A Note on Haemerythrin

A NOTE ON HAEMERYTHRIN

BY GUY FREDERIC MARRIAN.

(From the Marine Biological Laboratory, Plymouth, and The Department of Physiology and Biochemistry, University College, London.)

{Received loth December 1926.)

(With Three Text-figures.)

INTRODUCTION.

THE occurrence of a red pigment in the corpuscles in the body fluid of the Gephy-rean Sipunculus nudus was first observed by Ray Lankester (1872). This pigment was later investigated by Krukenberg in 1880, who gave to it the name Haemery-thrin. In the oxidised state a solution of the substance has a madder pink colour, and when reduced is colourless. Haemerythrin occurs in all species of Sipunculus, in Phascolosoma, in Phymosoma, and, according to Benham (1896), in the Annelid

Magelona.

The present communication was primarily intended to deal with the dissociation of oxyhaemerythrin obtained from Phascolosoma. However, during the progress of the work, certain interesting observations were made which may possibly be of some value in the elucidation of the chemical nature of the pigment. The difficulty of obtaining large quantities of Phascolosoma and the fact that no method of pre-venting decomposition of the pigment has yet been devised, have rendered a systematic examination impracticable at this stage of the investigation. It was thought, however, that these observations were of sufficient interest to justify their inclusion in this preliminary note. It is hoped that a more complete investi-gation into the nature of haemerythrin will be presented in a later communication.

EXPERIMENTAL.

Preparation of Material. As soon as possible after collection, the worms were

cut at one end and the body fluid squeezed out into centrifuge tubes. On spinning the tubes the corpuscles readily settled and the supernatant liquid could be readily pipetted off. The corpuscles were washed twice with sea water or an isotonic solution of NaCl (-57 N), and then laked in distilled water. After filtering to remove the stromata of the corpuscles a perfectly clear madder red solution showing

^rked blue fluorescence was obtained.

^Dissociation of Oxyhaemerythrin. The simple colorimetric technique devised by

358

colour of a totally reduced solution of the pigment could be easily obtainec tinting some distilled water with Orange G and methylene blue. The faint escence was imitated by the addition of a little egg white. This solution was used to dilute successive quantities of oxyhaemerythrin solution at 15° C. so that the colours corresponded to 100,80, 60, 50,40, 30,20 and 10 per cent, saturation of the pigment with oxygen. The assumption was made that at atmospheric pressure at 150 C , the pigment was totally saturated with oxygen. This assumption would

100

90

63" C

= volume of Os measured at N.T.P. that 1 volume of water dissolves at a certain, temperature at a partial pressure of O2 of 760 mm. Hg.

= partial pressure of O2 recorded.

op

Fig. 1. Effect of temperature on the dissociation of oxyhaemerythrin.

appear to be quite justified, since at o° C , where the oxygen affinity is greatly in-creased, the intensity of the colour at atmospheric pressure was no greater than the 100 per cent, standard.

Effect of Temperature on the Dissociation of Oxyhaemerythrin. 5 c.c. of a solution

of oxyhaemerythrin buffered with 2 c.c. of Palitzch's borate-boric acid buffer to />H y o were used. To the standard tubes were added 2 c.c. of water to allow for the volume of the buffer in the experimental tube.

the partial pressure corrected for the solubility of the gas at the various eratures.

Heat of Formation of Oxyhaemerythrin. From a consideration of the temperature

dissociation curves of oxyhaemocyanin, Hogben (1925) derived a figure for the heat of reaction between oxygen and haemocyanin. In.a similar manner a value for the heat of formation of oxyhaemerythrin has been derived.

Let the reaction be

m Hr + h O2 = /HrO2.

At the point of 50 per cent, saturation of the pigment with oxygen: [Hr] = [HrOJ,

• -iH-^=C (2)

provided that the total concentration of haemerythrin is constant throughout, as was the case. Hence from (1) and (2)

where a = vol. of O2 dissolved in 1 vol. of water, measured at N.T.P. when the

partial pressure of the O2 = 760 mm. Hg.

px = partial pressure of O2 at 50 per cent, saturation point at temperature t° C.

t° C. = temperature of water

Log, K = h . Log, at px + Log, C.

Now applying the integrated form of the Van 't Hoff Isochore

Log, Ki - Loge K2 = —¥ LL _ .i.)

— O I 1 1 \

h . Log, atlpx - h . Logeat2p2 = - ^ - ^ ~ f.) '

From the curves at 15° C. and 250 C. O — 10,572/; calories. And from the curves at 250 C. and 35° C. Q = 10,130A calories. Mean value Q = 10,350/? calories.

This is the value when h gramme-molecules of O2 are involved. Hence when

1 gramme molecule of O2 combines with haemerythrin to form oxyhaemerythrin,

10,350 calories are involved.

In Fig. 2 the linear relationship between Loge ap^ and 7^ is shown.

Effect of pH on the Dissociation of Oxyhaemerythrin. A preliminary experiment

360 GUY FREDERIC MARRIAN

At pH 3-0 and 4-0 the red colour immediately disappeared and the turned deep lemon yellow. At pH 5-0 the same phenomenon was observed 2 hours and at />H 6-o after 12 hours. After 2 days the tubes at />H 8-o and 9*0 still retained their red colour, while that at pH j-o was distinctly yellow. The tube at pH io-o was decolorised but no sign of a yellow coloration had appeared. The decolorisation in alkaline solution appeared to be irreversible, since the red colour

did not reappear on titrating back with acid.

It appeared evident, therefore, that the red colour of oxyhaemerythrin was most stable at/>H 8-o and/>H 9-0.

-•50

-1-0

I

1

-1-5

- 2 0

•0032 •0033 •0034 •0035

T Fig. 2.

It was obviously futile to attempt to study the dissociation of the pigment at hydrogen-ion concentrations other than those at which the red colour persisted for several hours. Accordingly it was decided to carry out experiments at pH's 6-o, 7-0, 8-o, 9-0, io-o.

At this point reference must be made to the effect of stronger acid on solutions of oxyhaemerythrin. On the addition of several drops of dilute CH3C00H or of HC1, all colour disappeared from the solution and a white precipitate was thrown down. The clear supernatant liquid gave a distinct Prussian blue coloration with K3FeCy6. It would appear from this experiment that the iron in the haemerythrin molecule is quite loosely bound, certainly less firmly than is the case with haeij globin.

Variation of pH within the range over which the pigment is stable appeared to

served were scarcely greater than the possible experimental error of the method, and Hogben (1925) recorded a somewhat similar result for the haemocyanin

o^Helix. In a later communication, however, Hogben showed that a small but

definite change in the shape of the dissociation curves occurred over a compara-tively narrow range of pH. Thus until further experiments have been carried out at intermediate />H's it would be unwise to draw any conclusions.

The Effect of Oxidising Agents on Oxyhaemerythrin. Reference has already been

made to the yellow colour that is produced in slightly acid solutions of oxyhae-merythrin. It was suggested that possibly a change analogous to the conversion of oxyhaemoglobin to methaemoglobin was occurring. It was therefore of interest to study the effect of oxidising agents on solutions of oxyhaemerythrin and reduced haemerythrin.

On the addition of a drop of a dilute solution of K3FeCy6 to a solution of oxy-haemerythrin, the colour immediately changed to a deep lemon yellow. Exactly the same effect was produced by the addition of a small quantity of hydrogen peroxide.

In order to show that the yellow colour produced was actually a product of oxidation (either aerobic or anaerobic), the following experiments were carried out.

A small tube containing a few c.c. of acetate buffer at pH 4-0 was placed in a# larger tube containing a solution of oxyhaemerythrin. The tube was then evacuated until the pigment was totally reduced. On shaking the buffer solution with the pigment, no change of colour was observed. However, on admitting air to the tube, the solution immediately turned yellow without any intermediate formation of the red colour of oxyhaemerythrin.

In a similar manner it was shown that reduced haemerythrin could be oxidised anaerobically by K3FeCy6 to the yellow compound. Only a very small volume of a very dilute solution of K3FeCy6 was used, so that the colour of the oxyhaemery-thrin would not be masked by the yellow colour of the oxidising agent itself.

If, as appeared probable, there was a methaemerythrin corresponding to methae-moglobin, it was thought that it would be possible to reduce the yellow solution with a suitable reagent and then to re-oxidise the solution with air to oxyhaemery-thrin.

Accordingly a small quantity of sodium hydrosulphite was added to a solution of "methaemerythrin" buffered to pH 9-0 to obtain the necessary alkalinity. The solution rapidly decolorised and on shaking vigorously with air, the red colour of oxyhaemerythrin appeared.

Robert (1903) noted that no particular spectrum was produced in a solution of oxyhaemerythrin by the addition of a crystal of K3FeCy6, and on this evidence concluded that no methaemerythrin was formed. However, the above evidence would appear to favour the supposition that a methaemerythrin somewhat analogous to methaemoglobin is formed by the action of oxidising agents on haemerythrin ^ B oxyhaemerythrin. The spontaneous change of oxyhaemerythrin to methae-merythrin appears to be most rapid at about pH 3-O-4-O.

362. Guy FREDERIC MARRIAN

preliminary examinations of solutions of oxyhaemerythrin and of " thrin" merely showed general absorption in the blue violet end of the

spectrum, the extent of which varied with the concentration of the solution. It was decided, however, that it would be advisable to carry out rather more delicate experiments before any conclusions concerning the absorption spectra of these substances could be drawn. Accordingly solutions of oxyhaemerythrin and of methaemerythrin were placed in Baly tubes and photographs of the spectra were recorded over a wide range of dilution, using a Tungsten Arc as the source of light. Solutions of oxyhaemerythrin were first examined. These were then converted into "methaemerythrin" by means of K3FeCy6 and the resulting solutions re-examined over an identical range of dilution. In order to gain some knowledge of the approximate concentrations of the solutions examined, 5 c.c. of the original solution of oxyhaemerythrin were evaporated to dryness and the solid residue weighed. No claim is made that the dried residue represents pure haemerythrin, but it was felt that some data relevant to the concentration of pigment in the solution was necessary. No sharply defined bands could be observed, but the photographs indicated that there was a certain amount of selective absorption in the solutions examined. These spectra are shown diagrammatically in Fig. 3. It will be observed that a higher concentration of pigment is necessary to show a band in the oxyhaemerythrin solution than is necessary in the case of the "methae-merythrin" solution.

The "methaemerythrin" band was visible over a fairly wide range of dilution, whereas in the case of oxyhaemerythrin, the selective absorption was less marked and was observed over a small range of dilution only.

The author wishes to express his thanks to Mr L. C. Baker for carrying out the photographic work.

Attempts to show the presence of Haem. Several experiments have been carried

out in the endeavour to demonstrate the presence of haem in the haemerythrin molecule.

Various attempts were made to obtain a haemochromagen spectrum by reduc-tion of oxyhaemerythrin in alkaline solureduc-tion. Solureduc-tions varying in alkalinity from

pW 8-o upwards have been reduced by simple evacuation, ammonium sulphide

and by sodium hydrosulphite, but in no case was any sign of haemochromagen spectrum visible.

It has already been mentioned that slight acidification promotes the formation of the " M e t " compound, and that stronger acidification causes precipitation and loss of colour from the solution. It was not surprising therefore that no coloured ethereal extract was obtained by the Schultz separation.

An attempt to prepare haemin by the Schalfejeft' method yielded a clear colour-less solution, no trace of a solid product being formed.

The significance of these results is discussed in the next section of the

The Action of H2SO4 on Oxyhaemerythrin. On the addition of an equal

•

always observed in such solutions. At first it was thought that the presence porphyrin in the pigment had been demonstrated, as the superficial resemblance between the product obtained and that obtained in a similar manner from haemo-globin was very marked.

On spectroscopic examination this solution showed a rather broad, ill-defined band in the yellow, Fig. 3. Acid haematoporphyrin obtained from haemoglobin shows two distinct bands, one in the orange and the other in the yellow-green.

AngstrOms

OO O 0 0 O 10 O io 10 10 *

1 I I o o o

CO

o

8

i

utio

n

'•S " S.S 55 052%

•258%

io

n

' solut

i

«•* o

Dept

h

8 cms.

5 cms.

OXYHAEMERYTHRIN

•052% 8 cms.

' METHAEMERYTHRIN

concentration unknown

H,S04 PRODUCT

Fig. 3.

Several observations have been made on various preparations, but it has not been possible to observe the second band that would show the similarity of this product with haematoporphyrin.

If a solution prepared as above is carefully neutralised with ammonia or caustic soda, the colour changes to a pale straw yellow. Several of such solutions have been examined spectroscopically, but no sign of the four-banded spectrum typical of alkaline haematoporphyrin could be observed.

364 GUY FREDERIC MARRIAN

complex analogous to the copper-protein compound haemocyanin. The failure to demonstrate the presence of haem would rather seem to lend support to ^ A view. However, it is felt that the inconclusive nature of the experimental evidence does not justify any conclusions being drawn at this stage.

Peroxidase reaction. Robert showed that haemerythrin did not cause a blueing

of guaiacum in the presence of hydrogen peroxide. This observation has been confirmed. One must conclude that haemerythrin, like haemocyanin, cannot act as a peroxidase.

SUMMARY.

1. The effect of change of temperature on the dissociation curve of oxyhae-merythrin is shown. The heat of combination between haeoxyhae-merythrin and oxygen has been calculated to be 10,350 calories per gramme molecule of oxygen.

2. It has been shown that oxyhaemerythrin is only stable over a small range of pH. The pigment appears to be most stable at pH 8-o and 9-0. Variation of hydrogen-ion concentration between pH 6-o and pH io-o appears to have little effect on the dissociation curve of oxyhaemerythrin.

3. Oxyhaemerythrin can be converted to a yellow " methaemerythrin" by the action of oxidising agents. The change occurs spontaneously at slightly acid hydrogen-ion concentrations.

4. Attempts to show the presence of haem in the haemerythrin molecule have been unsuccessful.

5. The nature of the purple colour produced by the action of concentrated H2SO4 on a solution of oxyhaemerythrin is discussed.

6. Photographs of the absorption spectra of oxyhaemerythrin, methaemery-thrin and the H2SO4 product of oxyhaemerymethaemery-thrin are shown.

The Author wishes to express his thanks to Mr C. F. A. Pantin for suggesting the above work and also for his helpful advice and continued interest in its progress. Thanks are also due to Professor J. C. Drummond for much helpful advice and to the Medical Research Council for a personal grant.

REFERENCES BENHAM, W. B. (1896). Quart. Journ. Micr. Set. 39, i. HOGBEN, L. T . (1925). Brit. Journ. Exp. Biol. 3, 225. K.OBERT, R. (1903). Pfliiger's Archiv. 98, 411.

KRUKENBERG (1880). Vgl.-physiol. studien, 1 Reihe, 3 Abth. S. 66. LANKESTER, E. RAY (1872-73). Proc. Roy. Soc. 21, 70.