

1-7 Man (T.R.P.)106 3-2 4-0 Man (G.S.)152 2-97 1-2 Man (H.F.)168 3-01 1-28 Man (Dr. F.)170 3-04 6-78 Man (G.S. A.)31 2-85 2-68 Horse undialysed78 3 0 7 1-3 Horse undialysed

126 3-09 2-4 Horse dialysed55 2-85 4-8 Ox laked

184 2-80 2-21 Sheep laked211 2-88 3-77 Sheep ether method222 2-83 2-82 Sheep crystallized

* 7r = osmotic pressure per 1 per cent, haemoglobin, f Grams Hb per 100 c.c. solution.

The theoretical interpretation of the pressure will be given in a later communication . Preliminary calculations indicate that, with the outer liquid used in these experiments (M/10 KC1 -j- M/15 Na2H P04, . 8'08), more than 90 per cent, of the pressure is due to protein, and less than 10 per cent, is due to the distribution of salts across the membrane. Deducting 10 per cent, of the pressure in Experiment 222 leaves 2'54, which corresponds to a molecular weight of 67,000.

10. Summary and Conclusions.A brief account is given of the causes of variations in osmometer readings,,

and experimental methods and sources of error are discussed.Particular attention is given to the criteria which may serve to distinguish

true osmotic pressures from temporary diffusion pressures and false equilibria-Experimental proof is given that in certain solvents, the osmometric observa

tions on haemoglobin satisfy the three criteria—permanence, reversibility and reproducibility, therefore they may be regarded as true osmotic pressures.

Readings remained constant within 6 per cent, for nine weeks, and no products of protein breakdown could be detected in the outer liquids.

With rigorous control of conditions and with adequate criteria for equilibrium,, the osmometer method proved thoroughly reliable ; and when certain corrections were applied, it proved capable of giving results accurate to 0*1 mm. of



Measur ing Osmotic Pressure o f Haemoglobin. 637

mercury, which corresponds to about 1 /100,000th of a degree in depression of the freezing point.

This work would have been almost impossible without the facilities of the Low Temperature Station placed at my disposal.

REFERENCES.

Adair, Barcroft and Bock, ‘ J, Physiol.,’ vol. 55, p. 332 (1921).Austin and Van Slyke, ‘ J. Biol. Chem.,’ vol. 41, p. 345 (1920).Bayliss, £ Roy. Soc. Proc.,’ B, vol. 81, p. 269 (1909).Bayliss, 4 Roy. Soc. Proc.,’ B, vol. 84, p. 229 (1911).Briggs, ‘ J. Biol. Chem.,’ vol. 53, p. 13 (1922).Eggerth, ‘ J. Biol. Chem.,’ vol. 48, p. 203 (1921).Ferry, ‘ J. Biol. Chem.,’ vol. 57, p. 819 (1923).Graham, 4 Phil. Trans.,’ vol. 151, p. 183 (1861).Heidelberger and Landsteiner, 4 J. Exp. Med.,’ vol. 38, p. 561 (1923).Htifner u. Gansser, 4 Arch. f. Anat. u. Physiol.,’ p. 209 (1907).Loeb, 4 Proteins and the Theory of Colloidal Behaviour,’ New York (1922).Morse, ‘ The Osmotic Pressure of Aqueous Solutions,’ Washington (1914).Reid, 4 J. Physiol.,’ vol. 31, p. 438 (1904).Reid, 4 J. Physiol.,’ vol. 33, p. 12 (1905).Sorensen, 4 Comptes-Rendus Lab. Carlsberg,’ vol. 12 (1917).Starling, 4 J. Physiol.,’ vol. 24, p. 317 (1899).Stoddard and Adair, 4 J. Biol. Chem.,’ vol. 57, p. 437 (1923).



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Measuring Osmotic Pressure of Hcemoglobin. 627rspa.royalsocietypublishing.org/content/royprsa/108/748/627.full.pdf · Measuring Osmotic Pressure of Hcemoglobin. 627 radiation emitted