THE ACTION OF INHIBITORY NERVES ON CARBON DIOXIDE PRODUCTION IN THE HEART GANGLION OF LIMULUS

It has been shown in this paper that stimulation of the inhibitory nerves of the neurogenic heart of Limulus, which correspond to the vagus nerves of the vertebrate heart, results in a marked diminution of CO₂ production in the heart ganglion, while stimulation of the ganglion, leading to increased activity of the heart, leads also to increased CO₂ production by the ganglion. This shows that inhibition of the automaticity of this ganglion by the action of its inhibitory nerves consists, not in a process of blocking, but in a diminution of those chemical reactions in the ganglion cells which give rise to the production of CO₂.     

DYNAMICS OF NERVE CELLS : I. THE TEMPERATURE COEFFICIENT OF THE NEUROGENIC RHYTHM OF THE HEART OF LIMULUS POLYPHEMUS.

In the case of the heart of Limulus polyphemus the same magnitude and variation of the temperature coefficient (Q ₁₀) is obtained from the whole heart as from the ganglion alone. From the magnitude of the temperature coefficients and their variation with changes of temperature we may conclude that the rate of the heart beat is determined by alteration of chemical processes in the ganglion cells.     

TEMPERATURE AND FREQUENCY OF CARDIAC CONTRACTIONS IN EMBRYOS OF LIMULUS

Temperature characteristics for frequency of myogenic heart beat in Limulus embryos, before the onset of nervous control of the heart, were found to be 11,500; 16,400; 20,000; 25,500. The two first values are the best established. The different values pertain to the hearts of different individuals outwardly similar, and to the hearts of single embryos in different parts of the temperature range. These values differ from that known in connection with the control of the heart beat through the cardiac ganglion. The occurrence of critical temperatures, also, is not the same in all embryos. These facts are employed in a discussion of temperature relations in pulsating explants of chick myocardium.     

OBSERVATIONS ON THE TRANSPORT OF CARBON DIOXIDE IN THE BLOOD OF SOME MARINE INVERTEBRATES

Although the results we have recorded merely serve to indicate the possibilities of this interesting field of investigation, we have sufficient data to enable us to draw certain general conclusions. In the first place it is evident that the bloods of the more highly developed marine invertebrates, such as the active Crustacia and the Cephalopods, are specially adapted for the carriage of carbon dioxide. The quantity of carbon dioxide taken up by the blood of Maia, Palinurus, or Octopus at any given tension of the gas is, in general, about twice or three times as great as that which is taken up by sea water under the same conditions. On the other hand, the blood of a slow, creeping form, such as Aplysia, or of a sessile animal such as the ascidian Phallusia shows no more adaptation for the carriage of carbon dioxide than does sea water. But our estimations of the CO₂ content of the blood as it circulates in the bodies of these more active invertebrates show that the conditions of transport of this gas differ considerably in some respects from those which obtain in mammals. For the invertebrate blood in the body contains only a relatively small quantity of carbon dioxide, averaging in the forms we examined from 3 to 10 cc. per 100 cc. of blood. This forms a marked contrast with the condition found in mammals where even the arterial blood contains about 50 cc. of CO₂ per 100 cc. of blood. The invertebrate, therefore, works at a very low CO₂ tension. There is a twofold significance in this circumstance. In the first place, it means that only the first portion of the carbon dioxide dissociation curve is in use in the respiratory mechanism. Now an inspection of our curves will show that at these low carbon dioxide tensions the dissociation curves tend to be steeper than at higher tensions. As we intend to show in a later paper it can be proved mathematically that, other things being equal, a blood with a carbon dissociation curve of moderate steepness, i.e. one in which the carbon dioxide content of the blood increases fairly rapidly with increase of carbon dioxide tension, is a more efficient carrier of the gas from the tissues to a respiratory surface than a blood in which the dissociation curve is either steeper or flatter. It would seem as if the active invertebrates avoid the use of too flat a part of their CO₂ dissociation curves by working over the initial steeper portion. Furthermore, it is seen that over the range of this initial steep portion of the curves the changes of reaction produced by the uptake of carbon dioxide are much smaller than at higher tensions of the gas; for these initial portions of the curves are more nearly parallel to the lines of constant reaction calculated for a temperature of 15°C. according to Hasselbalch''s method (10) on the assumption that the whole of the combined CO₂ is in the form of sodium bicarbonate. It is evident also that on this assumption the hydrogen ion concentration of the blood of invertebrates (with the exception of the tunicates) would appear to be practically the same as that of the warm-blooded vertebrates—a conclusion confirmed by the direct measurements of Quagliariello (9). On the other hand, our measurements do not lend support to the idea put forward by Collip (4) that in order to maintain an appropriate faintly alkaline reaction an invertebrate needs to retain carbon dioxide in its blood at a comparatively high tension. This idea was based on the observation that at comparatively high CO₂ tensions the blood of invertebrates contains considerably more sodium bicarbonate than does sea water. But our curves show that this is no longer true at the lower values of carbon dioxide tension, the amount of sodium bicarbonate falling off more rapidly in the blood than in the sea water with diminution of the carbon dioxide tension so that in order to maintain an appropriate reaction in the blood only a comparatively small tension of CO₂ is required. The largest amount of carbon dioxide that we found present in the circulating blood of any of the types examined was 9.7 cc. per 100 cc. of blood in the case of Maia, and in most cases the amount was considerably less. But even this lowest value corresponds to a tension of CO₂ of only about 3 mm., so that the tension gradient across the gill membrane must be even less than this. We would emphasize rather the circumstances that as the portion of the dissociation curve over which the reaction is approximately constant is of but small extent, it is necessary that in an active form like Octopus the carbon dioxide produced should be removed rapidly lest an accumulation of it should cause the limits of normal reaction to be exceeded; and this need is correlated with the extreme efficiency of the respiratory apparatus in this animal. It is interesting to notice that the mammal which, in order to obtain an appropriate reaction in the blood, has to work at relatively high carbon dioxide tensions where the dissociation curve is comparatively flat, secures a steeper physiological CO₂ dissociation curve in the body, and with it a more efficient carriage of carbon dioxide and a more constant reaction in the circulating fluid, in virtue of the effect of oxygenation on the carbon dioxide-combining power of its blood (3, 6). Returning now to the consideration of the actual form of the dissociation curves we have obtained—it is a significant fact that it is in those forms such as Maia, Palinurus, and Octopus whose bloods are rich in proteins—particularly hemocyanine—that the initial steep portion of the curve is observed. This suggests that in these forms the blood proteins act as weak acids and expel carbon dioxide from the blood at the low tensions which include the physiological range, just as in vertebrates the hemoglobin similarly displaces carbonic acid from its combination with alkali metal. On the other hand the cœlomic fluid of Aplysia contains no pigment and only 0.00672 per cent of protein nitrogen (Bottazzi (11)) and shows no initial rapidly ascending portion of the CO₂ dissociation curve. This is supported by the observation of Quagliariello (9) that the acid-neutralising power of the blood of an invertebrate is roughly proportional to its protein content. It seems as if the proteins of invertebrate blood like the blood proteins of vertebrates, exist in the form of sodium salts which are capable of giving up sodium for the transport of carbon dioxide as sodium bicarbonate. That this is so in the case of hemocyanine follows from the fact that the isoelectric point of this pigment occurs at a hydrogen ion concentration of 2.12 x 10^–5 N, i.e. at a pH of 4.67 (Quagliariello (12)) so that in the alkaline blood of the invertebrates possessing it, hemocyanine will act as a weak acid. It is probable that the initial steep portion of the carbon dioxide dissociation curves which we have found to be of such importance in the respiration physiology of Octopus, Palinurus, and Maia is produced by the competition of this acid with carbonic acid for the available sodium of the blood.     

REDUCTION OF CARBON DIOXIDE COUPLED WITH THE OXYHYDROGEN REACTION IN ALGAE

1. Unicellular algae possessing a hydrogenase system (Scenedesmus and other species), and having been adapted by anaerobic incubation to the hydrogen metabolism, reduce oxygen to water according to the equation O₂ + 2H₂ → 2H₂O. 2. The oxyhydrogen reaction proceeds undisturbed only in the presence of carbon dioxide, which simultaneously is reduced according to the equation CO₂ + 2H₂ → H₂O + (CH₂O) = (carbohydrate). 3. The maximum yield of the induced reduction is one-half molecule of carbon dioxide reduced for each molecule of oxygen absorbed. 4. Partial reactions are recognizable in the course of the formation of water and it is with the absorption of the second equivalent of hydrogen that the carbon dioxide reduction appears to be coupled. 5. The velocity of the reaction increases in proportion to the partial pressure of oxygen, but only up to a certain point where any excess of oxygen causes the inactivation of the hydrogenase system. The reaction then ends prematurely. 6. During the oxyhydrogen reaction little or no oxygen is consumed for normal respiratory processes. 7. Small concentrations of cyanide, affecting neither photosynthesis nor photoreduction in the same cells, first inhibit the induced reduction of carbon dioxide and then lead to a complete inactivation of the hydrogenase system. 8. Hydroxylamine, added after adaptation, has either no inhibitory effect at all, or prevents solely the induced reduction of carbon dioxide without inactivating the hydrogenase system. 9. Dinitrophenol prevents the dark reduction of carbon dioxide while the reduction of oxygen continues to the formation of water. 10. Glucose diminishes the absorption of hydrogen, probably in its capacity as a competing hydrogen donor. 11. The induced reduction of carbon dioxide can be described as an oxido-reduction similar to that produced photochemically in the same cells.     

THE RESPIRATION OF LUMINOUS BACTERIA AND THE EFFECT OF OXYGEN TENSION UPON OXYGEN CONSUMPTION

1. The respiration of luminous bacteria has been studied by colorimetric and manometric methods. 2. Limulus oxyhaemocyanin has been used as a colorimetric indicator of oxygen consumption and indicator dyes were used for colorimetric determination of carbon dioxide production. 3. The Thunberg-Winterstein microrespirometer has been used for the measurement of the rate of oxygen consumption by luminous bacteria at different partial pressures of oxygen. 4. The effect of oxygen concentration upon oxygen consumption has been followed from equilibrium with air to low pressures of oxygen. 5. Luminous bacteria consume oxygen and produce carbon dioxide independent of oxygen pressures from equilibrium with air (152 mm.) to approximately 22.80 mm. oxygen or 0.03 atmosphere. 6. Dimming of a suspension of luminous bacteria occurs when oxygen tension is lowered to approximately 2 mm. Hg (0.0026 atmosphere) and when the rate of respiration becomes diminished one-half. 7. Pure nitrogen stops respiratory activity and pure oxygen irreversibly inhibits oxygen consumption. 8. The curve for rate of oxygen consumption with oxygen concentration is similar to curves for adsorption of gasses at catalytic surfaces, and agrees with the Langmuir equation for the expression of the amount of gas adsorbed in unimolecular layer at catalytic surfaces with gas pressure. 9. A constant and maximum rate of oxygen consumption occurs in small cells when oxygen concentration becomes sufficient to entirely saturate the surface of the oxidative catalyst of the cell.     

THE EFFECT OF CARBON DIOXIDE TENSION ON THE METABOLISM OF CEREBRAL CORTEX AND MEDULLA OBLONGATA

Manometric measurements were made of oxygen uptake (Q _OO2) and aerobic lactic acid output (Q_G) by slices of cerebral cortex and medulla oblongata of the cat in the presence of mixtures of 1, 5, and 20 volumes per cent of carbon dioxide in oxygen. The concentrations of NaHCO₃ and NaCl in the medium were varied to maintain constant pH and sodium ion concentrations. The calcium ion concentration was 0.0002 M. At pH 7.5 under these conditions, an increase in carbon dioxide from 1 per cent to 5 per cent doubled the Q_G of both tissues but did not alter Q _OO2; an increase from 5 per cent to 20 per cent carbon dioxide had no further effect on Q_G in either tissue or Q _OO2 of cortex, but did depress the Q _OO2 of medulla. At pH 8.1, an increase in carbon dioxide from 1 per cent to 5 per cent raised the Q _OO2 and Q_G of cortex by about 60 per cent. Measurements at low oxygen tension carried out previously in phosphate medium were repeated in bicarbonate medium to obtain data for the combined output of lactic acid and carbon dioxide (Q_A). When the oxygen in the gas phase was decreased from 95 to 3 volumes per cent, the lactic acid output as measured colorimetrically increased by 114 mg./gm. in cortex and by 8 mg./gm. in medulla; Q_A increased from 12.3 to 13.5 in cortex and decreased from 5.1 to 3.8 in medulla.     

AN EXPERIMENTAL SEPARATION OF OXYGEN LIBERATION FROM CARBON DIOXIDE FIXATION IN PHOTOSYNTHESIS BY CHLORELLA

Using intact cells of Chlorella pyrenoidosa it is possible to obtain oxygen by the reduction of certain reducible materials other than carbon dioxide. Of these, benzaldehyde was studied in some detail. This reduction does not involve the production of carbon dioxide from the benzaldehyde. Stoichiometrical relationships as expressed by the following equation: 2C₆H₅CHO + 2H₂O → 2C₆H₅CH₂OH + O₂ are somewhat difficult to obtain because the benzaldehyde can disappear from the reaction mixtures by dark reactions. The technique is now available which permits detailed studies of the oxygen-liberating mechanisms in photosynthesis.     

THE RELATIVE IMPORTANCE OF pH AND CARBON DIOXIDE TENSION IN DETERMINING THE CESSATION OF CILIARY MOVEMENT IN ACIDIFIED SEA WATER

The length of time that cilia from the gills of Mylilus continue to beat in acidified sea water depends to some extent on the pH of the solution but to a greater extent on its carbon dioxide tension.     

THE RESPIRATORY QUOTIENT OF FROG NERVE DURING STIMULATION

1. By means of a differential volumeter the increased oxygen consumption and the increased carbon dioxide output of frog nerve during and after stimulation have been observed. 2. Measurements of the R.Q. of nerve by this method are complicated by the retention of carbon dioxide. Attempts were made to avoid this (a) by studying the nerves at high CO₂ tensions to make the retention small and (b) by calculating the amount of CO₂ retained from the carbon dioxide dissociation curve of nerve and applying this value as a correction. 3. The results of both those methods when averaged together give an R.Q. of the excess metabolism of 1.19 and an R.Q. of the resting nerve of 0.97. 4. Observations on the time course of the gas exchange during stimulation indicate a delay in the appearance of the extra carbon dioxide output relative to the oxygen intake. 5. Very similar time curves can be calculated from the diffusion coefficients and the solubilities of the oxygen and the carbon dioxide.     

REGULATION OF THE HYDROGEN ION CONCENTRATION AND ITS RELATION TO METABOLISM AND RESPIRATION IN THE STARFISH

The normal reaction of the cœlomic fluid in Patiria miniata and Asterias ochraceus is pH 7.6, and of the cæca, 6.7, compared with sea water at 8.3, all without salt error correction. A medium at pH 6.7–7.0 is optimum for the cæca for ciliary survival and digestion of protein, and is maintained by carbon dioxide production. The optimum pH found for carbon dioxide production is a true one for the effect of hydrogen ion concentration on the tissue. It does not represent an elimination gradient for carbon dioxide. Because the normal excised cæca maintain a definite hydrogen ion concentration and change their internal environment toward that as an optimum during life, there exists a regulatory process which is an important vital function.     

A LATENT PERIOD IN THE ACTION OF COPPER ON RESPIRATION

1. When copper chloride is allowed to act on Aspergillus niger there is at first a period during which there is no change in the rate of the production of carbon dioxide, following which the rate of respiration falls. The interval of no change is called the latent period. 2. When the copper is removed from the external solution before the end of the latent period this interval is prolonged. The rate of respiration then falls to a new level below the normal level. 3. Experiments on Nitella and on Valonia indicate that the copper penetrates the cell almost immediately. 4. The length of the latent period varies inversely as a constant power of the concentration of the copper. 5. These results are explained by assuming that the copper is made active in the respiration system by means of a reversible reaction. By using appropriate velocity constants the experimental curves can be duplicated by calculated curves.     

THE EFFECT OF POTASSIUM ON THE ACID METABOLISM OF SURVIVING SKELETAL AND CARDIAC MUSCLES OF THE FROG

The potassium contraction of skeletal muscle and relaxation of cardiac muscle have been correlated with the carbon dioxide and total acid production of these tissues. 1. The immersion of surviving sartorius muscles of the frog in isotonic potassium chloride solution causes a marked increase in the rate of acid production. 2. It is probable that carbon dioxide is the principal acid involved in the above effect. 3. The immersion of surviving cardiac muscle of the frog in isotonic potassium chloride solution causes a pronounced depression in the rate of survival acid production. 4. Reasons are given for believing that these changes in metabolism may be independent of the stimulation and inhibition of contraction which potassium simultaneously produces in these tissues.     

COMPARATIVE STUDIES ON RESPIRATION : XXIV. THE EFFECTS OF CHLOROFORM ON THE RESPIRATION OF DEAD AND OF LIVING TISSUE.

1. Chloroform in low concentration (0.25 per cent) causes an increase in the rate of production of CO₂ in Ulva; this is followed by a decrease. In higher concentration (0.5 per cent) only a decrease is observed. 2. Assuming that the normal oxidation depends on the action of peroxide and peroxidase, experiments were made by placing Ulva in 1.0 per cent H₂O₂ and in Fe₂(SO₄)₃ (which acts like a peroxidase). The former diminishes the rate, the latter increases and subsequently decreases it. 3. When Ulva is killed in such a manner as to destroy the oxidizing enzymes, no CO₂ is produced unless H₂O₂ and Fe₂(SO₄)₃ are present. If to this mixture chloroform is added, the effect depends on the concentration of the iron. If the concentration is low there is an increase in the production of CO₂ followed by a decrease. If the concentration is high the rate appears to decrease from the start.     

THE EFFECT OF CARBON DIOXIDE TENSION ON TISSUE METABOLISM (RETINA)

The metabolism of rat retina was found to be sensitive to the concentration of the carbon dioxide-bicarbonate buffer system. Increasing the carbon dioxide from 1 per cent to 5 per cent at constant pH nearly doubled both respiration and glycolysis. Increasing the carbon dioxide at constant pH from 5 per cent to 20 per cent had no effect on glycolysis, but depressed the Q _OO2 from 31 to 19. In a medium containing glucose and the 1 per cent carbon dioxide-bicarbonate buffer, the addition of succinate increased the Q _OO2 from 12 to 26, without affecting glycolysis. In a medium containing glucose and phosphate, succinate had no significant effect.     

COMPARATIVE STUDIES ON RESPIRATION : V. THE EFFECT OF ETHER ON THE PRODUCTION OF CARBON DIOXIDE BY ANIMALS.

1. The experiments on frog tadpoles show that with 0.15, 0.37, and 0.55 per cent ether solutions there is a decrease in CO₂ output. The effect is reversible. With these concentrations the breathing movements and body movements remained normal during the experiment. In 3.65 and 7.3 per cent ether there is a decrease of respiration followed by an increase which in turn is followed by a decrease. The increase may reach about three times the normal rate. The increase in the CO₂ output is accompanied by the peeling of the skin. The effect is irreversible. 2. Experiments on an aquatic insect, Dineutes assimilis Aube, show that in 7.3 per cent ether there is a decrease followed by an increase which in turn is followed by a decrease. There is no apparent disintegration of structures in the organism accompanying the increase. The effect is irreversible. 3. The experiments on frog eggs with 7.3 per cent ether show a result similar to that found in aquatic insects. 4. Experiments on Fundulus embryos show that with 0.73 per cent ether there is a reversible decrease in the rate of CO₂ production. In 3.65 per cent ether there is a temporary decrease followed by an increase, after which the rate begins to fall off. In 7.3 per cent ether there is an immediate increase amounting to 307 per cent which is followed by a decrease. The increase in the 3.65 and 7.3 per cent ether is accompanied by irreversible changes leading to death. The decrease found in 0.73 per cent ether is not sufficient to cause narcosis, as is shown by experiments on which the same decrease is produced by lowering the temperature. 5. These experiments show that narcosis is not due to asphyxia. The action of anesthetics is due to some other cause than the effect on respiration. There is a difference between the animals studied and the plants described in this series of articles, since in animals the increase in the CO₂ output is accompanied by irreversible changes leading to death, while this is not necessarily the case in plants. The reversible (narcotic) action of ether on the animals studied was accompanied by a decrease in the carbon dioxide output; in plants this is not ordinarily the case. These facts are of considerable interest, but their interpretation must be left to future investigation.     

SOME EFFECTS OF DERIVATIVES OF VITAMIN K ON THE METABOLISM OF UNICELLULAR ALGAE

Vitamin K₁, 2-methyl-3-phytyl-1,4-naphthoquinone, is a substance found in all plant chloroplasts. It is, therefore, interesting to know whether it has any influence upon the metabolism of plants. Experiments made with the phytol-free derivatives like 2-methyl-1,4-naphthoquinone or the corresponding 3-oxy compound, phthiocol, gave the following results. These substances accelerate the respiration of Chlorella or Scenedesmus in a way similar to the action of the dinitrophenols. They inhibit photosynthesis and the compensation of respiration in the light strongly like hydroxylamine. In Scenedesmus they hinder the adaptation to the anaerobic utilization of hydrogen. If given after adaptation in amounts sufficient to stop photosynthesis they do not prevent photoreduction but rather stabilize this reaction against reversion. Their presence destroys the coupling between the reduction of carbon dioxide in the dark and the oxyhydrogen reaction in adapted algae. One can expect, therefore, that the natural vitamin K present in plants in concentrations of about 10^–3 M takes part in some metabolic reaction as a catalyst or regulator.     

Metabolic flux distribution and thermodynamic analysis of green fluorescent protein production in recombinant Escherichia coli: The effect of carbon source and CO 2 partial pressure

Increasing recombinant protein production yields from bacterial cultures remains an important challenge in biotechnology. Acetate accumulation due to high dissolved carbon dioxide (pCO₂) concentrations in the medium has been identified as a factor that negatively affects such yields. Under appropriate culture conditions, acetate could be re-assimilated by bacterial cells to maintain heterologous proteins production. In this work, we developed a simplified metabolic network aiming to establish a reaction rate analysis for a recombinant Escherichia coli when producing green fluorescent protein (GFP) under controlled pCO₂ concentrations. Because E. coli is able to consume both glucose and acetate, the analysis was performed in two stages. Our results indicated that GFP synthesis is an independent process of cellular growth in some culture phases. Additionally, recombinant protein production is influenced by the available carbon source and the amount of pCO₂ in the culture medium. When growing on glucose, the increase in the pCO₂ concentration produced a down-regulation of central carbon metabolism by directing the carbon flux toward acetate accumulation; as a result, cellular growth and the overall GFP yield decreased. However, the maximum specific rate of GFP synthesis occurred with acetate as the main available carbon source, despite the low activity in the other metabolic pathways. To maintain cellular functions, including GFP synthesis, carbon flux was re-distributed toward the tricarboxylic acid cycle and the pentose phosphate pathway to produce ATP and NADH. The thermodynamic analysis allowed demonstrating the feasibility of the simplified network for describing the metabolic state of a recombinant system.     

COMPARATIVE STUDIES ON RESPIRATION : XXV. THE ACTION OF CHLOROFORM ON THE OXIDATION OF SOME ORGANIC ACIDS.

1. Organic acids when treated with H₂O₂ and Fe₂(SO₄)₃ produce CO₂ at a rate that can be measured by the indicator method. 2. In the case of acids containing a double bond, the rate of production of CO₂ can be varied by the addition of an anesthetic. The changes in the rate of production of CO₂ under the influence of a typical anesthetic, such as chloroform, show a striking resemblance to the reaction of the organism.     

THE CHLOROPHYLL-CARBON DIOXIDE RATIO DURING PHOTOSYNTHESIS

Using a rapid spectrographic method of carbon dioxide measurement previously described by McAlister (1937) further studies on the time course of photosynthesis in the higher plant, wheat, variety Marquis, are herein reported. Of major importance in this work is the discovery of a pick-up of carbon dioxide in darkness immediately following a high rate of photosynthesis (see Figs. 3 and 4). This pick-up is believed to be due to the action of a carbon dioxide-combining intermediate; i.e., the "acceptor molecule" for carbon dioxide in photosynthesis. The conditions under which this phenomenon has so far been observed indicate that the intermediate is formed in relatively large quantities during the actual process of photosynthesis and not before. That the intermediate is chlorophyllous in nature is suggested by a simple stoichiometry of the order of unity that is found to exist between the number of carbon dioxide molecules taken up and the total number of chlorophyll molecules present in the plant. This is in opposition to the idea of a large photosynthetic unit of some 2000 chlorophyll molecules operating together in the reduction of 1 carbon dioxide molecule. Further studies of the induction phase under various conditions of previous dark rest and of carbon dioxide and light limitation are herein described. Employing the simple hypothesis that the number of carbon dioxide molecules not reduced during the induction period (induction loss) gives a measure of the number of elementary photosynthetic cycles unoperative or compensated for during induction together with the experimental fact that this induction loss is of the order of the total number of chlorophyll molecules present, these latter studies also indicate, in a less direct manner, that chlorophyll participates in photosynthesis as an individual molecule and not as part of a very large multimolecular chlorophyll unit. The fast dark reaction lasting about 1 minute (Fig. 7) required to reproduce both (a) the phenomena of induction in carbon dioxide assimilation and (b) the recovery of fluorescence of chlorophyll in leaves in darkness as observed by Franck and Wood (1936), demonstrates a close relationship between the fluorescence of chlorophyll and induction in photosynthesis. The rate of respiration (carbon dioxide production) of the higher plant, wheat, was measured under intense illumination and in the absence of carbon dioxide (to suppress assimilation). This value was found to be identical with the dark respirational rate measured before and after the light period, indicating very positively the absence of any direct effect of light on respiration.