intracellular oxidation-reduction studies -...

229

INTRACELLULAR OXIDATION-REDUCTIONSTUDIES

II. REDUCTION POTENTIALS OF MARINE OVAAS SHOWN BY INDICATORS1

BY ROBERT CHAMBERS, HERBERT POLLACKAND BARNETT COHEN2.

(From the Eli Lilly Research Division, Marine Biological Laboratory, Woods Hole,Mass.; Department of Anatomy, Cornell University Medical College, NewYork City; and Hygienic Laboratory, Washington, D.C.)

{Received 14th September 1928.)

A NEW approach to the problems of cellular metabolism was opened by J. and D.Needham(8) who used the micro-injection of reversible indicators to determine theoxidation-reduction intensity of living protoplasm. Prerequisites for this workwere the elaboration of a series, as yet incomplete, of such indicators, and theelectrometric standardisation of the position of each dye on the scale of reduction(or oxidation) electrode potential by Clark, Cohen and their co-workers (1, 4,5. 7).Discussions of the various theoretical phases of oxidation-reduction and theirpossible applications to the study of life processes are found in the papers of Clarkand Cohen (s).

The method used by us for determining the intracellular reduction potential,consisted in observing the extent of decoloration undergone by aqueous solutionsof reversible oxidation-reduction indicators when injected into the protoplasm ofliving cells.

To yield valid interpretation, the results obtained with marine ova requiremore than the usual careful checking indicated in the first paper, which reportsthe work on the reduction potential in Amoeba together with a detailed descriptionof the method used (6). Of the twenty-five oxidation-reduction indicators usedon Amoeba, sixteen of the more important dyes and two additional ones (brilliantcresyl blue and ethyl Capri blue), were selected for a similar study of marine ova.The injections were made in atmospheres of air and nitrogen. Results from theimmersion of eggs in solutions of the dyes will be published in a forthcoming paper.

Material. The ova used in these experiments were those of the starfish (Asteriasforbesii) and the sanddollar (Echinarachnius parma). The eggs averaged 0-15 mm.

1 The expenses connected with this investigation were in part defrayed by grants from theElla Sachs Plotz Foundation and the Committee on Drug Addictions.

2 Published with the permission of the Surgeon-General of the United States Public HealthService.

230 R. CHAMBERS, H. POLLACK and B. COHEN

in diameter (approximate volume, o-ooo6 cu. mm.). The ova of the sanddollarare very suitable for colorimetric work because they are translucent and lack anyappreciable quantity of natural pigment. The ova of the starfish are slightly moreopaque and contain a pale yellow pigment which, however, is appreciable onlywhen the eggs are massed together in large quantities. Both types of ova are highlygranular, the greater translucency of the sanddollar egg being due to the cleareraspect of its more prominent granules, the macrosomes.

From the large number of published experiments in which these ova havebeen punctured or torn with microneedles, there seems to be no question that theinjury thereby produced is never more than of a quickly transitory nature unlessthe puncture or tear is followed by a visible disintegration of the protoplasm.Evidence supporting this view is provided by the recorded fact (3) that mechanicalinjury to the protoplasm of these eggs produces a localised acid reaction whichdisappears almost immediately provided that a distinctly visible disintegrationdoes not occur.

The eggs were injected in the following stages of their development: for thestarfish egg (a) full-sized, immature eggs (with a germinal vesicle), and (b) thoseundergoing maturation (during polar body formation): for the sanddollar egg(a) unfertilised mature eggs within half an hour after removal from the ovary,(b) fertilised eggs within 5 to 30 minutes after insemination, and (c, d) eggs imme-diately before and after the first segmentation. Some injections were made intoblastomeres after the second cleavage.

The immature starfish egg was used for experiments on the nucleus.Microscopic equipment. The cytoplasmic injections were all performed under

a Leitz No. 5 objective and a x 10 ocular. For nuclear injections a 3 mm. apo-chromatic objective was used. The No. 3 Leitz double demonstration eyepiecewas also employed and the observations were made simultaneously by twoobservers. No record was kept unless both observers agreed upon the findings.Artificial illumination with "daylight glass" was used exclusively(3).

The micromanipulative technique. This is the same as that previously described (2,6).To insure success and to control delivery of the solution injected into the ovait is well to use a micropipette of the quick tapering type, preferably with a tipabout 30 to 40 micra long and an aperture less than a micron in diameter.

A complete description of the method used for injections under strict anaero-biosis is given in our previous paper. The hermetic moist chamber was providedwith a mercury seal through which the shanks of the microneedle and micropipettepassed into the chamber. Rubber connections were reduced to a minimum andthe success of the various precautions taken to prevent the diffusion of oxygeninto the chamber is evidenced by the fact that hanging drops of the colourlessreductants of the most readily oxidisable dyes suspended from the roof of thechamber remained uncoloured during the period of the longest experiment. Itwas found that the use of unpurified tank nitrogen, owing to the presence ofresidual oxygen, completely vitiated the results.

The protoplasmic pU. The pH of the cytoplasm of sanddollar and starfish eggs

Intracellular Oxidation-Reduction Studies 231

has been found to be 6-8 ±0*1, that of the nucleus of the immature starfish eggto be 7-6 to 7-8 (3). These values were obtained by the injection of aqueous solutionsof acid-base indicators, all of which assumed the colours indicative of a constantpH irrespective of the actual pH of the dye solution injected. This fact points tothe existence of an appreciable buffering power in the protoplasm, a feature whichhas more recently been studied in greater detail (9). It was, therefore, consideredunnecessary for present purposes to buffer the oxidation-reduction dye solutions,since these solutions, upon injection, must operate in a protoplasmic mediumhaving a constant and buffered pH.

The pH of the cytoplasm of starfish and sanddollar eggs under anaerobicconditions (1 hour in the nitrogen chamber) as determined by the injection ofbrom cresol purple and phenol red was found to be the same as that in air.

Indicators and reagents. The compounds listed in the table (p. 11) were prepared1

at the Hygienic Laboratory. Opposite each compound is given its Eo' value (thepotential on the hydrogen scale) at^>H 7-0, i.e. the electrode potential of an equi-molecular mixture of oxidant and reductant at pH 7-0. These values may beconverted into r¥L values which are independent of pH. The compounds are thusplaced on a graduated scale of reducing (or oxidising) intensity irrespective of theirother chemical characters. The potentials of the hydrogen and theoretical oxygenelectrodes are included as orienting reference points. Compounds on the positiveend of the scale are "more oxidising" than those on the negative end and, con-versely, those on the negative end are "more reducing."

Potassium ferricyanide is included in the table because we have dependedupon injections of fresh solutions of this oxidising agent to restore the colour ofthe intracellularly reduced dyes, thereby making sure that the dye under investiga-tion was still present in the cell and potentially available. The cytoplasm of theovum tolerates the injection of a small amount of the aqueous 1 per cent, potassiumferricyanide. In eggs not previously injected with a dye, the ferricyanide solutionimparts a distinct yellow colour to the cytoplasm. There is never a visible trace of bluesuch as might possibly occur with the formation of a compound like Prussian blue.

Ova treated with dyes under anaerobic conditions were also tested for theintracellular presence of the reduced dye by a subsequent exposure to air of thehanging drops containing the eggs.

The influence of a time factor on recording a positive or negative result for theintracellular presence of the dye proved to be even more serious in the experimentson the ova than in those on Amoeba. When necessary, therefore, precautionswere taken to have a drop of the fresh ferricyanide available in a second micro-pipette so that it could be injected promptly after decolorisation of the indicator.

In our previous publication is given a discussion of the dyes, the preparationof their reductants and the way in which the reduced dyes were delivered in thenitrogen-filled moist chamber.

Briefly stated the procedure for injecting under anaerobiosis was as follows:on the coverslip, which was to roof the hermetic moist chamber, a series of hanging

1 Brilliant cresyl blue and phenosafranin were purified commercial preparations.

232 R. C H A M B E R S , H . P O L L A C K andB. C O H E N

drops were placed within circles marked off with paraffin rings. These were usuallyone or two drops of distilled water for washing the micropipette, two or three ofsea-water containing the ova to be injected, one a solution of the oxidant to beinjected, and one the ferricyanide solution to be used for testing the presence ofthe reductant in the eggs. One or two spaces on the coverslip were left vacant forthe reductant after anaerobic conditions had been established. The coverslip wasthen inverted over the hermetic chamber and sealed in place with vaseline andweighted, after which a steady stream of purified nitrogen was passed through thechamber during the entire course of an experiment. By experience it was foundthat the air in the chamber was sufficiently replaced with deoxygenated nitrogenby 10 to 15 minutes of vigorous flushing. When this had taken place, the reduceddye was delivered into the chamber by means of a fine-tipped delivery tube which,together with the microneedle and micropipette, already projected into the chamberfrom the beginning of the experiment. By raising the curved tip of the deliverytube a drop of clear reductant was deposited in its proper place on the undersurfaceof the coverslip of the sealed moist chamber.

Solutions. The aqueous indicator solutions were always freshly prepared, andunless otherwise stated were of a maximum concentration of 1 per cent, of thesodium salt.

Amounts injected. In one respect the marine ova were more advantageous towork with than the freshwater Amoeba. A real source of error in the micro-injection method, in its present stage of development, is the inability of gaugingaccurately the amount of fluid injected into the cell. This error is difficult to controlwhen injecting non-toxic substances into an Amoeba because of the apparentimpunity with which the cell tolerates varying quantities. The ova, on the otherhand, are far more susceptible to injury and only minimal quantities of the injectedsolutions can be introduced without causing disintegration and cytolysis. Therefore,when a dye was found to be toxic, successful injections were often only madepossible by diminishing the concentration of the solution used.

The amounts injected are sometimes recorded as "small," "moderate" or" large." These are relative terms. By a " small" amount is meant that the injectionceased and the pipette was removed from the cell at the first appearance of colouredsolution exuding from the microtip. The area visibly affected by such injection didnot exceed one or two micra in diameter. A "moderate" amount was consideredto be introduced when pressure on the plunger of the syringe was maintained untilthe introduced dye was seen to spread over an area of about 5 micra in diameteraround the tip of the pipette. More than this was reckoned as a "large" amountwhich always resulted in visible cytolysis of the immediate area injected.

A dye is recorded as "toxic" if visible cytolysis usually occurs after the injec-tion of a "small" amount. If cytolysis occurs at the instant of injection, thesurrounding healthy cytoplasm practically never becomes permeated with the dyeand the injection is recorded as unsuccessful.

In no case was the amount injected sufficient to produce an appreciable increasein the volume of the egg. In the dividing egg the injection occasionally caused


the cleavage furrow to disappear, a phenomenon apparently not due to the amountof fluid injected but to a disturbance of obscure factors. As a rule, fertilised eggswere easier to inject than unfertilised eggs.

The aqueous solutions of all the dyes always spread as a uniform colorationfrom the site of injection through the homogeneous matrix of the protoplasm.In the case of certain dyes possessing basic properties and then only after anappreciable time has elapsed or when too great a concentration has been introduced,there is evidence of selective accumulation of the dye on or in the intra-protoplasmicgranules.

Viability of starfish and sanddollar eggs in an oxygen-free nitrogen atmosphere.It was found that both starfish and sanddollar eggs tolerate anaerobic conditionsfor at least 1 hour without altering their, cytoplasmic pH or changing their normalappearance. The germinal vesicle of the immature starfish egg undergoes thechanges characteristic of prematuration within the first half hour in the nitrogenchamber. This is about the normal time for this process in air. Mature starfisheggs, after 1 hour in the chamber and then returned to air, were capable of formingtypical fertilisation membranes upon insemination but no cleavage occurred.

Sanddollar eggs placed in the nitrogen chamber 10 to 40 minutes after fertilisa-tion and kept under anaerobic conditions for 40 minutes developed swimmingblastulae after being returned to air.

If the eggs were allowed to develop to the stage just before the first cleavageand then were placed in the nitrogen chamber, cleavage proceeded but at a lowerrate than normal. A small percentage completed the division. However, a peculiarfeature of these eggs, when returned to air, was that they always reverted to theone-celled stage, with two nuclei. Subsequently, segmentation proceeded againwith the formation of abnormal swimming larvae.

EXPERIMENTAL RESULTS.

I. CYTOPLASMIC INJECTIONS.

A I1. Phenol m-sulfonate indo-2, 6-dibromophenol.

Aerobic injections. The blue oxidant, injected into fertilised sanddollar eggs,decolorised instantly. The dye was slightly toxic. The injected eggs, when torn soas to induce cytolysis, developed a red colour characteristic for the oxidant at thepH of the acid of injury, viz. pR 5-4. The colour usually faded within a few seconds.Frequently, it persisted for more than a minute and then changed from red to blue.

Anaerobic injections. The oxidant decolorised instantly. Frequently, the injuryincident to the injection caused cytolysis of the egg. When this occurred thecytolysed region took on a red colour which usually faded in 10 to 20 seconds, but

1 The dyes are listed here under the same designations as in the first paper of this series (6).Since not all the dyes were used here the lettering is incomplete.

234 R- C H A M B E R S , H . P O L L A C K and B. C O H E N

could not be brought back with ferricyanide. Some portions of the cytolysed debrisretained a pink colour for as long as 20 minutes.

C. m-Bromophenol indophenol.Aerobic injections. The blue oxidant gave a pink flash which decolorised instantly

in the cytoplasm of mature, unfertilised and fertilised starfish and sanddollar eggs.The dye was relatively non-toxic. Injection of ferricyanide into these eggs causedthe appearance of a pink colour, the colour of the oxidant at a pH more acid than7-7(7). A pale pink colour also developed in the cytolysed regions of eggs injectedwith the dye and then injured by tearing. The colour faded within 5 to 10 secondsand could not be brought back by the injection of ferricyanide.

H. Phenol indo-2, 6-dichlorophenol. I. Phenol indo-2, 6-dibromophenol.

Aerobic injections. The blue oxidant of both these relatively non-toxic dyesdecolorised almost immediately in unfertilised, fertilised and dividing sanddollareggs. Small amounts were decolorised in less than one second. Moderate amountstook several seconds. A subsequent injection, 20 minutes later, of ferricyanidebrought back the colour. Cytolysis by mechanical injury also brought back thecolour but less intensely.

Injection of dye H into the astral centre of the segmenting sanddollar eggresulted in the spread of a flash of blue throughout the hyaline area followedimmediately by complete decolorisation.

A sanddollar eggy just beginning segmentation 70 minutes after insemination,was injected with a moderate amount of the dichloro compound. The blue colourfaded immediately. Twelve minutes later the egg segmented into two blastomeres.Ferricyanide was then injected into each blastomere separately with a distinctreturn of the blue colour in both.

Anaerobic injections. The blue oxidants decolorised instantly.

L. o-Cresol indo-2, 6-dichlorophenol.

Aerobic injections. The blue oxidant decolorised almost instantly in both un-fertilised and fertilised eggs of the sanddollar and starfish. Ferricyanide broughtback the colour.

The non-toxicity of the dye is indicated by the development of a typicalamphiaster in the fertilised sanddollar egg after injection.

Anaerobic injections. The blue oxidant, injected into five immature starfish eggs,decolorised almost immediately.

M. i-Naphthol-2-sulfonate indophenol m-sulfonate (disodium salt).

This slightly toxic preparation contained 50 per cent, sodium chloride and wasrather pale in the 1 per cent, solution. Therefore, a 2 per cent, concentration was used.

Aerobic injections. The red oxidant, injected into immature starfish eggs, andfertilised arid dividing sanddollar eggs, decolorised almost immediately. Fiveminutes later an injection of ferricyanide restored the colour.


Cytolysing areas coloured pink when injected with the oxidant and graduallychanged to purple and then blue as the more alkaline sea-water permeated thecytolysed mass.

Anaerobic injections. The oxidant, injected into immature and mature starfisheggs, decolorised almost immediately.

N. m-Toluylene diamine indophenol chloride.

Aerobic injections. The red oxidant, injected into fertilised sanddollar eggs,decolorised almost immediately. The injection of ferricyanide brought back thecolour. Eggs which had been injected with the dye were torn with needles to inducecytolysis whereupon the colourless reductant in the cytolysed material took onthe pink colour of the oxidant. This faded within 5 to 10 seconds and could notbe made to return with ferricyanide.

Anaerobic injections. The red oxidant decolorised instantly in the cytoplasm ofboth unfertilised and fertilised sanddollar eggs. It is toxic.

The colourless reductant was distinctly less toxic and remained colourless inthe cytoplasm. Injections of ferricyanide brought out the colour of the oxidant.Exposure to air of eggs injected with the reductant occasionally brought out afaint pink colour. This was probably caused by the injection into the cell of anexcess of the relatively non-toxic reductant, which, in the presence of air, wasmore than the protoplasm could maintain in the completely reduced state.

0. i-Naphthol-2-sulfonate indophenol.

Aerobic injections. The relatively non-toxic red oxidant decolorised completelyin the cytoplasm of unfertilised, fertilised and dividing sanddollar eggs within1 to 4 seconds. Whenever cytolysis occurred at the time of injection the cytolysingdebris coloured pink. Ferricyanide restored the red colour in the surviving cyto-plasm.

Anaerobic injections. The oxidant was almost instantly decolorised. The re-ductant remained colourless. On exposure to air no return of colour could bedetected. A distinct colour was obtained by an injection of ferricyanide.

P. i-Naphthol-2-Na sulfonate indo-2, 6-dichlorophenol.

Aerobic injections. The relatively non-toxic blue oxidant always decolorisedcompletely within 2 to 5 seconds when injected into a large number of unfertilised,fertilised and segmenting starfish and sanddollar eggs. In some cases the injectioncaused local cytolysis, the debris of which coloured blue. A subsequent injectionof ferricyanide brought back the colour not only in healthy eggs but also in thesurviving portions of partially cytolysed eggs.

Anaerobic injections. The oxidant was almost instantly reduced. The yellowreductant was not oxidised upon injection. Upon exposure to air there was noappearance of a blue colour which, however, immediately showed up when ferri-cyanide was injected.


Q. Toluylene blue chloride.Aerobic injections. The purple oxidant of this basic dye was quite toxic and,

in the concentration used, resulted in cytolysis of the eggs within 2 to 7 minutesafter injection. The injected dye was reduced in 2 to 5 seconds. Ferricyanidebrought back the colour of the oxidant.

Anaerobic injections. The oxidant completely decolorised within 2 to 5 seconds.Occasionally, traces of colour on granules persisted for several minutes beforedisappearing.

Q 1. Brilliant cresyl blue chloride.Aerobic injections. The blue oxidant of this basic dye was toxic. A small quantity

injected into fertilised sanddollar eggs was reduced within 2 to 5 seconds. Ferri-cyanide brought back the colour.

Anaerobic injections. The oxidant injected in small quantities was reducedwithin 2 to 5 seconds. Ferricyanide brought back the colour.

R. Methylene blue chloride.Aerobic injections. The blue oxidant, injected very gradually and in small

amounts, diffused through the cytoplasm and decolorised within 10 to 30 seconds.Usually, however, a localised deep blue coagulum resulted at the site of the injectionfrom which the colour diffused through the cytoplasm. The diffuse blue colourdisappeared within half a minute or so while the colour of the coagulum graduallyfaded and disappeared only after 4 to 10 minutes. Injection of ferricyanide intothe surviving cytoplasm brought back the colour.

If the injection of the oxidant was performed gradually, a considerable amountcould be introduced into the cell without appreciable cytolysis. There was thena distinct fading of the blue colour beyond the site of the injection within thefirst half minute, after which the effect of gradual fading was accompanied by theaccumulation of the residual oxidant in the cytoplasmic granules about the siteof the injection. In one such fertilised sanddollar egg, segmentation proceeded insuch a way that one blastomere contained all the blue granules while the otherwas colourless. Injection of ferricyanide into each of these blastomeres resultedin a diffuse blue coloration which was much deeper in the one originally containingscattered blue granules.

Anaerobic injections. The relatively toxic oxidant decolorised within 2 to 5seconds except when excessive amounts caused cytolysis. Ferricyanide, injectedwithin 1 minute after decoloration, brought back the blue colour. The less toxic,colourless reductant, when injected, remained reduced, and a blue colour developedwith ferricyanide. Frequently, on exposure to air, the injected eggs coloured blue.However, when a sufficiently small quantity of the reductant was injected, exposureto air resulted in no colour which, nevertheless, could be subsequently producedby an injection of ferricyanide.

The reductant is troublesome to work with owing to the tendency of therelatively insoluble leuco-compound to form crystals which clog the pipette.


S. K4 indigo tetrasulfonate.Aerobic injections. The purple oxidant was not appreciably decolorised. Fre-

quently the pipette produced a local cytolysis in spite of which the survivingcytoplasm readily took on a blue colour.

The dye is somewhat toxic but after many attempts it was possible to securea number of injected sanddollar eggs which were sufficiently viable to form typicalfertilisation membranes upon subsequent insemination. Fertilised eggs afterinjection were observed to commence cleavage but not to complete it. Wheninjected into the cytoplasm of starfish eggs the colour was retained for at least5 minutes, after which cytolysis set in. In one case an immature egg retained theblue colour for half an hour before cytolysis occurred.

Anaerobic injections. The oxidant decolorised in 2 to 3 seconds. The yellowreductant, when injected, remained reduced. Injection of ferricyanide or a returnof the eggs to air brought back the colour.

That the reductant is less toxic than the oxidant was indicated by the factthat eggs, injected with the reductant, tend to undergo more or less extensivecytolysis as they turn blue on exposure to air.

S1. Ethyl Capri blue nitrate.This is the symmetrical tetra-ethyl oxazine analogous in structure to the

thiazine, methylene blue. It is one of the oxazines recently synthesised andstudied at the Hygienic Laboratory by Preisler and Cohen. (The electrometricdata on these compounds are as yet unpublished.)

Aerobic injections. The non-toxic blue oxidant, injected into fertilised anddividing sanddollar eggs, was partially decolorised to a pale greenish blue whichdiffusely coloured the cytoplasm. Injection of ferricyanide frequently intensifiedthe colour to a deeper blue. Cytolysis caused by tearing with a microneedleoccasionally resulted in a deepening of the colour in the cytolysed region.

Anaerobic injections. The injected oxidant decolorised within 2 to 5 seconds.Injection of ferricyanide brought back the blue colour. Eggs which had reducedthe injected oxidant in the nitrogen chamber took on a pale greenish blue colourwithin 15 seconds after being exposed to air.

T. K3 indigo trisulfonate.Aerobic injections. The blue oxidant was not decolorised. Injected dividing

eggs of the sanddollar continued cleavage without completing it. The dye seemedto be less toxic than the tetrasulfonate.

Anaerobic injections. The oxidant decolorised within 2 to 3 seconds. An in-jection of the yellow reductant containing the faintest tinge of green gave no evidenceof oxidation. Injection of ferricyanide or exposure to air quickly developed a bluecolour in the eggs.

U. K2 and Na2 indigo disulfonates (indigo carmine).Aerobic injections. The blue oxidant was not decolorised. The dye is somewhat


toxic since the majority of the injections caused at least some cytolysis within aminute after the injection.

Anaerobic injections. The oxidant was decolorised in 2 to 3 seconds. Thereductant, when injected, remained reduced. An injection of a moderate amountof either resulted almost always in complete cytolysis within a few minutes. Ferri-cyanide restored the colour. Eggs injected with small amounts and exposed after5 to 10 minutes to air showed a distinct blue colour within 4 to 8 minutes.

V. Neutral red iodide.This basic dye does not seem to possess an easily reversible oxidation-

reduction equilibrium at pH 7*0.The orange-red oxidant injected aerobically or anaerobically coloured the

cytoplasm a diffuse rose. Within a few minutes the colour became restricted togranules distributed throughout the cytoplasm.

X. Phenosafranin.

This system is also not quickly reversible at pK 7-0.The toxic, red oxidant of this basic dye, when injected either aerobically or

anaerobically, caused a diffuse pink coloration which persisted until cytolysisoccurred 10 to 15 minutes after the injection. The equally toxic, yellow reductantgave no sign of being oxidised in the egg under anaerobic conditions. Injectionwith ferricyanide brought out a red colour.

Fertilised and segmenting sanddollar eggs were injected with the reductantanaerobically. Upon exposure to air the eggs took on a distinct pink colour within45 minutes.

II. NUCLEAR INJECTIONS.

The immature egg of the starfish was selected for the nuclear injectionsbecause of the relatively large size of the nucleus (30 to 40/z, in diameter). Thenucleus is much more susceptible than the cytoplasm to mechanical injury butfrom previous experiments on the injection of pH indicators (3) we know that thenucleus can survive the effects of the manipulation and show positive signs ofviability for at least an hour. With the injection of the oxidation-reduction dyeswe were unable to obtain conditions for the nucleus to maintain its normal appear-ance longer than several seconds. The first visible sign of impending disintegrationof the nucleus is a change in the nucleolus. The latter changes shape, swells andfades from view or becomes permanently fixed with a change in its refractiveappearance. The nucleus also swells and then shrinks into a spherical remnantwhile the surrounding cytoplasm undergoes more or less extensive cytolysis.

The following are the experimental results of the nuclear injections:

C. m-Bromophenol indophenol.

The blue oxidant, injected aerobically and anaerobically, spread through thenucleus and immediately changed to a pink colour which persisted during the

Tab

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T to

Res

ults

of i

njec

ting

oxi

dati

on-r

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tion

ind

icat

ors

into

the

cyto

plas

m o

f san

ddol

lar

and

star

fish

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phen

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240 R. C H A M B E R S , H . P O L L A C K and B. C O H E N

3 to 5 seconds before the nucleus and the surrounding cytoplasm underwentcytolysis. If the dye was made to exude from the micropipette during the passageof the pipette through the cytoplasm it was possible to secure an almost simultaneousinjection of both nucleus and cytoplasm. In such cases the difference between thecytoplasm and the nucleus was strikingly shown, the nucleus persisting as a pink-coloured body while the cytoplasm showed a flash of pink which instantly dis-appeared.

The red colour assumed by the blue dye in both cytoplasm and nucleus is thecolour of the acid range of the dye whose turning point is near pH 7-7.

L. o-Cresol indo-2, 6-dichlorophenol.

The blue oxidant, injected aerobically or anaerobically, remained blue in thefour nuclei injected during the 3 to 4 seconds before they disintegrated.

M. i-Naphthol-2-sulfonate indophenol m-sulfonate (disodium salt).

The blue oxidant, injected aerobically, coloured the nucleus blue. The nuclei,however, disintegrated too rapidly to afford definite results.

O. i-Naphthol-2-sulfonate indophenol.

The red oxidant, injected aerobically, kept its colour within the nucleus for5 seconds before cytolysis set in, after which the colour faded from the nuclearremnant in 30 seconds.

R. Methylene blue chloride.

The blue oxidant, injected aerobically, coloured the nucleus blue for 5 seconds,after which the nucleus broke down. Before this occurred a diffusion of thecolour became apparent spreading from the periphery of the nucleus into thesurrounding cytoplasm, where the colour faded to a pale green and then dis-appeared. Diffusion of the colour from the nuclear remnant continued and finallycoloured the cytolysed cytoplasm blue. The nucleolus usually persisted within thenuclear remnant.

The colourless reductant, injected anaerobically, produced no colour in thenucleus, which broke down within a few seconds. In some cases the nucleolus wasdriven to one side of the nucleus by the injection.

S. K4 indigo tetrasulfonate.

The blue oxidant, injected anaerobically, retained its colour for 5 seconds. Thenuclei then broke down whereupon the colour faded. In several injections someof the dye entered the cytoplasm where it faded out completely while the nucleuswas still blue and normal in appearance.

The yellow reductant, injected anaerobically, remained yellow. The three nucleiinjected broke down after 4 seconds, whereupon the yellow colour disappeared.


T. K3 indigo trisulfonate.

The reductant, injected anaerobically, gave no sign of colour during the fewseconds that the nuclei appeared normal.

U. K2 indigo disulfonate.

The oxidant injected anaerobically remained blue during the several secondsbefore the nucleus broke down. Some of the dye injected at the same time into thecytoplasm disappeared completely while the nucleus still remained blue.

The yellow reductant, injected anaerobically, was not oxidised.

V. Neutral red iodide.

The red dye, injected aerobically, coloured the nucleus an orange yellow.Within a few seconds the dye gradually diffused into the surrounding cytoplasmwhere it changed to a rose pink colour. There was no apparent reduction either inthe nucleus or in the cytoplasm.

III. EXPERIMENTS ON THE REVERSIBILITY OF THE INTRA-CELLULAR

OXIDATION-REDUCTION REACTION OF THE DYE SYSTEMS IN PROTOPLASM.

The ability of the dyes to undergo repeated reversible changes in the protoplasmof the ova can be demonstrated by varying the oxygen content of the environmentwithin certain limits.

An indication of this has already been brought out in the injection experiments.For example, under anaerobiosis Amoeba^) and the marine ova (p. 9) reducethe three indigo sulfonates which, upon subsequent exposure of the cells to air,revert to the blue colour of the oxidants.

With indigo trisulfonate and with ethyl Capri blue nitrate this reversibilityhas been shown to occur repeatedly within the same living cell. Unfertilised andfertilised sanddollar eggs, after being injected with the oxidant under anaerobiosiswhere complete reduction took place, were exposed alternately to air and to thedeoxygenated nitrogen atmosphere three times in succession. With each exposureto air the blue colour returned, and as consistently disappeared with each exposureto the nitrogen atmosphere. The return of the blue colour in air was always morerapid than its disappearance in the nitrogen chamber.

The reversibility of the reaction can also be shown with methylene blue whichreadily penetrates starfish and sanddollar eggs until the accumulation of the dyein the cytoplasm passes beyond the threshold of the cytopiasmic reducing capacityunder aerobiosis. The eggs thereupon take on a distinct blue coloration. Eggsstained in this way with this dye decolorised on prolonged exposure to the nitrogenatmosphere. Upon return to air the colour returned. Experiments on the use ofan oxygen atmosphere with dyes which are reduced in protoplasm under normalaerobic conditions were inconclusive.

242 R - CHAMBERS, H. POLLACK and B. COHEN

DISCUSSION.


In a study of the oxidation-reduction potential (intensity factor) of protoplasmbased on the effect of introducing solutions of indicators into protoplasm one mustbear in mind the possibility of errors arising from variations in the amount of thedye introduced (capacity factor) and the rate at which the dye is reducible oroxidisable (rate factor). The existence of these factors has been discussed in aprevious paper. In the case of the marine ova the capacity factor seems to bepartially under control because the high susceptibility of the egg-protoplasm todisintegration when foreign substances are introduced prevents the injection ofmore than minimal quantities of the dyes.

A feature which must chiefly be taken into account is the peculiarity of certainbasic dyes, if given sufficient time, to stain the cytoplasmic granules. When thisoccurs there is a distinct delay in the disappearance of colour of the reducibledyes from the granules. The precaution was therefore taken, in the case of suchdyes, to use greater dilution or to perform the injection very gradually so as toallow possible reduction before the dye accumulated on the granules.

Regarding the rate factor it is to be noted that the speed of reduction wasgreatest for the most electropositive dyes and tended to be less for those dyeswhose potential approached that of the egg protoplasm.

When we consider the intensity factor we find some difference in the resultsreported for Amoeba and those given in this report on the marine ova. For Amoebathe dyes listed as partly reduced after injection under aerobic conditions, viz. toluy-lene blue and methylene blue, are the ones which stain the cytoplasmic inclusions,a complication which has been more carefully taken into account in the case of themarine eggs. In the report on the Amoeba, toluylene blue and methylene bluewere tabulated as being "reduced partly." This is an unfortunate statement inso far as it does not give the true meaning intended to be conveyed. Actually theexperiments on the Amoeba showed that these dyes were completely reducibleprovided the precaution was taken to prevent the capacity factor from obscuringthe result. In this respect our results on the marine ova are the same. A dis-crepancy which is apparently real concerns the intracellular action of potassiumindigo tetrasulfonate which, in the marine ova under aerobic conditions, gave nosign of reduction while in the Amoeba there seemed to be a suggestion of somereduction as evidenced by a paling of the blue colour of the injected dye. However,no positive significance should have been attached to this phenomenon since theinjection of ferricyanide was found ((6), p. 597) to have resulted in no increase incolour.

In the marine ova under aerobic conditions it was found that methylene blueappears to be completely reduced while potassium indigo tetrasulfonate remainsapparently unreduced. On the other hand, the new indicator, ethyl Capri blue,seems to be only partially reduced in the cytoplasm of the ova. No matter how


small the injections under aerobic conditions, the colour of this dye never com-pletely disappeared. This seems to be a real partial reduction and, for the present,we may, therefore, place the aerobic reduction potential of the starfish andsanddollar eggs close to an rH of 12.0, which is that of ethyl Capri blue nitratewhen 50 per cent, reduced at/>H 7-0.

Under anaerobic conditions these eggs develop a reduction potential which ishigh enough to reduce completely all the reversible oxidation-reduction indicatorsdown to and including indigo disulfonate. The latter when 99 per cent, reducedcorresponds to rH 7-9 atpH 7-0.

Attempts to utilise potassium indigo monosulfonate whose rH, when 99 percent, reduced, is 6.8 proved to be inconclusive owing to the unsuitability of thisinsoluble compound.

The apparent non-reduction of indigo tetrasulfonate while the adjacent indica-tors in the series were completely or partially reduced by the aerobic ova representsthe first anomaly encountered in our studies. Theoretically, if ethyl Capri blue isreduced, then the more electropositive tetrasulfonate should also be reduced, Wesuspect that the explanation for this anomaly lies in the toxicity of the tetra-sulfonate functioning, perhaps, as an " antireductant." Except for this, our resultshave been uniformly consistent both for the freshwater Amoeba and for themarine ova. The experiments still leave one point to be determined; althoughwe have found the direction we have not yet determined the lower limit of theanaerobic, intraprotoplasmic reduction potential owing to the lack at present ofsuitable indicators.

These values for the aerobic rH do not agree with those reported by J. and D.Needham(8) and Rapkine and Wurmser(io) who place the aerobic rH at 19 to 22for several species of marine ova and the salivary gland cells of some insects.

J. and D. Needham, in their work on the Amoeba, imply that all aerobic cellshave an rH which is well poised and probably independent of wide variations inthe external oxygen pressure. This conclusion is contradictory to our findings inwhich we show that the removal of O2 from the environment very definitely resultsin a shift of the reduction potential of the aerobic cells used toward very low values.What happens when pure O2 is employed still remains to be determined, ourexperiments in this direction being inconclusive.

The reliability of applying the dye systems to a study of protoplasmic reactionsmight be criticised because of the possibility that the dye in the protoplasm maynot behave reversibly as it does in vitro. However, the reversibility of the reactionshas been demonstrated by the alternate transference of injected eggs fromanaerobic to aerobic conditions and back again. This is well shown, for example,with eggs injected with K3 indigo trisulfonate and then exposed to nitrogen andreturned to air three times in succession. The colour disappeared and returnedwith each change.

When a dye is injected into the cytoplasm and the colour disappears it canreadily be shown that the dye is still present but in the form of the colourlessreductant. Under anaerobic conditions this is done by the intracellular injection of

244 R - C H A M B E R S , H . P O L L A C K and B . C O H E N

an oxidising agent, such as potassium ferricyanide or potassium chromate. Infact this was one of our chief criteria for differentiating between intracellularreduction and the possible loss of colour by other means such as chemical de-struction of the dye or outward diffusion. Under aerobic conditions it was sometimespossible, in addition to the ferricyanide test, to demonstrate the intracellularpresence of dye reductant by inducing cytolysis through mechanical injury uponwhich the reductant in the cytolysed region would be oxidised by exposure to air.It is significant, however, that the intensity of colour produced by cytolysis neverreached that produced by injecting ferricyanide into the intact egg. The colourfaded within a few seconds to several minutes and could not be brought back withferricyanide.

The cytolysing material may still possess some reducing power but the finalcomplete fading of the colour must be ascribed to the washing out or possiblythe destruction of the dye.

It is known that the reducing ability of tissues is not entirely destroyed atdeath. In order to determine this for the marine ova a mass of starfish eggs wascytolysed with distilled water in a test tube and the suspension tested by introducinga few drops of an easily reducible dye (phenol m-sulfonate indo-2,6-dibromophenol).

Quick reduction took place after repeated additions of the indicator althoughthe suspension was kept thoroughly aerated by shaking. That the decoloration wasdue to reduction and not decomposition of the compound was shown by the factthat the addition of ferricyanide promptly restored the colour of the oxidant.Similar observations on various tissue juices and macerates have been reported byCohen, Gibbs and Clark(7), and Cannan, Cohen and Clark d).

There is still the question whether the poising ability of the injected indicatorsystems has a disturbing effect on the intracellular reduction potential. This isprobably a second order effect. At any rate the results are consistent for the wholeseries of reversible indicators, irrespective of their chemical constitution, wheninjected under anaerobic conditions; and, in response to the presence and absenceof oxygen in the environment, all the dyes give consistent indications of a cha-racteristic shift in intracellular reduction potential.

All the experimental results recorded in this and our previous paper point tothe conclusion that the reduction potential in the aerobic cells studied is a functionof the presence or absence of oxygen in the environment.


In addition to determining the oxidation-reduction potential of the cytoplasm,an attempt was made to determine that of the cell nucleus in so far as the materialand the difficulties in technique permitted. It must be stated that our findingsleave much to be desired in respect to definiteness and detail. The technical diffi-culties were such that it was impossible to apply our customary critical chemicaltest (the injection of ferricyanide) for the detecting of the available reduced dye.

In the first paper of this series it was reported that the nucleus of an Amoeba


was frequently coloured when certain oxidants (I, N, P, Q, R, S, U, V and X)were injected into the cytoplasm. In every instance in which reduction of theoxidant took place in the cytoplasm the fading of colour from the nucleus wasmuch slower. The fact that the colour faded from the nucleus does not necessarilymean that the nucleus reduced the dye, for it is equally possible that the dyesimply diffused out of the nucleus into the reducing cytoplasm just as the dye hadpreviously passed from the cytoplasm into the nucleus when the injection wasfirst made.

J. and D. Needham(8) made a few attempts to inject the nucleus of the immaturestarfish egg and Rapkine and Wurmser(io) made similar experiments on the nucleiin a variety of cells. The latter claim from their results that the oxidation-reductionpotential of the nucleus is the same as that of the cytoplasm.

The extreme difficulty of dealing with the nucleus in the living state is broughtout in the experimental part of this paper. All the oxidation-reduction indicatorsproved to be distinctly toxic when injected into the nucleus of the starfish egg. Inonly a few instances did the injected nucleus maintain its normal appearance foras long as 30 seconds after the injection. In the majority of cases the nucleussuccumbed within a few seconds. That the inability of the nucleus to survive longeris not due to technical difficulties but to the toxicity of the dyes in question seemsto be indicated by the fact that we have been able to inject pH indicators witha subsequent normal survival of the nucleus (3).

Because of the very brief survival of the nucleus after injection, it is unjustifiableto draw any definite conclusion regarding the reduction potential of the nucleus.However, it is possible to make surmises from the fact that in every case theoxidants (C, L, M, O) which are all instantly or almost instantly reduced in thecytoplasm remain oxidised within the nucleus under both aerobic and anaerobicconditions for the several seconds that the nucleus survived and also after itexhibited signs of injury.

Moreover, all the reductants injected anaerobically remained reduced.We have no definite interpretation to offer at present regarding the oxidation-

reduction potential of the nucleus. There is the fact that the cytoplasm of thestarfish egg quickly reduces certain injected dyes, while its injected nucleus remainscoloured both under aerobic and anaerobic conditions even with a very easilyreducible dye, namely C. Is this to be regarded as evidence that the nucleus doesnot exert the same high reducing intensity as the cytoplasm or merely that thebrief interval during which the observation could be made permitted a capacityfactor to obscure the phenomenon?

SUMMARY.

A selected series of eighteen oxidation-reduction indicators was used in theinjection experiments on the ova of the Echinoderms, Asterias forbesii and Echin-arachnius parma.

The oxidants of many of the dyes and several of the reductants were alsoinjected into the nucleus of the immature starfish egg.



1. Under aerobiosis the ova completely reduced all the indicators listed in thetable down to and including methylene blue. Potassium indigo tetrasulfonate, atoxic dye, is not noticeably reduced. Ethyl Capri blue was only partially reduced.The rest of the indicators from potassium indigo trisulfonate down were notreduced. On this basis aerobic rH is placed at approximately 12*0 (— 0*06 volts).

2. Under anaerobiosis the egg cytoplasm reduced all the available reversibleoxidation-reduction indicators down to and including indigo disulfonate. Thereductants of none of these dyes were oxidised. Therefore, the anaerobic rH liessomewhere below 7-9 (calculated on the basis of 99 per cent, reduction of indigodisulfonate).

3. No difference could be found in the apparent reduction potentials of theunfertilised, fertilised, and segmenting ova of the two species.

4. Reduction was more rapid under anaerobiosis than under aerobiosis.5. The most toxic of the dyes appear to be the basic compounds and those

which are not reduced in aerobiosis. The reductants of the dyes tend to be lesstoxic than their oxidants.

6. There is a definite, reversible shift in the intracellular rH in response to thepresence and absence of oxygen in the environment. In oxygen-free nitrogenatmosphere it moves toward that of the hydrogen electrode.

7. By injecting an oxidising agent, i.e. potassium ferricyanide, into living ovait was possible to detect the intracellular presence of dyes which previously hadbeen introduced and had been reduced to the leuco-form. Under aerobic conditionsit was also frequently possible to bring back the colour of the oxidant by theinduction of cytolysis in eggs containing the reductant. Under anaerobic conditionsthis never occurred. The colour appearing in the cytolysing eggs never approachedthe intensity of that produced by the injection of ferricyanide into the intact egg.


8. All the oxidants injected into the nucleus of the immature starfish eggremained oxidised within the nucleus under both aerobic and anaerobic conditionsfor the several seconds that the nucleus survived the injection and also after signsof injury became apparent. All the reductants injected anaerobically remainedreduced.

III. REVERSIBILITY.

9. The oxidation-reduction indicator dyes do not lore the reversibility of theirreaction within the cytoplasm. This has been demonstrated by varying the environ-mental oxygen content within limits.

Grateful acknowledgment is made to the U.S. Bureau of Fisheries Station,Woods Hole, Mass., for the use of their facilities in connection with theseinvestigations.


BIBLIOGRAPHY.

(1) CANNAN, R. K., COHEN, B. and CLARK, W. M. (1926). "Studies on oxidation-reduction, X."U.S. Public Health Reports, 55.

(2) CHAMBERS, R. (1928). Micromanipulative technique in Gatenby and Cowdry's Microtomist'sVade-Mecum, J. and A. Churchill, London; and in McClung's A Handbook of MicroscopicTechnique, P. Hoeber, New York (1929).

(3) CHAMBERS, R. and POLLACK, H. (1927). " Micrurgical studies in cell physiology. IV. Colori-metric determination of the nuclear and cytoplasmic pH in the starfish egg." Journ. Gen.Physiol. 10, 739.

(4) CLARK, W. M. (1920). "Reduction potentials of mixtures of indigo and indigo white, andof mixtures of methylene blue and methylene white." Journ. Washington Acad. Sci. 10, 255."Reduction potential in its relation to bacteriology." Abstr. Bad. 4, 2.

(5) CLARK, W. M. and COHEN, B. (1922). "Some elementary aspects of the putrescibility andsimilar tests." Abstr. Bact. 6, 3.

(1923). "Studies on oxidation-reduction, I, II, III." U.S. Public Health Reports,38,443,666,933.

(1925). "Indicators of anaerobiosis." Abstr. Bact. 9, 11.(6) COHEN, B., CHAMBERS, R. and REZNIKOFF, P. (1928). "Intracellular oxidation-reduction

studies. I. Reduction potentials of Amoeba dubia by microinjection of indicators." Journ.Gen. Physiol. 11, 585.

(7) COHEN, B., GIBBS, H. D. and CLARK, W. M. (1924). "Studies on oxidation-reduction, V."U.S. Public Health Reports, 39, 381.

(1924). "Studies on oxidation-reduction, VI." U.S. Public Health Reports,39, 804.

(8) NEEDHAM, J. and D. M. (1925). "The hydrogen-ion concentration and the oxidation-reductionpotential of the cell-interior; a micro-injection study." Proc. Roy. Soc. London, B, 98, 259.

(1926). "The hydrogen-ion concentration and oxidation-reduction potential ofthe cell-interior before and after fertilisation and cleavage: a micro-injection study onmarine eggs." Proc. Roy. Soc. London, B, 99, 173.

(1926). " Further micro-injection studies on the oxidation-reduction potential ofthe cell-interior." Proc. Roy. Soc. London, B, 99, 383.

(1926). "The oxidation-reduction potential of protoplasm: a review." Proto-plasma, 1, 255.

(9) POLLACK, H. (1928). " Intracellular Hydrion Concentration Studies. I II . The buffering actionof the cytoplasm of Amoeba dubia and its use in measuring the pH." Biol. Bull. 55, 383.

(10) RAPKINE, L. and WURMSER, R. (1926). "Sur le potentiel de reduction du noyau et les oxyda-tions cellulaires." Compt. rend. Soc. bioL9^, 989.

(1927). " On intracellular oxidation-reduction potential." Proc. Roy. Soc. London,B, 102, 127.

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