Intracellular Oxidation Reduction Studies

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INTRACELLULAR OXIDATION-REDUCTION

STUDIES

II. REDUCTION POTENTIALS OF MARINE OVA AS SHOWN BY INDICATORS1

BY ROBERT CHAMBERS, HERBERT POLLACK

AND BARNETT COHEN

2

.

(From the Eli Lilly Research Division, Marine Biological Laboratory, Woods Hole, Mass.; Department of Anatomy, Cornell University Medical College, New York 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 the oxidation-reduction intensity of living protoplasm. Prerequisites for this work were the elaboration of a series, as yet incomplete, of such indicators, and the electrometric 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 their possible applications to the study of life processes are found in the papers of Clark and Cohen (s).

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

To yield valid interpretation, the results obtained with marine ova require more than the usual careful checking indicated in the first paper, which reports the work on the reduction potential in Amoeba together with a detailed description of the method used (6). Of the twenty-five oxidation-reduction indicators used on Amoeba, sixteen of the more important dyes and two additional ones (brilliant cresyl 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 the immersion 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 (Asterias forbesii) 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 the Ella Sachs Plotz Foundation and the Committee on Drug Addictions.

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

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

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

The eggs were injected in the following stages of their development: for the starfish egg (a) full-sized, immature eggs (with a germinal vesicle), and (b) those undergoing 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 into blastomeres 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 eyepiece was also employed and the observations were made simultaneously by two observers. 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 ova it is well to use a micropipette of the quick tapering type, preferably with a tip about 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 provided with a mercury seal through which the shanks of the microneedle and micropipette passed into the chamber. Rubber connections were reduced to a minimum and the success of the various precautions taken to prevent the diffusion of oxygen into the chamber is evidenced by the fact that hanging drops of the colourless reductants of the most readily oxidisable dyes suspended from the roof of the chamber remained uncoloured during the period of the longest experiment. It was found that the use of unpurified tank nitrogen, owing to the presence of residual oxygen, completely vitiated the results.

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

The pH of the cytoplasm of starfish and sanddollar eggs under anaerobic conditions (1 hour in the nitrogen chamber) as determined by the injection of brom 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 (the

potential 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 be converted into r¥L values which are independent of pH. The compounds are thus placed on a graduated scale of reducing (or oxidising) intensity irrespective of their other chemical characters. The potentials of the hydrogen and theoretical oxygen electrodes are included as orienting reference points. Compounds on the positive end 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 depended upon injections of fresh solutions of this oxidising agent to restore the colour of the intracellularly reduced dyes, thereby making sure that the dye under investiga-tion was still present in the cell and potentially available. The cytoplasm of the ovum tolerates the injection of a small amount of the aqueous 1 per cent, potassium ferricyanide. In eggs not previously injected with a dye, the ferricyanide solution imparts a distinct yellow colour to the cytoplasm. There is never a visible trace of blue such as might possibly occur with the formation of a compound like Prussian blue. Ova treated with dyes under anaerobic conditions were also tested for the intracellular presence of the reduced dye by a subsequent exposure to air of the hanging drops containing the eggs.

The influence of a time factor on recording a positive or negative result for the intracellular presence of the dye proved to be even more serious in the experiments on the ova than in those on Amoeba. When necessary, therefore, precautions were 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 preparation of their reductants and the way in which the reduced dyes were delivered in the nitrogen-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

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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 usually one or two drops of distilled water for washing the micropipette, two or three of sea-water containing the ova to be injected, one a solution of the oxidant to be injected, and one the ferricyanide solution to be used for testing the presence of the reductant in the eggs. One or two spaces on the coverslip were left vacant for the reductant after anaerobic conditions had been established. The coverslip was then inverted over the hermetic chamber and sealed in place with vaseline and weighted, after which a steady stream of purified nitrogen was passed through the chamber during the entire course of an experiment. By experience it was found that the air in the chamber was sufficiently replaced with deoxygenated nitrogen by 10 to 15 minutes of vigorous flushing. When this had taken place, the reduced dye was delivered into the chamber by means of a fine-tipped delivery tube which, together with the microneedle and micropipette, already projected into the chamber from the beginning of the experiment. By raising the curved tip of the delivery tube a drop of clear reductant was deposited in its proper place on the undersurface of the coverslip of the sealed moist chamber.

Solutions. The aqueous indicator solutions were always freshly prepared, and unless otherwise stated were of a maximum concentration of 1 per cent, of the sodium salt.

Amounts injected. In one respect the marine ova were more advantageous to work 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 gauging accurately the amount of fluid injected into the cell. This error is difficult to control when injecting non-toxic substances into an Amoeba because of the apparent impunity with which the cell tolerates varying quantities. The ova, on the other hand, are far more susceptible to injury and only minimal quantities of the injected solutions can be introduced without causing disintegration and cytolysis. Therefore, when a dye was found to be toxic, successful injections were often only made possible 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 injection ceased and the pipette was removed from the cell at the first appearance of coloured solution exuding from the microtip. The area visibly affected by such injection did not exceed one or two micra in diameter. A "moderate" amount was considered to be introduced when pressure on the plunger of the syringe was maintained until the introduced dye was seen to spread over an area of about 5 micra in diameter around the tip of the pipette. More than this was reckoned as a "large" amount which 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 injecinjec-tion, the surrounding healthy cytoplasm practically never becomes permeated with the dye and the injection is recorded as unsuccessful.

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

The aqueous solutions of all the dyes always spread as a uniform coloration from the site of injection through the homogeneous matrix of the protoplasm. In the case of certain dyes possessing basic properties and then only after an appreciable 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-protoplasmic granules.

Viability of starfish and sanddollar eggs in an oxygen-free nitrogen atmosphere. It was found that both starfish and sanddollar eggs tolerate anaerobic conditions for at least 1 hour without altering their, cytoplasmic pH or changing their normal appearance. The germinal vesicle of the immature starfish egg undergoes the changes characteristic of prematuration within the first half hour in the nitrogen chamber. This is about the normal time for this process in air. Mature starfish eggs, after 1 hour in the chamber and then returned to air, were capable of forming typical 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 condifertilisa-tions for 40 minutes developed swimming blastulae after being returned to air.

If the eggs were allowed to develop to the stage just before the first cleavage and then were placed in the nitrogen chamber, cleavage proceeded but at a lower rate than normal. A small percentage completed the division. However, a peculiar feature of these eggs, when returned to air, was that they always reverted to the one-celled stage, with two nuclei. Subsequently, segmentation proceeded again with 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 so as to induce cytolysis, developed a red colour characteristic for the oxidant at the pH 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 injury incident to the injection caused cytolysis of the egg. When this occurred the cytolysed region took on a red colour which usually faded in 10 to 20 seconds, but

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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 debris retained 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 caused the appearance of a pink colour, the colour of the oxidant at a pH more acid than 7-7(7). A pale pink colour also developed in the cytolysed regions of eggs injected with the dye and then injured by tearing. The colour faded within 5 to 10 seconds and 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 dyes decolorised almost immediately in unfertilised, fertilised and dividing sanddollar eggs. Small amounts were decolorised in less than one second. Moderate amounts took several seconds. A subsequent injection, 20 minutes later, of ferricyanide brought back the colour. Cytolysis by mechanical injury also brought back the colour but less intensely.

Injection of dye H into the astral centre of the segmenting sanddollar egg resulted in the spread of a flash of blue throughout the hyaline area followed immediately 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 colour faded immediately. Twelve minutes later the egg segmented into two blastomeres. Ferricyanide was then injected into each blastomere separately with a distinct return 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 un-fertilised eggs of the sanddollar and starfish. Ferricyanide brought back the colour.

The non-toxicity of the dye is indicated by the development of a typical amphiaster 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 was rather pale in the 1 per cent, solution. Therefore, a 2 per cent, concentration was used.

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

Anaerobic injections. The oxidant, injected into immature and mature starfish eggs, 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 the colour. Eggs which had been injected with the dye were torn with needles to induce cytolysis whereupon the colourless reductant in the cytolysed material took on the pink colour of the oxidant. This faded within 5 to 10 seconds and could not be made to return with ferricyanide.

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

The colourless reductant was distinctly less toxic and remained colourless in the cytoplasm. Injections of ferricyanide brought out the colour of the oxidant. Exposure to air of eggs injected with the reductant occasionally brought out a faint pink colour. This was probably caused by the injection into the cell of an excess of the relatively non-toxic reductant, which, in the presence of air, was more 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 completely in the cytoplasm of unfertilised, fertilised and dividing sanddollar eggs within

1 to 4 seconds. Whenever cytolysis occurred at the time of injection the cytolysing debris 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 be detected. 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 decolorised completely within 2 to 5 seconds when injected into a large number of unfertilised, fertilised and segmenting starfish and sanddollar eggs. In some cases the injection caused local cytolysis, the debris of which coloured blue. A subsequent injection of ferricyanide brought back the colour not only in healthy eggs but also in the surviving portions of partially cytolysed eggs.

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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 minutes after injection. The injected dye was reduced in 2 to 5 seconds. Ferricyanide brought 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 before disappearing.

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 reduced

within 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 injection from which the colour diffused through the cytoplasm. The diffuse blue colour disappeared within half a minute or so while the colour of the coagulum gradually faded and disappeared only after 4 to 10 minutes. Injection of ferricyanide into the surviving cytoplasm brought back the colour.

If the injection of the oxidant was performed gradually, a considerable amount could be introduced into the cell without appreciable cytolysis. There was then a distinct fading of the blue colour beyond the site of the injection within the first half minute, after which the effect of gradual fading was accompanied by the accumulation of the residual oxidant in the cytoplasmic granules about the site of the injection. In one such fertilised sanddollar egg, segmentation proceeded in such a way that one blastomere contained all the blue granules while the other was colourless. Injection of ferricyanide into each of these blastomeres resulted in a diffuse blue coloration which was much deeper in the one originally containing scattered blue granules.

Anaerobic injections. The relatively toxic oxidant decolorised within 2 to 5

seconds except when excessive amounts caused cytolysis. Ferricyanide, injected within 1 minute after decoloration, brought back the blue colour. The less toxic, colourless reductant, when injected, remained reduced, and a blue colour developed with ferricyanide. Frequently, on exposure to air, the injected eggs coloured blue. However, when a sufficiently small quantity of the reductant was injected, exposure to air resulted in no colour which, nevertheless, could be subsequently produced by an injection of ferricyanide.

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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 surviving cytoplasm readily took on a blue colour.

The dye is somewhat toxic but after many attempts it was possible to secure a number of injected sanddollar eggs which were sufficiently viable to form typical fertilisation membranes upon subsequent insemination. Fertilised eggs after injection were observed to commence cleavage but not to complete it. When injected into the cytoplasm of starfish eggs the colour was retained for at least 5 minutes, after which cytolysis set in. In one case an immature egg retained the blue colour for half an hour before cytolysis occurred.

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

That the reductant is less toxic than the oxidant was indicated by the fact that eggs, injected with the reductant, tend to undergo more or less extensive cytolysis 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 and studied at the Hygienic Laboratory by Preisler and Cohen. (The electrometric data on these compounds are as yet unpublished.)

Aerobic injections. The non-toxic blue oxidant, injected into fertilised and dividing sanddollar eggs, was partially decolorised to a pale greenish blue which diffusely coloured the cytoplasm. Injection of ferricyanide frequently intensified the colour to a deeper blue. Cytolysis caused by tearing with a microneedle occasionally 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 reduced the injected oxidant in the nitrogen chamber took on a pale greenish blue colour within 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 seemed to 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 evidence

of oxidation. Injection of ferricyanide or exposure to air quickly developed a blue colour in the eggs.

U. K2 and Na2 indigo disulfonates (indigo carmine).

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

Anaerobic injections. The oxidant was decolorised in 2 to 3 seconds. The

reductant, when injected, remained reduced. An injection of a moderate amount of either resulted almost always in complete cytolysis within a few minutes. Ferri-cyanide restored the colour. Eggs injected with small amounts and exposed after 5 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 to granules 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 cytolysis occurred 10 to 15 minutes after the injection. The equally toxic, yellow reductant gave no sign of being oxidised in the egg under anaerobic conditions. Injection with ferricyanide brought out a red colour.

Fertilised and segmenting sanddollar eggs were injected with the reductant anaerobically. Upon exposure to air the eggs took on a distinct pink colour within 45 minutes.

II. NUCLEAR INJECTIONS.

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

The following are the experimental results of the nuclear injections: C. m-Bromophenol indophenol.

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Table .

T

to Results of injecting oxidation-reduction indicators into the cytoplasm of sanddollar and starfish (Oxyge n electrode ) Potassiu m ferricyanid e .. . A i : Pheno l m-sulfonat e indo -3,6-dibromopheno i C : w-Bromopheno l indopheno l H : Pheno l indo-2 , 6-dichloro -pneno i I: Pheno l indo-3 , 6-dibromo -pneno i L : o-Creso l indo-3 , 6-dichloro -pheno l M : i-Naphthol-3-sulfonat e indo -pheno l m-sulfonat e N : m-Toluylen e diamin e indo -pneno i cmoria e O : i-Naphthol-3-sulfonat e indo -pheno l P : i-Naphthol-3-sulfonat e indo -3,6-dichloropheno l Q : Toluylen e blu e chlorid e Q i : Brillian t cresy l blu e chlorid e R : Methylen e blu e chlorid e S : K 4 indig o tetrasulfonat e S i : Ethy l Capr i blu e nitrat e T : K 3 indig o trisulfonat e U : K 2 indig o disulfonat e V : Neutra l re d iodid e (Hydroge n electrode ) X : Phenosafrani n 2?o ' at^ H 7-0 (volts ) +o-8 i +O-4 3 O-37 3 0-34 8 0-31 7 0-21 7 0-18 1 0-13 5 0-12 7 0-12 3 0-11 9 0-11 5 0-04 5 +O-OI I —0-04 6 -o-o o —0-08 1 -0-12 5 —0-3 0 ^approx. ^ —0-42 1 -0-53 5 (approx. ) 41-0 38-4 33' i 33-3 31-3 31-3 3O' I i8-s 18-3 18-1 18-0 17-9 15-5 14-4 I3' O n* 3 9' 9 4-0 O' O -3. 0 In aerobiosi s Oxidan t Reduce d instantl y „ Almos t instantl y „ r-4 sec . 3-5 sec . „ 5-1 0 sec . No t reduce d Partiall y reduce d No t reduce d

„

No t reduce d In anaerobiosi s Oxidan t Reduce d instantl y

M 55 „ „

Almos t instantl y 3-5 sec . „ 2-3 sec . 2-5 sec . 2-3 sec . „ No t reduce d _ _ No t reduce d Reductan t — — — — — — No t oxidise d M No t oxidise d No t oxidise d — No t oxidise d 5 5 __ _ No t oxidise d Toxicit y o f oxidant f Slightl y toxi c Non-toxi c „ Slightl y toxi c Toxi c Non-toxi c Toxi c 5 ) 5> Toxi c Non-toxi c Slightl y toxi c Toxi c Slightl y toxi c __ _ Toxi c * O f a mixtur e o f 5 0 pe r cent , reductan t an d 5 0 pe r cent , oxidan t a t 30

0 C.

; fo r a mixtur e o f 9 9 pe r cent , reductan t an d 1 pe r cent , oxidan t th e r H i s decrease d b y 2-0 . f O f al l th e toxi c dye s th e reductan t tend s t o b e les s toxi c tha n th e oxidant .

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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 underwent cytolysis. If the dye was made to exude from the micropipette during the passage of the pipette through the cytoplasm it was possible to secure an almost simultaneous injection of both nucleus and cytoplasm. In such cases the difference between the cytoplasm 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 the colour 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 the four 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 for 5 seconds before cytolysis set in, after which the colour faded from the nuclear remnant 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 the colour became apparent spreading from the periphery of the nucleus into the surrounding cytoplasm, where the colour faded to a pale green and then dis-appeared. Diffusion of the colour from the nuclear remnant continued and finally coloured the cytolysed cytoplasm blue. The nucleolus usually persisted within the nuclear remnant.

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

S. K4 indigo tetrasulfonate.

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

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T. K3 indigo trisulfonate.

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

U. K2 indigo disulfonate.

The oxidant injected anaerobically remained blue during the several seconds before the nucleus broke down. Some of the dye injected at the same time into the cytoplasm 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 cytoplasm where it changed to a rose pink colour. There was no apparent reduction either in the 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 protoplasm of the ova can be demonstrated by varying the oxygen content of the environment within 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) reduce the 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 reversibility has been shown to occur repeatedly within the same living cell. Unfertilised and fertilised sanddollar eggs, after being injected with the oxidant under anaerobiosis where complete reduction took place, were exposed alternately to air and to the deoxygenated nitrogen atmosphere three times in succession. With each exposure to air the blue colour returned, and as consistently disappeared with each exposure to the nitrogen atmosphere. The return of the blue colour in air was always more rapid than its disappearance in the nitrogen chamber.

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242 R- CHAMBERS, H. POLLACK and B. C O H E N

DISCUSSION.

In a study of the oxidation-reduction potential (intensity factor) of protoplasm based on the effect of introducing solutions of indicators into protoplasm one must bear in mind the possibility of errors arising from variations in the amount of the dye introduced (capacity factor) and the rate at which the dye is reducible or oxidisable (rate factor). The existence of these factors has been discussed in a previous paper. In the case of the marine ova the capacity factor seems to be partially under control because the high susceptibility of the egg-protoplasm to disintegration when foreign substances are introduced prevents the injection of more than minimal quantities of the dyes.

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

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

When we consider the intensity factor we find some difference in the results reported for Amoeba and those given in this report on the marine ova. For Amoeba the dyes listed as partly reduced after injection under aerobic conditions, viz. toluy-lene blue and methytoluy-lene blue, are the ones which stain the cytoplasmic inclusions, a complication which has been more carefully taken into account in the case of the marine eggs. In the report on the Amoeba, toluylene blue and methylene blue were tabulated as being "reduced partly." This is an unfortunate statement in so far as it does not give the true meaning intended to be conveyed. Actually the experiments on the Amoeba showed that these dyes were completely reducible provided the precaution was taken to prevent the capacity factor from obscuring the 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 potassium indigo tetrasulfonate which, in the marine ova under aerobic conditions, gave no sign of reduction while in the Amoeba there seemed to be a suggestion of some reduction 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 the injection of ferricyanide was found ((6), p. 597) to have resulted in no increase in colour.

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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 and sanddollar eggs close to an rH of 12.0, which is that of ethyl Capri blue nitrate when 50 per cent, reduced at/>H 7-0.

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

Attempts to utilise potassium indigo monosulfonate whose rH, when 99 per cent, reduced, is 6.8 proved to be inconclusive owing to the unsuitability of this insoluble 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 represents the first anomaly encountered in our studies. Theoretically, if ethyl Capri blue is reduced, then the more electropositive tetrasulfonate should also be reduced, We suspect that the explanation for this anomaly lies in the toxicity of the tetra-sulfonate functioning, perhaps, as an " antireductant." Except for this, our results have been uniformly consistent both for the freshwater Amoeba and for the marine ova. The experiments still leave one point to be determined; although we have found the direction we have not yet determined the lower limit of the anaerobic, intraprotoplasmic reduction potential owing to the lack at present of suitable 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 22 for 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 cells have an rH which is well poised and probably independent of wide variations in the external oxygen pressure. This conclusion is contradictory to our findings in which we show that the removal of O2 from the environment very definitely results

in 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, our

experiments in this direction being inconclusive.

The reliability of applying the dye systems to a study of protoplasmic reactions might be criticised because of the possibility that the dye in the protoplasm may not behave reversibly as it does in vitro. However, the reversibility of the reactions has been demonstrated by the alternate transference of injected eggs from anaerobic to aerobic conditions and back again. This is well shown, for example, with eggs injected with K3 indigo trisulfonate and then exposed to nitrogen and

returned to air three times in succession. The colour disappeared and returned with each change.

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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. In fact this was one of our chief criteria for differentiating between intracellular reduction 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 sometimes possible, in addition to the ferricyanide test, to demonstrate the intracellular presence of dye reductant by inducing cytolysis through mechanical injury upon which 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 never reached that produced by injecting ferricyanide into the intact egg. The colour faded within a few seconds to several minutes and could not be brought back with ferricyanide.

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

It is known that the reducing ability of tissues is not entirely destroyed at death. In order to determine this for the marine ova a mass of starfish eggs was cytolysed with distilled water in a test tube and the suspension tested by introducing a few drops of an easily reducible dye (phenol m-sulfonate indo-2,6-dibromophenol). Quick reduction took place after repeated additions of the indicator although the suspension was kept thoroughly aerated by shaking. That the decoloration was due to reduction and not decomposition of the compound was shown by the fact that the addition of ferricyanide promptly restored the colour of the oxidant. Similar observations on various tissue juices and macerates have been reported by Cohen, Gibbs and Clark(7), and Cannan, Cohen and Clark d).

There is still the question whether the poising ability of the injected indicator systems has a disturbing effect on the intracellular reduction potential. This is probably a second order effect. At any rate the results are consistent for the whole series of reversible indicators, irrespective of their chemical constitution, when injected under anaerobic conditions; and, in response to the presence and absence of 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 to the conclusion that the reduction potential in the aerobic cells studied is a function of 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 material and the difficulties in technique permitted. It must be stated that our findings leave 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 chemical test (the injection of ferricyanide) for the detecting of the available reduced dye.

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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 the oxidant took place in the cytoplasm the fading of colour from the nucleus was much slower. The fact that the colour faded from the nucleus does not necessarily mean that the nucleus reduced the dye, for it is equally possible that the dye simply diffused out of the nucleus into the reducing cytoplasm just as the dye had previously passed from the cytoplasm into the nucleus when the injection was first made.

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

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

Because of the very brief survival of the nucleus after injection, it is unjustifiable to 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 the oxidants (C, L, M, O) which are all instantly or almost instantly reduced in the cytoplasm remain oxidised within the nucleus under both aerobic and anaerobic conditions for the several seconds that the nucleus survived and also after it exhibited 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 the starfish egg quickly reduces certain injected dyes, while its injected nucleus remains coloured both under aerobic and anaerobic conditions even with a very easily reducible dye, namely C. Is this to be regarded as evidence that the nucleus does not exert the same high reducing intensity as the cytoplasm or merely that the brief interval during which the observation could be made permitted a capacity factor to obscure the phenomenon?

SUMMARY.

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

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

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

3. No difference could be found in the apparent reduction potentials of the unfertilised, 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 less toxic than their oxidants.

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

7. By injecting an oxidising agent, i.e. potassium ferricyanide, into living ova it was possible to detect the intracellular presence of dyes which previously had been introduced and had been reduced to the leuco-form. Under aerobic conditions it was also frequently possible to bring back the colour of the oxidant by the induction of cytolysis in eggs containing the reductant. Under anaerobic conditions this never occurred. The colour appearing in the cytolysing eggs never approached the 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 egg remained oxidised within the nucleus under both aerobic and anaerobic conditions for the several seconds that the nucleus survived the injection and also after signs of injury became apparent. All the reductants injected anaerobically remained reduced.

III. REVERSIBILITY.

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

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(2) CHAMBERS, R. (1928). Micromanipulative technique in Gatenby and Cowdry's Microtomist's

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

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

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