Principles of General Physiology

These results are very suggestive in view of what has been said above with regard to the phenomena of optical sensitisation, in which we find frequently a similar activation of oxygen, associated with the partial decomposition of the sensitiser. Although it appears that the product of the action of light on a dye acts, in the ordinary cases, as a catalyst, it does not seem necessary to assume this in the case before us, since the results can be explained by the oxidation of part of the pigment by the peroxide produced, with the production of an aldehyde. At the same time, there is no reason to deny the existence of a catalytic process as part of the phenomenon ; in fact, that part with which we are now concerned is not one in which energy is stored.

The point to which we have arrived seems to be this. The aldehyde which is split off from the chlorophyll system must have been derived from some constituent of the chlorophyll, probably the phytol, since it is formed by light irrespective of the presence of carbon dioxide. The experiments just related, therefore, give us no information as to the most difficult part of the problem, namely, how formaldehyde is produced from carbon dioxide. As to this, Hoppe-Seyler (1881, p. 139) suggested the hypothesis that chlorophyll combines with H2CO3 ( = CO2 + H2O) ; this compound is supposed " to fall apart, under the influence of light, in such a way as to yield chlorophyll (or the catalyst contained therein), oxygen and a third product, either sugar or a substance from which sugar may be formed." We have seen, on the other hand, that the main process, as a whole, is not a catalytic one, and the assumption of a chemical compound of chlorophyll with carbon dioxide is, at present, devoid of proof. We may perhaps regard the association of carbon dioxide and water with chlorophyll as some kind of a physico-chemical system analogous to that of muscle. By the taking up of light energy, this system is converted into one in which the chemical potential of the carbon dioxide plus water is raised to that of formaldehyde plus oxygen, or hydrogen peroxide. The formaldehyde may possibly be combined chemically with the chlorophyll, or it may be merely associated in some way, such that it is split off by the subsequent action of light and oxygen. Of course, the processes proceed simultaneously under natural conditions. We may represent it thus, in diagram, putting (C) for chlorophyll : —

It must be assumed that the oxygen is molecular, inactive, oxygen and given off as gas, so that the formaldehyde escapes oxidation. The first stage does not appear to have been obtained outside the living leaf and it probably requires the complex mechanism of the chloroplast. What this mechanism is requires further investigation. Willstatter and Stoll (1913, p. 2o) make the following suggestion. Carbon dioxide, attracted to the chlorophyll system by virtue of the magnesium it contains, is reduced by chlorophyll-a (by the agency of light energy), which itself becomes chlorophyll-''. A* \v saw, chlorophyll-/* is an oxidation product of chlorophyll-a, containing one atom more oxygen. Two molecules of chlorophyll-fr might then give off a molecule of oxygen, and become chlorophyll-a again. It is further suggested that this removal of oxygen may be the function of carotin, which thereby becomes xanthophyll. A reducing enzyme finally convert* xanthophyll into carotin again. But this naturally is, at present, purely hypothetical.

It will be seen how far we are from understanding the process. Usher and Priestley (1906) think that hydrogen peroxide is the immediate source of the oxygen given off in photo-assimilation ; this peroxide is decomposed by catalase, present in all green leaves, with evolution of molecular oxygen. It is possible, however, that the hydrogen peroxide detected by them is only the product of the second stage of our hypothetical process, which takes place in rifr» and is concerned with the splitting off of formaldehyde from its temporary ass. >ciation with chlorophyll. It may even have nothing to do with the real carbon dioxide assimilation, being possibly concerned with the action of the chlorophyll as an optical sensitiser. It is clear, in any case, that, in the reduction of carbon dioxide, oxygen must be dealt with in some way, and the final net result is that a volume of oxygen equal to that of the carbon dioxide is given off.

If formaldehyde is taken up in any way by chlorophyll, the latter is shown to be a chemical sensitiser, as well as an optical one. Fenton (1914) finds that, under appropriate conditions, formaldehyde and hydrogen peroxide combine to form a compound 2H.CHO.H2O.,, which is crystalline and fairly stable at ordinary temperatures. It takes fire if brought into contact with reduced iron or platinum black. It is decomposed by sunlight. After all, it seems most likely that the aldehyde which results from the action of light plus oxygen on chlorophyll in vitro may come from actual decomposition of the molecules of chlorophyll, as it does from other organic substances. In such a case it would have no relation to the photo-synthetic process, and be a purely artificial phenomenon. Curtius and Franzen (1912) obtained from leaves afjhexylene-aldehyde, CH3 - CH2 - CH2 - CH = CHO. The production of such higher aldehydes suggests a possible origin from the phytol of chlorophyll.

Since chlorophyll is an optical sensitiser and these act by formation of catalysts (Weigert, 1911, pp. 64-70), the possibility must not be disregarded that it may have no other function. If this be so, the formation of formaldehyde from carbon dioxide and water would only be possible in the complex system of the chloroplast, as already suggested above. Although there are certain difficulties in this view, such as the peculiar chemical nature of chlorophyll itself, as an organic magnesium compound, it seems by no means unlikely that it may turn out to be the correct one. If so, the photo-chemical reaction by which carbon dioxide and water are converted into formaldehyde and oxygen, with the taking up of light energy, is effected by other constituents of the chloroplast, perhaps with the aid of iron, as pointed out by Moore (1914), and that the use of the optical sensitiser, chlorophyll, is to enable a sufficient supply of light energy to be available.

As to the further change of formaldehyde into sugar and starch, this readily takes place under ordinary chemical conditions, as stated above (page 564). At the same time, if the process were as simple as this in the leaf, it would seem that formaldehyde should serve as a means of formation of starch, independent of light. Now, experiments by Miss Baker (1913) show that this is not so, formaldehyde does not serve as carbon food for plants in the dark, although it does so in the light. It is probable, therefore, that light accelerates the polymerisation, so that there is never much free formaldehyde present at one time, thus avoiding the wellknown toxic effects of this substance. The energy change is small in the process • it' polymerisation of formaldehyde and a catalyst may be produced in the chlorophyll system under the action of light, thus adding another factor to the complex system.

Timiriazeff (1903, p. 455) has made an interesting calculation, on the basis Of the measurements of Horace Brown and others, to be given presently, of the actual amount of light energy absorbed by chlorophyll. The result is that, if all the light energy were converted into heat in the chlorophyll itself, a temperature of 6,000° C. would be obtained. Of course, the energy is converted into chemical work, without passing through heat, but the calculation gives us an idea of the intensity of the energy changes brought into play. It may be noted that carbon dioxide is dissociated into carbon monoxide and oxygen at about 1,200°, but this fact would not assist the comprehension of the photo-chemical process, even if we admit that such a temperature might be attained locally in the chloroplast, since the union of carbon monoxide and hydrogen to produce formaldehyde requires the action of light.

The reduction of carbon dioxide can, nevertheless, be effected by certain photoplasmic systems by the aid of chemical energy without light, so that the photosynthetic process is not to be regarded as an altogether singular one. Other forms of energy, besides that of light, can be utilised by certain organisms for the purpose. For example, there are some bacteria which can use the energy obtained by the oxidation of hydrogen to reduce carbon dioxide for their own carbon needs. Suppose we grow these bacteria in a closed vessel, containing hydrogen and oxygen, we find that combustion of the gases to form water takes place. If carbon dioxide is also present, it simultaneously disappears and the carbon is assimilated by the bacteria. If hydrogen and carbon dioxide alone are present, there is a slight disappearance of both, but very little.

Electrical Changes. — Such have been described in the green leaf in consequence of illumination. The work of Haacke (1892) and of Waller (1900, 2) may be mentioned. The effects appear to be connected with the photo-synthetic process, since they are absent if the light has already been deprived of the rays absorbed by chlorophyll by passing through another green leaf previously. These effects are nearly as great when red light is used as when white light is used. Waller has shown that removal of carbon dioxide abolishes the response, and that it can be brought back by adding carbon dioxide to the atmosphere in which the leaf is situated. The fact of an electrical response is of interest in connection with the electronic theory of photo-chemical change, described above (page 552), but cannot as yet be explained. Harvey Gibson and Titherley (1908) have suggested an electrochemical theory of chlorophyll assimilation on the basis of these electrical effects.

Red and Brown Seaweeds. — The absorption of light by chlorophyll, as we have seen, is such as to make the best use of the light available. But a green pigment is, of course, transparent to the green rays, which preponderate under water, so that it would be inefficient in that situation. Accordingly, as Engelmann has pointed out (1882, 2), we find, in the seaweeds, red and brown pigments corresponding to chlorophyll and having the same function, but able to absorb effectively the green light available. For example, the red seaweeds show a maximum of carbon assimilation in the green and, spectrophotometrically measured, they show the greatest absorption in the same region, although there is also considerable absorption between the lines B and C, where the chief band of chlorophyll lies. The minimum of absorption is in the orange between C and D (p. 220 of the paper referred to). This fact serves to illustrate the function of chlorophyll as an optical sensitiser ; the same effect is produced by light of various wave lengths, provided that it is absorbed.

In certain cases, to which Engelmann has given the name of " complementary chromatic adaptation" we find that a pigment is actually formed under the action of coloured light and that the pigment has a colour which is complementary to that of the light to which the organism is exposed, so that this light is then absorbed. The alga, Oscillaria sancta, as shown by the work of Gaidukov (1902), occurs in several colours between reddish purple and blue-green. If cultures are made, say, of the reddish-purple variety, we find that under red light a green pigment is produced. If we take the green variety, it becomes reddish under green light, brownish yellow under blue light, and so on. The general colour of a mixed culture thus tends to become complementary to that of the light under which it is grown. The work was done with great care and spectro-photometer curves of the various pigments were compared with those of the light under which they made their appearance.

The phenomena seem to be related to the colours assumed by Wiener's (1895) phvlo<-hlori<l< * under the action of light of various colours ; but the case with which we are concerned shows the opposite effect, not the production of a similar colour. The production of a substance of a colour similar to that of the light acting is well shown by Stobbe's (1908) fufyide* ; or;i!i;:r light produces an orange dye, blue light, a blue one. Thus, a substance is formed which does not absorb the light acting, so that no further change takes place ; although, if the product is exposed to light of a different wave length from that under which it was produced, further change is effected since the light is absorbed.

Temperature. — A pure photo-chemical process has a low temperature coefficient, like that of physical phenomena, as we saw above (page 42). The photo-synthesis of carbon dioxide, not being a simple photo-chemical process, has a high one, especially at low temperatures; it is 6 for the 10° between -5° and -I- 5°, but only 1'76 between 20° and 30°. Certain "limiting factors," to be referred to below, complicate the measurements, so that, under ordinary conditions, the rate of the reaction is the same between 3° and 30°.

The coagulation of the chloroplast by heat takes place at a lower temperature than that of protoplasm in general. A high temperature coefficient at low temperatures, although only a more pronounced effect of a general phenomenon (see page 42), is of frequent occurrence in complex physiological processes. For example, that of the geotropic reaction is 6 "5 for the interval from - 10° to 0°. Anaesthetics. — The complex nature of the process is shown by the fact that chloroform, even in traces, stops it ; 0'002 c.c. per litre of air suffices.

Limiting Factors. — The importance of these factors as regards the velocity of the reaction has been emphasised by Frost Blackman (1905). They may be temperature, light, or access of carbon dioxide. It will be clear that, if the amount of carbon dioxide present is less than the system can deal with under a certain intensity of illumination, no increase in rate will be obtained by increasing the light, but will be obtained if the carbon dioxide is increased. This is, in fact, the usual state of affairs. Under ordinary conditions of good illumination, the leaf can deal with considerably more carbon dioxide than is able to diffuse to the chloroplasts through the stomata and intercellular passages (see Brown and Escombe, 1905).

The effect of accumulation of sugar is to cause the stomata to close and cut off the supply of carbon dioxide. First of all we require to know the amount of energy necessary to convert 1 c.c. of carbon dioxide to hexose. This can be calculated from the heat of combustion of hexose, and amounts to 5 -02 calories. To obtain the maximal efficiency, it is necessary to take account of the fact that the amount of light can be diminished nearly twelve times without affecting the rate of synthesis with the usual carbon dioxide tension of the atmosphere (Brown and Escombe, 1905, p. 86). Of the total radiation falling on the leaf, 65 to 78 per cent, is retained by it ; the rest is transmitted or radiated to the surroundings. The greater part of that retained is used for purposes such as transpiration, that is, for evaporation of water. To find out how much is actually used for photo-synthesis, Brown and Escombe compared the amount absorbed by the white and green parts of a variegated leaf. In a particular case the white part absorbed 7 4 '5 per cent, and the green, 78-7 per cent., so that 4-2 per cent, was absorbed by the chlorophyll. Now, it was found that, in Tropceolum majus, a total amount of incident light equal to 0-041 calorie per sq. cm. per minute caused the decomposition of 0-03034 c.c. of carbon dioxide per sq. cm. per minute. Since the energy stored in the conversion of 1 c.c. of carbon dioxide to sugar is 5-02 calories, that stored in 0-00034 c.c. is 0-00034 + 5-02 = 0-001 7 calorie per sq. cm. per minute. This is equal to 4'1 per cent, of the total incident radiation. We have just seen that 4'2 per cent, of the total incident radiation is absorbed by the chloroplasts, so that,

as Weigert points out (1911, p. 106), we obtain the astonishing efficiency of 98 per cent. But it must be remembered that the experiments were not made on the same leaf and also, according to Blackman, the position and distribution of the chlorophyll should be taken into account, which make the percentage of incident light absorbed by it 10 per cent, instead of 4 '2 per cent, and the efficiency is reduced to 41 per cent. In any case, the maximum efficiency is a high one and is, of course, only to be obtained under exceptional conditions. The usual one appears to be about 20 per cent.

The greater number of the constituents of living cells are colourless, that is, they do not absorb rays of the wave length of visible light. Many of them, however, absorb ultra-violet light so that it is not surprising to find that radiations of this kind have a very powerful effect on living cells as a rule. The use of the absorbing power for ultra-violet of some constituents of living cells for the purpose of photographing them has been referred to above (page 9), as also the use of the fluorescence excited in them by ultra-violet light absorbed.

A series of important researches on ultra-violet light is at present being carried on by Victor Henri with several coadjutors, the results of some of which have been published (see Mme. V. Henri, Victor Henri, J. Larguier des Bancels, and R. Wurmser, 1912). The first part of this work consisted in the determination of the wave lengths of the light emitted by various sources of ultra-violet light and of the absorption of screens. For the purpose of investigation it is clearly useful to have screens which will cut off ultra-violet and transmit visible light and others which will cut off the visible rays and transmit the ultra-violet rays. The most useful of the former was found to be " euphos " glass, which, in a thickness of 0'75 mm., cuts off very little of the visible spectrum, but only allows a very small amount of ultraviolet to pass. For the latter purpose, a colloidal solution of silver, prepared by the electrolytic method, is valuable. In a thickness of 20 mm., this allows no visible rays to pass, but is fairly transparent to ultra-violet, even waves as short as 219 fjifj, are slightly transmitted. In 10 mm. thickness, about 5 per cent, of the red, yellow, and green rays pass, but as much as 30 per cent, of the ultra-violet, in its middle region. Lehmann's (1910) modification of Wood's filter is much used. This consists of a double cell of Jena " uviol " glass, of 2 mm. thickness, that is, 6 mrn. of the glass in all. The depth of each chamber is 5 mm. One is filled with saturated copper sulphate, the other with a solution of nitroso-dimethylaniline in a strength of one part in 12,000. The filament of an incandescent lamp is just visible through it, while it transmits a considerable amount of ultra-violet.

The absorption of egg- and serum-albumin was next studied. In the former the absorption is feeble for rays longer than 300 /x/x, increases to a maximum absorption band at 280 /x/x, has a minimum again at 250 /x/x, and then rises again; so that, for the extreme ultra-violet, the extinction coefficient exceeds 1,000. It may be noted that the mercury arc in a quartz tube sends out rays, of moderate intensity, as short as 220 /tx/x, for which the absorption coefficient of albumin is over 1,000, and intense rays as far as 238 /x/x, for which the absorption coefficient is nearly 200, so that it is not surprising that protoplasm is very sensitive to the extreme ultra-violet of this lamp, as we shall see.

It has long been known that various small animals, such as those tiny Crustacea found in fresh water, flee from places illuminated by ultra-violet light, and Mme. V. Henri and Victor Henri (1912, pp. 12-21) have found that Cyclops is very useful for experiment. If it be illuminated for two to five minutes with the quartz mercury arc, it first shows great agitation, then becomes immobile. In this state, if kept in the dark, it remains for some hours motionless, but very sensitive to renewed illumination, to which it responds by a vigorous movement. It is thus easy to make exact measurements with it. After a day in water, it has become normal again.

A certain minimal duration of illumination is necessary for a reaction to take place. This duration is less, the greater the proportion of ultra-violet in the light. Thus, through a quartz screen, the time is about two seconds ; through 10 mm. of colloidal silver it is only increased to twenty-five seconds, although the ultra-violet is much diminished by the screen ; through " euphos " glass, no reaction was produced in 200 seconds. There is also a minimum intensity of illumination below which no reaction is obtained however long the exposure. There is also a certain intensity of illumination at which the necessary time of exposure is the shortest, an intensity greater or less than this requiring a longer time.

It is interesting to note that there is also a phenomenon of summation of sub-minimal stimuli, repeated at intervals, similar to that which we have seen in spinal reflexes. A remarkable fact is that the excitability to ultra-violet rays is independent of temperature, a fact which shows that the exciting cause of the reflex movement is a photo-chemical reaction, whose products, no doubt, excite receptors of some kind in the outer surface of the animal. These receptors then excite nerve endings.

Fatigue can be produced to ultra-violet light in Cyclops by very short duration of radiation, if the peripheral receptors have been modified by prolonged previous radiation or by cocaine, but not if the anaesthesia is of central origin, by ether, for example. In this latter case, the peripheral organs are left intact. These facts show that the reaction studied is really due to photo-chemical changes at the periphery. Since the absorbing power of protoplasm is very great, especially for the shorter wave lengths of ultra-violet, the effect can penetrate for only a short distance. In such small organisms as bacteria, however, it may affect the whole organism, so that the action, which is a lethal one in such cases, obeys the laws of photo-chemical reactions. In larger organisms, we have to take account of the diffusion of the products of photo-chemical change, and their action at places remote from that where they are formed. The Jaws are, therefore, more complex, and the effects last longer than the actual exposure.

The following table (from Victor Henri, etc., 1912, p. 33) gives the thickness of a layer of protoplasm which reduces ultra-violet light of different wave lengths to one-tenth its value, or absorbs nine-tenths of it. Action on Bacteria, Tissues, etc. — An important practical question is that of the lethal action of ultra-violet light on micro-organisms, since it has been used for sterilisation of water. Equally important is its destructive effect on the tissues of higher animals, as used in the therapeutic method of Finsen.

Hertel (1905) has studied in some detail the effect of the magnesium line of 280 ftfj. on bacteria, protozoa, and some toxins, etc. Bacteria were found to be killed in from fifteen to sixty-five seconds. Mme. V. Henri and V. Henri (1912, pp. 29-31) find that there is no particular wave length which is specially lethal, but that the effect increases more and more the shorter the wave length, as far as tested, that is, up to 214 /x/z. Protozoa are killed in about the same time as bacteria. according to Hertel. The body swells, watery drops appear on the surface, and finally disintegration occurs. Rotifers, nematodes, and molluscs are also killed. On the tadpole, the effect is swelling of the epithelium, followed by migration of pigment and nuclear division, together with stasis of blood in the capillaries. On the cells of Elodea, slowing of the protoplasmic movement was noted, which was much less if illuminated by ordinary light at the same time. A longer exposure is required for tissue cells than for bacteria.

Diphtheria toxin was destroyed in five minutes, but the antitoxin was not destroyed in thirty minutes. Various enzymes were also rendered inactive. Hertel regards the effect as due to reduction processes for three reasons : — 1. The effect is less in the green Hydra than in the colourless one, presumably due to the oxygen afforded by chlorophyll. 3. If alizarin blue is injected into the veins of a rabbit, the brain is blue, but, if acted on by ultra-violet light, the dye is reduced to its colourless derivative.

An interesting point is that, if rays of equal energy (as measured by the thermopile) are taken, one of 440 /X/M, the other of 280 /*//,, Hertel found that rotifers are killed by the short waves in fifteen seconds, by the long waves only after four or five hours. This fact shows strikingly that it is not a question merely of energy, but of the actual wave length, no doubt because the short waves are absorbed by the protoplasm. We have seen how large a number of photo-chemical reactions are brought about by ultra-violet light, so that we may expect to find it active also on protoplasmic systems. But the nature of these reactions in the latter case is still unexplained. One of the great difficulties in the therapeutic application of ultraviolet rays to stop the growth of malignant cells is the very rapid absorption by the superficial cells, so that the active rays do not penetrate more than a short distance. Considerable success has attended the treatment of superficial skin growth, such as lupus, by ultra-violet light. Since haemoglobin has so active an absorption for ultra-violet (see especially the data of Victor Henri, etc., 1912), Finsen (1901, p. 70) uses a method of compressing the blood out of the area of tissue to be acted on by light.

The great effect of ultra-violet light on the skin is familiar to every one in the inflammation (erythema solare) called sunburn, which results in a brown coloration. . It may be pointed out that this is not an effect of heat, in fact it is more liable to occur in cold surroundings, probably owing in part to the fact that the heat of the sun's rays is not noticed and no means taken to protect the skin from their action. It is, no doubt, due to the products of some photo-chemical reaction, acting on the arterioles, and it would be interesting to know whether, like the effect of oil of mustard, it is an axone reflex in sensory nerve fibres.