Principles of General Physiology

When a ray of light falls upon a metallic electrode, the potential of this latter is changed. The discharge of a charged electroscope by ultra-violet light, the Hallwachs effect, will be referred to later. The change in the resistance of selenium on exposure to light has been made practical use of in the transmission of pictures by electric current. Two kinds of explanation have been given of the origin of the potential difference in photo-electrical cells. One may say that the light which falls upon an electrode of silver chloride raises the tension of chlorine, and secondarily the potential of the electrode, or that electrons are torn off from their combination by the increased kinetic energy.

For further information the reader may consult the book by Allen (1913). We are now in a position to discuss with more profit the action of chlorophyll in the decomposition of carbon dioxide and evolution of oxygen, perhaps the most interesting of all natural phenomena. Priestley (1774, pp. 89-92) observed that air " spoilt " by mice, that is, incapable of supporting animal life, was made good again by allowing green plants to remain in it for some time.

Ingenhousz (1780) showed that this action of green plants only takes place in the light. He says : " The light of the sun is alone capable of producing in the leaves that movement which can develop dephlogisticated air " (that is, oxygen) : "as soon as the light ceases to act on the leaves, their operation ceases at the same time, and another of a different nature commences." (Translated from p. 17 of the French edition.) He also shows clearly that it is not the heat that is responsible for the result (p. 38). In Fig. 177 I have reproduced his little allegorical initial picture of how light destroys noxious things.

Senebier (1783, see pp. 410-442 of his book, 1788) showed that the chemical change involved is the conversion of fixed air into dephlogisticated air, that is, carbon dioxide into oxygen. De Saussure (1804, Chap. 2) investigated the phenomena quantitatively. General Nature of the Reaction. — As already mentioned, the process, taken as a whole, results in a large storage of light energy, and is one of those complex reactions which are the most difficult to investigate. We have to deal with several reactions chemically coupled, some sensitive to light, others apparently not; together with both optical and chemical sensitisation.

As is well known, it is the presence of the pigment, chlorophyll, which enables the reactions to take place. Those parts of variegated leaves which are devoid of chloroplasts, although otherwise similar to the green parts, are incapable of photosynthesis, as it may be called for convenience. The Chemistry of Chlorophyll. — Although much very interesting work has been done on the chemical constitution of chlorophyll, especially by Willstatter, it must

be confessed that, valuable as it is, it has not, as yet, thrown much light on the problem before us. The brief account which follows is taken from the book by Willstatter and Stoll (1913). The method of preparation will be found on p. 133 of the book. The most important point is the first extraction of dried leaves (say of nettle or elder) with 80 per cent, acetone, which does not extract wax and fatty substances. Stokes (1864) had already pointed out that what is usually known as chlorophyll consists of a mixture of two green substances, and that it is accompanied in leaves by two yellow pigments. Willstatter confirms this statement, and calls the two chlorophylls a and b : these are identical in all plants, and contain magnesium and nitrogen, but neither iron nor p h o s- phorus. The yellow pigments, carotin and xanthophyll, are free from nitrogen.

these four substances to be obtained from leaves, we find the following data : From 1 kg. of dried elder leaves ( = 4 kg. of the fresh leaves) were obtained : — FIG. 177. ALLEGORICAL PICTURE REPRESENTING THE EFFECTS OF THE LIGHT OF THE SUN ON GREEN PLANTS AND THE PURIFICATION OF THE The two chlorophylls are separated by partition between methyl alcohol and petroleum ether. The latter takes up a-chlorophyll ; the former, 6-chlorophyll, since it is insoluble in petroleum ether. Even a-chlorophyll is difficult to dissolve in pure petroleum ether. It has a blue-green colour, with red fluorescence. By rapid dilution with water a colloidal solution is obtained, devoid of fluorescence. Chlorophyll-6 has a green or yellow-green colour in solution. There is a slight difference between their absorption spectra, as will be seen later. The action of acid on the a-substance gives an olive-green derivative, on the 6-substance, a redbrown one. Chlorophyll-6 is an oxidation product of the a-substance, containing an extra oxygen atom in place of two hydrogen atoms. Both are micro-crystalline. They are adsorbed by charcoal, and cannot be extracted again by petroleum ether, but by pyridine, no doubt a question of relative lowering of surface tension. Their behaviour to reagents is essentially similar, and the word chlorophyll will be used below to include both.

Further information of their constitution is obtained by treatment with reagents. The Action of Acid on chlorophyll directly is to separate magnesium from it, forming a derivative called " phaeophytin." The series of derivatives devoid of magnesium are called in general "phytins." The magnesium in chlorophyll is in organic combination, and by treatment of the magnesium-free phaeophytin with Grignard's reagent (magnesium-methyModide), the magnesium is replaced and chlorophyll obtained again.

On saponification by alkali, phseophytin shows itself to be an ester, the acid contains nitrogen and has thirty-four carbon atoms, while the alcohol (called "phytol") is free from nitrogen and is a monatomic alcohol of the composition Phytol appears to contain a number of groups — CH — in a chain. It is colourless, and of less interest than the coloured acid component. Chlorophyll is, therefore, a phytol-ester of a nitrogenous acid chlorophyllin, which contains magnesium in organic combination. The acid of the phaeophytin from chlorophyll a is olive-green in neutral solvents, and called phytochlorin. That from chlorophyll 6 is red in neutral solution, and called phytorhodin. Since it is an ester, it is not surprising to find that chlorophyll is accompanied in the leaf by an enzyme (a lipase or esterase), which is active in alcoholic solution. In extracting leaves with alcohol, " alcoholysis " of the chlorophyll takes place, and ethyl takes the place of phytyl, forming an ethyl-chlorophyllide. The enzyme acts synthetically, as would be expected, and forms chlorophyll in a concentrated solution of phytol and the chlorophyll acid. The mono-carboxylic acid, which is split off by alkali from chlorophyll, is called " chlorophyllin," and contains magnesium. Its derivatives are the "phyllins." These latter are produced by further action of alkali, which splits off carboxyl groups. These phyllins still contain the magnesium, which is combined with the nitrogens of four pyrrol groups. To split off the magnesium from them acid is necessary, and we then obtain the " porphyrins." Thus : —

Magnesium + pb.<eophy tin a phytol + chlorophyllin (contains Mg) The porphyrins are of interest, because they serve to bring into connection chlorophyll and hcemoglobin. The blood pigment seems to be a derivative of a substance which contains iron united to four pyrrol groups in a way similar to the magnesium of chlorophyll (see Kiister's paper, 1908, and the book of Willstiitter and Stoll, 1913, pp. 42 and 39). Hoppe-Seyler (1880, p. 201) described a compound, which he called " phylloporphyrin," obtained from chlorophyll, which gave the same absorption spectrum as the haematoporphyrin derived from haemoglobin. Similar pyrrol derivatives have been obtained from both.

If one of the porphyrins, obtained by the action of acid on phyllins, be heated with soda-lime, " jetioporphyrin " is formed ; it turns out to be the same substance as that which is formed from haematoporphyrin by similar treatment. Now haematoporphyrin is formed from haematin, which is haemoglobin minus its protein component, by the removal of iron by acid, as magnesium is removed from phyllin by acid. To form haemoglobin, haematin combines with a protein, globin ; to form chlorophyll, phyllin, or a carboxylic acid derived from it, combines with an alcohol, phytol, to form an ester. Chlorophyll, however, loses its magnesium more readily than haemoglobin does its iron.

As regards the pyrrol constituents of the two pigments, information is to be obtained by oxidation. Kiister (1900) obtained, by oxidation of haemin ( = haematin hydrochloride), an imide of an acid, which he called htematinic acid. Willstiitter finds that chlorophyll behaves in a similar way. The porphyrins from it gave an imide of haematinic acid, together with methyl-ethyl-maleic imide. Further, Nencki and Zaleski (1901) found that haemin, on reduction, gave hcemo-pyrrol, which is, according to Kiister (1908) and Piloty (1909), a dimethylethyl-pyrrol. Willstatter obtains this substance from chlorophyll also, and finds it to be a mixture in both cases of three isomeric dimethyl-ethyl-pyrrols.

From information given me by Prof. Willstatter, I understand that he does not regard the similarity in constitution between chlorophyll and haemoglobin as being of any great significance. The mother substances were probably at hand, and compounds with the properties of the two pigments respectively being FIG. 178. ABSORPTION SPECTRA OF CHLOROPHYLL AND ASSOCIATED PIGMENTS.— Scale of wave lengths at top and bottom. Fraunhofer lines marked at the top. The colours of the regions of the spectrum indicated at the bottom.

1 Nettle leaf, living. Chlorophyll in colloidal state (Willstatter und Stoll, p. 62). " a-chlorophyll in ether (do. , p. 170). required, if one may use the expression, these pyrrol derivatives were made use of. In the leaf, according to Willstatter, chlorophyll probably exists as an adsorption compound with a colloid, but not combined with a lipoid, as some have stated. The two yellow pigments are both unsaturated and autoxidisable, that is, they spontaneously oxidise in the air. They are nitrogen-free. Both pigments have two absorption bands in the blue and blueviolet (see Fig. 178).

The one, carotin, is an unsaturated hydrocarbon (C40H5C). It is soluble in petroleum ether and is identical with the " lutein " of the corpora lutea of mammals, It is isomeric with the " lycoperdin " of the tomato. As we shall see later, it may play a part in the decomposition of carbon dioxide by the chloroplast system. Xanthophyll is an oxide of carotin (C40H56O2). It is insoluble in petroleum ether, but soluble in methyl alcohol. It is isomeric with the " lutein " of the fowl's egg, which has no relation to cholesterol, as had been supposed.

In Fig. 178 the absorption spectrum of chlorophyll, in its two forms, is given. The most striking and characteristic appearance is the dark band in the red, which is divided into two in chlorophyll-6. As will be noted, the chief absorption is in the longer wave lengths and is practically in the position of the maximum energy of the solar spectrum, during the greater part of the day. S. P. Langley's measurements of the position of maximum energy gave 650-666 /M/I for high sun. The latter number is easy to remember, as Timiriazeff points out, being the " number of the beast." The middle of the chief band of chlorophyll-a is, in solution in ether,

at 662 fifji. In colloidal solution in 1 per cent, acetone, the band is shifted towards the red, so that its maximum, is at 678 /I/M. This is the same as that of its natural state in the leaf. The maximum energy of solar radiation, also, would be for the greater part of the day nearer the red than the figure of Langley. Chlorophyll has a considerable absorption in the blue also, but practically none in the infra-red, nor in the yellow-green, not much in the ultra-violet.

It is remarkable that some of the earlier observers believed that their experiments showed that the maximum photo-chemical change occurred in the j'ellow-green region, in which the absorption of light energy is minimal. This would be a difficulty in view of Grotthus's law, and later observations, especially by Engelmann, showed it to be due to incorrect estimation of the absorption of the screens used. A striking demonstration of the fact that the maximum evolution of oxygen is at the place of the greatest (Engelmann, 1882, 1, p. 195.) absorption of light was given by

by the use of a bacterium, which was very sensitive to oxygen. Water containing these organisms was placed, along with a thread of a green alga, on the stage of a microscope. In the same plane, and along the thread of alga, a minute spectrum was projected by means of a spectroscopic arrangement beneath the stage. It was seen that the bacteria accumulated precisely at the places where the absorption bands of chlorophyll were situated (see Fig. 179). Another experiment, showing the same fact, is due to Timiriazeff (1903). A leaf on a plant is deprived of its stored starch by being kept in the dark. A small spectrum is projected on to its surface and, after some time, the leaf is decolorised by alcohol and treated with iodine. The absorption bands of chlorophyll are then found to be mapped out by the action of the iodine on the starch, which has only been formed in these places (Fig. 180). Measurements have also been made, spectrophotometrically, of the absorption of light in different regions of the spectrum and compared with the photo-chemical effect. There are two maxima shown, but, when the curve is corrected for the normal spectrum, in which equal abscissae correspond to equal differences of wave length, the second maximum in the blue end is found to be comparatively unimportant. From

CHLOROPHYLL. Part of a nlament of Cladophora in water containing motile bacteria, of considerable avidity for oxygen. a, Spectrum of sunlight, indicated by the position of the Fraunhofer lines, is projected from below to lie along the filament. The absorption of light by the chloroplasts, which practically fill the cells, is seen l>etween B and C and at the violet end. The accumulation of bacteria at places of absorption, especially in the red, shows that oxygen is being produced there. Magnified 188 times.

Engelmann's results, it appears that, in proportion to the light absorbed, the efficiency of the various regions of absorption is the same. In other words, so long as light is absorbed, it does not seem to matter what the wave length is. Kniep and Minder (1909), also, have compared the carbon assimilation with the relative amount of energy of the light absorbed in different parts of the spectrum and state that it is in direct proportion. This fact seems to suggest that the actual pigment itself is merely an optical sensitiser, since there is no relation between its particular absorption bands and the photo-chemical change.

Lasareff (1907) similarly showed that the bleaching of certain dyes is in proportion to the light energy absorbed, whatever the colour of the light. It is obvious, however, that, in so complex a system as the living cell, an exact agreement is not to be expected ; the oxygen, for example, may be partly used up by the protoplasm, and structures other than the chloroplasts absorb light. We may take it, then, that the maximal effect of the chlorophyll system is in relation to that part of the spectrum which is absorbed most.

In view of the results obtained by various observers with solutions of chlorophyll extracted from the leaf, it is important to remember that, in situ, this pigment is closely associated with other substances in the granules known as chloroplasts. It appears to form a thin, highly concentrated layer on the surface of these bodies and is practically solid (see the paper by Timiriazeff, 1903, p. 455), or in the colloidal state, since it shows no fluorescence. As we saw (page 562), its spectrum in the leaf is the same as that of the colloidal solution. Owing to its close association with the complex system of the chloroplast, it is scarcely to be expected that it would be possible to obtain the complete photo-chemical change in preparations containing chlorophyll alone. Miss Irving (1910) found that, if a seedling be grown in the dark and then placed in light, chlorophyll may be produced in the cells before they have developed the power of photosynthesis. At the same time, it is obviously of interest and importance to commence with the action of pure chlorophyll and, if possible, add the other constituents of the system later.

In this connection, we may remember the importance of the structure of the cell, not only for oxidation processes, about which we shall have to speak later, but also for the re-establishment of lactic acid in the contractile system of muscle with addition of energy, a process more analogous to that of photo-synthesis. The final result of the process may be represented by an equation such as : — a£!O2 + a;H2O + light energy = C^H^O^ + aO2, but this naturally gives us no indication as to how it is brought about.

Hydrangea leaves, still attached to the plant, have been deprived of starch by keeping in the dark. They have then projected upon them a small solar spectrum for five to six hours. Subsequent treatment with iodine, in the usual way, shows a picture of the absorption spectrum of chlorophyll in the blue "compound" of iodine and starch. The lower piece of leaf has been partiallv covered with a screen, represented below it, in such a way that the wider part of the aperture corresponded with the region of the spectrum between the lines B and C.

The fact that light energy is stored up shows at once that we have to deal with a process that is not a catalytic one. The reduction of carbon dioxide at ordinary temperatures is effected against chemical forces. As Weigert points out (1911, p. 99), this indicates that chlorophyll itself takes part in the reaction and that the considerable increase in potential which occurs is due to the interaction with other parts of the chloroplast, as indicated above. This raising of potential is a common phenomenon in physiological processes (see the paper by Weigert, 1908, p. 464).

In living organisms, as we know, the process is a reversible one, since carbohydrate is oxidised with production of carbon dioxide and water, but it is not necessary that the same intermediate stages should be passed through ; in fact it does not seem probable that they are. If, however, we take the simplest form of the equation given above, making x=\, formaldehyde is one of the products on the right-hand side, and this is oxidised by oxygen, at all events by " active " oxygen, giving out light as a phenomenon of chemi-luminescence (see page 557 above). According to Trautz (1905, p. 101), this light is of a reddish colour, in fact, of the same wave length as the position of the main chlorophyll absorption band. Thus, the equation might be written, with the inclusion of light as a part of the reversible system : —

CO2 + H2O + light energy of definite wave length ~ — > HCHO + O2. It was suggested by von Baeyer (1870) that formaldehyde is the first product of photo-synthesis, and Usher and Priestley (1906) found that an aldehyde is to be detected as a product of the action of light on films of chlorophyll in the presence of moist carbon dioxide. There is also reason to expect formaldehyde to be produced, since Bach (1893) obtained formic acid by the action of light on carbon dioxide in presence of solutions of uranium salts, and Moore and Webster (1913) have obtained formaldehyde by the action of ultra-violet light upon colloidal solutions of uranium hydroxide or ferric hydroxide. Moreover, formaldehyde is readily polymerised to higher carbohydrates. Loew (1889) obtained, by the action of magnesium oxide and lead on formaldehyde at 60°, a sugar which he called formose • this was afterwards shown by Emil Fischer (1890) to be inactive fructose. Berthelot and Gaudechon (1910) obtained formaldehyde by exposing a mixture of carbon dioxide and hydrogen, or water and carbon monoxide, to ultra-violet light, and Walther Loeb (1905) by exposing moist carbon dioxide to the silent electric discharge. In both cases, a series of intermediate reactions took place, and the conditions are, perhaps, rather far from those of the green leaf, although, as we have seen, the photo-chemical process, in Luther's view, is fundamentally an electric one. The alternating electric field of the silent discharge is not very far removed from that of light, but, of course, the frequency of the alternations is very much less.

Now, if the aldehyde in Usher and Priestley's experiments were actually derived from the carbon dioxide present, a great step would have been taken, but we have already seen reason to doubt whether such a reaction is possible by the aid of chlorophyll alone. Schryver (1910) showed that an aldehyde is only to be obtained from chlorophyll after it has been exposed to light, but that the production does not depend on the presence of carbon dioxide. Recent work by Wager (1914) and by Warner (1914) confirm this result as to the production of some aldehyde from chlorophyll by light in the absence of carbon dioxide ; they find also that oxygen is necessary, and that it is used up. Wager is doubtful whether the aldehyde formed is formaldehyde, since the colour given with Schryver's reagent is different from that given by pure formaldehyde. The aldehyde produced under these conditions is a result of the photo-chemical oxidation of chorophyll itself, which becomes bleached. Wager shows that the amount of aldehyde produced is proportional to the amount of absorption in the different regions of the spectrum. He could not detect any disappearance of carbon dioxide when chlorophyll films were exposed to light in its presence. but admits the possibility that his method might not have been delicate enough to detect a minimal disappearance. An important fact shown is that, when

chlorophyll is oxidised by such reagents as hydrogen peroxide or potassium permanganate, an aldehyde is formed. Similar results were arrived at by Warner independently. Both investigators also found that an oxidising substance is produced at the same time as the aldehyde. This oxidising agent is of a peroxide nature, and Warner makes the statement that the bleaching of chlorophyll is due to hydrogen peroxide, as had. been stated by Usher and Priestley previously. Wager, on the contrary, was unable to obtain any of the usual reactions of hydrogen peroxide ; but the experimental results of Usher and Priestley are difficult to interpret otherwise. They coated a plate with gelatine containing the enzyme, catalase, obtained from the liver. This enzyme decomposes hydrogen peroxide with evolution of gaseous oxygen, and so far as is known, does not so decompose other peroxides. There is, however, another enzyme, peroxidase, which decomposes other organic peroxides, as well as hydrogen peroxide, but does not cause the production of gaseous oxygen, so that it could not account for the following result. The film of gelatine, containing catalase, was coated again with a film of chlorophyll, and exposed to light in the presence of carbon dioxide. The gelatine film, after a time, was found to be full of bubbles of gas, while the chlorophyll remained unbleached. The fact can only be explained by the rapid diffusion of hydrogen peroxide into the gelatine film, and its decomposition there before it had time to affect the chlorophyll to any perceptible extent.