reduced hæmatin and hæmochromogen - royal...

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Reduced Hcematin and Hcemochromogen.By R. H ill , M.A., 1851 Exhibition Senior Student.

(Communicated by Sir Frederick Hopkins, F.R.S.—Received March 30, 1929.)

(From the Biochemical Department, Cambridge University.)

CONTENTS.

1. Introduction ........ .*.....................2. Apparatus and Materials ...........3. Pyridine Hsemockromogen .......4. Nicotine Hsemochromogen .......5. Cyan-Haemochromogen ...........6. Cyan-reduced Haematin...............7. Nicotine Cyan-Haemochromogen8. Discussion ..............*....................9. Summary.......................................

10. References ...................... ...........

Page112114117120120121123124 130 130

1. Introduction.

Haemochromogen, which originally was known only as an artificial degradation product of haemoglobin, has since been found to occur in almost all organisms.* Helicorubin, a haemochromogen occurring in the intestinal fluid of snails (such as Helix pomatia) was described by Krukenberg (1884), and its properties examined by Dhere (1917). Cytochrome has been shown by Keilin (1925) to be a mixture of at least two, possibly three, haemochromogens. Just as haemoglobin differs in its behaviour towards oxygen from any of the other compounds of haematin at present known, so most of the naturally occurring haemochromogens differ in properties from any of the artificial haemochromogens. Ordinary haemochromogen, obtained directly from haemoglobin, combines with carbon monoxide to give. CO-haemochromogen, and is rapidly oxidised under all conditions by free oxygen to haematin. The haemochromogens of cytochrome do not combine with carbon monoxide, and with the exception of component b (Keilin, 1929) are not rapidly oxidised by free oxygen over definite ranges of hydrogen-ion concentration.

Originally haemochromogen was only a name for the spectrum of reduced

* Cf. Barcroft, 1928.

on May 30, 2018http://rspb.royalsocietypublishing.org/Downloaded from

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Reduced Hcematin and Hcemochromogen. 113

haematin under special conditions. Zeynek (1920) had shown by an analysis of solid pyridine liaemochromogen that it contains 2-2 molecules of pyridine. Anson and Mirsky (1925) were the first to show that this particular type of hsematin spectrum is due to chemical combination of reduced haematin with substances containing nitrogen, and that each nitrogen compound gave its •characteristic haemochromogen. They showed that these compounds were dissociable in solution, and also that the various nitrogen compounds were in equilibrium with the haemochromogen according to their relative affinities. Later I (1926) measured the minimum quantity of pyridine required to change completely the spectrum of reduced haematin into haemochromogen, using- strong solutions of haematin, and found that 2 molecules per molecule of reduced haematin were required, which agreed with Zeynek’s analysis.

In a recent paper Anson and Mirsky (1928) describe a new type of haemochromogen, obtained by the action of potassium cyanide on reduced haematin in dilute solution. Using a similar method to that which I used for pyridine haemochromogen (R. Hill, 1926) they found that the compound is produced from 1 molecule of alkali cyanide per molecule of reduced haematin. In view of the fact that this new substance containing one molecule of cyanide has a spectrum-like typical haemochromogen, while the ordinary cyan-haemochromogen obtained by using an excess of cyanide has a rather different one, the above writers consider that typical haemochromogen is always represented by a combination of haematin with one molecule of another substance. Now in pyridine haemochromogen there appeared from experiments to be 2 molecules of pyridine per molecule of haematin. Such a conclusion, however, seemed to be so strongly against the views expressed by Anson and Mirsky (1928) that I felt it necessary to present some more experimental data on haemochromogen and allied compounds. In each case, as will be seen from the experiments described below, combination occurs with 2 molecules of the nitrogen compounds to 1 molecule of reduced haematin. In my previous publication haemochromogen was represented as a normal co-ordination compound of iron with the porphyrin, and with pyridine arranged so that the co-ordination number of iron was six. Such an arrangement would be the most likely one to deduce from the work of Anson and Mirsky, who first showed that haemochromogen is a dissociable molecular compound of reduced haematin with substances containing nitrogen. As the porphyrin will account for four of the co-valencies of the ferrous iron, we should naturally expect that the addition-compounds of the iron-porphyrin would involve two extra combining- molecules. In other words, most of the addition-compounds of reduced



114 R. Hill.

haematin would be of the type Htn A B, where A and B can be the same compound or different compounds.

Carbon monoxide reduced haematin, containing 1 molecule of carbon monoxide, does not appear to fit in with this scheme ; but it very easily takes up another molecule of a nitrogenous compound, forming CO-haemochromogen (R. Hill, 1926). The new haemochromogen of Anson and Mirsky will, as will be shown later, take up, either another molecule of alkali cyanide, giving ordinary cyan-haemochromogen, with the characteristic spectrum, or a molecule of nicotine, giving nicotine cyan-haemochromogen, the spectrum of which is similar to ordinary nicotine haemochromogen. Also the single molecule of cyanide seems to be replaceable by carbon monoxide, because carbon monoxide- reduced haematin, not a CO-haemochromogen, is formed by the action of carbon monoxide.* On account of the analogy between the cyan-haemochromogen of Anson and Mirsky and CO-reduced haematin the name cyan- reduced haematin would be more appropriate for the former substance. In this case the name cyan-haemochromogen can be retained for the substance containing 2 molecules, to which it has always been attached.

2. Apparatus and Materials.In a mixture of haemochromogen and free reduced haematin the relative

proportions of these two substances can be estimated spectroscopically. The simplest method is to view the spectrum of the mixture and compare it with that given by the two pure substances contained in equal concentrations in a double wedge trough. The thickness of the liquid examined in relation to total haematin concentration must be equivalent to the sum of the thicknesses of the two wedges, along the trough.

An apparatus found convenient for the purpose was arranged as is shown schematically in fig. 1. A Zeiss microspectroscope (a) was fixed above one of the sides of a Dubosq colorimeter in place of the rhomb and eyepiece. The double wedge trough (b) was mounted in front of the comparison prism aperture (c) of the spectroscope. The illumination of the trough was supplied by a small four-volt bulb (d) and the colorimeter cell (e) was illuminated by a pointolite lamp at a suitable distance from the mirror (/). The trough rested on a length of wood, carrying a scale. Both the trough and the spectroscope were supported on a rigid frame of wood (g) screwed on to the base of the colori-

* The existence of a compound such as CO-cyan-haemochromogen is not excluded, but so far it has not been detected. Experiments are being continued on the reactions with carbon monoxide.



Reduced Hcematin and 115

Fig. 1.—Schematic figure of apparatus showing spectroscope, colorimeter cell and double wedge trough (one half actual size).

meter. The two standard solutions of reduced haematin, with and without the haemochromogen-forming substance, were put into the double wedge trough and the cell adjusted to an equivalent thickness by means of the vernier scale (h) attached to the plunger (i ) of the colorimeter. The thickness of the trough

VOL. CV.— B. K



116 R. Hill.

was 2*3 cm., hence the thickness to be observed in the cell, if the trough fluid is diluted 2-3x times and the fluid in the cell y times, is y/x cm. The length of the trough was 15 cm. and the depth 2-5 cm.

As the same light source is not used for the trough and the cell, the light intensity must be adjusted or it is impossible to estimate the relative strengths of the absorption bands by moving the trough. The intensity of the light is adjusted so that at a given position of the trough the two spectra, that of the mixture and that of the standard, match in intensity throughout their length ; and there is only one position of the trough where this is possible. Then from the position of the trough the relative concentrations of the two coloured substances present in the mixture are known. If the intensity of light is not adjusted then at no position of the trough will the two spectra match over their whole length. The difference in distribution of energy in the spectrum between the bulb used and the pointolite was not sufficient to be noticeable over the range of visible spectrum transmitted. This method has the advantage of neutralising differences in general absorption due to comparing different thicknesses of solution with solid in suspension. These differences otherwise would make a spectro-colorimetric method inapplicable in the case of the nearly insoluble reduced hsematin in equilibrium with hsemochromogen. The method, of course, can only be used when the solution being analysed contains the same two compounds as are separately contained in the trough. If a different compound having selecti ve absorption is present in the cell, no position of the trough or adjustment of the illumination will give equality of illumination in the two spectra.

The hsemin was obtained from unwashed corpuscles of ox blood by the method of Schalfejeff. I t was washed with water, concentrated HC1, alcohol and ether. The solutions of hsemin were prepared by weighing the required amount of the substance in a 25 c.c. standard flask, dissolving in 3 equivalents of sodium hydroxide and making up to 25 c.c. with distilled water. The pyridine was dehydrated over caustic soda and the fraction distilling at constant temperature (114-115° C.) used for making up the solution. Pure nicotine was purchased from an established chemical firm, and as it was nearly colourless it was used directly. This pure nicotine was found to give the same result with hsematin as a sample of 90-95 per cent, technical nicotine tried in some preliminary experiments. The potassium cyanide was estimated by titration with silver nitrate in the presence of potassium iodide. The sample of cyanide used was equivalent to 96 per cent. KCN. The reducing agent employed was sodium hydrosulphite.




3. Pyridine Hcemochromogen.

i The following solutions were made up :—2 • 63 gm. of pyridine were made up to 1 litre with water, giving a 0 • 033 molar solution (pyridine = 79). 0 • 542 gm. of hsemin were dissolved in 2-5 c.c. N.NaOH and made up to 25 c.c., giving a 0-033 molar solution (hsemin = 650). The reducing solution contained 10 gm. of sodium hydrosulphite in 100 c.c. of 2N sodium carbonate. 1 c.c. portions of the hsemin solution were mixed with different volumes of the pyridine solution, and water, and 0 • 5 c.c. of the reducing solution added finally, so that the total volume was 4 c.c. in each case. The double wedge trough contained the solution of hsemin diluted 2*3 X 160 times, hence the thickness of the fluid to be examined in the cell was 0*025 cm. The following table shows the results obtained :—

Table I.

Cubic centimetre pyridine solution for 1 c.c. hsemin solution.

Percentage reduced hsematin uncombined.

0-5 751 0 491-5 252 0 Trace2-5 Zero

This would indicate that two molecules of pyridine are required to form pyridine hsemochromogen. But it is possible that at this dilution the hsemochromogen is dissociated so that this experiment only gives the minimum quantity of pyridine required to convert reduced hsematin into hsemochromogen at this dilution. However, if we estimate the percentage of free reduced hsematin for mixtures of hsematin and pyridine containing one, two and ten molecules of pyridine per molecule of hsematin at different dilutions of the solution, it is possible to see what allowance must be made for dissociation, and the true ratio of pyridine to hsematin in the hsemochromogen determined. The following is an example of the type of experiment carried out for this purpose.

0*271 g. of hsemin were dissolved in water with three equivalents of NaOH and made up to 25 c.c., this gives a 0 *0167 M. solution of hsemin. The pyridin- solution was 0*033 M. The reducing solution contained 10 g. of sodium hydrosulphite in 100 c.c. 2N sodium carbonate. The mixtures were made up as follows :—X c.c. of the pyridine solution were mixed with 2 c.c. of the reducing solution and diluted with 14 — 2X c.c. of water. To this mixture was then



118 R Hill.

added X c.c. of the hsemin solution with thorough mixing. This gives a total volume of 16 c.c. in which the total hsematin is one-half the total pyridine concentration and each has been diluted X/16 times. This fluid was then placed in the colorimeter cell. For the fluid to be used in the double wedge trough the hsemin solution was diluted 2-3 X 200 times for both wedges. For the reduced hsematin wedge the diluted hsemin solution contained the reducing fluid to the extent of 25 per cent. For the hsemochromogen wedge the diluted hsemin solution contained 25 per cent, of the reducing fluid and in addition 25 per cent, of the 0*033 M. solution of pyridine. In making these dilutions the hsemin solution was added last, so that it could become reduced as rapidly as possible. The thickness of the fluid in the cell was adjusted by calculation from the dilutions of hsematin as has been previously explained.

Table I I , HsematinPyridine

i2*

Cubic centimetres pyridine and

cubic centimetres Htn.

Dilution in terms of original

pyridine solution.

Logxo molecular dilution of pyridine.

Percentagefree

reducedhsematin.

Thicknessin

centimetresexamined.

2*00 8 2-38 Zero 0-041-33 12 2-56 11 0-061-00 16 2*68 34 0-080*62 24 2-86 86 0-120-50 32 2-98 92 0-160-25 64 3*28 98 0-32

The effect of dilution is best seen if percentage of free reduced hsematin is plotted as ordinate and log10 dilution of total pyridine as abscissa. This gives a curve showing the effect of dilution on the ratio of hsemochromogen to reduced hsematin when hsematin is reduced in the presence of pyridine, the molecular ratio of total hsematin to total pyridine being constant over the whole range. Three curves for proportions of hsematin to pyridine of 1 :1 , 1:2 , and 1 : 10 respectively, are given in fig. 2. The dilution of total pyridine at any point can be obtained directly from the value of the abscissa, while the dilution of total hsematin is obtained by multiplying the dilution of pyridine by 1, 2 and 10 according to the curve in question. From fig. 2 it is seen that until the concentration of total pyridine is less than 10“2'4 M. the curves can only be interpreted by assuming that the dissociation of the hsemochromogen is almost inappreciable. In Table I when 25 per cent, of hsemochromogen is present and half an equivalent of pyridine per equivalent of reduced hsematin,




the minimum concentration of pyridine was 4 (0-5 X 0-033) M. = 10 2-38 M. If our assumption is justified the results given in the table actually represent

Fig. 2.—Pyridine hsemochromogen.

the combination of reduced haematin with pyridine, the amount of free pyridine being small compared with the total haemochromogen present.

I t is not possible to get reproducible results with pyridine haemochromogen unless the haemin solution is freshly made up ; up to 8 hours, however, there is no appreciable ageing effect. Fig. 3 shows the same dissociation curves for

is!$<50**

/0020 2-5 3 0 3 J 4 0

fog jo dilution total pyridineFig. 3.—Pyridine hsemochromogen with an old solution of hsematin.

pyridine haemochromogen after the solution had stood in a stoppered flask for 4 days. I t will be noticed that the curves are less steep than with fresh haematin. This is evidently connected with the fact that while with a fresh solution of haematin, free reduced haematin is sparingly soluble and immediately separates as a crystalline precipitate when the alkaline solution of haemin is reduced, with an old solution of haemin precipitation occurs slowly and the solid is mainly amorphous. The same is true also of the haemochromogen. I t can be seen that with a greater solubility of both derivatives of reduced



120 R Hill.

haematin the curves representing the effect of dilution are less steep. In the actual formation of the haemochromogen it must be assumed that in this case two molecules of pyridine are required, showing that haematin suffers no appreciable decomposition on standing. All the other measurements have been made with fresh haematin solutions. The solutions were at room temperature 16° C. ± 1° C. in all the experiments.

4. Nicotine Hcemochromogen.A solution of nicotine 0 • 033 M. was made up, containing 5*40 gm. per litre

(nicotine = 162) and the experiments carried out as in the case of pyridine. As nicotine has a higher affinity for reduced haematin than has pyridine the haemin and nicotine solutions were diluted 10 times for making up the more dilute mixtures. The haemin solution contained 0-271 gm. in 25 c.c. For the double wedge trough the haemin solution was diluted 2 • 3 X 192 times and for the more dilute mixtures the original haemin solution was diluted 2 • 3 X 1920 times. The dissociation curves of nicotine haemochromogen are shown in fig. 4. I t will be seen that two molecules of nicotine per molecule of haematin

JO 3 5 4 0f°£/o oftZufio7i o / /o /a l n ic o tin e

Fig. 4.—Nicotine hsemochromogen.

are required to form the haemochromogen. As nicotine itself has two nitrogen atoms it must be supposed that in nicotine haemochromogen the nitrogen in the pyridine ring is in combination with the iron atom of the haematin, the methyl pyrrolidine nitrogen atom not entering into the reaction over the range of dilutions examined.

5. Cyan-hcemochromogen.The affinity of cyanide for reduced haematin appears to be greater the more

alkaline the solution, and as these experiments depend on having as little dissociation as possible, cyan-haemochromogen was examined in a solution of



sodium hydroxide instead of sodium carbonate. The experiments were carried out in a similar manner to those with pyridine and nicotine. In this case reduced hsematin and cyan-hsemochromogen were the only substances present in appreciable quantities over nearly the whole range of dilutions. It was only at the extreme dilutions of hsematin that the third compound cyan- reduced hsematin began to interfere with the measurements of the ratio of reduced hsematin and cyan-hsemochromogen. The solution of hsemin used contained 0*542 gm. hsemin in 25 c.c. and was 0*033 M. The solution of cyanide was 0*033 M. The reducing solution contained 10 per cent, sodium hydrosulphite in 8 per cent, sodium hydroxide and was present in the final mixture to the extent of 33 per cent. Thus the concentrations of salts in the mixtures examined were 3*3 per cent, of sodium hydrosulphite and 2*6 per cent, of sodium hydroxide. The hsemin solution for the double wedge trough was diluted 2 • 3 X 80 times for the more concentrated mixtures, and 2 • 3 X 400 times for the more dilute mixtures. The results are shown (as circles) together with those obtained in section 6 (as crosses) in fig. 5.

Reduced Heematin and Hcemochromogen. 121

3-0 3 5 Jf-0 4 .5 5 0fog/o drfafion 0/ /ofal A'C/Y.

Fig. 5.—KCN hsemochromogen and KCN reduced hsematin.

6. Cyan-reduced Hcematin.I t was found that cyan-reduced hsematin could only be produced in solutions

containing a relatively low concentration of dissolved salts. This fact is not recorded by Anson and Mirsky (1928), but the principal effect of salts is to precipitate reduced hsematin from a solution of cyan-reduced hsematin. If then the concentration of cyanide is increased, cyan-hsemochromogen is formed rather than cyan-reduced hsematin. The dissociation curve for 1 molecule of cyanide per molecule of reduced hsematin was repeated in the absence of excess salts. The hsematin and cyanide solutions used for making up the mixtures were both 0*0033 M. The hsemin solution for the trough was

Aeh

r.



122 R Hill.

diluted 2-3 X 40 times. By trial it was found that the maximum amount of cyan-reduced haematin at this dilution of hsematin was formed by adding 2 equivalents of cyanide, and this was used as the standard, being reduced with the minimum quantity (12 mg. in 24 c.c.) of solid sodium hydrosulphite. The mixtures were made up as follows : to about 19 c.c. of 0-04 per cent, sodium hydroxide cyanide and hsematin were added in equivalent amounts so that the total volume was 20 c.c. The mixture was then reduced by adding 10 milligrammes of solid sodium hydrosulphite, and the relative amounts of cyan-reduced and reduced hsematin estimated.

In fig. 5 the 1 : 1 Htn to cyanide curve is different according to the amount of salt present, the points obtained in the experiments described in this section being shown by crosses. Where the curves are dotted the two cyan compounds are present. I t is seen that under the conditions of the experiment it is not possible to obtain the compound with 1 molecule of cyanide at a concentration where dissociation is negligible, in a solution containing equivalent amounts of haematin and cyanide. If the solution is made more concentrated, reduced haematin and the ordinary cyan-haemochromogen which contains 2 molecules of cyanide appear. Over the range of dilution where only reduced haematin and cyan-reduced haematin are present in significant quantities, the values obtained agree within the error of the experiments with the dissociation curve for Htn + KCN = HtnKCN with a dissociation constant of 1 • 3 X 10“5 at 16° C. The curve is calculated from the equation K& — {a — x)2 where x = cyan-reduced haematin a = total cyanide and total haematin. This curve is represented by the fine continuous line.

I t will be seen that these curves are in agreement with the experiments of Anson and Mirsky (1928) on the compound which they describe containing 1 molecule of cyanide. They started with a 2 X 10“~4 M. solution of cyanide and of haematin (represented as log dilution by 3-70) where they found there was but little dissociation. Then, on using more dilute solutions, more than 1 molecule of KCN has to be added to cause the disappearance of reduced haematin. I t is remarkable that 2 X 10“4 molar seems to be the strongest solution of the compound containing 1 molecule that can be obtained under usual conditions, and also it is apparently the strongest solution mentioned by Anson and Mirsky in connection with this substance. I t can also be seen from the figure that to produce ordinary cyan-haemochromogen at this dilution of haematin the cyanide concentration must be increased at least ten times.



7. Nicotine Cyan-Hcemochromogen.If nicotine is added to a solution of cyan-reduced hsematin a spectrum

similar to that of nicotine hsemochromogen is produced at a concentration of nicotine insufficient to produce more than a trace of nicotine hsemochromogen in the absence of cyanide. The same spectrum is produced if a strong solution of hsematin (10-2 M.) containing 1 equivalent of cyanide and 1 equivalent of nicotine is reduced. This spectrum is not that of an optical mixture of nicotine and cyan-hsemochromogens. I t would appear that this spectrum is due to a compound of reduced hsematin with both cyanide and nicotine. The a band is less intense than that of nicotine hsemochromogen and the (3 band more nearly equal to the a band. The dissociation of this substance was examined under the same conditions as pyridine and nicotine and cyan- hsemochromogens. The hsematin solution used was 0*0033 M. The hsematin solution for the double wedge trough was diluted 2*3 X 40 times. The hsemochromogen standard contained 4 molecules of KCN and 4 molecules of nicotine per molecule of hsematin. In the dissociation curves shown in fig. 6

Reduced Hcematin and Hcemochvomogen. 123

3 0 3 J JfOtyio dilution of /CC/t -f- nicotine .

Fig. 6.—KCN nicotine hsemochromogen.

the molecular dilutions of cyanide and nicotine were equal in every case, and are represented by dilutions of nicotine and cyanide. For example, with the curve representing the dissociation of 1 molecule of reduced hsematin to 1 molecule of the nitrogen compound, the latter is represented by 0 • 5 molecules of nicotine + 0*5 molecules of cyanide. This method was chosen to give results directly comparable with those obtained with pyridine, nicotine and cyanide alone. Over the range of experiment recorded in fig. 6, in the curves represented by continuous lines, reduced hsematin and nicotine cyan- hsemochromogen were the only substances present in appreciable quantities. Where the curves are dotted appreciable amounts of other possible compounds, such as cyan-hsemochromogen and cyan-reduced hsematin, were present.



124 R. Hill.

8. Discussion.The object of the experiments described in this paper is to show the minimum

number of molecules of a nitrogen compound which must combine with reduced haematin to produce typical haemochromogen, as defined by its spectrum. Five cases have been studied : pyridine, nicotine, KCN, nicotine and KCN together, and pyridine with modified haematin. The figures (2 to 6) give the experimental results. The curves are drawn freehand through the determined points, and show the effect of dilution on the ratio of free haematin to that in the form of haemochromogen, expressed as a percentage of the total haematin, when it is reduced in the presence of a definite molecular proportion of a nitrogen compound. The values given on the diagrams represent at the higher concentrations pigment in solution together with pigment as a precipitate. The percentage of reduced haematin is plotted as ordinate and the Iogi0 of the molecular dilution of total nitrogen compound is plotted as abscissa. The percentages of haemochromogen are read on the ordinates in the reverse direction to the percentages of reduced haematin. The dilution of total haematin is known at any point, because it bears a constant ratio to the total nitrogen compound for any one curve. This method of plotting was used because it is the most convenient one for showing the results on a single diagram. As it is a somewhat unusual method of dealing with dissociable compounds a figure showing a simple dissociation is given, plotted in the same way as the experimental results. The curves given in fig. 7 show calculated values for an equilibrium of the type K . [HtnA2] = [Htn] [A]2, where Htn could represent

Fig. 7.—Theoretical dissociation curves for a reaction of the type HtnA2 ^ Htn + 2A, with dissociation constant of K = 4-8 X 10 ~ 8 corresponding to the experimental curves.

reduced hsematin and A a nitrogen compound, the value of K being 4 - 8 x 1 0-8. These curves show the effect of dilution on the relative concentrations




of Htn and HtnA2 in three mixtures of haematin and nitrogen compound in the molecular ratios 1 : 1, 1 : 2, and 1 : 10. In “ zero dilution ” the 1 : 1 curve will have become parallel with the abscissa at a value of 50 per cent. HtnA2, while the 1 : 2 and 1 : 10 curves will have reached the value of 100 per cent. HtnA2, both pigments being assumed to be infinitely soluble. In each case the five experimental series of curves become sensibly parallel to the abscissa at a concentration of hsematin less than 10"2 M., and here the values correspond in each case to the values at “ zero dilution ” of the theoretical curve given in fig. 7. As in these cases we have different haemochromogens possessing different solubilities the only inference we can make is that for some reason the haemochromogens are practically undissociated in a concentration of total haematin of 10"2 M., and that in each case therefore haemochromogen is a compound of the type HtnA2.

The experimental technique is arranged so that the nitrogen compound can react with reduced haematin at the moment of its formation from the haematin by the action of sodium hydrosulphite. At the higher concentrations of haematin the haemochromogen may at once precipitate. Now the precipitated or aggregated haemochromogen can be shown in the case of pyridine haemochromogen, by the simple method of adding it to water containing some sodium hydrosulphite and sodium carbonate, to be very slow in dissociating, several days being required before complete dissociation. A sample of reduced haematin which has been allowed to aggregate for the same length of time as the haemochromogen will react at once with pyridine solution (10” 2 M.) in about 1 second to give haemochromogen. If the solution of haemochromogen is dilute enough to avoid aggregation it will dissociate quite rapidly. This means that haemochromogen once precipitated is very slow in passing into solution. The result of this peculiarity of haemochromogen is that when the mixtures are prepared by the method described here the nitrogen compound will appear to have a very high affinity for the reduced haematin in a strong solution. In my previous paper (1926, p. 429) I mentioned the fact that the dissociation curve of pyridine haemochromogen had a strongly S-shaped form, and it is the property of haemochromogen when aggregated which makes this appear to represent a reaction of a high order. Hence it can be seen that beyond a certain concentration dissociation of the haemochromogen may be negligibly small.

Mirsky and Anson (1929) have used the facts that both reduced haematin and haemochromogen aggregate, in attempting to show the impossibility of deciding the true composition of pyridine haemochromogen from my experiments (1926). I t seems, however, that it is the aggregation of the haemo-



126 R. Hill.

ckromogen which makes the experiment possible in the case of pyridine. That pyridine haemochromogen becomes aggregated even in very dilute solutions can be seen by observing its spectroscopic changes by the method introduced by Keilin (1926) for the study of pigments related to haematin. A freshly prepared dilute solution (10-5 M.) of pyridine haemochromogen shows its absorption bands much nearer to the violet end of the spectrum than a solution which has stood for some minutes.

Cyan-haemochromogen is more soluble than pyridine, nicotine or nicotine cyan-haemochromogens. I t can be seen from the diagram representing the effect of dilution on the cyanide compounds in presence of salts that the three curves of haematin to cyanide in the ratio of 1 : 1, 1 :2 and 1 : 10 are more widely separated than in the case of the other haemochromogens. If we suppose that cyan-haemochromogen contains two molecules of KCN and calculate for the different dilutions the free haematin in solution from the equation

[Htn] = K [Hchr] [KCN]2 ’

choosing K = 4 • 8 X 10"8, Htn representing reduced haematin, it is found to be constant over a certain range of dilution. This concentration of reduced haematin would represent the solubility of the substance under the conditions of the experiments. As soon as any haemochromogen precipitates, as total haemochromogen is actually measured the calculated value of the reduced haematin will rise; from this we can infer the solubility of cyan-haemochromogen. When the haemochromogen precipitates we find that the calculated value of the free KCN in solution begins to fall which is in accordance with the property of aggregated haemochromogen mentioned in connection with pyridine. These results are shown in Table III. The mean value of the solubility of reduced haematin is 1*8 X 10“5 and 1*0 X 10”5 in the two experiments. The solubility of cyan-haemochromogen is exceeded at 5*3 X 10“4 M. in one experiment and 5*6 X 10”4 in the other where the concentration of free cyanide is respectively 9 • 5 X 10”4 M. and 8 • 6 X 10“4 M. The agreement, however, is sufficient to justify application of the equation. In Table II I the values for free reduced haematin and haemochromogen are given in parenthesis when the haemochromogen is beginning to precipitate, and the calculated value for the free haematin rises beyond its limit of solubility.



Reduced Hcematin and Hcemochromogen.

Table II I .—Haematin : KCN = 1:2.

127

Log dilution of total KCN.

PercentageHchr.

Free KCN X 10“5.

Free Htn.x io -6.

Hchr X io -‘.

4-08 5 7-9 1-6 0-213-78 12 14-6 2-2 1 03-58 15 22-3 1-6 2*03-48 20 26-5 2*2 3-33-28 21 41 1*6 5-5318 24 50 1*5 8-02-98 31 73 1-5 162-88 41 78 2-1 272-70 52 95 (2*7) (53)2-56 62 89 (5*7) (93)

Haematin : KCN = 1 : 1 .

3-58 4 24 0*9 1053-28 8 44 1*0 4-22-98 13 78 1 1 13-52-68 27 86 (3-6) (56)2*38 42 69 (38) (174)2-08 49 18 (600) (407)

In the case of cyanide two definite compounds are formed with reduced haematin, and one of them, cyan-haemochromogen, behaves in a similar way to pyridine and nicotine when the effect of dilution is studied in the presence of salts. The mixed compound of KCN and nicotine closely resembles nicotine haemochromogen. Where the haematin solution is sufficiently dilute the value

[Htn] [Ncpd]2 Hchr

is nearly constant.* Table IV shows this value calculated from the experiments on the mixtures of haematin to nitrogen compound in the ratio of 1 : 10. The constant for nicotine cyan-haemochromogen was obtained from the relation

^H tii]^ = ([Nicotine] + [Cyanide])2,

where the concentrations of nicotine and cyanide are equal. This gives a number directly comparable with the other constants while the actual dissociation constant will be J of this = 0-38 X 10"8.

* [Ncpd] represents the molecular concentration of pyridine, nicotine, KCN, or nicotine + KCN.



128 R Hill.

Table IV.

Logmolecular dilution of nitrogen

compound.

Percentagehsemo-

chromogen.

(Ncpd)2 x io -7.

Htn h I (Nc^X 10-7 = K

x io -7.Hchr

Pyridine .................. 3-28 3 2-8 32 902-97 11 11 8-1 89

Nicotine .................. 4-18 4 0 044 24 1 03-88 12 0 174 7-33 1-23-58 41 0-692 1-44 1-0

Cyanide...................... 3-78 37 0-28 1-7 0-473-48 69 11 0 0-45 0-503-18 90 4-37 0-11 0-48

Cyanide and 4-08 37 0-069 1-70 0-12nicotine 3*78 58 0-28 0-73 0-20

3-48 89 1-10 0-12 0-13

The dissociation of KCN-nicotine hsemochromogen is smaller than either of those for cyan-haemochromogen or nicotine haemochromogen, as would be expected if the mixed compound is the principal haemochromogen formed in the presence of cyanide and nicotine. These values of the equilibrium constants are, of course, only approximate, as quite a small error in the determination of the percentage of reduced haematin and haemochromogen produces a large variation in the constant. They are merely given to show the relative affinities of reduced haematin for the different substances under ordinary conditions, and to show that reduced haematin and haemochromogen behave in solution like the simple molecules Htn and HtnA2.

In the presence of salts cyan-haemochromogen behaves in the same way as the other haemochromogens, being in equilibrium with free reduced haematin ; cyan-reduced haematin, the compound containing 1 molecule of KCN, does not appear except to a very small extent. As this is true for solutions where reduced haematin appears to be in solution there must be two effects due to salts on the cyanide equilibrium. The principal effect is due to a precipitation of reduced haematin, but there must be a second effect without precipitation of the haematin. As cyan-reduced haematin does not aggregate at higher concentrations, and there is the possibility of forming cyan-haemochromogen which does aggregate, cyan-reduced haematin does not appear in strong solutions under any conditions. Here the reaction proceeds in the direction of the arrow

2HtnKCN Htn (KCN) 2 + Htn




because reduced haematin precipitates and at high concentrations Htn.KCN2 will also precipitate. A solution of cyan-reduced haematin of a concentration of 10-4 M. is dissociated by warming and also dissociated by cooling to 0° C. owing to the separation of reduced haematin. The same effect is produced by the addition of sodium chloride or sodium hydroxide in excess. In a similar solution containing a slight excess of cyanide where both cyan-haemochromogen and cyan-reduced haematin are present the equilibrium can be shifted in favour of the former compound without showing any visible precipitation of reduced haematin if small amounts of sodium chloride are added.

While in the case of cyanide there are the two compounds cyan-reduced haematin and cyan-haemochromogen, nicotine and pyridine give only the one type of haemochromogen which resembles cyan-haemochromogen. Cyan- reduced haematin will combine with nicotine giving a haemochromogen containing both cyanide and nicotine, and this behaves in a similar way to cyan- haemochromogen. In the case of the four typical haemochromogens, they are, in order of increasing solubility, pyridine, nicotine, nicotine and cyanide, and finally cyan-haemochromogen, the corresponding dissociation constants are respectively 8-9 X 10~6, 1-1 X 10-7, 1-5 X 10-8 and 4*8 X 10~8. Hence

'the solubility does not necessarily depend on the affinity for the nitrogen compounds but rather on their nature. By the action of nicotine and cyanide on reduced haematin we can obtain four different compounds all possessing different spectra and properties, these compounds are represented as

Nicotine /Nicotine /KCN /(H 20)H tn ' H tn ( H tn( H tn(

^Nicotine XKCN XKCN XKCN

where Htn = reduced haematin. The first three are to be considered as typical haemochromogens and show the property of aggregating in strong solutions, while the fourth can only be obtained in solution, and might reasonably be supposed to contain a molecule of water.

The conclusion I wish to present is that the haemochromogen spectrum is due in general to a combination of reduced haematin with two molecules of some other compound. The two extra molecules are co-ordinated with the ferrous iron atom, and in passing from reduced haematin to haemochromogen it must be supposed that the pigment itself undergoes some slight change in structure to account for the marked alteration in type of absorption bands. In the varied conditions in which we find haematin in nature its physiological properties seem to be connected with the possibility of forming molecular compounds whether it is as haemoglobin or haemochromogen. From a chemical



130 Reduced Hcematin and

point of view the hsematin-haemochromogen equilibrium, which can be studied by simple spectroscopic methods, has an intrinsic interest for our knowledge of co-ordination compounds.

9. Summary.(1) A method suitable for estimating spectroscopically haemochromogen and

reduced haematin over a large range of concentration is described.(2) Reduced haematin has a solubility of 10~4 M. to 10“5 M., depending on the

salts present in the solution.(3) Pyridine, nicotine and cyan-haemochromogens contain two molecules of

pyridine, nicotine and alkali cyanide per molecule of reduced haematin.(4) These haemochromogens behave as simple substances of the type

HtnA2, but readily aggregate in strong solutions.(5) The approximate values of the dissociation constants at 16° C. were

found to be pyridine 8-9 X 10-6, nicotine I d X 10“ 7, cyanide 4-8 X 10-8.(6) A haemochromogen containing 1 molecule of cyanide and 1 molecule of

nicotine per molecule of reduced haematin can be formed having a dissociation constant of about 0 • 38 X 10-8.

(7) The new haemochromogen described by Anson and Mirsky (1928), which they found contained 1 molecule of KCN per molecule of reduced haematin, is shown to be analogous to carbon monoxide reduced hsematin and the name “ cyan-reduced hsematin 5 5 is proposed for it, reserving the name “ cyan- hsemochromogen 55 for the compound containing 2 molecules of cyanide. The dissociation constant of cyan-reduced hsematin is approximately 1*3 X 10~5.

I wish to thank Sir Frederick Hopkins for his interest, and Dr. D. Keilin for the help he has given me in this work.

10. REFERENCES.Anson, M. L., and Mirsky, A. E., 4 Journ. Physiol.,5 vol. 60, p. 50 (1925).Anson, M. L., and Mirsky, A. E., 4 Journ. Gen. Physiol.,5 vol. 12, p. 273 (1928).Barcroft, J., ‘ The Respiratory Function of the Blood. Part II—Haemoglobin,5 University

Press, Cambridge (1928). •Dh6re, C., and Vegezzi, G., ‘ Journ. Physiol, et Path, generale,5 vol. 17, p. 44 (1917).Hill, R., ‘ Roy. Soc. Proc.,5 B, vol. 100, p. 419 (1926).Keilin, D., 4 Roy. Soc. Proc.,5 B, vol. 98, p. 312 (1925).Keilin, D., 4 Roy. Soc. Proc.,5 B, vol. 100, p. 129 (1926).Keilin, D., 4 Roy. Soc. Proc.,5 B, vol. 104, p. 206 (1929).Krukenberg, C. F. W., 4 Vergleichend-physiol. Studien 5 (2nd series), Part II, p. 63 (1884). Mirsky, A. E., and Anson, M. L., 4 Journ. Gen. Physiol.,5 vol. 12, p. 581 (1929).Zeynek, R. v., 4 Hoppe-Seyler’s Zeit. Physiol. Chem.,5 vol. 70, p. 224 (1920).



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