The oxidases and other oxygen - catalysts concerned in biological oxidations

Front matter

Public Health and Marine-Hospital Serve

Walter Wyman, Surgeon-Gei (eral

A o

Chapter II: The oxidizing ferments

The guaiacum reaction

Oxygen-exciters and oxygen-carriers

Oxidizing ferments (general)

Oxidases

Classification of the oxidases

Oxidase and peroxidase reagents....

Laccase gg

Anti-laccase g_

Tyrosinase ^

Anti-tyrosinase .

Oenoxidase „„

Maloxidase „

Spermase __

Oxidin (cerealin)

Schinoxidase

Olease ^

Purpurase ^

Luc if erase

Aldehydase

Indophenol oxidase (the Rohmann-Spitzer reaction)

Purin oxidases (Xanthin-oxidase)

Chapter III: 1Q8 Peroxidases and catalases

Sources of peroxidase, and methods of preparation H

Nature and mode of action of oxidases, peroxidases and catalases, and

the chemical relationships existing among these substances 112

The oxygen-catalysts of the blood - - ^0

Iron, copper, and manganese in their relation to the oxidizing fe rments; artificial peroxidases, etc 122

Peroxidase accelerators and auxiliary oxygen carriers 131

Catalases ^

References to the literature 141-161

THE OXIDASES AND OTHER OXYGEN-CATALYSTS CON- CERNED IN BIOLOGICAL OXIDATIONS."

Chief, Division of Chemistry,

By Joseph H. Kastle,

Hygienic Laboratory, U. S. Public Health and Marine- Hospital Service.

INTRODUCTION.

Durin°- the past eight years I have devoted a considerable part of my time to a study of the oxidases and of certain phases of biological oxidation, with the result that I have come to appreciate as never before the need of a somewhat more comprehensive and complete treatment of this subject than is ordinarily to be found in most treatises on fermentation. Furthermore, a study of the earlier literature of the subject has revealed certain rather curious mistakes, which probably found their way into the literature originally as the result of typographical errors, but which pervade the whole subject with an annoying persistency. During the past four years, as the lesult of easy access to the departmental libraries in this city, I lone had unusual facilities for familiarizing myself with the literature of this subject, and it was partly with the view of giving to other workers m this field the benefits of my opportunities along this line, that this monograph has been written. No special claims are made for originality either in the mode of treatment of the subject, or in some cases even with regard to phraseology, my only object being to present the subject as accurately and comprehensively as possible within the time at my disposal. To this end I have made free use of such treatises on fermentation as “The soluble ferments and fermentation,” by J. Reynolds Green (Cambridge Natural Science Manuals, London, 1899); “Die Fermente und ihre Wirkungen,” by Carl Oppenheimer, (Leipzig, 1900); “Traite de Microbiologie,” by E. Duclaux (Paris, 1898); “Biochemie der Pflanzen,” by Fred. Czapek (Jena, 1905); “Theorie der Ferment wirkungen,” by Moritz Traube (Berlin, 1858); “Les Enzymes et leurs Applications,” by Effront (translation by Prescott, New York, 1902); “Recent advances in physiology and biochemistry,” by Leonard Hill and others (London, 1906); “Das SauerstofT-Bediirfniss des Organismus,” by Paul Ehrlich (Berlin, 1885); “Les Oxydations de l’Organisme,” by E. Enriquez and J.-A. Sicard (Paris, 1902); “Die Bedeutung der Katalyse fur die Medicin,” by H. Schade (Kiel, 1907) ; and “Kritische Studien fiber die Vorgange

der Autoxydation ,” by C. Engler and J. Weissberg (Braunschweig, 1904). I am especially indebted for much valuable information to a dissertation by Pierre See, entitled '‘Contribution a l’Etude des Applications Therapeutiques des Oxydases et des Metaux Ferments” (These, No. 239, Faculte de Medicine de Paris, 1905), and to recent articles by Engler and Herzog, "Zur chemischen Erkenntnis biologischer Oxydationsreaktionen” (Hoppe-Seyler’s Zeitschrift fur physiologische Cliemie, 1909, vol. 29, pp. 327-375), and to Moore and Whitley, ' ' The properties and classification of the oxidizing enzymes and analogies between enzymic activity and the effects of immune bodies and complements” (Bio-Chemical Journal, 1909, vol. 4, pp. 136-167). In many instances in dealing with original contributions to the subject I have adhered as closely as possible to the words of the author in order that the exact sense and meaning of the original article might be preserved to at least as great an extent as possible within the limits of the present communication. Furthermore, no claim is made for the completeness of this production. It has been impossible, through lack of time and space, to refer to all original communications on the subject, even in the bibliography. Even the current numbers of the journals contain many contributions bearing on the subjects treated of in the following pages, and out of this vast and constantly growing literature I have been compelled to select what seemed to me the most essential to the proper presentation of the subject.

In the writer’s opinion the subjects considered in the following pages are strictly germane in their general scope to the work of the Hygienic Laboratory. The oxidases and related oxygen-catalysts belong to the same group of bodies as the serums, viruses, toxins, and antitoxins. Any facts throwing light on the one class of substances will doubtless assist ultimately to a better understanding of this whole group of biologically active substances. Portier is of the opinion that the oxidases play an important role in the defense of the organism against pathogenic micro-organisms, and according to Sieber they are able to destroy toxins, such as the toxins. of diphtheria and tetanus. Furthermore, they are of importance as enabling us to form correct conclusions regarding the condition and character of certain foodstuffs — as to whether they are cooked or raw — and in enabling us to determine, within certain limits of temperature, whether a given sample of milk has been pasteurized or not. For the biological chemist, the physician, and the sanitarian, all of these matters are of considerable importance.

I am greatly indebted for much valuable material to various authors, whose works I have freely consulted and made use of in the preparation of this monograph, and also to Mr. F. A. McDermott, one of the assistants in the division of chemistry of the Hygienic Laboratory, for much valuable assistance.

Chapter I. THEORIES OF OXIDATION

In the period which elapsed between the classic researches of Lavoisier on Combustion (1774-1785) and the discovery of ozone by Schoenbein in 1840, a number of interesting and important observations had accumulated in the literature concerning the influence exerted by one substance on the oxidation of another by air or oxygen. Thus in 1806 D6sormes and Clement (137) pointed out that nitric acid is not the principal agent concerned in the complete oxidation of the sulfur in the leaden-chamber process for the manufacture of sulfuric acid, but its ''base,” nitric oxide (le gaz nitreux), which takes oxygen from atmospheric air in order to ofler it to the sulfurous acid in a condition capable of accomplishing its oxidation. A few years later it was observed by Vogel _(443) that hydrogen and oxygen combine at low temperatures under the influence of charcoal. Then followed the numerous and interesting observations by Sir Humphrey Davy (134), Edmund Davy (l32-138), Erman (167), Pleischl (328), and others on the remarkable power of platinum and other metals of the platinum group, such as iridium, osmium, and palladium, to effect the slow combustion, or under certain conditions even the actual ignition, of combustible gases such as hydrogen, carbon monoxide, etc., in an atmosphere containing oxygen, and the rapid conversion of alcohol into acetic acid, observations which culminated in Davy’s lamp without flame ( see Erman 167), Doebereiner’s lamp (141), and the rapid method for the production of acetic acid.

The effect of various substances on the oxidation of combustible gases and vapors was also exhaustively investigated by Dulong and Thenard (151~152), with the result that various metals, such as iridium, palladium, rhodium, gold, silver, mercury, nickel, cobalt, and iron, in spongy or pulverulent form, were found to accomplish the same changes as platinum, and that to a degree at least such changes can also be brought about by certain nonmetallic substances, such as carbon, pumice stone, porcelain, glass, and quartz crystals, at temperatures under 350° 0., while salt, fluorspar, and marble did not appear to act to a sensible extent within these limits. These changes, they concluded, could not be ascribed entirelj’ to electrical phenomena, as was at first supposed, and they explained them on the assumption of the condensation of large volumes of gas on the surface of the substance capable of causing the oxidation— as, for example, the condensation of oxygen upon platinum, whereby heat was liberated and the body (platinum) attained the temperature necessary to accomplish the oxidation.

The discovery of ozone by Schoenbein (307) in 1840 and the finding of this remarkable substance among the oxidation products of readily oxidizable substances gave a new impulse to the study of oxidation phenomena and led ultimately to a more careful study of the whole subject of induced oxidations and of the phenomenon of oxygen carrying. This distinguished observer seems to have been the first to clearly recognize that in the final products of chemical combination, particularly those in which oxygen is concerned, we see, as he put it, only the closing scene of a chemical drama which is in reality composed of several intermediate acts, and that for a correct understanding of such processes it is quite as essential to know these intermediate acts as it is to know the beginning and the end.

To the investigation of ozone and to the task of learning more of the initial and intermediafe acts of the oxygen drama, Schoenbein set himself with remarkable assiduity and success. It is beyond the scope of this communication to attempt anything like a chronological account of the numerous investigations on the subject of ozone, which in themselves form one of the most interesting chapters in the history of chemical science. (See Engler and Weissberg161.) It is sufficient to say in this connection that from the time of its discovery until its composition and nature were finally settled through the labors of Odling, Soret, and Andrews and Tait, Schoebein himself held different notions at different times respecting its composition. As the net result of his many researches on the subject, Schoenbein came to look upon ozone and the ozonides as containing an atom of negatively polarized oxygen, which in its conduct toward oxidizable substances was far more active than oxygen in its ordinary form, and as we would interpret these phenomena at the present day, he accounted for its production from ordinary oxygen through the action of electricity upon the supposition that as a result of the electrical discharge the ultimate particle (molecule) of oxygen is resolved into two atoms, one of which is negatively polarized and the other positively polarized, thus :

The negatively polarized atom them combines with a particle of ordinary oxygen to form ozone, 030, whereas the positively polarized atom (antozone) combines with water to form hydrogen peroxide, II20.©. These two substances, ozone and hydrogen peroxide, were regarded as the prototypes of a whole series of compounds to which Schoenbein gave the name ozonides and antozonides, respectively.

n

According to Hagenbach (203), Schoenbein did not hold that the oxygen molecule consisted of two atoms. Therefore, according to Schoenbein, the chemical polarization of neutral oxygen is no true decomposition, but only a calling into being of two oppositely active

states of the element

The © combines with water to form II202, and the 0 goes partly to form ozone, but in greatest part to oxidize the metal or phosphorus.

Like ozone, the ozonides were believed to contain a part ot then oxygen in a negatively polarized condition, whereas the antozonides were supposed to contain a part of their oxygen m a positive y polarized condition. Like ozone itself , the ozonides were supposed to contain a part of their oxygen in an intensely active condition. On the other hand, in antozone and the antozonides the antozomc oxygen was supposed to be less active chemically than the active oxygen m ozone and the ozonides, at least in most of its chemical relations, and to exhibit chemical activities of a different order. The following is a list of some of the common ozonides and antozonides included in

this classification :

PbO.0, lead peroxide.

MnO.O, manganese peroxide.

HC1.0, hypochlorous acid, etc.

©, antozone.

II20.©, hydrogen peroxide.

BaO.©, barium peroxide, etc.

This view regarding the nature of ozone and antozone was appaiently in harmony with a great many facts, among which may be mentioned :

1. Modes of formation from ordinary oxygen by the action of electricity and as a result of the autoxidation of readily oxidizable substances such as phosphorus, its production in the electrolysis of water and as the result of heating various oxides and peroxides and in the decomposition of highly oxygenated compounds by acids.

2. The greater oxidizing power of ozone and the ozonides as compared with the oxidizing power of antozone and the antozonides. Thus it was proven by Schoenbein that ozone and the ozonides can accomplish the oxidation of a number of metals, guaiacum, etc., which are not acted upon by hydrogen peroxide or other antozonides.

3. The mutual decomposition of ozone and the ozonides by antozonides resulting in the formation of ordinary oxygen. Such decomsitions are those met with in the decomposition of hydrogen peroxide by lead and manganese dioxides and by potassium permanganate, and also the remarkable decomposition of ozone itself by hydrogen peroxide, whereby water and ordinary oxygen are formed.

4. The apparent occurrence of antozone in nature in certain varieties of fluorspar, and its apparent formation (or the actual formation of hydrogen peroxide, an antozonide) by the action of an acid on an antozonide like barium peroxide.

While the differences existing between the so-called ozonides and antozonides and also their mutual decomposition, can be explained upon grounds ° other than those involving the idea of oppositely electrified oxygen atoms, Schoenbein’s theories respecting the nature of this inteiesting gioup of substances were more or less in harmony with the dualistic theory of Berzelius and with the fact that the molecule of oxygen consists of two atoms. Their principal interest in this connection is that erroneous as they were in some particulars, they afforded a satisfactory explanation of oxygen activation. A few examples will serve to make this clear: When a solution of indigo blue is exposed to oxygen or atmospheric air it suffers no change. On the other hand, as is well known, phosphorus is readily oxidized under these conditions. And now, what is more remarkable, if indigo be brought in contact with slowly oxidizing phosphorus, not only does the phosphorus continue to be oxidized but under these new conditions the indigo is oxidized as well. In other words, oxidizing phosphorus has the power to excite or induce the oxidation of a substance which alone is incapable of undergoing oxidation by simple contact with oxygen or atmospheric air. As shown by Schoenbein, however, when phosphorus is slowly oxidized in the air ozone is produced and it is the latter substance which oxidizes the indigo. When a solution of potassium iodide and starch or a suspension of guaiacum resin in water is shaken with air, no change of color occurs. If, however, as shown by Schoenbein (38°), a drop or two of oil of bitter almonds be added to these solutions and the solutions afterwards shaken with air, each of them becomes blue in color, due in the first case to the oxidation of the potassium iodide with the setting free of iodine and the formation of the blue iodide of starch, and in the second case to the oxidation of the guaiacum with the production of guaiacum blue. According to Schoenbein, the oil of bitter almonds ozonized the air and the resulting ozone oxidized these substances which are not oxidizable by oxygen in its ordinary form.

He also made the interesting observation that certain of the higher fungi, as well as other plants, contain substances which apparently have the power of ozonizing the air to a remarkable degree, thereby accomplishing the oxidation of substances not oxidizable by ordinaiy

“ Mendelejeff (299) divides the peroxides into two classes, 1, superoxides, in which the oxygen atoms are directly united to one another as well as to the oxidized element, and 2, polyoxides, in which the oxygen atoms are not directly united with one another. The superoxides yield hydrogen peroxide when dissolved in acids; the polyoxides do not. Similar views are held by Traube and by Richarz (349).

oxv-en and in certain instances resulting in the development of characteristic vegetable colors (378). He proved, further, that just as a We number of substances have the power of activating the oxygen of the air, so also an even larger number have the power of activating the oxygen of hydrogen peroxide and other antozomdes. Thu while hydrogen peroxide and old oil of turpentine are without action on guaiacum or a solution of potassium iodide and starch, the oxidation and bluing of these reagents are readily accomplished by these oxidizing agents through the action of platinum black, extract of malt, the red coloring matter of the blood, and the juices and extracts of many plants, for the reason that, according to Schoenbem ( ), they are converted into ozonides, in the same way that an ozonide, like lead peroxide, results from the action of lead acetate on hydrogen

peroxide. _ „

He also proved that ozone is formed during many processes of combustion, an observation which led to the conclusion that every combustion is accompanied by the formation of ozone, and finally to the view that no oxidation can proceed of itself without the previous conversion of the common inactive oxygen of the air into active oxygen or ozone. In this manner he readily accounted for the phenomenon of oxygen carrying, and arrived at the further conclusion that through the agency of living tilings and the organic matter on the surface of the earth, the inactive oxygen of the air is constantly being transformed into ozone. This gradual formation of ozone through these agencies was deemed sufficient to account for the slow oxidations continually taking place on the surface of the earth, and in speaking of the oxygen-carrying power of certain substances contained in the blood of animals, which like phosphorus and oil of bitter almonds can activate the inactive respired oxygen, he says that “as a matter of fact, without the presence of such substances as convert ordinary oxygen into ozone, animals would be suffocated in the midst of an ocean of the purest but inactive oxygen as quickly as in a vacuous space. ” (Schoenbein (380).)

Indeed, upon every hand, apparently, facts and observations multiplied, indicating the importance of ozone in the economy of nature. Hare (205) observed its production as the result of rubbing a piece of flint. Forexample, theodorof ozone isvery evident when flint is struck with a piece of steel. Brame (93) detected ozone in rain water. Scoutetten (394 > 395 > 396’ 397) observed its production in the evaporation of impure waters. As a matter of fact ozone became the chemical fad of the day, with the result that its importance in nature was greatly overestimated. Thus during the i^ears 1866-67 daily observations were made on the amount of ozone in the atmosphere of Paris and other localities, and everywhere men were busily engaged in studying its relation to health and disease. Its absence from the atmosphere of certain localities, or its presence therein in diminished amounts was associated with several violent outbreaks of Asiatic cholera that occurred during this period, and according to Doctor Moffatt (303), an English physician, the approach of an ozone period, during which the quantity of ozone in the atmosphere suffers a considerable increase, is followed by a corresponding increase in the luminosity of the glowworm and of certain phosphorescent protozoa, and even of phosphorus itself, when exposed to the air; and in man by a decided increase in the output of phosphates in the urine. Further, according to Doctor Moffatt, the advent of an ozone period was found to be marked by the approach of thunderstorms and unsettled weather conditions generally, and by a marked increase in the number of cases of toothache, neuralgia, apoplexy, etc. Finahy, as the result of a large number of observations extending over a period of ten years, the most exact agreement was established between the warnings of the admiralty cautionary telegrams, as the British weather forecasts were then called, and the readings of the Doctor’s ozonometer. The Doctor himself had evidently fallen a victim to ozone.

Despite, however, the great number of faulty and imperfectly controlled observations on this remarkable substance and the erroneous theories arising therefrom, the fact remains that this remarkably active form of oxygen is produced under a great variety of conditions, and that it is responsible for the oxidation of many substances not ordinarily oxidized by atmospheric oxygen {see Wurster (464)) ; and the fact that it is formed as a by-product in certain processes of autoxidation affords a simple explanation of oxygen carrying on the part of such substances as give rise to ozone during autoxidation.

From the ozone theory of Schoenbein as a point of departure, our theoretical conceptions regarding processes of autoxidation and the phenomenon of oxygen-carrying have been developed mainly along three different lines. Briefly stated these conceptions are: (1) The ionization theory of Yan’t Hoff; (2) the theory of Hoppe-Seyler ; and (3) the peroxide theory of Traube, Engler, Bach, and Manchot.

Yan’t Hoff’s theory of oxygen-activation rests upon the assumption that in slow oxidation, such as that met with in the gradual oxidation of phosphorus, the oxidizable substance enters into combination, not with molecular oxygen, but with the very small amounts of atomic (ionic) oxj^gen which are constantly being produced from molecular oxygen in the sense of the equilibrium 02=20, and that in a gaseous system such as that furnished by acetic aldehyde and gaseous oxygen, the velocity of the reaction should be exactly proportional to the square root of the oxygen pressure.

The idea that molecular oxygen dissociates as the result of electrification and under the influence of heat and chemical action, is by no means a new one, nor did it originate with Van’t Hoff As a matter of fact, it had been promulgated by Clausius (»" ■ 12<\ ) upon theoretical grounds and as affording a simple explanation of the differences between active and common oxygen, as early as 1857 to 1863, and has since been employed by other physicists (among them Schuster, Von Helmholtz, Richarz, Thomson, and others) as affording a simple explanation of the phenomena observed m the electrification of gases, including oxygen, and in the condensation of aqueous vapor in the steam jet as the result of the oxidation of various substances in close proximity thereto. Thus Clausius (“») in a communication entitled ‘ ‘Ueber die Art der Bewegung, welche war Warme nennen, had put forward the suggestion that in common oxygen the atoms are not detached, but combined in twos to form molecules, a conclusion which also derived support from the views of Gerhardt on the constitution of gaseous molecules. He reached the conclusion therefore that the active oxygen which common oxygen sometimes contains and which at this time was not distinguished from ozone, consists not of atoms combined in pairs, but of single atoms distributed among the molecules of the element in its ordinary form, and in a later communication on the difference between active and common oxygen (121) this author reiterates the view that common oxygen consists of paired atoms, and active oxygen of unpaired atoms, and further that the two atoms which go to form ordinary oxygen are in oppositely electrified states. Hence, according to Clausius, the molecule of ordinary oxygen is diatomic, containing one electro-positive and one electro-negative atom. On the other hand, active oxygen consists of unpaired atoms which may exist eithei fiee oi loosely bound together ( lose gebunden). If electro-negative, these atomk form ozone; if electro-positive, antozone.

Long afterwards it was pointed out by Schuster (392) in connection with his investigations on the electrification of gases that the passage of electricity from one molecule to another in a gas is always accompanied by an interchange of atoms composing the molecule. According to this author, physicists of the Faraday-Maxwell school had long considered it probable that the conduction of electricity through gases is due to something similar to the electrolytic conduction in solutions, or, in other words, to the migration of ions under the influence of the current.

Similarly, according to Von Helmholtz and Richarz (208) the interesting phenomena observed during the electrification of a steam jet and the influence of certain chemical reactions on condensation occurring in the jet, can only be explained on the supposition that molecular oxygen or the gaseous substances in the immediate vicinity of the jet are dissociated to a greater or less extent into free atoms or ions, or into molecular groups containing free valences, and that these are responsible for the phenomena observed. It has been observed by these and other investigators that when a jet of steam emerges from a small orifice into dust-free air, there is very little if any condensation to be observed within half an inch or so of the orifice. " On the other hand, if an electrode from which electricity is escaping be placed near the origin of the jet, dense condensation occurs right up to the orifice, and the jet itself appears colored, the color being due to the scattering of light by a great number of very small particles of water, the diameter of which is very small compared with the wave length of light.

According to J. J. Thomson (422) , the electricity which escapes into the gas is carried by the charged atoms of the gas, and since in the region immediately around these atoms there will be a very intense electric field there will be a tendency for the steam to deposit in these regions. Thus around these charged atoms there will be very small drops of water, which mil scatter the blue light more than the red, so that the jet when seen by transmitted light will appear reddish.

Von Helmholtz and Richarz (208) found further that condensation in the steam jet is also brought about by chemical action going on in its neighborhood. The reactions investigated were of the most varied character, involving such processes as the combination of nitric oxide and oxygen, ammonia, and hydrochloric acid, the oxidation of phosphorus, sulphur, and other readily combustible substances. All of these were found not only to effect the condensation of live steam, but also to cause the condensation of other vapors, such as those of alcohol and acetic acid. As a matter of fact so universal is this influence that Von Helmholtz and Richarz proposed to employ the steam jet as a means of detecting ionic dissociation in gases. Thus Von Helmholtz (207) found that condensation is effected in the steam jet by various glowing metals, such as platinum foil which had been heated to redness and which had not been artificially electrified. Similarly other metals such as silver, iron, copper, etc., after heating in the flame, color the steam jet for a long time. This is also shown in a high degree by all glowing organic bodies, such as wood, paper, tobacco, and especially by glowing sulphur. It was also proved that the production of smoke has nothing to do with the phenomenon observed. Tobacco smoke, especially, was observed to be without influence. Von Helmholtz and Richarz (208) found that condensation in the steam jet could be brought about by a piece of glowing phosphorus at a distance of several decimeters away, and that at this distance it acted remarkably powerfully, but less actively, on the vapors of alcohol, anilin, formic, and acetic acids. They also proved that the thick white clouds which are produced when moist phosphorus is exposed to the air or oxygen do not exhibit this phenomenon when the glowing phosphorus is quenched. So, also, the oxidation products of phosphorus already prepared had no influence on the condensation.

On the other hand, the active agent produced during the slow oxidation of phosphorus diffuses with considerable velocity, even against a current of air. Potassium and sodium burning in contact with water also exert an influence on the steam jet through great distances. As has been observed in the case of phosphorus, the resulting cloud, which consists of finely divided potassium or sodium oxide or hydroxide, is of itself inactive. It is also known that potassium and sodium glow on their freshly cut surfaces as the result of autoxidation. Hence all these phenomena are analogous to those observed in the burning o

phosphorus. . .

According to these observers, it can readily be understood that m

all oxidations occurring in atmospheric air there are produced so many substances that it is often difficult or impossible to obtain experimental proof by the exclusion of all other causes, that the action on the steam jet is only brought about by ions, especially those of active oxygen. Of the three substances, however, which are always produced during combustions in air, namely ozone, nitrous acid, and hydrogen peroxide, none were found to have any action on the steam jet.

In this connection it is of interest to note that Meissner f297) as early as 1863 had observed that under the excitation of oxygen by electrical induction there is produced, besides ozone, still another substance, which exerts a highly remarkable action on water vapor. F urther investigations led him to believe that this substance was Schoenbein’s antozone.

According to Von Helmholtz and Richarz C08), in all such oxidation processes as effect a condensation of aqueous vapor in the steam jet, the oxygen is dissociated into single atoms (ions), 0£ and 0_, or into groups of atoms such as — O — 0 — . In terms of the electro-chemical theory both of these kinds of particles have free valences and are carriers of an excess of positive or negative electricity, and hence serve to conduct the current through the gas during electrification, or, as explained by Thomson (see p. 16), they cause a condensation of aqueous vapor when they come in contact with the steam jet. According to Von Helmholtz and Richarz (208), chemists have ascribed to active oxygen only a momentary existence; on the other hand, Clausius (l21) , on purely physical grounds, postulated the existence of dissociated atoms of oxygen at least as a transitional form enduring certainly for a short time, and if we judge from their conduct toward steam these particles of active oxygen exist for a sensible interval. By means of tetramethyl-p-phenylene-diamin, which is colored blue by contact with monatomic oxygen (antozone), Wurster (4<!4) was able to obtain evidence of the existence of the dissociation products of oxygen in the vicinity of gas flames. Thus Wurster’s reagent is instantly colored intensely blue in the Bunsen burner flame; more slowly, but still visi- 1167C — Bull. 59—10 2 bly , in the luminous zone of flames and combustible gases, and also in the flame of burning alcohol. Similarly it was observed by Von Helmholtz (-07) a that Wurster’s paper is colored blue in the neighborhood of a glowing platinum spiral, pointing to the presence of active oxygen in the surrounding atmosphere.