On the correlation between the spectra of various ...rspb.royalsocietypublishing.org/content/royprsb/97/680/61.full.pdf · tions be made from human and Gastrophilus haemoglobin respectively,

61

On the Correlation between the Spectra of various andtheir Relative Affinities for Oxygen and Carbon Monoxide.

By M. L. Anson, J. B arcroft, F.R.S., A. E. Mirsky, and S. Oinuma.

Received July 29tli, 1924.

(From the Physiological Laboratory, Cambridge.)

The spectra of the haemoglobins from different animals, and even different mammals, are measurably different. The relative affinities of these haemoglobins for oxygen and carbon monoxide are also different, and it turns out that certain properties of the chemical affinities and of the spectra are so related that by the use of a simple equation they may be deduced from one another.

That the spectra of the haemoglobins of all forms of life are not the same was discovered by Sorby (1), and has since been verified by Vies (2), who has carefully mapped out the spectra of a number of animals, notably Arenicola and the horse. The particular difference with which we are concerned in this paper is in the position of the a-band, which was found by J. and H. Barcroft (3) to occupy a place in Arenicola 18 Angstrom units from that which it occupies in man. The considerations which have led us to undertake the present research are to be found in their paper.

Vies laid stress rather on the difference in the character of the bands than in that of their positions of maximum intensity. This difference is very striking as between such forms as man and Chironomus or Gastrophilus. If preparations be made from human and Gastrophilus haemoglobin respectively, such that the a-oxy-haemoglobin bands appear to be of equal but faint intensity, and if now the preparations are treated with carbon monoxide, the human haemoglobin will show a much less faint band than the Gastrophilus haemoglobin. In fact, the latter may be very difficult to see.

For this and other reasons we have confined ourselves in the present paper to the study of vertebrate blood. Even in the philum, where the bands are more uniform in appearance, there are differences in the position of maximum intensity of the a-bands of both oxy- and carboxy-haemoglobin. These differences exist as between species and species, and also in the same species as between individual and individual, as a following table shows. The data in it have all been obtained with the Hartridge reversion spectroscope (4). The strength of haemoglobin solution used has been usually that which is equivalent in colour

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to a solution made by dissolving 1 c.c. of human blood in 50 c.c. of distilled water. The solution has usually been slightly alkaline, but changes in hydrogen- ion concentration have been found by Hartridge and Houghton to make no appreciable difference to the position of the bands. This observation we have verified. The positions of the bands of other animals have all been referred to those of human blood, in which it has been assumed that the points of maximum density of the oxy- and carboxy-haemoglobin correspond to 5764 and 5710 Angstrom units respectively.

The following give the positions of the a-bands in various forms of life :—

62 M. L. Anson, J. Barcroft, A. E. Mirsky, and S. Oinuma.

Animal.

Position of Angstron

o 2m>.

a-Band in a Units.

COHb.

Span (S) in Angstrom Units

which separates the a-bands of CO and

0 2Hb.

Man ................................. 5764 5710 54Arenicola (mean of) ....... 5746 5698 48Lumbricus ............... ...... 5755 5720 35Planorbis......... ................ 5746 5708 38Chironomus ....................... 5777 5727 50Pigeon ............ ................. 5762 5710 52Carp ................................. 5762 5716 46Horse ...... ...... ................ 5764 5708 56Tortoise ........... .............. 5766 5717 49Fowl.......... ...................... 5769 5718 51Lizard ......... ........... ...... 5762 5715 47

The Calibration of the Reversion Spectroscope.The facts already recounted in this paper, together with those in the former

paper on Arenicola, make it clear that a calibration curve relating the CO content of the blood to the scale readings of the instrument, is required not only for each instrument but for each species, and even for the blood of each individual. Probably in some species, such as man, the individuals differ little, but in others, such as the mouse, in which the “ s p a n m a y vary very much, the whole relation of the scale readings (which are proportional to the Angstrom units) to the saturation of the haemoglobin with carbon monoxide must also change.

At first we made it a matter of routine to make out a calibration curve for each sample of blood observed. This system proved very unsatisfactory. In the first place, there was often too little haemoglobin for the “ wedge- trough ” method of calibration which Hartridge uses. We therefore used another method, to which Hartridge gives less prominence, namely, to take



Correlation between the Spectra o f various 63

two ordinary troughs such as are described below, A and B, each holding about 1 c.c. of fluid, the thickness of fluid traversed by the beam of light being the same in each, and proceed as follows :—

(1) Make a solution of human blood, 1 c.c. of blood to 5 of ammonia. (Thestrength of the ammonia is 1 c.c. of NH3 (s.g. '880) in 250 of distilled water.) This solution is made up to 50 c.c. with distilled water. The solution so made should be quite clear.

(2) Make up a similar solution of the blood in question, adding water tillthe intensity of tint matches that of the human solution. Call this solution X.

(3) Find the position of the oxy- and carboxy-hsemoglobin bands for the human solution and the solution X.

(4) Take a portion of X, dilute to precisely twice its bulk. Call this Y. Divide Y into two portions. Saturate one with CO and call this Z. Take the two troughs A and B, put solution Y in A and solution Z into B. Place both in the path of the light. Obtain the scale reading for an optical mixture, 50 per cent, oxy- and 50 per cent, carboxy-hsemoglobin. Lest A and B should be of different thicknesses, put a portion of solution Z in A and a solution of Y in B, and take another set of readings. The mean of the two sets of readings should give the correct point.

(5) To obtain other points solutions corresponding to Y and Z are made up,but of known concentrations, which differ from one another, the sum of the concentrations being equal to that of X. Thus, Y' might be two-thirds the concentration of X, and Z' one-third the concentration of X, in which case a point would be obtained for 66*6 per cent. Q2Hb and 33-3 per cent. COHb, and so on.

In most cases it is only possible on a given sample of haemoglobin, and in a short time, to determine a few points. Say the 100 per cent. 0 2Hb, 100 per cent. COHb, and 5 others. Even these may be unattainable if the haemoglobin falls short. In addition there is the practical and very real difficulty that the determination of the curve imposes an often too considerable strain upon the eyes, and that when the time arrives for the use of the curve the eyes are unduly tired.

When a great number of such curves had been obtained for different animals, different species, by different observers, and with different instruments, we were able to arrive at a procedure wrhich was more satisfactory than performing



a scanty calibration of the instrument afresh for each haemoglobin that was studied. A comparison of such curves revealed the fact that though the spans were different (i.e. the distance between the positions of maximum density of the fully saturated 0 2Hb and COHb a-bands) in each case the points for given percentage saturations differed in the same proportion as the spans. Thus, if A be the scale reading for oxy-haemoglobin, B that for CO haemoglobin, and C that for a mixture of 50 per cent, oxy- and 50 per cent. COHb, A—C is always between 32 and 33 per cent, of A—B, irrespective of the absolute value of A or B or A—B. A curve which, so far as we have tested it, is of general application can be obtained, in which the percentage of CO haemoglobin relative to the total haemoglobin is the ordinate and the percentage shift of the band from the oxy- position, relative to the span is the abscissa.

The following curve is as accurate as can be obtained for haemoglobin from the frog, Arenicola, rabbit, rat, mouse, sheep, guinea-pig and man, and is true for the eyes of seven different observers; being, however, quite empirical there is no assumption that it will be as correct for any individual who has not been tested as for those who have.

64 M. L. Anson. J. Barcroft, A. E. Mirsky, and S. Oinuma.

90 * 10010 . . . 20 30Per cent of “Span

F ig. 1.—Calibration curve for Hartridge reversion spectroscope. Ordinate — percentage of COHb ; abscissa = displacement of the band expressed as a percentage of the span.

The troughs used are made in the laboratory out of strips of plate glass of about 45 by 10 by 5 mm., which form the ends and bottom of the trough, cemented with Canada balsam to the sides which are made of glass, 2 mm. thick, cut to a size suitable to the capacity of the trough required. Thus, a trough may be made of 5 mm. internal thickness and any desired capacity. For small



Ord

inat

e fo

r A

B

quantities of fluid it may conveniently be V-sliaped. Any tendency to leak may be obviated by placing a little paraffin wax on the bottom.

Though we have regarded the curve shown in fig. 1 as quite empirical, and, indeed, there is no ascertained ground for any other opinion, its use has been simplified by a relationship which it possesses, so far as we know by chance. If the logarithm of the ordinate be plotted against the logarithm of the abscissa the result is, within the region of experimental error, a straight line, with the equation—

Log «/ = 0-77 + 0-56 log x,

Correlation between the Spectra o f various Hcemoglobins. 65

Abscissa for AB &.BCl > *■ 2

(continued at B)

20 B *25 30 35 40 45 50 60 20 80 90 100A bscissa for C'l)

F ig . 2.—Ordinate — percentage COHb. Abscissa = percentage movement of «-band. A—B, Ordinate, 5-7—10 per cent, to abscissa, 1—2-4. B'—C, Ordinate, 10—24 per cent, to abscissa, 2-4—10. C'—D, Ordinate, 24—100 per cent, to abscissa, 10—100.

VOL. XCVII.— B. F

Ord

inat

e fo

r BC

& C

D




where y in the percentage COHb and x is the displacement of the band expressed as a percentage of the span.

This relation is shown on fig. 2, plotted on logarithmic paper. The dots are the points taken from fig. 1. Along that portion of the line between 10 and 100 per cent, of the span the agreement is obviously very good. Below 5 per cent, of the span the discrepancy appears to loom rather large. I t must be remembered that 2 per cent, of the span is less than one Angstrom unit, which is about the experimental error of the apparatus.

The Interaction between Hcemoglobin, Oxygen and Carbon Monoxide.Preliminary discussion.—We have chosen for the subject of investigation

described in the present paper what seemed to be the relation which involved the most simple technique, namely, that between the span and K in the

equation K j = r7=rwrr> rather than a comparison of the actual positions L^2J L O 2 H.DJ

of the a-bands of 0 2Hb and COHb with the values of K' for the equations Hb 4- 0 2 IIb02 and Hb + 00 HbCO respectively.

For this there are two principal reasons :—(1) The a-band does not shift with changes in hydrogen-ion concentra

tion, while the value of K does. If the hydrogen-ion concentration is altered the degree of dissociation is altered also and in effect a different substance is produced. I t would therefore be necessary in order to institute a proper comparison between any two haemoglobins, to be sure that they were rigidly at the same hydrogen-ion concentration. This was not done at all exactly in the research on Arenicola already quoted, the hydrogen-ion measurements which were kindly carried out by Dr. Atkins, who was most particular to point out that on the material with which he was supplied they could at best be only of a rough nature. The blood used by Brown and Hill (10) is assumed to have the hydrogen-ion concentration of normal human blood.

(2) There is another difficulty. If the values of K are to be compared, as a measure of affinity, the curves must all have the same general form. In the language of Hill’s equation* they must all have the same value for n. Thus, to take an exaggerated case, if one curve was a hyperbola = 1 ) and the other an S-shaped curve (say n — 2-5) these might cross, and whilst each

* y _ Kxh1 0 0 — r+

y = percentage saturation, x = pressure of oxygen, K = equilibrium constant, and » = constant.




would have a certain value for K along its whole length, the blood with the hyperbolic curve would take up more oxygen pressure than that with the double inflection above the point of crossing and less below it. The comparison of the “ K’s ” would be difficult to interpret. I t so happens that the human and Arenicolan bloods had curves which were rather similar in their value of n, but it could not be claimed that n was at all accurately determined in the latter.

Experimental procedure.—For the determination of the span we diluted samples of blood in the way indicated. A portion, A, was set aside and a portion, B, was converted into carboxy-haemoglobin. The positions of the a-bands were measured, corresponding measurements being made for human blood. A was then put into a saturator containing air and B into another. 10 c.c. of 3 per cent, (approximately) CO in nitrogen was accurately measured and then put into each saturator, and the pair were shaken or rotated in a waterbottle at 15° C. for an hour. At the end of that time if the equilibration takes place strictly in the dark, the haemoglobin in each saturator will contain oxy- and COHb mixed in the same proportions as shown by the Hartridge spectroscope. The saturators being about 300 c.c. in capacity (the size of each being known to a cubic centimetre), the relative pressure of oxygen and CO can be calculated. I t is of the order of 1 per cent, which with most haemoglobins gives between 50 and 60 per cent, saturation with CO.

The results obtained from A and B (i.ewdiether the substance put into the saturator was oxy- or carboxy-haemoglobin) w'ere expected to agree, the amount of CO contributed to the gas mixture by B or abstracted from it by A being too small to be of significance. We found a good deal of variation in the time wdiich was required to insure this agreement, but an hour always sufficed.

In order to calculate the relative partial pressures of oxygen and CO in the presence of which the haemoglobin would be 50 per cent, oxy- and 50 per cent, carboxy-haemoglobin (or as we termed it, in conversation, the “ 50/50 point ”) it is necessary to be clear as to the shape of the equilibrium curve w7hich relates

[CO] [COHb][0 2] This curve has been observed by Haldane and his

[ 0 2H b]collaborators (5), (6), at various times. They have shown that it is in form a rectangular hyperbola in the case of the whole bloods of man, ox, and the mouse, and for the dilute laked bloods of the same animals. That being so, the curve is determined by its equilibrium constant. This is expressed



numerically as the relative concentration of 0 2/C0 at the “ 50/50 point,” which, in turn, may be calculated from the relative concentrations of the two gases in the atmosphere above the liquid.

The authors to whom reference has just been made have expressed their results usually in terms of the percentages of CO in air at the 50/50 point. In other places they have expressed the same fact in another way, namely, by stating the partial pressure of oxygen per unit pressure of CO. Thus human

blood is 50 per cent, saturated with CO when the : 400/1 at 15° C. and2?CO

about 250/1 at 37° C. The relative concentrations of 0 2/C0 dissolved in the fluid at 5° C. and 37° C. respectively are obtained by the multiplication of these figures by 1*35 and 1 • 28 respectively when the following figures are obtained : 540 and 320. These are the values which we have used in the present paper for the expression of K, and are the ordinates in such figures as fig. 5. Whether they, or the ratios of the partial pressures in the atmosphere, are more significant is one which may be reserved for further discussion.

Haldane, Lorrain Smith, Douglas and J. B. S. Haldane (6) have shown that the CO—0 2 equilibrium constant for human whole blood is approximately the same as for human dilute blood at a given temperature, that human blood, whole or dilute, differed from mouse blood and closely resembled ox blood, that the blood of different mice differed in their values for K to a very remarkable extent, and that the equilibrium constant, unlike those of the equations 0 2 + Hb — H b02 and CO -f- Hb = COHb, was uninfluenced by the addition of ammonia, that is, to change of hydrogen-ion concentration.

Krogh (7) observed that the bloods of the rabbit and the ox, respectively, differed in their relative affinities for oxygen and carbon monoxide.

The bloods of the dog and the pig have been compared in vivo and in vitro by Nicloux (8), who in an elegant research showed, among other things, that the two animals possessed haemoglobins of differing relative affinities for oxygen and carbon monoxide. It was necessary, over a wider range of the animal kingdom, to test the assumption that the reaction between oxygen, CO, and haemoglobin is of that simple character which is expressed by the formula

[HbCO] [OJ = K [Hb02] [COj,and which, therefore, is represented graphically by a rectangular hyperbola.

This we have done in the following animals : mouse (in which we verified the statements made by Douglas, Haldane and Haldane), frog, rabbit, Planorbis and Arenicola.




Fig. 3 shows the positions of six points observed on frogs’ blood, each determined independently bv two observers, and the average taken. All of these fall closely in the rectangular hyperbola. This figure may also give some idea of the experimental errors involved in the calculation of K by the use of the reversion spectroscope, as will be seen also from the tabulated results.


20

o 10

% CO in a i rFig. 3.

Of the twelve sets of observations made in the six points shown in fig. 3, eleven gave values for log K between 2'31 and 2*38. One observation was divergent, giving a value of 2'51. This was at the lowest CO pressure, 0*075 per cent, of CO in air, and wns probably due to some accidental circumstance.

A number of points were determined on rabbits’ blood ; in the case of one animal four values for log K fell between 2 • 43 and 2 • 36 ; in the case of another rabbit four determinations at different CO pressures gave values of log K which varied between 2-15 and 2-19.

f̂ye may take it, then, that the value of log K as observed may be O'04 on either side of the true value. The error in the determination of the span is 1 A.U. on either side of the correct reading.

The above data show : (a) That the dissociation curve of the haemoglobin CO equilibrium, in the case of dilute solutions, is a rectangular hyper

bola in the cases of the rabbit, the frog and Planorbis, as well as of the mammals already studied by other authors. There seems no reason to suppose that it is otherwise in any animal. J. and H. Barcroft (3) found some slight divergence in Arenicola, and it will be clear from what is said in this paper that the cause is to be attributed to the lack of rigid precautions to carry out the




equilibration in the dark. (6) That the experimental error in the value of log K for points measured at and above 1 per cent. CO pressure is about 0-035- 0-040. We have taken it at the latter figure.

We have repeated the curve on Arenicola, obtaining the equilibrium between the haemoglobin, the oxygen and the CO in the dark, with the results shown in fig. 4.

Fig. 4.—Curve representing the partition of Arenicolan haemoglobin between CO and 0 2. Ordinate = percentage of COHb. Abscissa = percentage CO in air.

We confined ourselves, in the present series of observations, to vertebrate blood for the reason stated at the commencement of this paper (para. 3).

I t appeared possible, therefore, that the haemoglobins of vertebrates were less comparable with those of invertebrates than with one another.

Relation of the Span to the Equilibrium Constant in Different Animals.If the span be plotted against the equilibrium constant of the reaction

COHb + 0 2 0 2HB + CO. the points fall on a very definite curve, as shown in fig. 5.

A more striking result is obtained by plotting the span for the blood of thirteen different kinds of vertebrates, not against K, but against its logarithm. This relation, at 15° C., is shown in fig. 6. It appears that the points still have a very definite relationship to one another, and over the range of the



Correlation between the Spectra Hcemoglobins. 71

•Horse

H an J* CatHowl •

P ig eo n •Mouse

-onse

i L izard Loacli-—

4 5 5 0 55S pan— A n g s tro m u n i t s

F ig . 5.—Ordinate = K in the relation concentrations of 0 2/C0 of gas dissolved in the haemo' globin solution. Abscissa = the distance in Angstrom units between the positions of maximum density of the a-bands for 0 2 and COHb respectively.

Tortoise -

Mouse 4-

j - i ..j i45 50 55S p an — A n g s tro m u n i ts

Fig. 6. Relating log K to the span. The heavy line shows the mean relation; the light ones indicate the limits of experimental error.



diagram they fall on a line which is indistinguishable from a straight line with the equation.

Log K = 0-05 (span in A.U.).

Some of the individual points in fig. 6 deserve a few words of comment; such will be considered from above downwards.

Horse.— Is the mean of this determination done independently by threeof us.

Mouse.—The points are those of individual mice. They have, as Douglas, Haldane and Haldane pointed out in the case of the whole blood of mice, very different values for K. There are considerable individual variations also in the haemoglobins of horses, mice and rabbits shown in fig. 6.

Cat. Fowl and Several Mice.—The span was determined by one person and the value of K independently by another.

Man.—The figure for K is almost exactly that given by Haldane. We have found little difference as between different persons.

Sheep.—Our figure for K confirms that obtained by Houghton.Frog.—Is the mean of estimations in six frogs.Rabbits.—The rabbits shown present an interesting problem as they conflict

with any obvious phylogenetic application of the position of the other points. Some other determinations suggest a good deal of variation in this span of rabbits’ blood. The points shown here were among the shortest spans observed and were determined independently by different observers.

We may here mention a source of trouble which delayed us very much in our work, but which in the end turned out to be rather a confirmation than otherwise of our general result, namely, the approximately linear nature of the relation between log K and the span, which, over the region observed, was not distinguishable from a straight line with the equation—

Log K — 0-043 x (AU).

From time to time we obtained points on another line.Only on one occasion did one appear to be in any situation other than

on one of these two lines. On that occasion it fell between them. The fact of obtaining two lines suggested the possibility of two different types of haemoglobin. This hypothesis broke down when it was observed that the blood of the same individual fell at one time on one line and at another time on the other line. The difference uras clearly one caused by some irregularity of technique, and this turned out to be the case. In the water bath in which the saturators were rotated there was an electric lamp for the purpose of regulating




the temperature of the bath. It was found that the points from the blood of a single individual would be obtained on either of the two lines at will, according as the saturators were or were not protected from the influence of this light. The effect of light had been pointed out by previous workers— Haldane and his collaborators and Hartridge—and has been studied in detail by Hartridge and Houghton, but we had not been alive to the fact that absolute darkness was so essential to the obtaining of a correct equilibrium.

The Significance of the Relation between the Span and log K.Having, as it seems to us, established a relation between (1) the distance

which separates the a-band of oxy- and carboxy-haemoglobin, and (2) the equilibrium constant of the partition of haemoglobin between the two gases, we have performed some experiments to test the significance of the relation. In the first place it was necessary to be sure that the relationship was really a property of the haemoglobin and was not conferred upon it by some condition of the solvent, some admixture with pigment, or some other adventitious circumstance. Miss Leich (9), for instance, working in Krogh’s laboratory, attributes this apparent difference in colour as between the blood of some invertebrate forms and that of man to the presence of a lipochrome discovered by Marie Krogh which alters the optical properties of the fluid. We, therefore, crystallised haemoglobin from the following vertebrates—rat, dog, horse and rabbit—and observed that the properties of the re-dissolved crystals did not differ from those of the original haemoglobin. The results are given below\

Correlation between the Spectra 73

Comparison of Hcemoglobin before and after Crystallisation.

Animal. TemSpan (A.U.). Log K.

perature.Before. After. Before. After.

R at.................................1

17° C. 52 52 2-54 2-54Horse ................... 19° C. 53 53 2*66

2*382-59

Rabbit ...................... 15— 10° c. 46 46 2-32Dog .................. 15° C. 55*5 55

2-41 2 • 76

2*392*79

The blood of the horse, dog and rat were crystallised by subjecting the corpuscles to ether (Bohr’s method), whilst that of the rabbit, which is impossible of crystallisation by ether, was treated by a method for which we have to thank Adair. The corpuscles were separated and washed with Ringer’s solution



they were then laked with a small quantity of ether. The ghosts were centrifuged off and the haemoglobin so obtained was reduced to 0° C. by being


4 0 45 50 55 60Span. — A n g s tro m u n i t s

F ig. 7.—Ordinate and abscissa as in fig. 6.

placed in a freezing mixture. Alcohol (97 per cent.) was also cooled to 0° C., and then added very slowly to the haemoglobin solution, care being taken that the temperature never rose above zero. The change in colour indicated the formation of the crystals which ultimately formed a solid mass. These were pressed between folds of blotting paper, after being partially dried on a Buchner filter. The crystals were examined microscopically in the case of each animal.

Chlorophyl exists in more than one form as does uric acid and many other substances of physiological importance. On the other hand, there is no positive evidence of more than one hsematin, and it would be quite in accord with our knowledge of other blood proteins to imagine that the globins were specific.

We tested the h semochromogens from various forms of life to see whether their spectra showed any differences, the haemochromogens being particularly easy to test, as they have very definite bands—much more definite even than those of oxyhaemoglobin.




The position of the haemochromogen band situated between D and E in the spectrum obtained from blood of each of the following seems to be identical:—

Mammalia. Birds, Reptiles. Fish. Amphibia. Insect larva.

Man Pigeon Roach Frog Chironomus.Ox Hen Gastrophilus.SheepPigHorseMouse

Lizard

It is not to be supposed that the haemochromogen of each of the above forms was compared with every other one ; they were taken at random in pairs, or in groups of three, and no difference greater than could be attributed to experimental error was ever found either between different forms of life or different individuals in the same species. This was so in the actual samples of haemoglobin whose spans and values for K differed markedly from one another.

Granting that the spectra of the haemochromogens from various animals are identical, there remains for consideration the power of the pigment to unite with gas.

Haemochromogen forms compounds with oxygen and with carbon monoxide ; of these the oxygen compound is insusceptible of investigation, is not reversible, whilst that of the carboxy compounds has been found by two of us—Anson and Mirsky—to be so. It is not possible therefore to investigate the partition of the haemochromogens of different species between oxygen and CO as has been done in the paper for the corresponding haemoglobins, but it is possible to determine the values of K for the reaction :—

Haemochromogen and CO ^CO-Haemochromogen.The reaction has been found by Anson and Mirsky (10) to be a simple one

represented by a rectangular hyperbola, as is also the case with the corresponding reaction for mammalian haemoglobin in dilute solution, so that there is every prospect of determining the relations of the position of the principal band in the spectrum of the carboxy compound to the value of K for the reaction of that compound with CO. This comparison is being made both for the haemochromogens and the haemoglobins of various forms of life.

No difference then can be found in the haemochromogens, but on the addition of the protein two differences in property appear—one in the power of uniting with gases (that power for which the pigment is of use to the animal), the other



in the spectrum, and the two are so closely connected that within limits either one may be estimated from an observation of the other.

Discussion of Results.The facts noted above indicate a relationship so novel that it is premature

to say much about its significance. Clearly it opens a whole field for research which is susceptible of investigation in many and widely different directions.

On the physical side, we must admit quite frankly that our knowledge of physical chemistry and of the constitution of matter is quite unequal to the task of discovering the mechanism by which a change in the affinity of a substance for this or that gas can be translated quantitively into a change in the position of this band of the spectrum. We would content ourselves with calling attention to the fact—pointed out to us by A. Y. Hill—that a variation in log K is a measure of the change in free energy of the system, and that therefore the relationship which we have discovered amounts to this, that a given addition to the free energy is associated with a given displacement of this a-band of the haemoglobin.

We may here point out two relationships which, whether accidentally or otherwise, bear a striking resemblance to the one which forms the subject of this paper. Two of us, Anson and Mirsky, have under investigation the relation of the absolute position of this a-band to the absolute value of the equilibrium constant in the reaction

COHb CO + Hb.

In a preliminary experiment the values were compared for the haemoglobins of the sheep and of the rabbit respectively.. Taking the position of the human oxyhaemoglobin band as 5764 A.U., the corresponding CO bands for the rabbit and the sheep, or rather for a certain rabbit and a certain sheep, had their maximum densities at 5718 and 5710 respectively, and their values for log K were for the rabbit 4 • 24 and for the sheep 5 • 80. As the curve which represents the equilibrium between haemoglobin and CO has been shown by Hartridge and Houghton to be a rectangular hyperbola, the values of K simply represent the concentrations of CO in the fluid when the haemoglobin is half saturated with CO, naturally the smaller the affinity the greater the value of K as so expressed, it is therefore desirable to express K as the reciprocal of the concentration of CO at 50 per cent, saturation, the values of log K for the rabbit and the sheep would then be 5*76 and 6-20 respectively, the sheep’s blood having the greater affinity for CO. It will be seen that a shift of 8 A.U. in




the band is associated with a shift of 0 • 44 in log K, or of 0 • 055 in log K per Angstrom unit. This relation is very close to that given in the equation relating the “ span ” to displacement of the band where each Angstrom unit corresponds to a change of 0-05 in log K.

Lastly, the displacement of the band due to temperature may be treated in the same way : taking Brown and Hill’s (11) curves for the effect of temperature on the dissociation curve of sheep’s blood, we may obtain values for the logarithm of the reciprocal of the concentration of oxygen in the fluid when the haemoglobin is one-half oxy- and one-half reduced haemoglobin at temperatures which range between 0. and 43° C. From the observations of Hartridge we learn that the a-oxyhaemoglobin band moves towards the violet approximately 41 A.U. (12), i.e., a shift per degree of 0-205 A.U., between room temperature and that of liquid air. The shift in the a-band appears to be somewhat different at different temperatures, and at those situated between 0° C. and 43° C. it is 0-28 A.U. per degree centigrade. Such being the case, let us consider the relation of temperature to the affinity of haemoglobin for oxygen. The first point to observe is that as the temperature gets lower, the affinity for oxygen increases and the band shifts towards the violet end of the spectrum. The second point is the quantitive relation between the degree of shift and the degree of change in affinity. This is expressed in the following table :—


Temperature.

(a)

0 2 pressure at half

saturation.

(b)

Solubility c.c. per

1 c.c. liquid.

(0

Concentration of 0 2 liquid

c.c. per c.c. liquid.

Logl/C. Displacement of band A.U.

0° c.mm.

3-5 0-049 0-00028 3-64 010 7-8 0-038 0-00039 3-41 2-820 12 0-031 0-00049 3-34 5-630 19 0-026 0-00065 3-18 8-438 29 0-023 0-00084 3-07 11143 36-5 0-022 0-00105 2-98 12-4

If the above figures be plotted on the same scale as fig. 6, it will be seen, firstly, that the points again come nearly on a line, this line having a slope very similar to that in fig. 6, the alteration in log 1/C50 per cent. being 0-049 for each increment of 1 A.U. To summarise these, so far as our present information goes, three ways of testing the shift in the band have been tried—(1) by replacing oxygen by CO in different bloods and testing the difference in the position of the band against the difference in log 1 /C50; (2) by testing



the affinity of different bloods for CO and testing the position of the band against log 1/C50, and (3) by altering both the position of the band and the value of log 1/C50 by change of temperature.


3-7 ° 3-6g 3*5•rH%h 3*4gi *'301 3-Z5? 3*1

H S° 3-0bO9 2-9

-f

- / •

-

/1 1 1 i I i i till^ 1!5 10 5 0

Shaft in band A.U.Warm-*— — *- Cold

Fig. 8.—Ordinate and abscissa are the last two columns in the above Table (p. 77).

Of these three ways the first has been tested exhaustively, the second and third only in a preliminary way, but the results come out as follows, the change in the value of log 1 /C50 for a change of 1 Angstrom unit is

Method (1) ............................................... 0-050Method (2) ............................................... 0-055Method (3) ............................................... 0-049

When one recollects that there was no a -priori reason to suppose that a relation existed at all, or that if it did exist it would be in the same sense, or even if it were in the same sense that there should be any quantitive similarity, one will admit that the coincidence seems more than a haphazard one.

To turn from the physical to the biological significance of the fact recited, it cannot fail to be a matter of satisfaction to the teacher of physiology to know that the spectral bands which hitherto he has presented as mere labels, have some definite place within the range of biologically significant facts. Thus, were other factors equal, the relative minimal lethal doses of carbon monoxide for various animals could be read from fig. 6, the gas exerting a more potent



Correlation between the Spectra o f various Hemoglobins. 79

effect on those whose blood had the longer span. It is possible that in healthy animals of the same species, the collateral factors might be sufficiently alike to admit of a decision as to individual resistance of carbon monoxide from spectroscopic examination of the blood.

One of us (Barcroft) in collaboration with Major McCleland, made a number of determinations of the minimal dose of carbon monoxide fafal to rats and was surprised to find occasional individuals which were very much less affected by the gas than were their fellows. On consultation with Dr. Haldane, he told us that he had had a similar experience. Such differences as are found in the value of K for different individuals of the same species might form an intelligible basis for the idiosyncracy. Thus, rabbits have been observed with spans as low as 43, and as high as 54 A.U. ; from fig. 5 it is evident that four times as great a percentage of CO in the air would be required to produce 50 per cent, saturation in the blood of the former as in that of the latter.

Before leaving the question of idiosyncracy, it may be well to note our ignorance of the sort of property in the protein which influences the affinity of the haematin for oxygen or CO. It may be a property quite specific to this reaction, or again it may be something more general, which will determine other reactions of the haemoglobin and may be linked with the properties of other proteins in the body. Thus the “ span ” may have no further significance than appears in the paper, but on the other hand, it may be evidence of a whole range of properties which confer idiosyncracy in various directions, but which unfortunately do not present themselves in a way which are so evident and so susceptible of measurement as the bands in the spectroscope or the affinity for a gas.

There is no evident phylogenetic significance about the length of the span, the variations between individuals looming larger than those between different species. In this connection, one must remember (1) that the span has no .relation to the absolute position of either the oxy-haemoglobin band or the CO band, and (2) that the affinity of haemoglobin for CO is not likely to have any significance either in the physiology or the evolution of the organism. More likely would it be that the absolute position of the oxy-haemoglobin band had an evident place in the scheme of nature. Thus in fig. 5 the shortness of span of some of the rabbits cuts across any deductions which might be made as regards the higher form of life being at the top of the picture, but this short span in the rabbit is due to an unusual position of the CO band ; the oxy-haemoglobin band of the rabbit is almost coincident with that of the sheep. Frogs’ haemoglobin is somewhat of a special case because its properties alter



very soon after it is shed, in an interesting way which demands investigation ; at present we must strike it out of our list. The animals which have been studied divide themselves into three categories :—

(1) Those with the a-band at about 5777 A.U. The larvae of two flies, Gastrophilus and Chironomus.

(2) Those with bands at 5764 ± 5 A.U. All the vertebrates observed, for instance


Horse .. 5764 Tortoise .. 5766Man .. 5764 Mouse .. 5768Rat .. .. 5762 Lizard .. 5766Pigeon .. 5761 Roach .. 5762Fowl .. .. 5768 Rabbit .. .. 5765

(3) Those with bands at or below 5755 A.U.Worms :

Lumbricus, 5755. Arenicola, 5746.Mollusc :

Planorbis, 5746.The present paper deals only with the span, and that must be regarded as an

emblem of specificity rather than of physiological adaptation or phylogenetic evolution. Two very interesting instances of specificity may be quoted. For the first we are indebted to Mr. Charles Stockman, who made the following observation. Leeches were starved until the alimentary canal was presumed to be empty of haemoglobin. The animals were then fed on the blood of one of our number (Barcroft), the spectroscope properties of which are pretty well established. After ten days the leeches were killed and two examined. The blood in the alimentary canal, which was abundant in quantity, could not be distinguished definitely from that of the donor, different observers being a t variance as to whether they could or could not detect a difference, but alt observed agreed about the distinctive character of the haemoglobin, which was withdrawn from the blood vessels of the leech. The second instance is one for which we have to thank Dr. Keilin. This refers to the larva of Gastrophilus, the bot-fly. The eggs, in which a minute larva is developed, are found on the hair of the horse’s legs a short distance above the grass. When the horse licks them the larvae become transferred to the tongue. They seem to make their way down the alimentary canal underneath the mucous membrane and develop in the stomach into larvae, approaching the size of acorns, which contain considerable quantity of haemoglobin. All this haemoglobin has



Correlation between the Spectra of various 81

been made witliin the horse, yet it is by no means merely horse’s haemoglobin transferred to the Gastrophilus, for it differs both in span and in the position of the bands.

Animal.

Position of a-band.Span.

0 2Hb. COHb.

Gastrophilus...................... 5776 5725 51Horse ............................. 5767 5711 55

That the haemoglobins of different species differ in crystalline form has long been known; indeed, many elementary text-books contain figures illustrating the fact, e.g., Schafer figures the crystals from human blood and those of the guinea pig, squirrel and the hamster as all being different. The most compendious study of this subject has been made by Keichert and Brown (13).

Nor is it merely this form of the crystals which differs, but the ease with which they may be obtained. Here it may be noted, though not stressed, that the forms of haemoglobin shown in fig. 6, which easily crystallise, are all found at the top of the figure, having long spans; but this converse is not true for some of the long-spanned forms, e.g., that of man are difficult to crystallise. It had generally been supposed that these differences were due to differences in the proteins of the various forms and therefore had no physiological significance. With the view that the haematin portion of the haemoglobin is identical in all vertebrate forms we are in agreement, but the burden of the present paper is to show that differences in the protein do plant their impress on the gas-binding, and therefore the physiologically significant, properties of the haemoglobin of which they form a part.

The specificity of the haemoglobins in different species had been demonstrated in the researches of Landsteiner and Heidelberger (14), who showed that the bloods taken from forms of life less closely allied than the horse and the mule yield haemoglobins which were independently soluble, whilst Dale has demonstrated the serological difference between the albumen of the duck and hen.

Had it not been for the fact that Landsteiner and Heidelberger, unlike some other observers, only find one form of haemoglobin in a single species, one would have been tempted to see in their observation a mechanism by which the corpuscle, by enclosing two or more haemoglobins of independent solubility held a much greater quantity of total pigment in solution than it could otherwise secure. I t might be claimed that our own results could be interpreted

VOL. XCVII.— B . Cr



on the hypothesis that there were only a limited number of haemoglobins which in different animals were mixed in different proportions. This view would be simple and in some way attractive, but it is not supported by the data shown in fig. 7. Suppose, to take the simplest explanation, that there were but two haemoglobins ( a and b) of which a, with a short span, preponderated in rabbit’s blood, and b, with a long span, in dog’s blood, it would be probable that a would crystallise with difficulty, as does rabbit’s haemoglobin, and 6, with ease, as does dog’s, the redissolved crystals from the rabbit’s blood would therefore contain a larger proportion of 6 than did the original blood. That, indeed, would be the case with all the bloods, but it would be most marked in the case of the bloods which contained most of a.

We would not seem to be justified in attempting to solve the puzzle of how the corpuscle manages to hold so much haemoglobin in solution by supposing that the haematin in a given corpuscle is united with more than one sort of globin, and thus more than one sort of haemoglobin is formed, each soluble independently of its neighbour. Nevertheless the protein is not a negligible factor in the mechanism of haemoglobin solubility. If one enquires into the physiological significance of the globin in architecture of the great respiratory pigment which has made life for the larger and more intense forms possible, one sees that on the globin depends the fact that the haematin is dissolved at all in considerable quantities at the hydrogen-ion concentration of the body, for haematin is rather insoluble in neutral solutions. One sees also that nature has contrived by uniting this with a globin whose iso-electric (15) point is somewhere about 8-1 to produce a body whose iso-electric point is 6-8 or thereabouts, and in general one sees the adaptation of a very crude material haematin which has the one essential property of being oxidised and reduced with ease to the multifarious needs of the body so exactly as to make haemoglobin, in the words of Lawrence J. Henderson, “ perhaps the most interesting substance in the world.”

Summary.1. A convenient calibration of the Hartridge reversion spectroscope is

described.2. For a number of different mammals the following relationship exists.

If A be the position of maximum intensity of the a-oxy-haemoglobin spectral band, B that of CO haemoglobin, and K the equilibrium constant of the equationCO + H b02 COHb + 0 2

Log K = 0-05 (A—B),A and B being measured in Angstrom units.





3. Log K is a measure of the change in free energy involved in the reaction.4. The value of (A—B) called in the paper “ the span,” varies from 43 to

56 Angstrom units in the mammals which we have observed. The variation in individuals in the same species is very marked.

5. The relation stated in § 2 is true of the recrystallised haemoglobins.6. The cause of variation seems to lie rather in the specificity of the globin

portion of the molecule than in the hsematin portion.7. Brown and Hill’s observations on the effect of temperature on blood

have been treated along similar lines. The reaction being taken as

Hb„ + » 0 2 5 ± (H b 0 2)„.If at temperatures Tx and T2, CT, and CT„ represent the concentrations of

oxygen in solution when the haemoglobin is half saturated in each case, and At , and AT.2 represent the position of maximum intensity of the a-bands,

Log ~ — log -049 (ATl — AT2).Ut,

The expenses of the above research were in part -defrayed by the Medical Research Council. We should like to thank Prof. A. V. Hill for his advice and help.

BIBLIOGRAPHY.

1. Sorby, ‘ Q.J.M.S.,’ vol. 16, p. 76 (1876).2. Vies, ‘ Arch, de Phys. Biol.,’ vol. 2, p. 2 (1922).3. Barcroft, J., and Barcroft, H., ‘ Roy. Soc. Proc.,’ B, vol. 104, p. 28 (1924).4. Hartridge, ‘Journ. of Physiol.,’ vol. 44, p. 1 (1912). Ibid., vol. 57, p. 47 (1922).5. Haldane and Lorrain Smith, Ibid., vol. 22, p. 231 (1897).6. Douglas, Haldane, J. B. S., and Haldane, J. S., ‘Journ. of Physiol.,’ vol. 44, p. 275

(1912).7. Krogh, * Skand. Arch.,’ vol. 23, p. 217 (1910).8. Nicloux, ‘Journ. de Physiol, et de Path. Gen.,’ vol. 16, pp. 64 and 145 (1914).9. Leich, ‘ Journ. of Physiol.,’ vol. 50, p. 370 (1915-16).

10. Anson and Mirsky, ‘ Physiol. Soc. Proc.,’ ‘ Journal of Physiol.’ (June, 1924).11. Brown and Hill, ‘ Roy. Soc. Proc.,’ B, vol. 94, p. 298.12. Hartridge, ‘ Physiol. Soc. Proc.,’ ‘ Journ. of Physiol.’ (June, 1924).13. Reichert and Brown, ‘Carnegie Institute of Washington Publications,’ vol. 116 (1909).14. Landsteiner and Heidelberger, ‘ Journ. Gen. Physiol.,’ vol. 6 (1923).15. Osato, * Biochem. Zeitschr.,’ vol. 132, p. 485 (1922).

VOL. XCV1I.— B. H



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On the correlation between the spectra of various ...rspb.royalsocietypublishing.org/content/royprsb/97/680/61.full.pdf · tions be made from human and Gastrophilus haemoglobin respectively,