THE OXIDATION-REDUCTION POTENTIAL OF THE LUCIFERIN-OXYLUCIFERIN SYSTEM

The oxidation-reduction potential of the Cypridina luciferin-oxyluciferin system determined by a method of "bracketing" lies somewhere between that of anthraquinone 2-6-di Na sulfonate (E_o ^'' at pH of 7.7 = –.22) which reduces luciferin, and quinhydrone (E_o ^'' at pH of 7.7 = +.24), which oxidizes luciferin. Systems having an E_o ^'' value between –.22 and +.24 volt neither reduce oxyluciferin nor oxidize luciferin. If the luciferin-oxyluciferin system were truly reversible considerable reduction and oxidation should occur between –.22 and +.24. The system appears to be an irreversible one, with both "apparent oxidation" and "apparent reduction potentials" in Conant''s sense. Hydrosulfites, sulfides, CrCl₂, TiCl₃, and nascent hydrogen reduce oxyluciferin readily in absence of oxygen but without luminescence. Luminescence only appears in water solution if luciferin is oxidized by dissolved oxygen in presence of luciferase. Rapid oxidation of luciferin by oxygen without luciferase or oxidation by K₃Fe(CN)₆ in presence of luciferase but without oxygen never gives luminescence.     

THE ACTIVITY OF YEAST INVERTASE AS A FUNCTION OF OXIDATION-REDUCTION POTENTIAL

The activity of yeast invertase as a function of oxidation-reduction potential has been investigated using a large number of oxidants and reductants. The activity is constant over the range of E_h from –270 to +600 mv., but above E_h = +600 mv. there is a sharp decrease in activity reaching 0 at E_h = +1,000 mv. The inhibiting action of strong oxidants is upon the enzyme rather than on the substrate and appears to be essentially irreversible Experiments indicate that the inhibiting action of strong oxidants on invertase is primarily related to their high oxidation-reduction potential rather than to a specific toxic action unrelated to E_h. The effects of oxidation-reduction potential upon invertase activity are independent of the purity of the enzyme, since they are the same for commercial invertases, fresh bakers'' yeast, powdered bakers'' yeast, brewers'' yeast, and highly purified invertase. Possible mechanisms involved in the inactivation of invertase by oxidants are discussed.     

THE KINETICS OF PENETRATION : XVI. THE ACCUMULATION OF AMMONIA IN LIGHT AND DARKNESS

The accumulation of ammonia takes place more rapidly in light than in darkness. The accumulation appears to go on until a steady state is attained. The steady state concentration of ammonia in the sap is about twice as great in light as in darkness. Both effects are possibly due to the fact that the external pH (and hence the concentration of undissociated ammonia) outside is raised by photosynthesis. Certain "permeability constants" have been calculated. These indicate that the rate is proportional to the concentration gradient across the protoplasm of NH₄ X which is formed by the interaction of NH₃ or NH₄OH and HX, an acid elaborated in the protoplasm. The results are interpreted to mean that HX is produced only at the sap-protoplasm interface and that on the average its concentration there is about 7 times as great as at the sea water-protoplasm interface. This ratio of HX at the two surfaces also explains why the concentration of undissociated ammonia in the steady state is about 7 times as great in the sea water as in the sap. The permeability constant P'''''' appears to be greater in the dark. This is possibly associated with an increase in the concentration of HX at both interfaces, the ratio at the two surfaces, however, remaining about the same. The pH of sap has been determined by a new method which avoids the loss of gas (CO₂), an important source of error. The results indicate that the pH rises during accumulation but the extent of this rise is smaller than has hitherto been supposed. As in previous experiments, the entering ammonia displaced a practically equivalent amount of potassium from the sap and the sodium concentration remained fairly constant. It seems probable that the pH increase is due to the entrance of small amounts of NH₃ or NH₄OH in excess of the potassium lost as a base.     

THE EXTRACELLULAR RELEASE OF ECHINOCHROME

A study was made of the diffusion of the red pigment echinochrome from the eggs of the sea urchin, Arbacia punctulata, into sea water. Unfertilized eggs retained their pigment, over periods of hours. Outward diffusion of pigment from unfertilized eggs normally is entirely negligible, or does not occur at all. Enchancing the calcium or potassium content of the artificial sea water (while retaining isosmotic conditions) did not induce pigment release. Under anaerobic conditions, unfertilized eggs release pigment in small quantities. Fertilization alone brings about echinochrome release. Fertilized eggs invariably released pigment, whether in normal sea water, or sea water with increased calcium or potassium. This diffusion of the pigment began during the first cleavage, possibly soon after fertilization. The pigment release is not a consequence solely of the cell''s permeability to echinochrome (or chromoprotein, or other pigment combination) but is preceded by events leading to a release of echinochrome from the granules in which it is concentrated within the cell. These events may be initiated by activation or by anaerobiosis. The phenomenon was not due to cytolysis.     

THE KINETICS OF THE BACTERIUM-BACTERIOPHAGE REACTION

The above data relating to the reaction between 16 hour cultures of S. aureus and antistaphylococcus bacteriophage in nutrient broth of pH 7.6 at 36°C. and with mechanical shaking to maintain a uniform B suspension, bring out the following points: (a) B growth in P-B mixtures does not differ from growth in controls without P except in the case of a very high initial P/B ratio as noted below. There is no evidence that lytic destruction of B begins shortly after mixing P and B nor that B growth is stimulated by P, for the B growth curves in the presence of ordinary [P]''s and in controls are identical. Only at the sudden onset of the rapid lytic process does the B curve of a P-B mixture deviate from the control curve. (b) B growth is an essential conditioning factor for P formation. (c) Both B growth and P production exhibit short lags. During this time P diffuses into or becomes adsorbed to B so rapidly that by the end of the lag period only 10 to 30 per cent of the total P present is extracellular, the remainder being associated with the B. (d) During the logarithmic B growth phase, P formation is also logarithmic but proceeds at a much faster rate. That is, d P/d t is proportional to a power of d B/d t. Consequently the statement that each time a B divides a certain amount of P is formed is not correct. (e) As B growth enters the phase of positive acceleration equilibrium between the extracellular and intracellular P fractions becomes established and is maintained up to the onset of lysis, extracellular [P] representing a small constant percentage of total [P]. The distribution of P on a constant percentage basis suggests the manner in which a relatively simple chemical compound would be distributed and is not at all typical of the distribution one would expect if P were a complex organized parasite. (f) When the value of log P/B = 2.1 lysis begins. Obviously, this limiting value for any initial [B] is reached sooner the higher the initial [P]. When log P/B at the time of mixing P and B is already 2.1 or greater, there is no growth of B and lysis soon occurs. (g) While there is good evidence that lysis is brought about by the attainment of a particular [P] per B and not by a certain [P] per ml., it is not clear at this time which of the ratios intracellular P/B, extracellular P/B or total P/B is the major conditioning factor for B lysis. (h) Experimentally the maximal [P]''s of lysates made by mixing a constant initial [B] with widely varying Po''s fall within a relatively narrow range. This fact is explained by the large value of d log P/d t as compared to d log B/d t. That is, the loci of points at which log P = 2.1 + log B (maxima-lysis begins) on the curves of log P against t originating in various [Po]''s will lie at a nearly constant level above the abscissa. Because of this same relationship the maximal [P]''s of such a series will be in the reverse order of magnitude of the Po''s, i.e., the larger the Po the smaller will be the maximal [P] attained during the reaction (cf. Fig, 16). (i) The lytic destruction of B is logarithmic with time, in this respect being similar to most death rate processes. The value -d log B/d t for a particular initial [B] is constant for various initial values of [P]. There is good evidence that cells need not be growing in order to undergo lysis. (j) During B lysis a considerable percentage of the total maximal P formed is destroyed, the chief loss probably occurring in the intracellular fraction. The major portion (70 to 90 per cent) of the final P present after the completion of bacteriophagy is set free during the brief phase of bacterial dissolution. (k) When the entire process of bacteriophagy is completed the lysates are left with certain [P]''s determined by the foregone P-B reaction. The destruction of P during lysis is sufficiently regular to maintain the relationship established at the maximal [P]''s. Therefore the final [P]''s have the same points in common that were noted in "h" as applying to the maximal [P]''s. That is, they all are grouped within a narrow range of [P] values, those having been made with high Po''s being of lower titre than those made with low initial [P]''s. (1) There is a significant difference in the temperature coefficients of P and B formation. Further, the temperature coefficients of P and B destruction during lysis differ in almost the same ratio. Consequently, while all experimental evidence postulates B growth as an essential conditioning factor for P formation, the temperature coefficient data suggest that the two processes are basically separate reactions. A similar interpretation holds in the case of B dissolution and P inactivation. (m) The major events in the complete process of "bacteriophagy" are mathematically predictable. The [B] at which lysis occurs under certain standard conditions for given values of Bo and Po may be calculated from the equation: See PDF for Equation Substitution of this value for log B in the equation: See PDF for Equation gives satisfactory agreement with observed values for t _(lysis). (n) The kinetic analysis of the P-B reaction predicts that the values of log Po plotted against t _(lysis) for a constant Bo will give a straight line. This plot is employed in a method for the quantitative estimation of P described in an earlier paper on the basis of experimental observation alone. Its use is made more rational by the facts given above.     

THE EFFECT OF OXIDANTS AND REDUCTANTS UPON THE BIOELECTRIC POTENTIAL OF NITELLA

Nitella cells were exposed to various oxidants and reductants, to determine their effect upon the bioelectric potential. These included five systems, with an E_h range from +0.454 v. to –0.288 v., a total range of 0.742 v. When proper regard was given to buffering against acidity changes, and concentration changes of Na or K ions in the oxidized and reduced forms, no significant effect upon the bioelectric potential was found: 1. When an oxidant or reductant (K ferri- or ferrocyanide) was applied instead of an equivalent normality of an "indifferent" salt (KCl). 2. In changing from a given oxidant to its corresponding reductant (ferri- to ferrocyanide; oxidized to leuco-dye, etc.). 3. When a mixture of 2 dyes, (indophenol with positive E''₀, and safranin with negative E''₀) was oxidized and reduced, to give better poising at the extremes. It is conduded that the outer surface of this cell is not influenced by the state of oxidation or reduction of the systems employed; at least it does not respond with a manifest change of bioelectric potential to changes in oxidation-reduction intensity of the medium. The cells continued to show, however, at all times their usual response to concentration changes of KCl, NaCl, etc., and to electrical stimulation.     

FURTHER STUDIES ON THE INDUCTION OF THE DRUG-HYDROXYLATING ENZYME SYSTEM OF LIVER MICROSOMES

Further studies of the induction of the liver microsomal drug-hydroxylating enzyme system by pretreatment of rats with various drugs are presented. The phenobarbital-induced increase in the microsomal content of CO-binding pigment and in the activities of TPNH-cytochrome c reductase and the oxidative demethylation of aminopyrine is proportional, within certain limits, to the amount of phenobarbital injected. Removal of the inducer results in a parallel decrease in the levels of CO-binding pigment, TPNH-cytochrome c reductase, and aminopyrine demethylation. Other inducing drugs have been investigated and shown to act similarly to phenobarbital. The early increase in these enzymes is found in the microsomal subfraction consisting of rough-surfaced vesicles, whereas repeated administration of the inducing drug results in a concentration of the enzymes in the smooth-surfaced vesicles. The phenobarbital-stimulated formation of endoplasmic membranes is reflected in increased amounts of the various microsomal phospholipid fractions as revealed by thin layer chromatography. There is no significant difference between the stimulated rates of P_i³² incorporation into phospholipids of the two different microsomal subfractions in response to phenobarbital treatment. The drug-induced enzyme synthesis is unaffected by adrenalectomy.     

ON THE VARIABILITY OF CRITICAL ILLUMINATION FOR FLICKER FUSION AND INTENSITY DISCRIMINATION

From the data of experiments with bees in which threshold response is employed as a means of recognizing visual discrimination between stripes of equal width alternately illuminated by intensities I ₁ and I ₂, it is shown that the detectable increment of intensity ΔI, where ΔI = I ₂ - I ₁, is directly proportional to σ_I2 (I ₁ being fixed). From tests of visual acuity, where I ₁ = 0 and the width of the stripes is varied, σ_I2 = kI ₂ + const.; here I ₂ = ΔI, and ΔI/I ₂ = 1. When the visual excitability of the bee is changed by dark adaptation, λI ≡ kΔI (= k'' σ_ΔI) = k'''' I + const. For the measurements of critical illumination at threshold response to flicker, σ_I2 (= σ_ΔI) = k I ₂ = k'' ΔI + const. The data for critical illumination producing threshold response to flicker in the sun-fish Lepomis show for the rods σ_I2 = K I ₂ for the cones σ_I2 = K''(I ₂ + const.). The data thus indicate that in all these experiments essentially the same visual function is being examined, and that the recognition of the production of a difference in effect by alternately illuminated stripes takes place in such a way that d (ΔI)/d (σ_I2) = const., and that ΔI is directly proportional to I (or "I ₂," depending on the nature of the experiment). It is pointed out that the curve for each of the cases considered can be gotten equally well if mean I or σ_I is plotted as a function of the independent variable involved in the experiment. Certain consequences of these and related facts are important for the treatment of the general problem of intensity discrimination.     

THEORY AND MEASUREMENT OF VISUAL MECHANISMS : X. MODIFICATIONS OF THE FLICKER RESPONSE CONTOUR,AND THE SIGNIFICANCE OF THE AVIAN PECTEN

1. When there is projected on the retina (man, monocularly) the shadow of a grid which forms a visual field in several distinct pieces (not including the fovea in the present tests), the ordinary properties of the flicker recognition contour (F vs. log I) as a function of the light-time cycle fraction (t_L) can be markedly disturbed. In the present experiments flicker was produced by the rotation of a cylinder with opaque vertical stripes. In the absence of such a grid shadow the "cone" segments of the contours form a set in which F_max. and the abscissa of inflection are opposite but rectilinear functions of t_L, while the third parameter of the probability integral (σ''_{log I}) remains constant. This is the case also with diverse other animals tested. In the data with the grid, however, analysis shows that even for low values of t_L (up to 0.50) there occurs an enhancement of the production of elements of neural effect, so that F_max. rises rather than falls as ordinarily with increase of t_L, although σ''_{log I} stays constant and hence the total number of acting units is presumed not to change. This constitutes valid evidence for neural integration of effects due to the illumination of separated retinal patches. Beginning at t_L = 0.75, and at 0.90, the slope of the "cone" curve is sharply increased, and the maximum F is far above its position in the absence of the grid. The decrease of σ''_{log I} (the slope constant) signifies, in terms of other information, an increase in the number of acting cone units. The abscissa of inflection is also much lowered, relatively, whereas without the grid it increases as t_L is made larger. These effects correspond subjectively to the fact that at the end-point flicker is most pronounced, on the "cone" curve, along the edges of the grid shadow where contrast is particularly evident with the longer light-times. The "rod" portion of the F - log I contour is not specifically affected by the presence of the grid shadow. Its form is obtainable at t_L = 0.90 free from the influence of summating "cone" contributions, because then almost no overlapping occurs. Analysis shows that when overlapping does occur a certain number of rod units are inhibited by concurrent cone excitation, and that the mean contribution of elements of neural action from each of the non-inhibited units is also reduced to an extent depending on the degree of overlap. The isolated "rod" curve at t_L = 0.90 is quite accurately in the form of a probability integral. The data thus give a new experimental proof of the occurrence of two distinct but interlocking populations of visual effects, and experimentally justify the analytical procedures which have been used to separate them. 2. The changing form of the F - log I contour as a function of t_L, produced in man when the illuminated field is divided into parts by a shadow pattern, is normally found with the bird Taeniopygia castenotis (Gould), the zebra finch. The retina has elements of one general structural type (cones), and the F - log I contour is a simplex symmetrical probability integral. The eye of this bird has a large, complex, and darkly pigmented pecten, which casts a foliated shadow on the retina. The change in form of the F - log I curve occurs with t_L above 0,50, and at t_L = 0.90 is quite extreme. It is more pronounced than the one that is secured in the human data with the particular grid we have used, but there is no doubt that it could be mimicked completely by the use of other grids. The increase of flicker acuity due to the pecten shadow is considerable, when the dark spaces are brief relative to the light. The evidence thus confirms the suggestion (Menner) drawn from comparative natural history that the visual significance of the avian pecten might be to increase the sensory effect of small moving images. It is theoretically important that (as in the human experiment) this may be brought about by an actual decrease of effective retinal area illuminated. It is also significant theoretically that despite the presence of shadows of pecten or of grid, and of the sensory influences thus introduced, the probability integral formulation remains effective.     

THE INFLUENCE OF AMMONIUM SALTS ON CELL REACTION

1. It may be shown by means of cells of the flowers of a hybrid Rhododendron which contain a natural indicator, by means of starfish eggs stained with neutral red, and by means of an "artificial cell" in which living frog''s skin is employed that increased intracellular alkalinity may be brought about by solutions of a decidedly acid reaction which contain ammonium salts. 2. These results are analogous to those previously obtained with the CO₂-bicarbonate system, and depend on the facts: (a) that NH₄OH is sufficiently weak as a base to permit a certain degree of hydrolysis of its salts; and (b) that living cells are freely permeable to NH₄OH (or NH₃?) and not to mineral and many organic acids, and presumably not at least to the same extent to ammonium salts as such.     

THE FLICKER RESPONSE CONTOURS FOR GENETICALLY RELATED FISHES. II

The flicker response contour has been determined for several species and types of the teleosts Xiphophorus (X.) and Platypoecilius (P.) under the same conditions. The curve (F vs. log I_m) is the same for representatives of each generic type, but is different for the two genera. Its duplex nature is analyzable in each instance by application of the probability integral equation to the rod and cone constituent parts. The parameters of this function provide rational measures of invariant properties of the curves, which have specific values according to the genetic constitution of the animal. The F ₁ hybrids (H'''') of X. montezuma x P. variatus show dominance of the X. properties with respect to cone F_max. and σ'' _{log I}, but an intermediate value of the abscissa of inflection (τ''). The rod segment shows dominance of σ'' _{log I} from P., but an intermediate value of F_max. and of τ''. The composite flicker curve involves the operation of two distinct assemblages of excitable elements, differing quantitatively but not qualitatively in physicochemical organization, probably only secondarily related to the histological differentiation of rods and cones because almost certainly of central nervous locus, but following different rules in hereditary determination, and therefore necessarily different in physical organization. The interpretation of the diverse behavior of the three parameters of the probability summation is discussed, particularly in relation to the physical significance of these parameters as revealed by their quantitative relations to temperature, retinal area, and light time fraction in the flash cycle, and to their interrelations in producing the decline of rod effects at higher intensities. It is stressed that in general the properties of the parameters of a chosen interpretive analytical function must be shown experimentally to possess the physical properties implied by the equation selected before the equation can be regarded as describing those invariant properties of the organic system concerned upon which alone can deduction of the nature of the system proceed. The importance of genetic procedures in furthering demonstration that the biological performance considered in any particular case exhibits constitutionally invariant features provides a potentially powerful instrument in such rational analysis.     

THE PIGMENT-PROTEIN COMPOUND IN PHOTOSYNTHETIC BACTERIA : I. THE EXTRACTION AND PROPERTIES OF PHOTOSYNTHIN

1. Photosynthetic bacteria in water suspension break open when treated with supersonic vibration thus liberating the cell contents, including a water soluble protein to which is attached the otherwise water insoluble pigments, bacteriochlorophyll and carotinoids. Both types of pigments appear to be combined with the same protein. 2. The protein pigment compound is insoluble in the region of pH 3.0 to 4.5 and in neutral solution can be completely precipitated by 0.5 saturated (NH₄)₂SO₄. It is soluble in distilled water and adsorbable on fullers'' earth. 3. Supersonic extracts of photosynthetic bacteria do not have the ability to carry on photosynthesis, but will act as a photocatalyst for the oxidation of ascorbic acid with visible or infrared radiation. The rate of the photochemical oxidation is proportional to the light intensity.     

THE GROWTH OF DUCKWEEDS IN MINERAL NUTRIENT SOLUTIONS WITH AND WITHOUT ORGANIC EXTRACTS

1. Detmer''s solution and a modified Knop''s solution are unfavorable culture media for the growth of Spirodela polyrhiza. 2. When the modified Knop''s solution was diluted to 10 times its volume, Spirodela polyrhiza and Lemna valdiviana grew and reproduced for periods of 26 months and 21 months, respectively. 3. Growth in the dilute Knop''s solution, which alone can support the growth of Spirodela indefinitely, was considerably stimulated over a period of 23 days by adding to every liter the water-soluble material of 0.4 gm. autolyzed yeast, or the material of 2.5 gm. peat soluble in a 1 per cent solution of NaHCO₃. 4. The nature of the stimulus or of the protection afforded by the organic material is as yet unknown. 5. The necessity of organic accessory foods (auximones) in the nutrition of green plants cannot be accepted as an established fact.     

TEMPERATURE CHARACTERISTICS FOR THE METABOLISM OF CHLORELLA : II. THE RATE OF RESPIRATION OF CULTURES OF CHLORELLA PYRENOIDOSA AS A FUNCTION OF TIME AND OF TEMPERATURE

The respiration of the green alga Chlorella pyrenoidosa, suspended in Knop''s solution, has been studied in the dark as a function of time and of temperature. The rates of oxygen consumption and of carbon dioxide production (at constant temperature) decline for about 25 hours to a low, constant level. From an analysis of the curves it is suggested that two substances, A and B, are utilized, whose respiratory quotients are 1 and 0.65 respectively. The values of the temperature characteristics were found to be: for oxidation of A, 19,500 (0.6 to 11.5°C.) and 3,500 (11.5 to 28°C.); for oxidation of B, 5,600 (23.4 to 0.6°C.).     

A KINETIC ANALYSIS OF THE ENDOGENOUS RESPIRATION OF BAKERS' YEAST

The process of endogenous respiration of two strains of bakers'' yeast, Saccharomyces cerevisiae, was examined kinetically. The rate of respiration with respect to time in a non-nutrient medium was found to exhibit two phases: (a) a period of constant rate of O₂ consumption and CO₂ production (R.Q. = 1) characteristic of cells with ample concentrations of stored material; (b) a first order decline in rate of respiration with respect to time, where the rate was proportional to the concentration of some substrate, S. (R.Q. = 1 throughout second phase.) The nature of this substrate was reexamined and the evidence summarized confirms the notion that it is a carbohydrate, probably glycogen. These phases of endogenous respiration were shown to depend upon the age of the culture and the amount of substrate available.     

INFLUENCE OF LIGHT OF VERY LOW INTENSITY ON PHOTOTROPIC REACTIONS OF ANIMALS

1. The negative phototropism of certain land isopods was investigated over a large range of intensities, especially low ones. The responses were determined quantitatively by measuring the angle through which an animal turned away from a line perpendicular to the rays of light. 2. In the absence of light the undirected movements set up by obscure stimuli were such as to compensate each other statistically, the average path being a movement in the direction in which the animal was headed. 3. Over a large range of intensities (0.0026 m.c. up) the average turning is maximal, about 55° (Oniscus). This maximal response is due to an anatomical peculiarity, in that the carapace cuts off the light on the eye after the animal has turned 50–60°. This peculiarity probably accounts for specific differences among land isopods. Any light, therefore, which is strong enough to turn an animal through this maximal angle in a radial distance of 10 cm. will give results whose mean will be maximal. 4. Below 0.0026 m.c. the amount of angular deflection becomes less and less, in proportion to the logarithm of the intensity, until at 0.00003 m.c. the movements are the same as in darkness. 5. This proportionality between amount of turning and the logarithm of the intensity indicates the photochemical nature of phototropism on the basis of Hecht''s work with Mya. As a result, Loeb''s theory of phototropism may then be stated in the mathematical form See PDF for Equation in which I ₁ and I ₂ are the two intensities, E ₁ and E ₂, their respective effects, and R, the muscular action set up by the difference in photochemical effect on the two sides.     

RELATION OF OXYGEN TENSION AND TEMPERATURE TO THE TIME OF REDUCTION OF CYTOCHROME

The time for the appearance of the cytochrome C absorption band after shaking a suspension of bakers'' yeast with various O₂-N₂ mixtures was determined at each of six temperatures. At each temperature a linear relation between this interval—called the reduction time—and O₂ tension was found. It was shown: 1. That under our experimental conditions, absorption bands of cytochrome were seen when the O₂ tension of the suspension was reduced to, or below, a certain pressure which was found to be specific for each temperature (this pressure is provisionally considered to be identical with or very near to the "critical O₂ tension" usually found in Q _OO2-O₂-tension relationships); 2. That the x-axis intercept obtained from the reduction time - O₂-tension plot gives the value of the "critical" O₂ pressure at each temperature; 3. That the O₂ tension within the suspension is reduced by the respiratory activity of the yeast cells. An equation describing these observations is given and is used in calculating rates of O₂ consumption from measurements of reduction time of cytochrome. The average difference between the calculated values and the manometric measurements of Q _OO2 was found to be 6.6 per cent. A rapid optical method of measuring rates of O₂ consumption based on the findings of these experiments is proposed for use with cytochrome-containing microorganisms.     

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.     

THE ACCUMULATION OF ELECTROLYTES : II. SUGGESTIONS AS TO THE NATURE OF ACCUMULATION IN VALONIA

It is suggested that K enters chiefly as KOH, whose thermodynamic potential (proportional to the ionic activity product (a _K) (a _OH)) is greater outside than within. As this difference is maintained by the production of acid in the cell K continues to enter, and reaches a greater concentration inside than outside. KOH combines with a weak organic acid which is exchanged for HCl entering from the sea water (or its anion is exchanged for Cl^-), so that KCl accumulates in the sap. Na enters more slowly and its internal concentration remains below that of K. The facts indicate that penetration is chiefly in molecular form. As the system is not in equilibrium the suggestion is not susceptible of thermodynamic proof but it is useful in predicting the behavior of K, Na, and NH₄.     

CONTRIBUTION TO THE THEORY OF PERMEABILITY OF MEMBRANES FOR ELECTROLYTES

On page 39, Vol. viii, No. 2, September 18, 1925, multiply the right-hand side of formula (2) by the factor See PDF for Equation. On page 44, immediately after formula (1) the text should be continued as follows: Let us suppose a membrane to be separated by two solutions of KCl of different concentrations K₁ and K₂ and these concentrations and the corresponding concentrations of K⁺ within the membrane, which are in equilibrium with the outside solutions, to be so high that the H⁺ ions may be neglected. When a small electric current flows across the system, practically the K⁺ ions alone are transferred and that in a reversible manner. Therefore the total P.D. is practically See PDF for Equation This P.D. is composed of two P.D.''s at the boundaries and the diffusion potential within the membrane. Suppose the immobility of the anions is not absolute but only relative as compared with the mobility of the cations, KCl would gradually penetrate into the membrane to equal concentration with the outside solution on either side and no boundary potential would be established. In this case the diffusion P.D. within the membrane is the only P.D., amounting to See PDF for Equation but, V being practically = 0, it would result that See PDF for Equation So the definitive result is the same as in the former case. Now cancel the printed text as far as page 48, line 13 from the top of the page, but retain Fig. 1. On page 50, line 19 from the top of the page, cancel the sentence beginning with the word But and ending with the words of the chain.