A Method for the Estimation of Iron in Biological Material Author(s): Robert Hill Source: Proceedings of the Royal Society of London. Series B, Containing Papers of a Biological Character, Vol. 107, No. 750 (Nov. 3, 1930), pp. 205-214 Published by: The Royal Society Stable URL: http://www.jstor.org/stable/81405 . Accessed: 07/05/2014 19:27 Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp . JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact [email protected]. . The Royal Society is collaborating with JSTOR to digitize, preserve and extend access to Proceedings of the Royal Society of London. Series B, Containing Papers of a Biological Character. http://www.jstor.org This content downloaded from 169.229.32.136 on Wed, 7 May 2014 19:27:18 PM All use subject to JSTOR Terms and Conditions
Transcript
A Method for the Estimation of Iron in Biological MaterialAuthor(s): Robert HillSource: Proceedings of the Royal Society of London. Series B, Containing Papers of aBiological Character, Vol. 107, No. 750 (Nov. 3, 1930), pp. 205-214Published by: The Royal SocietyStable URL: http://www.jstor.org/stable/81405 .
Accessed: 07/05/2014 19:27
Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at .http://www.jstor.org/page/info/about/policies/terms.jsp
.JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range ofcontent in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new formsof scholarship. For more information about JSTOR, please contact [email protected].
.
The Royal Society is collaborating with JSTOR to digitize, preserve and extend access to Proceedings of theRoyal Society of London. Series B, Containing Papers of a Biological Character.
http://www.jstor.org
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Estimation of Iron in Biological Material. Estimation of Iron in Biological Material.
3. The ease with which such agglutinins are formed seems to vary with the
degree of difference in character between the corpuscles of the injected fowl and those used for immunisation.
4. The red blood corpuscle must be regarded as a " multiple antigen " in the sense that it contains a large number of different antigenic units or ' receptors," which apparently behave as independent units when hereditarily
transmitted.
The writer wishes to record his indebtedness to the Medical Research Council, to Capt. S. R. Douglas, F.R.S., in whose department of the Institute the work was carried out, and to Major G. W. Dunkin, M.R.C.V.S., who very kindly
supervised the breeding and care of the fowls.
REFERENCE.
Todd, C. (1930). 'Proc. Roy. Soc.,' B, vol. 106, p. 20.
543 849:547 .828
A Method for the Estimation of Iron in Biological Material.*
By ROBERT HILL, Beit Memorial Research Fellow.
(Communicated by Sir Frederick Hopkins, F.R.S.-Received August 5, 1930.)
(From the Biochemical Laboratory, Cambridge.)
I. Introduction.
The intense red colour and the stability of the product of reaction between ferrous salts and ocx' dipyridyl has remained a striking instance of complex salt formation since the discovery of this dipyridyl by Blau (1888). Although for obvious reasons this compound would seem a good reagent for the colori- metric estimation of iron, it has never been used to any extent for the purpose, without doubt because there are other more sensitive and accessible reagents. It seems, however, to possess unique advantages for the study of iron in con- nection with living material, and on this account it was thought advisable to
bring aa' dipyridyl, as a reagent for iron, to the notice of biologists. * Part of this work was carried out during the tenure of a senior studentship of the
Exhibition of 1851.
3. The ease with which such agglutinins are formed seems to vary with the
degree of difference in character between the corpuscles of the injected fowl and those used for immunisation.
4. The red blood corpuscle must be regarded as a " multiple antigen " in the sense that it contains a large number of different antigenic units or ' receptors," which apparently behave as independent units when hereditarily
transmitted.
The writer wishes to record his indebtedness to the Medical Research Council, to Capt. S. R. Douglas, F.R.S., in whose department of the Institute the work was carried out, and to Major G. W. Dunkin, M.R.C.V.S., who very kindly
supervised the breeding and care of the fowls.
REFERENCE.
Todd, C. (1930). 'Proc. Roy. Soc.,' B, vol. 106, p. 20.
543 849:547 .828
A Method for the Estimation of Iron in Biological Material.*
By ROBERT HILL, Beit Memorial Research Fellow.
(Communicated by Sir Frederick Hopkins, F.R.S.-Received August 5, 1930.)
(From the Biochemical Laboratory, Cambridge.)
I. Introduction.
The intense red colour and the stability of the product of reaction between ferrous salts and ocx' dipyridyl has remained a striking instance of complex salt formation since the discovery of this dipyridyl by Blau (1888). Although for obvious reasons this compound would seem a good reagent for the colori- metric estimation of iron, it has never been used to any extent for the purpose, without doubt because there are other more sensitive and accessible reagents. It seems, however, to possess unique advantages for the study of iron in con- nection with living material, and on this account it was thought advisable to
bring aa' dipyridyl, as a reagent for iron, to the notice of biologists. * Part of this work was carried out during the tenure of a senior studentship of the
Exhibition of 1851.
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sGe' dipyridyl was first prepared by Blan (1888) by distilling the copper salt of a picolinic acid; the yield is small, but this method has the advantage of readily
yielding a pure product. Recently Hein and Retter (1928) have found a
relatively simple method of preparation by heating pyridine in a sealed tube with an oxidising agent such as ferric chloride, which removes hydrogen without the formation of water. This method though more economical needs greater manipulation to produce the substance in a state of purity. Smith (1926) isolated the'same substance by the oxidation of sodium pyridines with dry air above 100? C. The ferrous complex salts were examined by Werner (1912) who showed that they could be resolved into two optical isomers. The ferrous salts of oaa' dipyridyl have the formula [Fe (C10oHN2)3] X2 (Blau, 1898) where X is a monovalent acid radicle, and from the fact that the optical isomers do not racemize at once (Werner, 1912) dissociation into free ferrous salts must be
relatively slight. They differ from the normal ferrous salts in not being easily oxidised, in fact only powerful agents such as permanganate or chlorine oxidise them to a blue compound. This latter compound, which should be the ferric
complex salt, is not obtained directly from ferric salts and oc' dipyridyl and
passes very easily into the red ferrous salt. These properties at once suggest the use of dipyridyl not only as a reagent
for detecting iron in the presence of reducing agents where other reagents are unsuitable but as a reagent for inhibiting the catalytic reactions of iron. It is from these considerations that the experiments described in this paper have been carried out.
II. Preparation o, Oa' Dipyridyl.
The method based on that of Blau (1888) was adopted. 45 g. of oc-picoline (boiling point 126-132? C.) were heated under a reflux condenser with 173 g. of potassium permanganate dissolved in 4-25 1. of water for 7 hours (Weidel,
1879). The colourless liquid was filtered from the hydrated oxides of man-
ganese and the alkalinity reduced by passing in a current of CO2. The bulk of the water was then distilled off together with the unchanged bases and the residual fluid evaporated to dryness over a water bath. The residue was
dissolved in 150 c.c. of water and filtered. Glacial acetic acid was then added until the liquid was acid (pH 5-6) and then about 180 c.c. of 1 per cent. copper
sulphate solution was added slowly until there was no further precipitation of the blue-violet crystals of copper picolinate. The copper salt was filtered off after 15 minutes, washed with cold water in which it is sparingly soluble and dried, first at room temperature, then at 105? C.; 40 g. were obtained.
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The dry copper picolinate was destructively distilled in a slow current of dry CO2 from a retort, the receiver being cooled in ice, until a dry blackish residue was left. The liquid distillate, about 15 c.c., containing aa' dipyridyl and
pyridine was heated to 50? C. on a water bath and a current of air blown through until the pyridine had been largely removed. The residue which solidified on cooling was then distilled with steam after the addition of a few drops of 40 per cent. soda. 1-4 g. of oa.' dipyridyl were obtained on cooling the dis- tillate. The distillation was then continued until the fluid gave only a faint colour with ferrous sulphate. This distillate added to the previous mother
liquor was evaporated with an excess of hydrochloric acid to dryness on a water bath. The residue was dissolved in a little water, made alkaline with soda and distilled with steam. 1 2 g. were obtained. The ao.' dipyridyl was obtained as colourless shining plates, melting point 71? C. (when dried over
KOH). It is sparingly soluble in water; the saturated solution at 20? C. is 0-025 molar and has a pH of 7-2. It is appreciably volatile at ordinary temperature and the vapour has an aromatic odour recalling that of vanillin. It is very stable to acids and alkalies and is unaffected by mild oxidising and
reducing agents. III. Method of Estimation of Iron.
On adding aa' dipyridyl to a solution of a ferrous salt between pn 3 5 and 8-5 the intense red complex ferrous ion is produced. The presence of other metals unless in great excess over the iron does not influence the colour. For
instance, a copper salt gives a blue colour which is nearly invisible at a con- centration of copper 1000 times that of the concentration of iron needed to render the pink colour just perceptible. Zinc on the other hand gives a colourless compound sparingly soluble in water (Blau, 1889). If other heavy metals are present in appreciable quantities it is necessary to use an excess of the reagent, when testing for iron, so that all the metals may be combined with the dipyridyl.
Ferric iron unless present in high concentration gives no colour and does not interfere with the reaction with ferrous iron. Thus it is a, simple matter by adding dipyridyl and measuring the intensity of the pink colour before and after reducing the iron to estimate ferrous and ferric iron in a mixture.
The ferrous dipyridyl salts are soluble in the presence of most anions. They are less soluble in the presence of an excess of iodide and are precipitated by tungstates and the usual alkaloidal reagents. If pyrophosphates are present, owing to the stability of ferric pyrophosphate, it is necessary to use a reducing agent and as long as the iron is kept in the ferrous state pyrophosphate does
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not influence the colour. Thiocyanates, while giving the intense red colour
with ferric iron, give no colour with ferrous iron and do not affect the ferrous
dipyridyl salts.
aa' dipyridyl combines, however, with ferric thiocyanate in presence of
HC1 giving an intense bluish-red substance insoluble in water and only sparingly soluble in ether and amyl alcohol. With a copper salt and thiocyanate in
the presence of acid orc' dipyridyl similarly gives a green-yellow substance insoluble in both aqueous and organic solvents.
If oco' dipyridyl is added to reduced hoematin there is no change in colour or absorption spectrum; the same is true if it is added to pyridine hsmo-
chromogen. If hsmatin is precipitated with acetate buffer in the presence of dipyridyl no colour can be obtained in the supernatant fluid on adding
reducer. This shows that the iron of hsematin is not removed by dipyridyl. It therefore follows that to estimate the inorganic iron in a fluid between
pa 3-5 and 8 5 all that is required is to add dipyridyl and a reducing agent, and determine the intensity of the pink colour.
IV. Purification of Sodium Hydrosulphite.
For a reducing agent on the acid side of neutrality sodium hydrosulphite seems most suitable. As this substance always contains iron it must be
purified before use. This is accomplished most easily by the following method.
The sodium hydrosulphite is dissolved in warm water (40? C.) to give a nearly saturated solution and a small quantity of oco' dipyridyl added so that the
maximum red colour is obtained. The dark red solution is then rapidly filtered on a Buchner funnel and the salt precipitated from the filtrate by alcohol. After standing 20 minutes the salt is filtered off and washed with 70
per cent. alcohol till colourless, then with 97 per cent. alcohol. The solid is
then boiled with 97 per cent. alcohol for 10 minutes, filtered off at once while
hot and immediately transferred to a vacuum desiccator containing'sulphuric acid. This preparation was found to keep well and to give no red colour
either with iron or aoa' dipyridyl, and if the manipulations are conducted with
reasonable speed there is no serious oxidation of the hydrosulphite during its
purification.
V. Preparation of Standard Iron Dipyridyl.
For the estimation of the pink colour it is most convenient to use a series of
standard tubes similar to those used for the determination of pH by means of
indicators. By this method it is possible to determine the iron with an
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accuracy of 10 per cent. down to a concentration of 0 0003 mg. Fe per cubic
centimetre. The standard tubes are most conveniently made up as follows. Stock solutions of M/100 ferrous iron (0-392 g. ferrous ammonium sulphate in 100 c.c.) and 3M/100 coa' dipyridyl hydrochloride (0.468 g. of aa' dipyridyl and 6 c.c. of N. HC1 in 100 c.c.) were prepared. These solutions were then
equivalent from the point of view of the coloured complex ion. A solution of iron dipyridyl M/10,000 in acetate buffer was prepared containing twice the theoretical amount of ooc' dipyridyl and a trace of sodium hydrosulphite. 5 c.c. of this solution were taken as the first of the series of standards, and the others made up as follows. For preparing 5 c.c. of a standard the formula x 5f(n-1) was used, where x is the number of cubic centimetres of the
M/10,000 solution of iron dipyridyl, f is a factor for the dilution, and n is the number of the standard, the fluid being made up to 5 c.c. with acetate buffer in each case. The factor was 19/21; this gives a difference equal to 10 per cent. of the mean iron concentration of two consecutive standards. For example, in the case of standard No. 2, x = 5 X 9-12-,1 = 4.52; buffer to be added = -048 c.c. Standard No. 1 contains 0-0056 mg. Fe per cubic centimetre and standard No. 2 0 0051 mg. Fe per cubic centimetre. By this method a
sertes of 24 standards was prepared giving a range of concentration of iron from 0-0056 mg. per cubic centimetre to 0 00056 mg. Fe per cubic centimetre, the limits found most suitable for general use. These were sealed off in tubes
1P3 cm. in diameter and 8 cm. long containing 5 c.c. of the fluid. To ensure the greatest permanence some SO2 should be passed through the liquid to convert a little of the acetate into sulphite. Prepared in this way a standard of M/10,000 Fe made 3 years ago was found to be unaltered on comparing it with a fresh standard.
VI. Effect of pa on the Colour given by Ferrous Salts and Dipyridyl.
On adding a dilute mineral acid to a dilute solution of the iron dipyridyl salt the colour slowly fades, showing that acids dissociate the complex. The same effect is produced by adding alkali. In order to define the limits of Pu suitable for the estimation of iron some experiments were carried out in buffer
mixtures, the concentrations of dipyridyl iron being measured by the standard tubes. On the acid side of neutrality glycine HC1 and phthalate buffers were used. 10-4 M. iron was present and the theoretical amount of dipyridyl (3 times 10-4 M.). The experiments on the association and dissociation of the complex were in agreement, equilibrium being established in 10 to 15 hours. The dissociation followed a rather steep curve extending from pin
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1.7 to pa 3 7 where there was nearly complete association. The presence of
excess of oa' dipyridyl shifted the curve along the scale to a more acid range. As aa' dipyridyl is appreciably soluble in water an excess can a lways be present when using it as a reagent. Hence on the acid side if neutrality it is safe to
reach p, 3 5. On the alkaline side owing to the complication of the oxidation
and precipitation of the iron it was not possible to obtain precise results.
Some experiments were made in glycine NaOH buffers and cysteine used as
a reducing agent. At a concentration of 10- M. Fe the dissociation extended
from pH 8.5 to pi 10. Hence on the alkaline side of neutrality it is safe to
reach -pt 8-5. The range of pi- is thus from 3 5 to 8 5, when an excess of dipyridyl is used.
For controlling the pa it is usually sufficient to add to the fluid to be tested a
solution of sodium acetate (free from iron) containing a little acetic acid, as
the iron compound shows its maximum stability throughout the range of
the acetate buffer mixtures. The iron compound can be detected up to p,. 8.5 if a slight excess of dipyridyl is added and hydrazine hydrate used as a
reducer at 40? C.
VII. Adsorption of Dipyridyl Iron by Insolzuble Proteins.
Werner (1912) pointed out that iron dipyridyl will dye animal fibres red.
It will not dye vegetable fibres and is not adsorbed by filter paper to an
appreciable extent. It is, however, adsorbed by proteins hence when esti-
mating iron in the presence of insoluble proteins care must be taken that the
iron is not lost through adsorption. In the case of fresh yeast the adsorption of added dipyridyl iron was found to be negligible. In the case of boiled yeast, there was about 50 per cent. adsorption of the iron dipyridyl at pu 6, but by
passing 802 in the presence of acetate until the pa was 4 the adsorption was very slight and the colour could easily be removed by washing. The same was found
to hold in the case of the proteins of boiled egg yolk. In 30 per cent. alcohol
the iron compound is readily soluble and shows no adsorption on either animal
fibres or insoluble proteins if the fluid is acid; so that if the method of extrac- tion of the iron dipyridyl with acetate and sulphite fails alcohol can be used. In neutral or slightly alkaline solution the adsorption on the proteins is very marked and the iron compound is not always extracted even by alcohol.
VIII. Estimation of the Inorganic Iron in Bakers' Yeast.
20 g. of bakers' yeast were washed with glass distilled water until the wash-
ings gave no colour with dipyridyl and sodium hydrosulphite. The yeast was
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then suspended in dilute acetate buffer and dipyridyl added. No colour was produced on standing 15 minutes. A little iron-free sodium hydrosulphite was then added and the yeast immediately became pink. Hence in the washed yeast the iron is practically all in the ferric state. This is also true of unwashed yeast. The yeast was then washed until the liquid was colourless. The remaining residue of pink yeast cells was suspended in dilute acetate buffer and after the addition of a little more oaa' dipyridyl and hydrosulphite SO2 was passed through the suspension. This caused apparent damage to the cell membranes, because the colour diffused out into the liquid leaving the yeast practically colourless. The yeast was then washed. The iron
dipyridyl extracted in these three stages was made up to 100 c.c. in each case and the solutions reduced by adding hydrosulphite (to bleach certain yellow substances of the yeast cells). The iron was estimated colorimetrically with the standard tubes, and the results were as follows, the iron being expressed as a percentage on the original wet weight of the yeast. Washings, 0-00012 per cent.; removed from the yeast by dipyridyl, 0 0001.5 per cent.; extracted after damaging the cell membranes, 0-0013 per cent.; total 0-0016
per cent. It appears therefore that about 80 per cent. of the inorganic iron in this sample of yeast was actually inside the cells in the ferric form. The
remaining 20 per cent. being due to some dead cells and chance contaminations.
ac' dipyridyl in sodium hydrosulphite at pa 4 does not easily liberate the iron from heematin compounds, and as the intracellular haematin compounds (Keilin, 1925) remain in the yeast during the manipulations, the iron found
by oc' dipyridyl should represent the non-hTematin iron of the yeast.
IX. Estimation of Iron in Egg Yolk.
When egg yolk boiled or unboiled is suspended in acetate buffer and aa'
dipyridyl added, no red colour is produced, but on the addition of sodium
hydrosulphite a deep red colour is immediately developed. This would indicate that the iron is present as free ferric iron rather than in any stable
organic combination in the yolk. Bunge (1885) drew attention to the fact that the iron of egg yolk is not shown easily by the usual tests, but as the iron is so easily " unmasked " by a reducing agent, it would suggest that in the
egg yolk we have colloidal ferric hydroxide. The iron was therefore estimated in the yolk of egg (hen) by means of dipyridyl with and without incineration. For extracting the iron from yolk of egg without ashing the following method was used. An egg was boiled 15 minutes and cooled in water. The yolk was broken up into a homogeneous crumbly mass and 1 g. samples immediately
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weighed out. One sample was suspended in acetate buffer (5 c.c.) and treated with oca' dipyridyl and sodium hydrosulphite. After passing SO2 until the
proteins and fat were aggregated the mixture was filtered and the filtrate and
washings made up to 50 c.c. Another sample was treated in a similar way, alcohol being used (30 per cent.) instead of SO2 to prevent adsorption of the iron compound on the protein. Two further samples were incinerated with 5 c.c. concentrated sulphuric acid and 1 g. of potassium perchlorate (the iron content of which was determined) added with due precautions. After incinera- tion water was added and the solution was neutralised with sodium acetate and made up to 100 c.c. with the addition of sodium hydrosulphite and.
dipyridyl. The iron was determined in solution by the standard tubes. The results were as follows: by incineration the iron content was found to be 0-0086 and 0-0083 per cent. of wet weight; without incineration, using SO2,
0) 0080 per cent.; and using 30 per cent. alcohol, 0.0085 per cent. In order to see if the iron was liberated from some organic combination by acid and a
reducing agent a small quantity of the egg yolk was suspended in 10 per cent. ammonia solution and warmed with a little dipyridyl and hydrazine hydrate. The mixture became red showing that the iron is " unmasked " even by a
reducing agent in alkaline solution.
X. The Effect of aa' Dipyridyl on the Iron and Copper Catalysis of the Oxidation
of Cysteine at pH 7-3.
In each experiment a series of four separate Barcroft differential manometers
was used, each containing in the right-hand side 5 mg. cysteine hydrochloride (neutralised), 1 c.c. of phosphate buffer, the total volume of fluid being 3 c.c.
The left-hand side contained 3 c.c. of buffer. The control contained the
maximum quantity of oaa' dipyridyl used in an experiment and no metals. The
temperature was 20? C. In the following table are shown the results of two
experiments. The cysteine without metal absorbed about 4 mm.3 of oxygen
per hour.
Iron added = 0 0033 mg.
Molecular ratio dipyridyl oxygen uptake metal mnm.3 per hour.
Zero 119
3 52 6 Zero
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It is seen, as might have been expected, that oca' dipyridyl in low con- centration inhibits the catalysis by iron. It is, however, curious that while the colormetric measurements indicate complete association of iron and dipyridyl at p 7 3 (cysteine not influencing the colour) only about 50 per cent. inhibition is observed in the presence of the theoretical amount of dipyridyl. There is no appreciable effect on the copper catalysis at this range of dipyridyl cona centration. ooc' dipyridyl thus behaves in a corresponding way to pyro-
,phosphate, only it shows its effect at a lower concentration comparable with that required in the case of cyanide inhibitions.
XI. Summary.
1. ca' dipyridyl is proposed for a reagent for estimating inorganic iron in
biological material for the following reasons:--
(a) The colour is almost specific for ferrous iron, other metals such as copper giving but faint colours.
(b) There is no necessity for ashing, while other methods require it.
(c) The standards can be kept indefinitely and the fluid after adding
dipyridyl for estimation can be kept without fear of the colour fading.
(d) The colour is given quantitatively over a region of n) which covers the
physiological range. (e) It is possible to determine the ferrous and ferric iron in a mixture.
(f) The colour does not become yellowish in great dilution.
(g) The colour is stable to reducing agents such as sodium hydrosulphite.
(h) The sensitivity approaches that of the thiocyanate method, it being possible to detect iron in a concentration of 0-0002 mg. per cubic centimetre in a layer of fluid 1 3 cm. thick.
(i) Heematins and their various compounds do not give a reaction for iron with ca' dipyridyl.
2. The inorganic iron in yeast and egg yolk appears to be in the ferric form, and that in egg yolk resembles colloidal ferric hydroxide in properties, there
being no detectable organic iron analogous to a hematin compound. VOL. CVII.-B. R
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' Z. physiol. Chem.,' vol. 9, p. 49. Hein and Retter (1928). ' Ber. D. Chem. Ges.,' vol. 61, p. 1790. Keilin (1925). ' Proc. Roy. Soc.,' B, vol. 98, p. 312. Smith (1926). ' J. Amer. Chem. Soc.,' vol. 46, p. 416. Wiedel (1879). ' Ber. D. Chem. Ges.,' vol. 12, p. 1992. WVerner (1912). ' Ber. D. Chem. Ges.,' vol. 45, p. 433.
612 .742
The IHeat of Rigor of Marnnmalian lMuscle.
By E. C. SMITH, Low Temnperature Research Station, Cambridge.
(Communicated by Sir William Hardy, F.R.S.-Received August 6, 1930.)
The stiffening of muscle in rigor mortis is closely related to gelation of the muscle plasma (Smith, 1930). Neither the stiffening of the muscle (Hoet and
Marks, 1926) nor the gelation of the plasma is immediately due to an increase in the hydrogen-ion concentration of the muscle, 'but, apart from the formation of lactic acid, no reaction is known to occur post-mortem which might be held
responsible for the coagulation of the plasma. It was with a view to the detection of any such reaction that the follovwing measurements of the heat
production accompanying rigor mortis were made. The heat of rigor morrtis has not previously been measured, although A. V.
Hill (1912) measured the heat produced by frog's muscles undergoing heat and chloroform rigor. The result suggested tliat the conversion of glycogen into lactic acid accounted for almost the whole of the heat produced. This has been found to be the case in the muscle of a normal well-fed rabbit when
passing into rigor 1mor tis, and also in the case of fatigued or exhausted muscle, but starved animals produce a larger quantity of heat than can be accounted for by the lactic acid produced.
3. ax' dipyridyl resembles pyrophosphate in its inhibition of iron and not of copper in the oxidation catalysis of cysteine by these metals.
EIEFERENCES.
Blau (1888). ' Ber. D. Chem. Ges.,' vol. 21, p. 1077. Blau (1889). ' Mschr. Chem.,' vol. 10, p. 375 (from Werner). Blau (1898).
' Z. physiol. Chem.,' vol. 9, p. 49. Hein and Retter (1928). ' Ber. D. Chem. Ges.,' vol. 61, p. 1790. Keilin (1925). ' Proc. Roy. Soc.,' B, vol. 98, p. 312. Smith (1926). ' J. Amer. Chem. Soc.,' vol. 46, p. 416. Wiedel (1879). ' Ber. D. Chem. Ges.,' vol. 12, p. 1992. WVerner (1912). ' Ber. D. Chem. Ges.,' vol. 45, p. 433.
612 .742
The IHeat of Rigor of Marnnmalian lMuscle.
By E. C. SMITH, Low Temnperature Research Station, Cambridge.
(Communicated by Sir William Hardy, F.R.S.-Received August 6, 1930.)
The stiffening of muscle in rigor mortis is closely related to gelation of the muscle plasma (Smith, 1930). Neither the stiffening of the muscle (Hoet and
Marks, 1926) nor the gelation of the plasma is immediately due to an increase in the hydrogen-ion concentration of the muscle, 'but, apart from the formation of lactic acid, no reaction is known to occur post-mortem which might be held
responsible for the coagulation of the plasma. It was with a view to the detection of any such reaction that the follovwing measurements of the heat
production accompanying rigor mortis were made. The heat of rigor morrtis has not previously been measured, although A. V.
Hill (1912) measured the heat produced by frog's muscles undergoing heat and chloroform rigor. The result suggested tliat the conversion of glycogen into lactic acid accounted for almost the whole of the heat produced. This has been found to be the case in the muscle of a normal well-fed rabbit when
passing into rigor 1mor tis, and also in the case of fatigued or exhausted muscle, but starved animals produce a larger quantity of heat than can be accounted for by the lactic acid produced.
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