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ON MUSCLE-PLASMA'. BY W. D. HALLIBURTON, M.D.,
B.Sc., Assistant Professor of Physiology, University College, London.
(From the Physiological Laboratory, University College, London.)
OUR knowledge of the properties of muscle-plasma depends almost entirely on Kuihne's' researches on the muscle-plasma of frogs. In this investigation I have endeavoured to extend his observations to the warm-blooded animals, and to discover if possible the precursors of myosin in the muscle-plasma, as well as to ascertain more accurately the cause of the formation of myosin. Incidentally the research has included an investigation of the proteids of muscle-plasma, and of muscle- serum. The subject may conveniently be discussed under the following beads:- I. The influence of cold on the coagulation of the muscle-plasma of warm-blooded animals. II. The influence of neutral salts on the coagulation of muscle- plasma. III." The properties of saline extracts of muscles which have under-
gone rigor mortis.
IV. The properties of the muscle-clot. V. The development of acid during coagulation. VI. The preparation and properties of myosin-ferment. VII. The^ proteids of muscle-plasma and^ of^ muscle-serum.
I. THE INFLUENCE OF COLD ON THE COAGULATION OF MUSCLE-
PLASMA OF WARM-BLOODED ANIMALS.
Kiihne found that the muscle-plasma which he obtained from the
frog could be prevented from coagulating by a^ temperature below^ 0° C.;
about this temperature it clots slowly, and at a temperature of 400 C. (^1) The expenses in connection with this research have been (^) defrayed out of a (^) grant from the (^) Scientific Grants Committee of the British Medical Association. (^2) Kiihne, Protoplasma, Leipsig, 1864, and Lehrbuch der physiologische Chemie. pH. lil^11
113. D. HALLIBURTOK.
almost instantaneously; the muscle-plasma being a liquid of syrupy consistency, of a faintly alkaline reaction, and separating at a suitable temperature into a solid clot composed of the proteid substance called myosin, and a liquid residue which is squeezed out by the contraction of the clot and which has received the namne of (^) musele-serum. Kuhne was not able to employ the muscles of warm-blooded animals, as he found that they do not preserve their vitality sufficiently lono to permit of the plasma being removed before coagulation occurred. The first point therefore which it was necessary to settle before proceeding to the further quiestions involved in an investigation of the properties of mammalian muscle, was whether the facts above mentioned as true for the (^) muscle-plasma of (^) the frog are also true for the muscle- plasma of warm- blooded animals. These experiments together with several of the earlier ones which will be detailed under the next heading were performed in conjunction with Professor Schafer. Several unsuccessful attempts were made to obtain (^) muscle-plasma from the rabbit, but it is unnecessary to enter into a description of these; the causes of failure will be mentioned in the following description of the method which was found to be wholly successful (^) :-The animal was killed by bleeding from the carotids; the abdomen was quickly opened, and a cannula inserted into the abdominal aorta; the blood was then
washed out from the lower limbs by mearns of a stream of cold salt
solution (0i6 sodium chloride solution); it was found unadvisable to cool
the salt solution to 00C. as so cold a fluid caused such powerfuil con-
traction of the small vessels as to hinder the free flow of the fluid. It was found that a temperature of 5°C. was sufficiently low, and this was
produced in a very sinmple way by putting lumps of ice into the salt
solution; as they melted some dilution of the solution was produced, but this was immaterial. When the lower limbs were well swollen with
the saline solution the vena cava inferior was opened, and tlhe mixture
of blood and salt solution allowed to flow out; in about five minutes the fluid came through^ perfectly clear.^ The lower limbs^ were^ then^ quickly skinned, and pieces of the muscles which were still excitable were cut
off and plunged into^ a^ freezing mixture^ of ice and^ salt^ which had^ a
temperature of about -120C. These pieces if^ small become almost
instantly solid hard^ lulmps: the^ outside^ of each^ lump is^ necessarily
frozen first, this forms^ a protective case^ to^ the^ initernal^ part of the piece of muscle and effectively prevents (^) any but the snmallest
a(dmixture of the strong salinie^ solution^ with the^ muscular^ stubstance.
It is necessary to put the mnuscles in this way directly inlto the freezing
W. D. HALLIBURTON.
and squeezed; but in neither case did the expressed fluid undergo clot- ting, either at the temperature of the air or in the incubator at the temperature of 40° C. In both cases the expressed fluid was acid. In one experiment similarly performed to that just described an apparent exception to this statement occurred. Twelve hours after death, the muscles of the lower limbs of a rabbit freed from blood by a stream of salt solution injected by the abdominal aorta, were chopped up finely and wrapped in linen: this was squeezed and a strongly acid fluid
expressed. This did not undergo clotting at the temperatuire of the
atmosphere, but when kept in the incubator (^) at 400 C. and examined half-an-hour afterwards it was found to have become thick and opaque. This was produced by a fine flocculent precipitate which pervaded the liquid, which was removable by filtration, an.d which resembled in^ ap- pearance a heat coagulum muich more than a clot of myosin formed spontaneously. That it was a^ heat^ coagulum, was^ proved by the following experiments; (1) The precipitate was collected on a filter and (^) washed with (^) water, and was found to be insoluble except in strong
mineral acids. (2) This precipitation began immediately the tempera-
ture of the fluid reached 400 C., and gradually increased in amount. (3)
If the temperature of the incubator^ was^ lowered^ to^350 C. this^ precipita-
tion did not occur; while the coagulation of muscle-plasma occurs as
readily or almost as^ readily at^350 as at^ 400 C.^ In^ connection^ with^ this
explanation the question will naturally arise, why should .a heat coagu-
lum have occurred at^ so^ low^ a^ temperature as^400 C.^?^ This^ is^ however to be accounted for by the acidity of the liquid, which was extreme as
compared with that^ usually met^ with^ in fluids obtained from^ coagulated
muscle, and the reddening of litmus paper of a -decidedly blue shade was very marked.^ As^ will be^ seen^ later^ on, the lowest^ point at^ which^ any
proteid occurring in muscle coagulates is^470 C., but^ the acidity in this
case was presumably sufficiently great to account for the lowering by 70
of the temperature of^ coagulation.
The general conclusions that can be drawn from the foregoing experi- ments are as follows
1. That with a few slight modifications the facts previously dis-
covered by Kiihne^ in^ relation^ to^ the^ preparation of^ muscle-plasma from
frog's muscle, are true also with regard to mammalian muscle.
- That the^ preparation of^ muscle-plasma from^ mammalian muscle is by no means so difficult a process as has hitherto been supposed.
Indeed, by following the^ simple method^ we^ have^ described, it^ is^ an^ ex-
periment which can easily be demonstrated^ to^ a^ class.
MUSCLE-PLASMA.
3. That the coagulation of mammalian muscle-plasma, after it has
been subjected to a^ low^ temperature, is^ a^ process of^ longer duration than
that described by Kiih ne in the case of frog's, muscle-plasma.
II. THE INFLUENCE OF NEUTR0AL SALTS ON THE COAGULATION
OF MWSCLE-PLASMA.
The separation of muscle-plasma into a clot and mnuscle-serum is
very similar to what occurs in the blood-plasma after its removal from the body, and the resemblance is borne out by the fact that a low temperature prevents or^ hinders the^ separation into clot and^ serum^ in both cases. In the case of blood-plasma cold is not the only agent which prevents the occurrence of coagulation, but the same result is brought about by mixing with the plasma certain proportions of neutral salts, such as magnesium sulphate, sodium chloride, or sodium sulphate. An (^) important point then to determine is, whether the analogy between muscle-plasma and blood-plasma holds in this particular also; that is, whether it is possible to prevent the coagulation of muscle-plasma by mixing it with solutions of such salts as those just enumerated. In order to elucidate this point, the following method of procedure
was adopted. A rabbit was killed by bleeding,, a cannula inserted into
the abdominal aorta, and the blood washed out frorn the lower limbs by a stream of cold salt solution in the manner (^) already described. The lower limbs were then quickly skinned; the muscles of one limb (A) were (^) placed in the (^) freezing mixture, the muscles of the (^) other limb (B) not. When frozen the muscles of the limb A, were cut into fine slices,
by means of^ a^ cooled^ knife1; the^ pieces being kept frozen^ during this
proceeding by placing them on a^ cooled^ plate. The^ finely divided frozen
muscle was then divided into three parts; one portion was put into
100/°0 sodium chloride^ solution, the^ second^ into^5 0/0 magnesium sulphate
solution, and the third into a half-saturated solution of sodium sulphate; in all (^) cases these fluids (^) were kept at (^) the temperature of 00 C. or a few
degrees below. By means of a cooled pestle and mortar, the pieces of
muscle were (^) thoroughly crushed, and mixed (^) with the (^) fluids in (^) question; it was found that a very considerable amount of proteid went into solu-
tion; the fluids^ were^ then^ rapidly filtered, by using a^ large number^ of
small filters which^ were^ also^ kept cold^ as^ far^ as^ possible by means of cooled glass funnels. The filtrates were in all cases alkaline in (^) reaction, I (^) In some cases a freezing microtome was employed.
MIUSCLE-PLASMlAA.
five per cent. maonesium sulphate solution. These two latter mixtures kept either at the temperature of the air or in an incubator of the tem- perature of 370 C. did not (^) undergo coagulation. The sodium chloride plasma was alkaline in reaction, the magnesium sulphate plasma which was collected a few minutes later was neutral. (^) Moreover these specimens of salted plasma were found to contain the same proteids by methods shortly to be described as^ the^ extracts^ obtained^ by pounding the cooled muscle with the salt solutions, and in fact exhibited preciselv similar reactions, of which the most important was the fact (^) immediately to be ftully entered upon, that on dilution with water, coagulation occurred and an acid reaction was developed'. From this experiment I felt fully justified in concluding, that the extracts of cooled muscle were really specimens of salted (^) plasma, and that in^ them, as in the^ case^ where the^ plasma itself was used, the salts employed had prevented (^) coagulation. The former (^) method, that of
extracting frozen muscle with the salt^ solution, was^ the one employed in
most experiments, particulars of which will follow; it was the less troublesome process of the twvo, and (^) what was more imnportant gave a larger yield of salted muscle-plasma. The solutions (^) employed for extracting the plasma from the frozen muscle, are those that have already been mentioned: a ten per cent. sodiun
chloride solution, or a five per cent. maognesium sulphate solution, or a
half-saturated solution of sodium sulphate. A number of trials with solutions of these same salts of different strengths showed that the above (^) mentioned are the best to use, since they extract a larger
quanitity of proteid than either weaker or stronger solutions. Saturated
or stronD solutions of sodium (^) chloride, or (^) magnesium sulphate, extract very little proteid, for, as will be shown later on, many of the proteids of muscle-plasma are precipitated by (^) saturation with these salts: saturated solution of sodium sulphate has not this disadvantage to such an extent. Solutions of the salts weaker than those (^) mentioned do not with certainty prevent the formation of myosin, as will be seen by the dilution experiments next to be described. I have not employed ammonium chloride solutions in any of (^) my experiments, although it has been largely
used by others for dissolving myosin, for the reason, that in subsequent
saturation experiments with magnesium sulphate or sodium chloride, the
presence of a second salt would have complicated the reaction. 1 The sodium chloride plasma mentioned above was in this case an (^) exception; after clotting had occurred the reaction still remained (^) very faintly alkaline, though whether it was less alkaline than previously I did not have sufficient material to (^) investigate.
W. D. HALLlBUI?TON.
Following up the analogy between the coagulation of blood-plasma and muscle-plasma, the next point to investigate was one to which allusion has already been made; namely, whether dilution of the salted plasma with water produces coagulation in one case as in the other. Diluition of salted blood-plasma with water is supposed to produce coagulation by removing the inhibitory influence that a more concen- trated salt solution has upon the formation of fibrin. Dilution of salted muscle-plasma with water does cause the formation of a clot of myosin, and acts presumably in a similar way by removing the inhibitory influence that a more concentrated salt solution has upon the formation of myosin. The following are the notes of the observations made on the results of dilution of (^) the three extracts described as having been made from (^) the frozen muscle.
Extract 1; with 10 per cent. sodium chloride solution. Reaction alkaline; fluid slightly opalescent.
Time of obser- Result when allowed to Result when placed in the vation vation -^ Amount of^ dilution^ stand at the tempera-ture of the air (150 C.) warm chamber (tempera-ture 370 C.)
10 am. (^) a. Diluted with an equal amount of water
10 a.m. next^ No change Slight increase of
day opalescence
10 a.m. b. Diluted with Slight increase of Slight increase of three times its opalescence opalescence volume of water 10.30 a.m. No change Clot beginning to form 12.15 (^) p.m. No (^) change Clot (^) extends through-
out the liquid
12.30 p.m. No change Clot beginning to contract 2 p.m. No change Clot contracted and floats at the top a colourless clear fluid (^10) a.m. next No change No furtlher ehange day
140
TV. D. HALLIBUKR XO:\r.
The clotting which is described as occurring at various times in the precedinog tables was in appearar.ce very like that which occurs when blood-plasma is similarly diluted; there is at first a jellying throughout the liquid; this very rapidly contracts, and floats in a clear fluid. The consistency of the clot is however not nearly so great as that of fibrin, and the jelly on shaking breaks up into fragflments. In some instances, though not in the experiment^ just^ described, but when a very concen- trated muscle-plasma was uised, the jelly which formed was sufficiently firm to allow of the vessel in which it was contained, to be turned upside down without the contents being spilled. In all cases also the occur- rence of the clot was^ accompanied by the^ change of^ reaction of^ the^ fluid from alkaline to acid. On redissolTina the clot, it was found by its reactions to be in^ all cases myosin. One (^) point which is (^) very important in the determination of the clotting, is. the temperature; the temperature of 370 C. is seen to^ be miuchl more effectual in causing the coagulation of the myosin than the temperature of la, that of^ the atmosphere. The influence of^ tempera- ture is more strikingly seen in the following experiment; specimens of salted muscle-plasma were obtained from a rabbit in the way described; it underwent coagulation more rapidly than in the experitnent already detailed, and so comparative observations were more easily made. A preparation made with^ 10 per cent. sodium chloride was diluted with four times its bulk of water and divided into five portions, a, b, c, d, e.
a. Placed in^ a^ freezing mixture at a temperature of^ -^10 (C.^ Thawed after three hours; no clot, clotting commenced 50-60 minutes after the temperature of the air 13° C. was reached.
b. Kept at the temperature of melting ice 00 C. No coagulation after four hours.
c. Kept at the temperature of^20 C. After 70 miniutes a faint coagu-
lation began to^ appear throughout the^ liquid.
d. Kept at the temperature of the air 130C.: coagulation began in
25 minutes.
e. Kept at^ the^ temperature of 350^ C.:^ coagulation^ began^ in^10 minutes.
With a specimen prepared by means^ of^ a^ five per cent. soluition of
magnesium sulphate, and then similarly treated, similar results were obtained.
142
MIUSCLE-PLASMAL4.
a. (^) b. c. corresponded exactly to (^) a, b, and (^) c above. d. Coagulation began in 20 minutes. e. Coagulation began in 8 minutes.
The specimens of salted muscle-plasma obtained by mixingf the
sqtueezed out (^) plasma with salt solutions underwent precisely similar coagulation when diluted with water; at the temperature of the air the clot was very slowly formed (12-24 hours); at the temperature of (^370) C- it appeared rapidly (10-1 minuites).
So far then the analogy between the occurrence of coagulation in
salted muscle-plasma, and that in salted blood-plasma is very close,
especially in the influence of temperature, a low temiperature hindering,
a (^) temperature of 350-40' C. hastening the formation of a clot. The development of acid however does not appear to take place in the sepa- ration of fibrin from blood-plasma as it does in that of myosin from nmuscle plasma. The close resemblance in other points however stuggests a similar cause in the two cases; the formation of fibrin is believed to
be due to the action of fibrin-fernment, which like other enzymes acts
best at a temperature of 30° 40° C. and is inhibited by a low ternpera- ture: the question of a similar ferment action in the formation of myosin is one which will be entered into fully later on. The general conclusions which can be drawn so far with regard to the
influence of neutral salts on the coagulation of muscle-plasma are as
follows.
- That admixture with solutions of neutral salts is able to prevent
in uscle-plasma from undergoing coagulation.
- That dilution of the salted muscle-plasma brings about the coagulation (^) prevented by the more (^) concentrated salt solutions.
- That the coagulation of diluted salted muscle-plasma occurs
readily at^ temperatures between 300 and 400C. more slowly at lower
temperatures, and is wholly prevented by a temperature of 00 C.
- That with the exception of the formation of acid which occurs
sinmultaneously with the produietion of a clot of myosin, the phenomena
regarding the^ formation^ of^ myosin are closely similar to those^ which are
observed in the formation of fibrin from blood-plasma.
- This similarity suggests that the formation of^ myosin^ may be due to a ferment, in the same way that the formation of fibrin from the
fibrinogen of^ blood-plasma is^ dlue to^ the^ action of^ the^ fibrin-ferment.
14:
MJUSCLE-PLAAUiA.
Time of obser- Allowed^ to stand at^ Placed^ in the^ warm vation the^ temperature air 150 C.^ of the^ chamber^370 C.
11 a.m. b. Diltuted with Marked increase of Marked increase of three times its^ opalescence opalescece volume of^ water 12 noon No change Jellying through liquid
12.30 p.m. No^ chaaige^ Clot^ beginning^ to contract 5 p.m. Jellying beginning Clot contracted anid abundant 10 a.m. next Small^ clot which ditto.
day had^ contracted
Similar tables might be given of the behaviour of^ the^ sodium chloride, and sodium sulphate extracts, but the above will sufficiently indicate the results obtained. The coagulation was in appearance not
a simple precipitation, but consisted first in^ a^ jellying throughout the
liquid which suibsequiently contracted, squeezing out a colourless fluid, and this occurred more^ readily at^ the^ temperature of the body than at the temperature of the air. This experiment was the first performed with apparently dead muscle, and it was at first (^) thought that the result might be (^) explained on the supposition that the muscle^ in^ question was not really dead, or at least had not undergone rigor mortis; since it was removed fromi the body of the animal only one hour after death. Observations were then mnade in a similar way on extracts of muscles removed from the body, four, ten, and^ twenty-four hours after^ death;^ in^ the^ first^ two^ cases^ those removed four and ten hours after death the muscles^ were markedly rigid; in^ the third^ case,^ that^ removed^ twenty-four^ hours after^ death,^ the stiffness had passed off; in a fourth case, the limbs were kept in an incubator at^ the temperature of^400 C.^ for^ twenty-four^ hours,^ a^ condition which would favour the coagiilation of myosin in^ the muscles to the fullest extent; in^ this^ case^ the^ odour^ indicated^ that^ putrefaction^ had also commenced. But in all^ four cases, the result obtained was precisely
the same; i.e. the myosin after being, redissolved by the salt solution
underwent a re-coagulation when^ that salt solution^ was^ diluted.^ In a further experiment it was found that this re-coagulation was entirely
1 4-
14. D. [IA LLIB URTONX.
prevented by a temperature of 0°C., took place^ slowly^ a^ few degrees above that temperature, and occurred readily at the temperature of the body. I have been careful to speak of it as a recoagulation and not as a simple precipitation; the former term implying that^ the^ coagulum is of a similar nature and formation to the clot obtained from muscle-plasma or blood-plasma; whereas the term precipitation would^ simply imply that myosin, being a^ proteid^ of the^ globulin^ class,^ would^ naturally^ be precipitated by dilution, as all globulins are^ insoluble^ in very dilute solutions of^ salt.^ This^ latter^ statement^ is in fact that^ which^ one^ finds in the text books; the dropping of^ a^ solution^ of^ myosin into^ water^ is said to (^) produce a precipitation of the myosin. This statement is perfectly true, and'if one employs a^ large excess of water, as^ is^ implied in the above-mentioned statement, the clot-like character of the coagulum is lost.^ If^ one^ dilutes^ the muscle^ extract^ with^ ten^ or^ twenty times its bulk of water, a precipitation of the myosin occurs usually at the atmospheric winter^ temperature in less than^ an^ hour;^ and^ the precipitate is a flocculent one, which soon settles to the bottom of^ the vessel; but^ in^ this^ case^ also^ the^ precipitation^ is^ more^ rapid^ at^ the temperature of the body. A^ myosin clot^ under^ any circumstances is
never such a^ coherent^ coagulutm as^ one^ of^ fibrin,^ and^ when the dilution
of a muscle extract is so great as^ that described^ above, the^ stage of jellying is^ so^ transitory, (the clot^ being^ immediately^ broken up into flocculi when it contracts in^ various directions^ throughout a^ large volume of liquid,) that it is apt to be, and in fact generally is overlooked.
Still by very carefiul and continuous^ observation I have been able^ to
perceive it even in such cases as these. Buit it^ can be perfectly well observed in cases in^ which the dilution is^ slight,^ as^ in^ the^ first^ ex- periment detailed at the commencement of this section. The clot of myosin which is formed has exactly the same characteristics as the clot of myosin formed when salted^ muscle-plasma is^ first made to coagulate. The rabbit is not the only animal on which observations have been made by me; but in addition to some thirteen rabbits, I have observed an (^) exactly similar phenomenon in the muscles of two cats, and three pigeons. The appearance of the clot is however not the only ground on which I regard it as due to process of coagulation as distinguished^ from^ that of
a simple precipitation. The other grouinds on which this assertion is
based are^ the following:
146
18. D. IHALLIBURTON.
IV. THE PROPERTIES OF TffE MUSCLE-CLOT.
1. Solubility and re-coagulation. I desired in the first place to
determine whether myosin, the proteid substance of which the muscle- clot is (^) composed, undergoes a similar re-coagulation on dilution of its saline solutions. Myosin was prepared either from specimens of salted muscle-plasma, or from saline extracts of muscle which had undergone rigor in the following way. The saline solution was diluted with about twenty times its volume of distilled water. This dilution caused a precipi- tation of the (^) myosin, which settled into a flocculent deposit at the bottom of the vessel in which the operation was performed; it was then washed by decantation with distilled water three or four times until the superna- tant fluid gave only a faint indication of the presence of proteid. (It was found impracticable to wash it more than this, as after prolonged washing, the precipitate of myosin becomes quite insoluble in saline
solutions1.) The myosin was then dissolved by adding solid sodium
chloride, (^) or magnesium sulphate, as the case might be, until the strength of the salt solution reached ten or five per cent. respectively. The solu- tions so obtained were neutral in reaction, and after dilution with two or three times their volume of water underwent re-coagulation; this as in the previous cases was first a jellying through the liquid, the coagulum subsequently contracting and squeezing out a clear fluid; this occurred more readily at the temperature of the body than at lower temperatures; it was accompanied by the development of an acid reaction in the liquid, and lastly, as will be fully stated later on, (^) the addition of myosin-ferment
hastened the process. The myosin thus obtained was again washed and
redissolved in salt (^) solutions, which were again diluted, and once more underwent a precisely similar coagulation. I have repeated this process in two cases four times, and in one case five (^) times2. The clear liquid which is squeezed out by the contraction of the clot of myosin obtained from such (^) solutions, contains (^) only the faintest trace of (^) proteid, such as (^) miight arise from the fact that the mvosin was not for the reason stated washed perfectly free from other proteids. There seems therefore to be no other proteid formed during coagulation beyond the precipitate of myosin. Here is a difference between the formation of myosin and the formation of (^) fibrin; for Sch midt stated that formation of (^) the latter (^) from fibrinogen under the influence of the fibrin ferment is accompanied by the simul-
(^1) See below, p. 153. (^2) In (^) prolonged experiments such as (^) these, decomposition was prevented by the addition to the liquid of a few crystals of (^) thymol.
148
MUSCLE-PLA SJIIA.
taneous formation of a globulin (^) corresponding in its character to (^) serum globulin. We may call the (^) hypothetical precursor of the myosin in muscle,
myosinogen; this after death is converted into myosin; the passing off
of rigor mortis seems to be due to the reconversion (^) of myosin back to (^) the
former condition of myosinogen. This process occurs not only in the
muscles after death, but something very similar can be made to occur in artificial solutions (^) of myosin, the clotting and unclotting being brought about by alternate dilution and concentration of the salt solution (^) used to dissolve it. We have been (^) tracingf hitherto the analogy between the coagulation of blood and that of muscle; we have noted already (^) many resemblances; we have also come across certain differernces of which the most (^) important hitherto mentioned are the two following:-
1. The formation of myosin from myosinogen is accompanied by
the development of acid, whereas that of fibrin from fibrinogen is (^) not so far as we know.
- The formation of myosin from myosinogen is not accompanie(l by the formation of another globulin, whereas that of fibrin from fibrinogen is. We have now the question before us, have (^) we a third difference to deal with in the readiness with (^) which myosin is reconverted into myosin- ogen, which once again with suitable treatment will become myosin? or is it possible by similar (^) meains once more to reconvert fibrin into fibrin- ogen which in its turn will again become fibrin? (^) Stich an (^) experiment was performed in the (^) following manner. Fibrin (^) obtained by whipping from ox blood was first well washed under a tap with running water, and then with 10 per cent. sodium chloride solution to remove any adherent globulin. An extract of the fibrin was then made with 10 per cent. sodium chloride, and another with five per cent. magnesium (^) sulphate solution. Thymol was added to each to (^) prevent decomposition. After standing for two days in contact with these solutions, a (^) considerable amount of the fibrin was dissolved. These (^) solutions of fibrin were opalescent and gave a copious precipitate on heating; the heat coagulation (^) temperature of saline solutions of fibrin is stated1 to (^) be between 60° and 630. I tried the heat coagulation of the solutions mentioned above as well as in two others similarly prepared, the solution (^) being in all cases made faintly acid by weak acetic acid. The results of these (^) determinations may be stated mi the following table.
Gamgee, Physiological Cheemistry, p. 36.
PH. VIII. (^12)
MUSCLE-PLASM1A.
The effects of dilution upon the solutions of fibrin were (^) then tried. Different specimens of the 10 per cent. sodium chloride extract were diluted to two, four, and six times their volume (^) respectively; each (^) was divided into two parts, one of wlhich was (^) kept at the temperature of the air (^) (150 C.), the other at that of the body (360 C.). In those kept at the temperature of the air no change occurred after the (^) lapse of forty-eight hours: in those kept at the teinperature of the body there was no change after thirty-six hours: but after forty-eight hours a (^) slight precipitate formed. This precipitate (^) consisted of exceedingly fine
flocculi, and a stagre in which there was a jellying was neither in this
nor in similar experiments ever observed: the precipitate (^) represented a very small fraction of the total proteid dissolved, and there was no change in the neutral reaction of the (^) liquid. A similar experiment was performed with another sodium chloride extract, with two specimens of magnesium sulphate (five per cent.) extract, with one (^) specimen of an eight per cent. potassium nitrate extract, and with one made with half-saturated
solution of sodium sulphate. But in all a sirmilar result was obtained
to that already (^) stated. The different solutions differed somewhat in the length of time after dilution at which precipitation of a small amount of dissolved (^) proteid occurred; but it (^) was in all cases over twenty hours in the (^) cold, and over twelve hours at the temperature of 370 C. In some of the above-mentioned solutions dilution was (^) performed with water in some (^) cases, and to an equal extent with an aqueous extract of fibrin ferment in others. But it was never found that the presence of the fibrin (^) ferment had any influence in hastening the precipitation. I should not be inclined to regard it as a coagulation on (^) the following grounds:-
- It is (^) never, so far as could be ascertained, a jellying throughout the liquid.
- It is not hastened (^) by the addition of an aqueous solution of fibrin ferment. It is (^) however a curious fact, and one worth noting, that this pre- cipitation occurred rather more rapidly at the (^) temperature of the body than at the temperature of the air; and in this fact it resembles a ferment coagulation. The (^) coagulation in muscle thus differs from that in blood in a third particular which must be added to the two differences already (^) stated p. 149.
- (^) Myosin, the solid proteid formed when muscle coagulates, is 12-
151-
W. D. HALLIBURTON.
readily soluble in solutions of salt; on dilution of these a recoagulation
of the myosin occurs. Fibrin, the solid proteid formed when blood coagulates, is^ not^ so^ readily soluble in^ salt solutions; on dilution of these a small amount of reprecipitation of the dissolved fibrin occurs, but this does not appear to be a ferment (^) coagulation.
- Heat coagulation. The first property of the muscle-clot that we
have hitherto considered is that of recoagulation.
The next point we have to take up. and it is closely connected with the first question, is the phenomenon of heat (^) coagulation. By applying the^ process of fractional heat^ coagulation, which^ I^ have
fully described elsewhere', to salted muscle-plasma obtained by mixing the
plasma squeezed out from frozen muscle with five per cent. magnesium sulphate solution, or^ to^ that obtained^ by extracting frozen muscle with the same solution, or to that obtained by extracting muscle which had undergone rigor with the^ same^ solution, I in all^ cases^ obtained the same series of precipitates. I also obtained the proteids in a state of com- parative purity by saturating the^ muscle-plasma, or the extract of dead
muscle with ammonium sulphate; this precipitates all the proteids in the
solution. The (^) precipitate was collected on a (^) filter, washed with saturated solution of ammoniunm sulphate, and then^ dissolved again by the addition of water; in this solution the process was repeated, and to the solution finally obtained the^ process of^ fractional heat coagulation was applied; the result was precisely the same as that obtained from the muscle extract.
The series of precipitates obtained in all these cases was
470 C. A flocculent somewhat sticky precipitate.
560 C. A more abundant, and very sticky precipitate.
630 C. A finely flocculent (^) precipitate: not (^) sticky. 730 C. A^ finely flocculent^ precipitate: not sticky.
After filtering off the precipitate at 730, a small amount of proteid
remained in^ solution^ which had^ the^ characters of an albumose, but
which will be fully described later on.
The (^) point to which it is (^) necessary here to call marked attention, is
the fact that the series of precipitates in a muscle-plasma in which
coagulation has^ not^ occurred, is^ precisely the same as in an extract of
muscle in which rigor mortis has occurred.
-Tle beat^ coagulation of^ the fluid^ left after the separation of myosin
was brought about by dilution and filtration was found to be
I (^) This Journal, Vol. v. p. 157.
