The Ionic Nature of Haemoglobin

The Ionic Nature of HaemoglobinAuthor(s): Harold TaylorSource: Proceedings of the Royal Society of London. Series B, Containing Papers of aBiological Character, Vol. 96, No. 678 (Aug. 1, 1924), pp. 383-397Published by: The Royal SocietyStable URL: http://www.jstor.org/stable/81095 .

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383

The Ionic Nature of HIemoglobin.

By HAROLD TAYLOR.

(Communicated by Prof. A. V. Hill, F.R.S.-Received May 23, 1924.)

(From the Physiological Laboratories of Cambridge and Manchester.)

The study of the reaction between CO2 and hEemoglobin under the conditions

obtaining in the blood of man and animals had led to two opposite views being taken on the nature of the reaction. These views are :

(1) The haemoglobin in the corpuscles acts as an electrically inert substance, and either combines directly with the CO2 or adsorbs it, due to its colloidal

properties. (2) The haemoglobin in the corpuscles is partly in the form of a salt, as

sodium* haemoglobinate, which exists partly as sodium and haemoglobin ions and

partly as the undissociated salt. The action of the CO2 in this case would be the normal action of an acid acting upon the salt of a weak acid, and the

CO2 would combine with the base, forming a bicarbonate and the weakly acidic

haemoglobin. This theory requires that the whole of the CO2 in the corpuscles :is either in physical solution or in the form of bicarbonate.

The earliest workers on the subject seem to have had very varying views on the reaction, but this was due to their technique not being as good as that of later workers. They found out, however, that acid would drive the CO2 out from the blood, but disagreed as to whether CO2 could be pumped out by means of a vacuum. This was due to the fact that most of them could not

get a sufficiently high vacuum, but by means of a new pump Pfluiger (1864) succeeded in abstracting all the CO from blood. He also made the important observation that, in the absence of corpuscles, the CO2 could not be removed from the plasma without the aid of a strong acid, and from this concluded that, in the case where the CO2 had been removed from whole blood by pumping, the haemoglobin must have been the chief agency in driving it out.

From these facts Zuntz (1882) put forward his well-known theory as to the reaction between blood and CO2. This theory is essentially the same as the second given above and is held by the majority of physiologists to-day, i.e., that under the conditions existing in the body heemoglobin does not combine with CO2 and that all the " combined " CO2 is present as bicarbonate.

* Sodium here and in the sequel is taken, for shortness, to include potassium. 2 F 2



H. Taylor.

Zuntz's theory seems to explain most of the phenomena found about blood, and is supported by a large amount of evidence. The formulae of Hasselbalch

(1916), Henderson (1909), and Warburg (1922) for the hydrogen-ion concentration of the blood all depend upon the total amount of combined CO2 being present as bicarbonate, and the fact that these formulae do give reasonable

results, which agree closely with the results found by means of a hydrogen electrode, gives strong support to the theory.

The chief grounds on which this theory is attacked are that various reports have been made where haemoglobin has been said to combine directly with

CO2 in appreciable quantities. The best known experiments are those of Buckmaster (1917), but as he used CO2 pressures very much in excess of those found in the body, it is quite likely that the hydrogen-ion concentration of the solution was raised above the isoelectric point of haemoglobin, in which case it would combine with the CO2. Bohr (1904) also considers the possible formation of compounds between the CO2 and the basic groups that probably exist in the haemoglobin molecule. Various similar views have been put forward

by different investigators at different times, but these views seem to be of

the nature rather of possibilities than of what actually occurs. Bayliss (1919} is also of the opinion that the haemoglobin acts as a carrier of CO2, and considers the possibility of the CO2 being adsorbed by the haemoglobin. A complete review of the theories has been made by Warburg (1922), and he comes to the

conclusion that the theory as enunciated by Zuntz is the correct one.

All the above theories have been based on the amount of CO2 that could be

made to combine with the blood in question. In no case has any attempt been

made to ascertain the ionic nature of the hemoglobin supposed to be responsible for the carriage of CO2. Knowledge of the nature of the charge on the haemo-

globin is very important, since it affords an immediate decision as to which

theory is the correct one. If the haemoglobin be present as an anion, then the

action of the CO must necessarily be the same as that of another acid on the

salt of a weak one, and an alkali bicarbonate (or carbonate) will be formed along with the free acid, which will be mostly undissociated. If, on the other hand, the haemoglobin be present as a cation, then the reaction must be similar to

that of an alkali with CO2, the haemoglobin (e.g., HbOH) being a weak base

reacting with the CO2 to form haemoglobin bicarbonate.

If, however, no hamoglobin-ions be present, then the CO2 must have com-

bined with the haemoglobin directly. It would seem very unlikely that a

compound of an alkali metal with hemoglobin or h-emoglobin bicarbonate (if it exists) would remain completely undissociated. Thus some property must

384



The Ionic Nature of Haneoglobin. 385

be sought that depends upon the ionisation of the haemoglobin and the sign of its charge.

The Donnan Equilibrium.

The Donnan equilibrium offers a means of investigating the electrolytic

properties of heemoglobin. This is to be preferred to conductivity measure-

ments, as the high viscosity of solutions of haemoglobin renders it very difficult

to interpret conductivity measurements. Donnan (1911), when working on

the osmotic pressure of Congo-red solutions in the presence of sodium chloride, found that the osmotic pressure was less than the calculated value, and showed

that this was due to the unequal distribution of the chlorine-ions on the two

sides of the semi-permeable membrane. He also proved that this is a thermo-

dynamic consequence of the presence of one diffusible and one indiffusible ion.

He also showed that there must be an electrical potential difference between

the two sides of the membrane, and gave a formula for calculating it, depending on the concentrations of the diffusible ion on each side of the membrane.

More recently, Loeb (1921, 1922) has investigated the physical chemistry of gelatin and albumin solutions from the standpoint of the Donnan equilibrium. The potential difference observed across the membrane agrees very well with

that calculated from the hydrogen-ion concentrations of the protein solution

and the dialysate. The potential difference also is a minimum at the isoelectric

point of the protein in question and shows, as is to be expected, that the protein acts as an undissociated colloid at its isoelectric point.

Loeb takes the agreement he obtains between the calculated and observed

potential differences as proving that a Donnan equilibrium has been set up. While in Loeb's case this is very likely, it has been pointed out by A. V. Hill

(1923) that the formula found for the potential difference must hold for any case of an equilibrium which involves an unequal distribution of ions due to whatever cause. Hill also suggested that the potential difference could be

used, if the ionic concentrations on one side of a membrane were known, to obtain the concentrations on the other side.

Consider the equilibrium obtained between a protein salt NaR and sodium chloride with a collodion membrane between. In equilibrium we have on one side of the membrane sodium chloride, and on the other side a mixture of the

protein salt and sodium chloride, i.e.,

NaR + NaCl I] NaCl 4,

Membrane

When these two solutions are in equilibrium the membrane potential is given



H. Taylor.

by the formula E o= - Oge C1/C2 where C1 and C2 are the concentrations of nF

any diffusible ion of valency n on the two sides of the membrane. For sodium RT [Na']l and all cations this formula becomes E =- log[Na

Donnan's theory also demands that

[Na']i [CI']1 = [Na']z [C1']2, i.e. that

[Na]1 [C'1]2 [Na']2 [Cl']1

and we get for the potential difference formula

E_ T [Na'], RT [Cl']2 nF [Na] F loge [Cl'l'

In the case of hydrogen-ions the formula is the same as for sodium-ions, and we get

E RT [H']1 E= log, nF e[H12'

If we now give the necessary numerical values to R, T, n and F at 18? we get H"*1 E =0 058 1log1 [H' volts, or, assuming S6rensen's notation and placing [H']2

- log [H] = pH, the formula becomes E = 0 058 [pH.2 - pH1] volts. It will be seen in the scheme given above that if the indiffusible ion is a

cation there will tend to be a greater number of diffusible cations on the opposite side of the membrane, and this will mean that the potential difference will be of the sign opposite to that given by the formula. Thus the sign of the potential difference gives a direct test as to whether the indiffusible ion present is an anion or a cation. The sign of the potential difference is related to the sign of the

charge on the indiffusible ion in such a way that the side containing the indiffusible ion is positive when that ion is a cation and negative when it is

an anion. An idea as to the amount of protein ionized (i.e., as to the total charge

incapable of passing by diffusion through the membrane) can be obtained from (a) the potential difference, and (b) an analysis of the dialysate, since the concentration of diffusible ions in the protein solution is then obtained. It can be shown that the concentration of the protein ion, if an anion, is given by the formula

Concentration of Cone. of anions in ]

2 Cone. of anions in-2 protein ion ( in _ crystalloid solution J - protein solution J gram equivalents)-- Cone. of anions in protein solution

386



The Ionic Nature of Hemoglobin. 387

(This is true only when all the diffusible ions are monovalent, as in the case of

bicarbonate and chloride ions in blood corpuscles.) (Michaelis, 1922.) Hence we see that any potential difference obtained between laked corpuscles

and a crystalloid solution will give an indication as to the state of the haemo-

globin in the laked corpuscles and in the corpuscles themselves.

Description of Method employed.

It was proposed, in the present investigation, to obtain, if possible, a Donnan

equilibrium betweeL the hemroglobin of the blood corpuscles and a known

crystalloid solution. The corpuscles obtained from the whole blood can be

laked by freezing and thawing, and the resulting solution dialysed against a

dilute sodium chloride solution. If the heemoglobin be acting as a colloidal

electrolyte a potential difference will be set up across the membrane, and this

can be measured and will show in what manner the haemoglobin is acting. The technique used is very similar to that adopted by Loeb (1921, 1922) in

his estimations of the potential differences in the case of gelatin chloride solutions. In his case he placed the colloidal solution inside the collodion bag and suspended this in distilled water. This method, however, causes rather a

large dilution of the colloidal solution, owing to the osmotic pressure set up. To prevent this taling place the laked corpuscles employed in this investigation are placed outside the collodion membrane and the crystalloid solution placed inside the membrane. By this means the ratio of the volume of the laked

corpuscles to that of the crystalloid solution can be made as large as possible by increasing the amount of corpuscles, and the dilution of the corpuscles made so small that it can be neglected.

After equilibrium is attained the potential difference is measured by placing a saturated KC1 calomel electrode in each of the two solutions and employing a potentiometer. The cell employed is as follows :

Saturated Saturated Laked e Dilute Saturated Saturated KC1 KC1 corpuscles

. solution of KC1 KC1 calomel NaCl & calomel electrode NaHCO3 electrode.

To P/ To Potentiometer

In practice it is nearly always found that the potential of the two calomel



H. Taylor.

electrodes is not quite equal, but any difference between them can be determined and allowed for in the calculation.

Loeb in his work on gelatin solutions waited until the osmotic pressure had become constant before taking the potential difference. It is not, however,

necessary to wait until osmotic equilibrium has been attained as "diffusion

equilibrium " is set up long before then. Also to measure the potential difierence Loeb removes the osmotic pressure tubes and the solutions can therefore no longer be in osmotic equilibrium. The fact that the solutions are not in osmotic equilibrium can make little difference to the potential difference as is shown by the agreement Loeb obtains in his results. Donnan also works out the formula for the membrane potential on the assumption that there is a difference of osmotic pressure on the two sides of the membrane.

A few experiments were done on the passage of water and salt across a membrane under pressure and the potential difference obtained was negligible. Any potential due to the osmotic pressure as such must necessarily be of a similar nature and thus can be neglected.

"Diffusion " equilibrium between a salt solution and laked corpuscles is set

up in about 2} hours. This was determined by measuring the potential difference until it became constant. In most cases constancy was obtained in two hours and in all cases after three hours. This method assumes that there is no change in the reaction of the contents of the corpuscle on laking ; the laked corpuscles are treated as representing the inside of the corpuscle. This

assumption cannot at present be proved, as no evident line of proof presents itself. If the corpuscles be laked by freezing and thawing no other substance need be added to them and the reaction will not be altered.

It may also be asked whether the corpuscles and plasma, when in equilibrium with one another at any time, are under the same pressure of CO2. That they are both in equilibrium with the same pressure of CO2 may be proved thermodynamically as follows:-

Suppose the corpuscles and. the plasma are under different CO, pressures Pl and p2 when in

equilibrium with each other. Then we have the following arrangement:-

CO2 pressure CO2 pressure

1P1 Corpuscles Plasma p2

If An gram molecules of CO are taken from the corpuscles and expanded to any pressure Po then the work done will be

pv + SnRT log p_ = nRT [1 + log p1 . Po Po

Similarly if 8n gram molecules are taken from the plasma and expanded to the same pressure

p0 the work done will be 8nRT [1 + log P2]. These must necessarily be equal or else perpetual

motion is obtained by driving the CO2 back again into the system by the one which does less work. Hence we get p1 = P2.

388



The Ionic Nature of Hcemoglobin. 389

The only assumption made in this proof is that the corpuscular wall is freely permeable to CO.. This proof is necessary as in the actual experiment the laked corpuscles are equilibrated with the same CO2 pressure as was the original blood.

Detailed Description of Method.

A. Sheep's blood has been used exclusively for all experiments. The blood

obtained fresh is immediately treated with 0-5 per cent. sodium fluoride solution as recommended by Evans (1922) 1 c.c. of the fluoride being added to

200 c.c. of blood. This prevents the formation of lactic acid during the

experiment, whilst the amount added is too small to cause any appreciable difference in the ionic concentrations of the corpuscles and plasma. The

blood is then equilibrated with a known pressure of C02. This is done by placing approximately 150 c.c. of blood in a large bottle of 2? litres capacity and partly exhausting the bottle, the pressure being read off on a manometer. (The

pressure is generally reduced to about 300 mm. of mercury.) C02 is then run in until the increase in pressure is the amount required, the pressure being regulated by means of a spring clip to within one millimetre. The pressure read off on the gauge is taken as the correct pressure of 002, the correction due to the amount in the atmosphere being within the limits of error of the method used. Atmospheric pressure is then restored in the bottle by opening the tap and the whole then revolved in a revolving shaker for a half-hour. If the blood originally contains a large amount of C02 it is quite probable that the CO2 pressure has been altered. To prevent this the blood is again shaken for a

quarter of an hour with the same CO2 pressure as originally, the gas in the bottle

being withdrawn and replaced. The shaking also ensures that the blood is

fully oxygenated. B. The blood is then immediately centrifuged for about three-quarters of

an hour, at the end of which time a fairly good separation of the corpuscles from the plasma can be effected, the volume of the corpuscles being generally just under half of the total volume of blood. This separation is not absolute, as the centrifuge which had to be used had not the necessaly centrifugal force to cause an absolute separation. As the volume of the corpuscles is said generally to be about one-third of the volume of the whole blood, it is seen that there is a considerable amount of plasma in the separated corpuscles. It is

unlikely that this plasma will affect the qualitative results of the experiments, but the quantitative results obtained can only be regarded as approximations.

The plasma is then drawn off from the corpuscles and the corpuscles laked. This is done by freezing and thawing them, this method being preferred to



H. Taylor.

other methods of laking as it does not involve the addition of any other substance to the corpuscles and cannot therefore alter the reaction.

To get efficient laking by this method it is necessary to freeze the corpuscles into an extremely hard mass, and to do this the laking has to be done in small

quantities. The laking is done in lots of about 10 c.c., which are placed in a round flask and revolved in a freezing mixture of ice and salt. The freezing is continued until the colour of the solid in the flask is a dull light red. This is then thawed by immersing the flask in lukewarm water, when the liquid obtained should be perfectly clear. If the liquid is not clear the laking has not been thorough, and the whole is frozen again. The clearness of the liquid shows that there are very few corpuscles left as such in the liquid.

This method of laking the blood corpuscles involves a considerable amount of shaking, during which some CO2 is sure to be lost. To remedy this the laked corpuscles are shaken again with the same CO2 pressure as that with which the blood was originally in equilibrium; this should restore them to

the same pH as originally. It can make no difference, however, to the chlorine- ion content of the corpuscles.

C. The volume of the corpuscles after laking and shaking with CO2 is generally about 70 c.c.; the whole is placed in a small beaker, and a small fairly thin

collodion tube containing about 10 c.c. of a dilute salt solution inserted. The

concentration of the dilute salt solution is varied according to the reaction of

the blood, the concentrations being between 0 4 x N/10 and 0.8 x N/I0. This is corked up and the whole left for three hours until diffusion equilibriumi is set up. The potential difference between the outside and the inside of the

collodion tube is then determined by placing one calomel electrode in the

outside and one in the inside of the membrane (as in figure) and measuring the potential difference between them on a potentiometer. Any original

potential difference between the electrodes themselves is found by placing them in the same potassium chloride solution and measuring the potential difference on the potentiometer. This amount is then deducted from the

potential difference found between the two sides of the collodion membrane.

D. The aqueous solution in the collodion tube is then analysed for both

hydrogen and chlorine-ions. The hydrogen-ion concentration of the original blood has previously been determined by the method of Dale and Evans (1920). The blood is placed in a thin collodion tube which holds about 1 c.c. and is

dialysed against 1 c.c. of 0-85 per cent. saline solution. After dialysing for

about half an hour the hydrogen-ion concentration in the dialysate is taken

as being equal to that of the plasma. The Donnan equilibrium effect at the

390



The Ionic Ncture of Ilcemoglobin. 391

membrane between whole blood and saline solution has previously been proved to be negligible (Taylor, 1923). The hydrogen-ion concentration of the saline

Calomel Electrodes

==- ; Saturated1KC/

Th . --=

iNaMCO i i ________ L _e d i _

Potentiio metecr--=ry Potent/omtem Dl-al/-ysade- - ''----- lWre

NaHCO3 __ Laked _ i i I _ I Corpusc/es

is then obtained by comparing the colour of an indicator added to it with the colour of the same indicator in buffers of known lpH.

The dialysate from the laked corpuscles is treated in exactly the same way as the dialysate from the whole blood in the Dale and Evans method, and the

pH of the dialysate thus determined. This estimation is done immediately to prevent losses of C02, and also because a very small amount of protein matter always passes through the membrane and this may decompose. The

hydrogen-ion concentration of the laked corpuscles can then be calculated from the formula given, i.e.,

E = 58 [pH (dialysate) - pH (corpuscles)] millivolts.

The chlorine-ion concentration is estimated by titration against a standard silver nitrate solution, Volhardt's thiocyanate method being used. Five c.c. of the dialysate are added to 5 c.c. of a standard silver nitrate solution in the

presence of pure nitric acid. The excess of silver nitrate is then obtained by titration against a N/20 solution of potassium thiocyanate, iron alum being used as the indicator. This method is preferred to a direct titration as it is

applicable in extremely acid solution and prevents the precipitation of any



H. Taylor.

other silver compounds. The chlorine-ion concentration of the corpuscles is then calculated from the formula

E -58 log [C'] dialysate [CI' corpuscles

The collodion tubes used in the experiments are made on the outside of a

revolving test-tube, the method used being similar to that adopted by S6rensen

(1917). The test-tube is carefully cleaned and dried and revolved in a hori- zontal position. The collodion mixture used is a 5 per cent. solution in a mixture of 75 per cent. alcohol and 25 per cent. ether (by volume). This mixture is quite fluid and can be poured out quite easily. The test-tube is revolved fairly fast and the collodion poured over it in a fairly thick stream, after which the coat is allowed to dry whilst rotating at a lower speed. The time of drying is about 10 minutes, when another coat is added in exactly the same manner. When three coats have been added and the tube had been rotated until the collodion is too viscous to flow, the whole is allowed to dry for one hour. The membrane is then immersed in water for a few minutes and removed from the test-tube. To aid the removal of the membrane from the test-tube there is a small hole in the bottom of the test-tube which allows air to enter the space between the membrane and the test-tube as the membrane is removed. The collodion thimble thus obtained is either tied or "shrunk" on to a piece of glass tubing (or the top of a test-tube) of approximately the same diameter. The membrane thus obtained is fairly permeable to salts but quite impermeable to haemoglobin, although minute traces of plasma proteins may get through. The glass top is placed on the end to allow the membrane to be corked up and clamped easily.

The calomel electrodes are fitted up much after the style of those used by Loeb, the ends being drawn out to a fine capillary point to prevent loss of the KC1. The calomel used was made by the method advised by Clark (1922) i.e., by the electrolysis of dilute hydrochloric acid with a mercury anode. This gives a grey mixture of calomel and finely divided mercury which is very efficient for use in electrodes. The presence of the mercury in the mixture also

prevents the formation of any mercuric chloride. (The mixture was tested

qualitatively and no mercuric chloride found.) Saturated KC1 solution is used in the electrodes, the KC1 used being re-

crystallised before use. The potentiometer used to measure the potential difference was made by

Pye of Cambridge, and graduated to read to a thousandth of a millivolt. The

392



The Ionic Nature of Hcemoglobin. 393

galvanometer used with the potentiometer was a very sensitive high-resistance

galvanometer, made by the Cambridge Instrument Company. (Type L 115, resistance approximately 2100 ohms). The galvanometer was mounted in

paraffin blocks to insulate it from any external electrical effects.

The technique described has a disadvantage in that when a collodion tube

containing a dilute salt solution is placed in laked corpuscles, the greater osmotic pressure outside tends to cause the membrane to collapse. If the

membrane is a thin one it will collapse fairly soon, but a thicker membrane will

not collapse absolutely for several hours. Also with a very thin membrane the

aqueous solution is drawn into the colloidal solution very quickly and no liquid is left for analysis. In the membranes used in the experiments described the

amount of liquid left in is always between 7 and 8 c.c., the dilution of the outside

colloidal solution being at the most 3 c.c. This leaves quite a sufficient quantity for analytical purposes.

Results.

The following table (I) gives the results obtained for the chlorine-ion concen-

tration of the corpuscles. The dialysate is positive in the potential difference

readings. In the calculation of the normality of the protein anion the concentration of the bicarbonate-ion, as well as that of the chlorine-ion, has been taken account of in the formula given on page 386 above. The bicarbonate-ion concentration has, in some cases, been obtained by calculation from a knowledge of the pH and the pressure of C02, and in other cases directly by the Van Slyke apparatus, assuming that all the CO2, less that in physical solution, is present as bicarbonate-ions.

Table II gives the results obtained for the pH of the inside of the corpuscle. The first twelve experiments correspond to the first twelve experiments in the table given previously.

Discussion of Results.

The tables given show that an appreciable potential difference exists between laked corpuscles and a salt solution in equilibrium with it. This potential difference is much higher than that found in the case of whole blood in contact with salt solution across a membrane, as in the Dale Evans method of determin-

ing the pH of blood; there the plasma proteins are the ones which determine the

potential difference as has been shown previously (Taylor, 1923). This shows that the haemoglobin in the corpuscles must have much more strongly ionic

properties than the colloids of the plasma and must be ionised to a considerable extent. In every case investigated the sign of the potential difference has corre-



TABLE i.

No. of Experiment .... 2 3 4 5 6 7 8 9 10

Pressure of C02 (mm) .... .... 04 - - 41 43 60 30 42 34 Membrane potential (millivolts)

(Dialvsate lositive) ........ 635 7-2 9-7 3-9 6 5 8-3 6-1 4-5 6-2 2-6 [Cl'] in dialysate .... .... 070 x N/10 086 x N/1 0 0 49 x N/10 0-93 x N/10 0-81 x N/10 058 x N /10 0-78 xN/10 0-72 x N/10 0-65 xN/10 0-89 x /N10 [Cl'] in corpuscles ...... . 055 x -N/L10066xN /10 0-34xN/100O80 xN/10 063 xN10 0-42 xN/10 062 xN/10 060 xN/10 051 xN/10 0SOx /N10 Normality of protein-ion .... 034 N/10046 1037 xN/10 028 x 1 050 xN/10 0-44 xN/10 044 xN/10 029 xN/1 0-41 xN/30 021 x /N10

XN/~ xi0 jl046 O

N 0?3'7 xN/I10 ? x N/ 0? N/100'44xN/100'44XN/1010'29X N~/100-41 XN/100'21X/N10

No. of Experiment ....11 .12 13 14 15 16 17 18 19

Pressure of CO2 (mm) .... .... 40 28 34-5 30 0-4 61 20 40 23 Membrane potential (millivolts)

(Dialysate positive) .... .... 2-4 4-1 6-6 8'3 8-4 4-0 5-2 3-2 2-4 [Cl'] -in dialysate .... .... 063 x N/10 1 05xN/10 0-82 xN/10 0-60 xN/10 0-81 xN/101090 xN/10 0O94 xN/lO 0-94 xN/10 0-99 x N/j [Cl'] in corpuscles ..... 0-58 x N/10 0-91 x N/1 0-63 x N/10 0-43 xN/10 xN/0-5 N1077 x N/10 0'77 x N/10 0-83 x N/10 0-90 x N/10 Normality of protein-ion .... 013 xN/10 0-38 xN/10 048 xN/1O 0-47 xN/1 0-57 xN/10 0-33 x N/1010-45 x N/10 0-27 x N/10 027 x N/10

.~~~~03x/0'5Nt02

m

k-ir

0

3 _

o



The Ionic Nature of faemoglobin. 395

TABLE II.

No. of Experiment .... ... .... 1 2 3 4 5 6 7 8

Membrane potential (millivolts). (Dialysate positive) .... .... 6-35 7 2 9 7 3 9 6 5 8-3 6-1 4-5

pH of plasma ... ....... 7-53 7-36 7-59 7 15 7-43 7-20 7-11 7-20 piH of corpuscles .... .... .... 739 7-22 7-44 7-11 7-23 7.02 6-99 7-16

(corpuscles) .... .... 138 1-48 1-41 1-10 1.59 1-51 1.32 110 cH (plasma)

No. of Experiment .... .... .... 9 10 11 12 13 14 15

Membrane potential (millivolts). (Dialysate positive) .... .... 6-2 2-6 2-4 4-1 2-9 7-0 7-1

pH of plasma .... .......7 756 737 7-37 7-38 7-26 7-71 7-88 pH of corpuscles .... .... .... 7-31 733 7 29 7-28 7-20 7-60 7 76 cH '(corpuscles).... .... 178 1.10 1.15 1.26 1.15 129 132 cH (plasma)

sponded to the hcemoglobin acting as an anion, i.e., the haemoglobin solution has been negative to the crystalloid solution. This being so the reaction between the C02 and the hiemoglobin must of necessity bring about the formation of sodium bicarbonate and the undissociated haemoglobin, as this is what is found to occur always when an acid acts upon the salt of another acid, which is

practically undissociated in the free state. Hasselbalch and Warburg (1918) and Campbell and Poulton (1920) assume

that the haemoglobin is acting as an anion, and in fact this assumption is implied in all the formulae for the calculation of the pH of blood. These assumptions have been made entirely on the evidence of CO2 measurements with blood, which

give good ground for believing that the haemoglobin acts as an anion in all

physiological processes. The measurement of the potential difference furnishes a direct and independent proof that the hcemoglobin is so acting.

The method used gives a new means of analysis of the inside of the blood

corpuscles, since the composition of the dialysate depends entirely on that of the corpuscles, whilst the measurement of the potential difference gives the

necessary factor relating the two concentrations. Some quantitative measurements have been attempted, but at present can only be regarded as approximations, and are being repeated with more accurate apparatus.

It has been shown, however, that the inside of the corpuscle is appreciably more acid than the plasma. In the table given, in spite of individual variations,

,the ratio (orpuscle) is in every case above unity. The mean of the cHi (plasma)



H. Taylor.

numbers given is 1-33, i.e., the corpuscles are 33 per cent. more acid than the

plasma. This means that the pH of the corpuscles is on the average 0.12 less than the pH of the plasma. This agrees well with the curves which

Warburg (1922) works out from his formula. It is also of the order of magnitude to be expected from the oxygen dissociation curve of the corpuscles.

The chlorine-ion concentration also of the corpuscles has been estimated and is found to vary considerably. This was to be expected on theoretical grounds, since the taking up of CO2 by blood causes a shift of chlorine-ions from the

plasma to the corpuscles. The observations partly bear this fact out, the more

acid corpuscles as a general rule having the higher concentration of chlorine- ions. The results obtained, however, cannot be regarded as conclusive, as the

dilution by the plasma in laking will affect this result more than any other. From the chlorine-ion concentration the ionic normality of the colloidal-ion

has been calculated from the formula on page 386. This number is a measure of

the maximum amount of CO2 that the blood can possibly take up as bicarbonate.

The values obtained are all of the same order of magnitude as the chlorine-ion

concentration, and much too large to allow of the potential difference being

explained by any other proteins than haemoglobin that may be present. A few experiments were made to try to determine the isoelectric point of

haemoglobin, but were not successful, owing to the haemoglobin crystallizing out on the mixture becoming acid. The potential difference, however, did

change sign as the solution became acid, and this shows that the sign of the

potential difference depends on the reaction of the corpuscles. It shows also

that the potential difference measured is in all probability due to the presence of the hmemoglobin and is the true Donnar-potential difference.

Summary.

1. A method is given of ascertaining the ionic contents of the blood corpuscle and the magnitude and sign of the charge on the haemoglobin molecules existing there.

2. The hiemoglobin is acting as an anion over the whole range of physiological

importance: this implies that the haemoglobin will necessarily surrender base, and become an undissociated weak acid, when a stronger acid is added to it.

This agrees with the assumption commonly made as to the manner in which acid and CO2 are carried by the blood.

3. The inside of the corpuscle has, on the average, a hydrogen-ion concen-

tration about 33 per cent. higher than the plasma.

396



The Ionic Nature of Hamoglobin. 397

4. The chlorine-ion concentration of the corpuscle is variable, lying, however, between the limits 0 34 X N/10 and 0 91 X N/10.

5. The normality of the indiffusible protein-ion in the laked corpuscles is also variable, having a mean value of 0Q37 X N/10 over the particular range of C02 pressures investigated. This corresponds to a maximum further uptake of C02 of about 83 volumes per cent. of the laked corpuscles, or about 40 volumes per cent. of whole blood.

I wish to express my best thanks to the Lancashire County Council, and to the Scientific and Industrial Research Board, for grants which have made the research possible.

I must also thank Prof. A. V. Hill, at whose suggestion this work has been done, for much helpful advice and criticism.

REFERENCES.

Bayliss, W. M. ' J. Physiol.,' vol. 53, p. 162 (1919). Bohr, C. ' Centr. Physiol.,' vol. 17, p. 713 (1904). Buckmaster, G. A. 'J. Physiol.,' vol. 51, p. 105 (1917). Buckmaster, G. A. 'J. Physiol.,' vol. 51, p. 164 (1917). Buckmaster, G. A. 'J. Physiol.,' vol. 51; 'Proc. Physiol. Soc.,' p. i (1917). Campbell, J. H., and Poulton, E. P. 'J. Physiol.,' vol. 54, p. 152 (1920). Clark, W. M. "The Determination of Hydrogen Ions " (Baltimore) (1922). Dale, H. H., and Evans, C. L. 'J. Physiol.,' vol. 54, p. 167 (1920). Donnan, F. G. ' Zeitschr. fir Electrochemie,' vol. 17, p. 572 (1911). Evans, C. L. 'J. Physiol.,' vol. 56, p. 146 (1922). Hasselbalch, K. A. ' Biochem. Ztschr.,' vol. 78, p. 112 (1916). Hasselbalch, K. A., and Warburg, E. J. ' Biochem. Ztschr.,' vol. 86, p. 410 (1918). Henderson, L. J. 'J. Biol. Chem.,' vol. 7, p. 29 (1909). Hill, A. V. 'Roy. Soc. Proc.,' A, vol. 102, p. 705 (1923). Loeb, J. 'J. Gen. Physiol.,' vol. 3, p. 667 (1921). Loeb, J. 'Proteins and Theory of Colloidal Behaviour' (New York) (1922). Michaelis, L. 'Die Wasserstoffionenkonzentration' (Berlin) (1922). Pfliiger, E. F. W. 'Ueber die Kohlensaure des Blutes (Bonn)' (1864) (Quoted- from

Warburg). Sorensen, S. P. L. 'Travaux du Laboratoire de Carlsberg,' vol. 12, p. 28 (1917). Taylor, H. ' Biochem. Journal XVII,' vol. 3, p. 406 (1923). Warburg, E. J. ' Biochem. Journal XVI,' p. 153 (1922). Zuntz, N. 'Hermann's Handb. Physiol.,' Leipzig (1882).

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The Ionic Nature of Haemoglobin