The action of nitrite on haemoglobin in the absence of...

368 W. E. Bachmann and others

D escriptio n of P lates

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P la te 14F ig. 9—R a t A . Day 461. D ow ngrow th from epidermis resembling rodent ulcer,

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and non-squam ous (left) portions, x 35. (126/35).F ig. 12— R a t B . Colum nar arrangem ent of cells ju s t below epidermis, x 141.

547 • 963 • 4 : 546 - i73

The Action of Nitrite on Haemoglobin in the Absence of Oxygen

B y J . B r o o k s

Food Investigation Board of the Department of Scientific and Industrial Research, and Low Temperature Research Station, Cambridge

[Communicated by D. Keilin, F .R .S.— Received 23 April 1937)

Much work has been done on the action of n itrite on haemoglobin,* but there is little agreement on the course of the reaction. In the presence of a reducing agent H aldane (1901; H aldane and others 1897) detected only the form ation of NO-haemoglobin (the compound formed by the action of nitric oxide on reduced haemoglobin); in the presence of oxygen a m ixture of methaemoglobin and NO-haemoglobin was apparently obtained (cf. H aurowitz 1924; Meier 1925). On the other hand, H artridge (1920-1) believed th a t in the presence of oxygen one compound, nitrite-methaemoglobin, was formed. The constitution of NO-haemoglobin, usually assumed to be a ferro-compound of the same type as oxyhaemoglobin or C0- haemoglobin, is also doubtful. Anson and Mirsky (1925) state tha t NO-haemoglobin is a ferri-compound, the first product of the reaction

* A review of earlier work is given by Meier (1925).

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between NO and haemoglobin being methaemoglobin which then combines with NO.

The presence of oxygen probably complicates the reaction between nitrite and haemoglobin. I f the mechanism involves HNOa the presence of oxygen may result in an ill-defined concentration of NO. In addition, although the oxidation of haemoglobin to methaemoglobin by oxygen is relatively slow (Brooks 1931, 1935) n itrite m ay act as catalyst. Finally, in the presence of oxygen and excess n itrite compounds other than m ethaem oglobin and NO-haemoglobin are certainly formed (Meier). For these reasons the reaction was studied quantitatively in the absence of oxygen.

Material and Methods

Sodium nitrite was added to haemoglobin in a phosphate buffer (a) in the absence of oxygen, (6) in the presence of a reducing agent. The H artridge reversion spectroscope was used to identify and estim ate the products; later a gasometric method was found which confirmed the spectroscopic results.

Sodium Nitrite. A.R. NaNOa was twice recrystallized and solutions were freshly prepared before use.

Buffer Mixtures. The reaction mixtures were buffered with K 2H P 0 4 and K H 2P 0 4; the to ta l concentration of phosphate was 0-1 M. The pH was obtained from the data of Cohn (1927) and Green (1933) and by the glass electrode.

Reducing Agents. Sodium hydrosulphite and titanous ta rtra te solutions were prepared immediately before use. The 4 % N a2S20 4 solution contained enough NaOH or N H 4OH to make the pH roughly 7. The titanous ta rtra te solution (Conant, Scott and Douglass 1928) was made by adding 0*4 c.c. 15% titanous chloride solution (B.D.H.) to 1-6 c.c. of a solution saturated with sodium potassium ta rtra te and 0*2 M with respect to H 3B 03 and 1-8 M with respect to KOH.

Haemoglobin. Laked blood and solutions of pure haemoglobin were used. Defibrinated pig’s blood was laked by several freezings and thawings. Pure haemoglobin was prepared from pig’s blood by the method of Adair and Adair (1934). Both solutions were stored a t — 20° C. and portions thawed when required. The manometric apparatus of van Slyke and Neill (1924) was used to measure the following concentrations in the thawed solutions before u se :

(1) Haemoglobin—from the CO capacity determined by the method of van Slyke and Hiller (1928).

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370 J. Brooks

(2) Total pigm ent—from the CO capacity after reduction with Na2S20 4 determ ined by the m ethod of van Slyke and Hiller (1929).

(3) U ndenatured pigm ent—from the 0 2 capacity after reduction with titanous ta rtra te determ ined by a slight modification of the method of Conant, Scott and Douglass (1928).

The concentration of haemoglobin is given by (1), methaemoglobin by (3) minus (1), and denatured haemoglobin by (2) minus (3). There was never any denaturation during storage; there was, however, a very slow oxidation of haemoglobin to methaemoglobin a t — 20° C. Analyses of a solution of pure haemoglobin after preparation are given below. The results are expressed in vols. % , i.e. c.c. CO or 0 2 (0° C., 760 mm. Hg) combined w ith or liberated from the pigm ent contained in 100 c.c. The concentration in equivalents per litre is given by (vols. %) x 10/22,400 (a gram-molecule of haemoglobin containing four equivalents):

(1) Haemoglobin 28-93, 28-99 vols. %(2) Total pigm ent 30-12, 30-12 vols. %(3) U ndenatured pigm ent 30-03, 30-25 vols. % .

I t will be seen th a t during preparation about 4 % methaemoglobin was formed bu t there was no denaturation.

Nitric oxide. NO was prepared in an all-glass apparatus by the method of Noyes (1925).

Oxygen-free nitrogen. Cylinder N2 was freed from 0 2 by the method of K autsky and Thiele (1926).

The experim ents were usually carried out a t roughly 15° C .; the same final values were obtained a t 0° C.

Spectroscopic R esults

Comparison of NO-haemoglobin and nitrite-haemoglobinH aldane’s result was confirmed by comparing (1) the positions of the

mid-points of the two absorption bands of NO-haemoglobin with (2) those observed when excess N aN 02 was added to a haemoglobin solution containing N a2S20 4.

(1) Excess NO was adm itted into the evacuated vessel.* A (fig. 1) containing (a) laked blood *j* diluted in a phosphate buffer, (6) pure haemo-

* W ith th e H artridge spectroscope available it was difficult to obtain accurate readings unless a flat-sided vessel was used.

t The blood was used im m ediately after slaughter to avoid methaemoglobin form ation during storage.



The Action of Nitrite on Haemoglobin 371

globin in a phosphate buffer containing 0-1 % Na2S20 4 (any methaemoglobin present or formed was therefore changed to reduced haemoglobin). The vessel was evacuated (with a Cenco pump) and filled with 0 2-free N2 three times, followed in the case of (a) by shaking the solution vacuo for 30 min. Oxygen was previously removed from the NO-generating vessel B and its reagents by the passage of N2. The long thin-walled capillary C allowed A to be shaken. The position of the bands was the same in (a) and (b). No prior formation of methaemoglobin was observed in (a), none could occur in (6) (cf. Anson and Mirsky 1925). Similar results have recently been obtained by Keilin and H artree (1937).

F ig. 1— Action of NO on a haemoglobin solution.

(2) Excess N aN 02 was added to laked blood or pure haemoglobin in a phosphate buffer containing 0-1 % N a2S20 4. Mixing was first carried out in a sealed vessel filled with N2 bu t this was found to be unnecessary; it was sufficient to cover the m ixture w ith a thick layer of liquid paraffin. The positions of the only visible bands are given in Table I.

Banda-band/?-band

T able I

N O -haemoglo bin A

57535371

Excess N a N 0 2 added to haemoglobin in

the presence of N a2S20 4

A57505369

The values are the mean of several experim ents; different values varied by less than 3A. The position of the bands was independent of the p H (6-22-7-75). The only detectable product of the action of nitrite on haemoglobin in the presence of Na2S20 4 is apparently NO-haemoglobin.

Vol. CXXIII—B.



372 J. Brooks

In these and other experiments laked blood and pure haemoglobin gave the same results and no further distinction is made between them.

A calibration curve for m ixtures of reduced haemoglobin and N 0- haemoglobin was obtained w ith the usual double-wedge trough technique. The m ixtures showed one broad, ra ther ill-defined b a n d ; * the mid-point of this merged band for different proportions of the two pigments is given in fig. 2. The same curve was given a t different pH values. I t was difficult to measure the position of the band accurately and each value is the mean of twelve readings.

100

80a

'rO

S,bo 60os<Dc3•*? 40O'

^ 20

05560 5570 5580 5590 5600 5610 5620

M erged band (A)

F ig . 2— Position of m erged band of reduced haem oglobin and NO -haemoglobin.

A solution of N aN 02 was added to haemoglobin in a phosphate buffer containing 0*1 % f (or more) N a2S20 4. After covering the mixture with paraffin the position of the merged band was measured a t intervals until a final value was obtained. Some results a t p H 6*22 are given in Table I I ; each value is the mean of a t least six experiments.

W ithin the limits of the ra ther large experim ental error 1 mol. N aN 02 changed completely one equivalent reduced haemoglobin to NO-haemoglobin in the presence of N a2S20 4. The same result was obtained over the pH range 6-22-6-63, and a t p H 6-22 when the concentration of haemoglobin

* A t points corresponding to very high proportions of NO-haemoglobin the a- and /?-bands could be distinguished on the background of the single broad reduced haemoglobin band.

f Increasing the concentration of N a2S20 4 above this value had no effect on the results obtained.

The action of nitrite on haemoglobin in the presence of Na2S20 4

\<

N

k

7Z ~



was increased twentyfold (thinner flat-sided cells wrere used for the higher concentrations).


T able I I —2 x 10~6 E quivalents R educed H aemoglobin

in 10 c.c. B u f f e r ; pH 6-22; 15° C.Position. E quivalents

Mol. N aN 0 2 of band N O -haemoglobin N O -haemoglobinadded A % formed

0 5620 0 00-5 x 10-6 5603 25 0-5 x IQ-61 0 5581 50 1 01-5 5560 75 1*5

2-0 (a -b an d 5750 \/?-band 5370 100 2 0

The rate of reaction decreased writh increasing pH and was relatively very slow a t pH 7* 16-7*75. Over this range final values were not obtained in the presence of an equivalent am ount of N aN 02* in 6 hr. a t 15° C.

For other reasons similar measurements were made using a phosphate buffer containing NaCl. The to ta l concentration of phosphate was OT M and the ratio [H2PO t ]/[HPOT] was 4. A decrease in p H with increasing ionic strength would be expected. Values of the pH , determined by the glass electrode, are given in Table I I I ; they can be represented approximately by the following equation (cf. Cohn 1927; Green 1933):

pH = 7 -20 -1*5 yjfl

1 + 1*5 Vp + K s/i — log[H2P 0 4- ] [H P 0 4=] ’

where p is the ionic strength and K s is given the value —0-27.

T able I I IConcentration

of NaCl Ionic pTL pH.% streng th observed calculated

0 0 1 4 6-22 6-205 1-00 5-66 5-73

10 1-85 5-38 5-4315 2-71 51 5 5-15

The same result wras obtained in each buffer m ixture—complete change of one equivalent reduced haemoglobin to NO-haemoglobin by 1 mol. N aN 02 in the presence of N a2S20 4. The rate of reaction increased with decreasing pH (or increasing salt concentration). The times for 50 % change

* A t p H 7-75 reduced haemoglobin is changed to NO-haemoglobin com pletely in the presence of excess N aN 0 2.

2 c 2



374 J. Brooks

of reduced haemoglobin to NO-haemoglobin w ith the experimental conditions used were very roughly 10, 4 and 1 min. a t pH 5*66, 5-38 and 5*15 respectively and 15° C.

The action of nitrite on haemoglobin in the absence of a reducing agentIn these experim ents no reducing agent was u sed ; oxygen was removed by

evacuation. The haemoglobin solution contained therefore reduced haemoglobin and a small am ount of methaemoglobin (present initially in the stock solution).

Bulb A (fig. 3) contained haemoglobin in a phosphate buffer, bulb B an aqueous solution of•Jo

c

• a 0 , 8

F ig. 3—Vessel used for m ixing solutions of n itrite and haem oglobin.

N aN 02. A fter evacuating and filling with N2 three times the solutions were shaken vacuo for 30 min. and the vessel sealed off a t C. After mixing the solution was transferred to the cell D.

The following bands were finally observed at p H 6-22 on adding 2-18 x 10~6 mol. N aN 02 to 4-36 x 10-6 equivalents reduced haemoglobin (O lO xlO -6 equivalents methaemoglobin were initially present bu t not detectable spectroscopically*) :

B andA R em arks

6327 R ath e r fa in t band5752 —

5366 —

ca. 4910 Ill-defined band

The two outer bands have the same position as the and #-bands of methaemoglobin (pig’s methaemoglobin, p H 6*22) and the two inner bands as the cl- and /?-bands of NO-haemoglobin.| Methaemoglobin and NO- haemoglobin were therefore the only detectable products (the a-band of methaemoglobin was also observed by Meier when nitrite acted on haemoglobin in an atm osphere of hydrogen).

The presence of methaemoglobin was confirmed by the addition of Na2S20 4. The two outer bands disappeared and a broad merged band

* Before m ixing, the solution in A was transferred to D and no methaemoglobin bands were visible.

t Using the double-wedge trough and solutions of NO-haemoglobin and m ethaem oglobin it was found th a t th e position of these bands was the same a t different points along the trough. The /?- and y-bands of m ethaem oglobin were no t visible w ith the thickness of cell and the concentrations used.




(reduced haemoglobin plus NO-haemoglobin) appeared a t 5580 A, corresponding to 50 % of each compound (fig. 2). Roughly therefore 2-18 x 10~6 mol. N aN 02 formed 2-23 x 10-6 equivalents NO-haemoglobin and 2-13 x 10-6 equivalents methaemoglobin (since NO-haemoglobin is stable in the presence of Na2S20 4).

W ith smaller relative am ounts of N aN 02 two methaemoglobin bands and a merged band of NO-haemoglobin and uncombined reduced haemoglobin were seen. Let the position of the merged band correspond to a fraction of NO-haemoglobin in the m ixture of NO-haemoglobin and reduced haemoglobin. Methaemoglobin was then changed to reduced haemoglobin by the addition of N a2S20 4; let the position of the new merged band correspond to a fraction y of NO-haemoglobin. I f the initial am ount of reduced haemoglobin plus methaemoglobin was a, and the initial am ount of methaemoglobin was b equivalents, th e n :

Equivalents NO-haemoglobin formed = ay,Equivalents methaemoglobin formed ay/x .

The results of several experiments analysed in this way are given in Table IV, the jpH was 6*22.

T able IV

In itia l am ount to ta l pigm ent = 4-46 x 10-6 equivalents In itia l am ount m ethaem oglobin = 0-10 — 0-25 x 10~6 equivalents

Mols. N aN O a added

2-18 x 10-6 1-36 1-20 1-09

Equivalents N O -haemoglobin

formed2-23 x 10-61-26 1-21 1-09

E quivalentsm ethaem oglobin

formed2-13 x 10-6 1-50 1-30 0-90

Roughly therefore 1 mol. N aN 02 gives one equivalent NO-haemoglobin and one equivalent methaemoglobin. I t would be expected th a t the values in the last column would be less consistent than those in the second.

Gasometric ResultsAs spectroscopic analysis was rather unsatisfactory owing to the lack of

definition of the bands another method, using van Slyke’s manometric technique, was found.

W ith a m ixture of NO-haemoglobin, reduced haemoglobin and methaem oglobin, the CO or 0 2 capacity gives the concentration of reduced haemoglobin and the CO capacity after reduction with Na2S20 4 (or the 0 2 capacity after reduction with titanous ta rtra te) gives the sum of the concentrations



376 J. Brooks

of reduced haemoglobin and methaemoglobin if it can be assumed that NO-haemoglobin or a derivative is inert (as regards gas evolution or uptake) during both determ inations. The determ inations include an equilibration with CO or 02 and the action of an acid K 3Fe(CN)6 reagent.

The inertness of NO-haemoglobin to acid K 3Fe(CN)6* was first established. To 3-67 x 10-5 equivalents reduced haemoglobin in a phosphate buffer containing N a2S20 4 was added 3-67 x 10-5 mol. N aN 02. W hen only N0- haemoglobin was detectable spectroscopically, 2 c.c. of the mixture (total volume 14-5 c.c.) were analysed by the usual method for to tal pigment, except th a t shaking in vacuo for 5 min. was substituted for shaking with 150 mm. CO. A value of 0-18 vol. % was obtained; the control (from an analysis of a solution of the same concentration of phosphate and Na2S20 4 bu t containing no haemoglobin or n itrite f) was 0-16 vol. % . The initial concentration of reduced haemoglobin in the m ixture was 5-67 vol. %. Although NO-haemoglobin was precipitated in some unknown form there was no gas evolution or uptake. A similar result was obtained using titanous ta rtra te in place of N a2S20 4.

I t was found th a t shaking for 5 min. w ith 150 mm. CO apparently changed about 23% of the NO-haemoglobin present to CO-haemoglobin, this combined CO being liberated on adding acid K 3Fe(CN)6. As haemoglobin has a much smaller affinity for 0 2 than for CO or NO, air was then used for equilibration; w ith 5 min. shaking there was no detectable decomposition of NO-haemoglobin. A m ixture of NO-haemoglobin, reduced haemoglobin and methaemoglobin can therefore be analysed (if the total concentration of the three pigments is known) by determ ining:

(i) the 0 2 capacity—reduced haemoglobin,(ii) the 0 2 capacity after reduction w ith titanous ta rtra te J—reduced

haemoglobin plus methaemoglobin.

The action of nitrite on haemoglobin in the presence of titanous tartrateIn these experim ents titanous ta rtra te was used in place of Na2S20 4 in

the reaction m ixture. W hen no further change was observed spectro-

* The more acid reagent of van Slyke and H iller (1929) was used: 92 c.c. 32% K 3Fe(CN)6 solution, 20 c.c. lactic acid (sp. gr. 1-2).

f Free N aN O a gives a gas evo lu tion ; 3-67 x 10-5 mol. N aN 0 2 in 14-47 c.c. phosphate buffer (containing N a2S20 4) analysed as for to ta l pigm ent gave a value of 2-06 vol. %, corresponding to 54% of th e to ta l NO which would be liberated if the reaction 3 H N 02-> H + + N 0 ^ -f 2N 0 + H 20 w ent to completion.

J N a2S20 4 cannot be used when th e 0 2 capacity a fter reduction is to be determined since the oxidation products of N a2S20 4 m arkedly accelerate the oxidation of haemoglobin to m ethaem oglobin by oxygen. T itanous ta r tra te is free from this objection (Conant, Scott and Douglass 1928).




scopically 2 c.c. of the m ixture, 3 drops caprylic alcohol and 4-7 c.c. H 20 were shaken with air for 5 min. in the chamber of the van Slyke apparatus with the mercury meniscus a t the 50 c.c. m ark and the tap open. The air was expelled and 0*3 c.c. acid K 3Fe(CN)6 reagent added. After shaking in vacuo for 5 min. any C 02 present was absorbed by the admission of 1 c.c. air-free NaOH and the residual gas measured in the usual way.* The details of one experim ent are given below:

Total phosphate 0-1 M; p H 6*41; 15° C.; 7*74 c.c. reaction m ixture contained in itia lly :

1 c.c. titanous ta rtra te solution,3-36 x 10-5 equivalents reduced haemoglobin,1*74 x 10~5 mol. N aN 02.

Analyses (02 capacity) after 2 | hr. standing under paraffin:

(a) 4.63 vol. % .(b) 4-66 vol. % .

Equivalents uncombined reduced haemoglobin

4-65 x 7-74 100 x 22400

1-61 x 10-5.

Equivalents NO-haemoglobin formed = (3-36—l-61)x 10-5= 1*75 x 10~5,

Mol. N aN 02 added = 1*74 x 10-5.

Results are summarized in Table V ; each value is the mean of duplicate determ inations.

T a b l e V—p H 6-18-6*58; 15° C.

Mol. N aN 0 2 added

Equivalents reduced E quivalentshaemoglobin initially NO-haemoglobin

present formed3-91 x 10-5 1-96 100 1-74 1-00

3-91 x 10-53-913-913-363-52

3-88 x 10~6 2030- 981- 75 101

* Equilibrating w ith 760 mm. air in the cham ber of the van Slyke apparatus gives a large value for the control (about 6 vol. %) b u t this can be determ ined very accurately by repeating the whole of th e procedure (om itting haemoglobin and n itrite from the reaction m ixture b u t no t the titanous ta r tra te ) and standardizing rates and times of shaking etc. A correction factor for the effect of tem perature on the value of the control was also determ ined.



378 J. Brooks

The results agree w ith those described previously; in the presence of a reducing agent 1 mol. N aN 02 gives one equivalent NO-haemoglobin.

The action of nitrite on haemoglobin in the absence of a reducing agent

In these experiments no reducing agent was used. A similar vessel to th a t shown in fig. 3 was used (the flat-sided cell was absent) and oxygen was removed by evacuation etc. A fter sealing off a t C and mixing (in vacuo) the solution was distributed between bulbs A and B and the vessel was divided in half by sealing off a t E. One-half was used for duplicate determ inations of the 0 2 capacity as described in the previous section, the other for the 0 2 capacity after reduction w ith titanous ta rtra te . In the second case 2 c.c. of the m ixture, 3 drops caprylic alcohol, 4-3 c.c. H20 and 0-4 c.c. titanous ta rtra te solution were shaken with air for 5 min. in the cham ber of the van Slyke ap p ara tu s ; the rest of the analysis has been described.*

The details of one experim ent are given below. Two corrections were necessary: (a) separate experim ent showed th a t about 2 % of the haemoglobin was oxidized to m ethaem oglobinf during evacuation; (b) there was a decrease in volume caused by evaporation during evacuation, this was measured by weighings.

Total phosphate 0*1 M; pH 6*22; 15° C.

Amounts of pigm ent present before adding N aN 02:Total pigm ent = 6-72 x 10~5 equivalents.Reduced haemoglobin = 5-86 x 10-5 equivalents.Methaemoglobin = 0*86 x 10-5 equivalents (0-12 x 10~5 formed during

evacuation).

Total volume of solution after mixing = 17*22 c.c.

A nalyses:(1) 0 2 capacity 0*22, 0*18 vol. % .(2) 0 2 capacity after reduction with titanous ta rtra te 4*95, 5*01

vol. % .

* This is essentially a slight modification of th e m ethod of Conant, Scott and Douglass (1928) for undenatured pigm ent. The preparation of the titanous ta rtra te solution in an atm osphere of N 2 was found to be unnecessary. I t was also more convenient to equilibrate the reduced solution in th e cham ber of the van Slyke appara tus th an in a tonom eter.

f The ra te of oxidation of haemoglobin to methaem oglobin by oxygen increases w ith decreasing oxygen pressure (Brooks 1935).




After adding N aN 02:

Equivalents reduced haemoglobin = 0-20100 x 22400 x 17-22

= 0-15 x 10~5.

Equivalents reduced haemoglobin plus methaemoglobin

4-98= --------------- x 17-22

100 x 22400

= 3-83 x 10-5.

NO-haemoglobin formed = (6-72 — 3-83) x 10~5

= 2-89 x 10-5 equivalents.

Methaemoglobin formed = (3-83 — 0-15 — 0-86) x 10-5

= 2-82 x 10~5 equivalents.

N aN 02 added = 2-90 mol.

Results are summarized in Table V I ; each value is the mean of duplicate determinations.

T able V I—pH 6-22-6-63; 15° C.

Mol. N a N 0 2 added

E quivalents N O -haemoglobin

formed

E quivalentsm ethaem oglobin

formed1-44 x 10-51- 632 - 00 2-90

1- 46 x 10~5 1-6020 42- 89

1-43 x 10-5 1-641- 992- 82

I t will be seen th a t 1 mol. N aN 02 gives one equivalent NO-haemoglobin and one of methaemoglobin.

D iscussion

Conant and his co-workers (1923, 1924-5, 1926, 1928, 1932) have shown th a t reduced haemoglobin and methaemoglobin form a reversible oxidation- reduction system of similar type to the ferrocyanide-ferricyanide system. The change of haemoglobin to methaemoglobin requires one hydrogen- equivalent of oxidizing agent per gram-atom of iron in the haemoglobin molecule (i.e. per one equivalent haemoglobin). I f reduced haemoglobin



380 J. Brooks

is represented by (H3X)4, methaemoglobin is then (H2X)4.* W ith the conditions of pH , etc., used 1 mol. N aN 02 in the absence of oxygen or a reducing agent gives one equivalent NO-haemoglobin and one of methaemoglobin, i.e. the reaction goes to completion and no n itrate is formed. I t follows th a t the stoichiometric equation for the reaction can be w ritten :

2 (H3 X )4 + 4H N 02 ->(H3 X . NO )4 + (H2 X )4 + 4H20 , (1)

where (H3X .N O )4 represents NO-haemoglobin. The results show clearly th a t NO-haemoglobin is a ferro-compound of the same type as oxyhaemoglobin or CO-haemoglobin since the one hydrogen equivalent of oxidizing agent available per mol. of H N 0 2 is fully accounted for by the production of one equivalent methaemoglobin.

In the presence of a reducing agent methaemoglobin is changed to reduced haemoglobin and the net reaction can be w ritten :

(H3X)4 + 4H N 02 + 2 R(̂ H 3X . NO)4 + 2H20 + (2)

where R represents the reducing agent and RO its oxidation product.The increase in the ra te of reaction w ith decreasing p H observed quali

tatively suggests th a t the mechanism involves H N 0 2. Abel and his co-workers (1928, 1930) have shown th a t the decomposition of H N 02 is given by the net equation :

3 H N 02 ->H+ + NOy + 2NO + H 20 (3)

and consists of two s tep s :

4H N 02^ N 20 4 + 2NO + 2H20 , rapidly established equilibrium, (4)

N20 4 + H 20 - kH N 02 + H + + N 0 y , rate-determ ining step. (

Since no n itra te is formed in the reaction between nitrite and haemoglobin in the absence of oxygen it appears th a t the net reaction (3) can be neglected. This is consistent w ith the range of p H and tem perature used. Kinetic measurements would be necessary to determ ine the reaction m echanism; a possible one is as follows:

8H N 02^ 2 N 20 4 + 4NO + 4H20 ,

4NO + (H3X)4->(H3X .N O )4,

2N20 4 + 3(H3X)4-> (H3X .NO)4 + 2(H2X )4 + 4H2(X

(4)

* The representation of reduced haem oglobin and m ethaem oglobin by (HgX)4 and (H 2X )4 respectively is sim ilar to the convention used by Conant and Fieser (1924—5). I t shows form ally the difference in valence of the iron-containing radicals in th e 2 mol.




A study of the action of N20 4 on reduced haemoglobin would be interesting in this connexion.

The results throw no light on the reaction in the presence of oxygen. I t is clear th a t NO-haemoglobin would not be the sole product, bu t the question whether derivatives other than NO-haemoglobin and methaemoglobin are formed is still unanswered.

Summary

The reaction between N aN 02 and haemoglobin in the absence of oxygen was studied spectroscopically and gasometrically. Both methods gave the same results.

In the presence of a reducing agent a compound is formed with the same absorption bands as NO-haemoglobin prepared by the action of NO o n , reduced haemoglobin.

There was no prior form ation of methaemoglobin in the reaction between NO and reduced haemoglobin.

In the presence of a reducing agent 1 mol. N aN 02 changed completely one equivalent reduced haemoglobin to NO-haemoglobin a t pH 5-15-6*63. The rate of reaction decreased with increasing pH and was relatively very slow a t pH 7-16-7-75.

In the absence of both oxygen and a reducing agent 1 mol. N aN 02 combined with two equivalents reduced haemoglobin to give one equivalent NO-haemoglobin and one equivalent methaemoglobin.

The results show th a t NO-haemoglobin is a ferro-compound of the same type as oxyhaemoglobin and CO-haemoglobin.

R efer en c es

Abel, E . and Schmid, H . 1928a Z. phy Chem. A, 132, 55.--------19286 Z . phys. Chem. A , 134, 279.--------1928 c Z. phys. Chem. A, 136, 430.

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518.42 : 612.816

A Graphical Solution of a Differential Equation with application to Hill’s Treatment of Nerve Excitation

B y W. A. H. R ushton

Research Fellow of Emmanuel College, Cambridge

From the Physiological Laboratory, Cambridge

{Communicated by A . V. Hill, Sec. R .S.—-Received 27 April, 1937)

I — Graphical A nalysis

Value of the MethodLinear differential equations with constant coefficients are very common

in physical and chemical science, and of these, the simplest and most frequently m et is the first-order equation

+y=M,f1)where a is a constant, and f{t) a single-valued function of t. The equation signifies th a t the quantity y is removed a t a rate proportional to the amount present a t each instant, and is simultaneously restored a t a rate dependent only upon the instan t in question. Fam iliar examples of this equation are



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