THE STRUCTURE OF THE COLLODION MEMBRANE AND ITS ELECTRICAL BEHAVIOR : XII. THE PREPARATION AND PROPERTIES OF "MEGAPERMSELECTIVE" PROTAMINE COLLODION MEMBRANES COMBINING HIGH IONIC SELECTIVITY WITH HIGH PERMEABILITY

The technique of Abrams and Sollner for the preparation of electropositive dried protamine collodion membranes has been improved. Porous collodion membranes cast on the outside of rotating tubes are treated for 48 hours with a solution of 2 per cent protamine sulfate buffered at pH 11. After being washed thoroughly the membranes are dried in air for several hours, soaked in water for several hours, and removed from the tubes. Further drying in air but without support shrinks the membranes slightly. The resulting membranes are designated "permselective" or "megapermselective" protamine collodion membranes. These membranes regularly give characteristic concentration potentials of –52 to –53 mv. and (in 0.1 M KCl) resistance of 0.5 to 15 ohms per membrane of 50 cm.² area. This resistance is several orders of magnitude smaller than that of the conventional dyestuff- and alkaloid-impregnated positive membranes. The megapermselective protamine collodion membranes can be kept either dry or in water for prolonged periods without detectable deterioration. They are quite smooth, have a regular shape, and stand considerable handling without breakage. The megapermselective protamine collodion membranes are the electropositive analogues of the electronegative megapermselective collodion membranes described by Carr and Sollner.     

Water transport in invertebrate peripheral nerve fibers

Osmotic and diffusion permeabilities (P_f and P_d) of invertebrate nerve fibers to tritiated water were measured to determine what water flux studies could reveal about "the nerve membrane" and to directly test the possibility of active transport of water into or out of invertebrate nerve fibers. P_f/P_d ratios for lobster walking leg nerve fibers were found to be about 20 ± 7 at 14°C. P_d measurements were made for squid giant axons at 25°C. and found to yield a value of 4 x 10^–4 cm.^–1 sec.^–1. When combined with the data of D. K. Hill for P_f, a P_f/P_d ratio of 21 ± 5 is obtained. These P_f/P_d ratios correspond to "effective pore radii" of about 16 ± 4 angstrom units, according to theories developed by Koefoed-Johnsen and Ussing and independently by Pappenheimer and his colleagues. Variations of water flux ratios with temperatures were studied and apparent activation energies calculated for both diffusion experiments and osmotic filtration experiments using the Arrhenius equation, and found to be close to 3 to 5 cal. per mole of water transferred. Cyanide (5 x 10^–3 molar) and iodoacetate (1 x 10^–3 molar) poisoned lobster leg nerve fibers showed no appreciable change in diffusion or osmotic filtration water effluxes. Caution in interpreting these proposed channels as simple pores was emphasized, but the possibility that such channels exist and are related to ionic flow is not incompatible with electrophysiological data.     

THE BEHAVIOR OF THE NUCLEIC ACIDS DURING THE EARLY DEVELOPMENT OF THE SEA URCHIN EGG (ARBACIA)

The authors wish to correct an error in the paper "The behavior of the nucleic acids during the early development of the sea urchin egg (Arbacia)" (J. Gen. Physiol., 1947–48, 31, 203). Owing to an oversight, the figures for the amounts of various P fractions in a single Arbacia egg have been erroneously expressed in γ x 10^–3 units (Tables I and II, page 205; the last two lines of page 206). The figures should have been expressed in γ x 10^–5 units. Thus, the fertilized Arbacia egg contains an average of 20 γ x 10^–5 ribonucleic acid P and 0.7 to 1 γ x 10^–5 desoxyribonucleic acid P.     

THE STRUCTURE OF THE COLLODION MEMBRANE AND ITS ELECTRICAL BEHAVIOR : V. THE INFLUENCE OF THE THICKNESS OF DRIED COLLODION MEMBRANES UPON THEIR ELECTROMOTIVE BEHAVIOR

1. Experiments were carried out to decide whether or not the electromotive properties of dried collodion membranes depend upon their thickness. 2. A number of dried collodion membranes of varying thickness, 3–160 µ, were prepared from collodion preparations of different electrochemical activity. The characteristic concentration potentials across them were measured and the means of these values determined for each thickness. 3. The characteristic concentration potentials across dried collodion membranes are a function of their thickness. The thinnest membranes yield in all cases the lowest concentration potentials; increasingly thicker membranes give increasingly higher potential values, until a constant value is reached which is characteristic of the particular collodion preparation used. With electrochemically active collodion, characteristic concentration potentials approaching the thermodynamically possible maximum are obtained with membranes of only 10 µ thickness, thinner membranes giving appreciably lower values. With two rather inactive commercial collodion preparations the characteristic concentration potential increases from about 30 mv. for membranes 3 µ thick to about 42 mv. for 20 µ membranes; still thicker membranes do not show a significant increase in the potential values. With a highly purified collodion preparation the constant maximum value was found to be about 32 mv., 4 µ thick membranes giving only about 22 mv. 4. These results do not support the homogeneous phase theory as applied to the dried collodion membrane. They are readily compatible with the micellar-structural theory. Several special possible cases of the latter as applied to the dried collodion membrane are discussed.     

Osmosis in Cortical Collecting Tubules : ADH-Independent Osmotic Flow Rectification

The present experiments were designed to evaluate the effects of varying the osmolality of luminal solutions on the antidiuretic hormone (ADH)-independent water and solute permeability properties of isolated rabbit cortical collecting tubules. In the absence of ADH, the osmotic water permeability coefficient (cm s^–1) P_f^l→b, computed from volume flows from hypotonic lumen to isotonic bath, was 20 ± 4 x 10^–4 (SEM); the value of P_f^b→l in the absence of ADH, computed from volume flows from isotonic bath to hypertonic lumen, was 88 ± 15 x 10^–4 cm s^–1. We also measured apparent urea permeability coefficients (cm s^–1) from ¹⁴C-urea fluxes from lumen to bath (P_DDurea^l→b) and from bath to lumen (P_DDurea^b→l). For hypotonic luminal solutions and isotonic bathing solutions, P_DDurea^l→b was 0.045 ± 0.004 x 10^–4 and was unaffected by ADH. The ADH-independent values of P_DDurea^l→b and P_urea^b→l were, respectively, 0.216 ± 0.022 x 10^–4 cm s^–1 and 0.033 ± 0.002 x 10^–4 cm s^–1 for isotonic bathing solutions and luminal solutions made hypertonic with urea, i.e., there was an absolute increase in urea permeability and asymmetry of urea fluxes. Significantly, P_DDurea^l→b did not rise when luminal hypertonicity was produced by sucrose; and, bathing fluid hypertonicity did not alter tubular permeability to water or to urea. We interpret these data to indicate that luminal hypertonicity increased the leakiness of tight junctions to water and urea but not sucrose. Since the value of P_f^b→l in the absence of ADH, when tight junctions were open to urea, was approximately half of the value of P_f^l→b in the presence of ADH, when tight junctions were closed to urea, we conclude that tight junctions are negligible paracellular shunts for lumen to bath osmosis with ADH. These findings, together with those in the preceding paper, are discussed in terms of a solubility-diffusion model for water permeation in which ADH increases water solubility in luminal plasma membranes.     

THE COMBINATION OF GELATIN WITH HYDROCHLORIC ACID : II. NEW DETERMINATIONS OF THE ISOELECTRIC POINT AND COMBINING CAPACITY OF A PURIFIED GELATIN.

1. Cooper''s gelatin purified according to Northrop and Kunitz exhibited a minimum of osmotic pressure and a maximum of opacity at pH 5.05 ±0.05. The pH of solutions of this gelatin in water was also close to this value. It is inferred that such gelatin is isoelectric at this pH and not at pH 4.70. 2. Hydrogen electrode measurements with KCl-agar junctions were made with concentrated solutions of this gelatin in HCl up to 0.1 M. The combination curve calculated from these data is quite exactly horizontal between pH 2 and 1, indicating that 1 gm. of this gelatin can combine with a maximum of 9.35 x 10^–4 equivalents of H⁺. 3. Conductivity titrations of this gelatin with HCl gave an endpoint at 9.41 (±0.05) x 10^–4 equivalents of HCl per gram gelatin. 4. E.M.F. measurements of the cell without liquid junction, Ag, AgCl, HCl + gelatin, H₂, lead to the conclusion that this gelatin in 0.1 M HCl combines with a maximum of 9.4 x 10^–4 equivalents of H⁺ and 1.7 x 10^–4 equivalents of Cl^- per gram gelatin.     

THE COMBINATION OF CERTAIN PROTEINS WITH HYDROCHLORIC ACID

Electromotive force measurements of cells without liquid junction, of the type Ag, AgCl, HCl + protein, H₂, have been made at 30°C. with the proteins gelatin, edestin, and casein in 0.1 M hydrochloric acid. The data are consistent with the assumptions of a constant combining capacity of each protein for hydrogen ion, no combination with chloride ion, and Failey''s principle of a linear variation of the logarithm of the mean activity coefficient of the acid with increasing protein concentration. The combining capacities for hydrogen ion so obtained are 13.4 x 10^–4 for edestin, 9.6 x 10^–4 for gelatin, and 8.0 x 10^–4 for casein, in equivalents of combined H⁺ per gm. of protein.     

STUDIES ON PERMEABILITY OF MEMBRANES : VI. MENSURATION OF THE DRIED COLLODION MEMBRANE (CALCULATION OF DIMENSIONS AND OF RELATIONS TO CERTAIN BIOLOGICAL MEMBRANES).

The flat type of dried collodion membrane used by Michaelis and his associates in numerous investigations has been subjected to mensuration in order that the dimensions of these membranes may be placed on record. The membranes had a functioning area of about 30 cm., were approximately 0.1 mm. in thickness and were composed on the average of 87 per cent by volume of collodion and 13 per cent by volume of pores. In reviewing some of the previously reported results of diffusion experiments with non-electrolytes in the light of the calculated values for the total pore area for the same membranes additional evidence was presented to show that a smaller molecule (acetone) probably utilizes a much larger percentage of the total pore area for its diffusion than is available for a larger molecule (glycerol). By using the figures of Fricke and McClendon for the thickness of the membrane of the red blood cell some comparisons were drawn between the dried collodion membrane as a model for certain biological membranes and the red blood cell membrane. In these comparisons emphasis was placed on the exaggerated importance of small electromotive forces and very slight permeabilities when these were associated with membranes of such extreme thinness as the red blood cell membrane.     

FLUIDITY OF THE SURFACE OF CULTURED MUSCLE FIBERS : Rapid Lateral Diffusion of Marked Surface Antigens

Fluorescent antibody fragments of anti-muscle plasma membrane antibody bound as small fluorescent spots when applied by micropipetting to cultured myotubes. The spots were observed to enlarge with time. The rate of enlargement of fluorescent spots was greater when fragments were applied than when divalent antibody was used. It was also greater at 23°–25°C than at 0°–4°C. With glutaraldehyde-fixed cells no increase in the size of the spots was seen. The observations are consistent with the spread of fluorescent spots due to diffusion of surface protein antigens within the plane of a fluid membrane. From measurements of spot size against time, a diffusion constant of 1–3 x 10^-9 cm² s^-1 can be calculated for muscle plasma membrane proteins of mol wt approximately 200,000. This value is consistent with other observations on the diffusion of surface antigens and of labeled lipid molecules in synthetic and natural membranes.     

AMPHOTERIC BEHAVIOR OF COMPLEX SYSTEMS : IV. NOTE ON THE ISOELECTRIC POINT AND IONIZATION CONSTANTS OF SULFANILIC ACID.

From the solubility minimum the value of the basic ionization constant of sulfanilic acid is shown to lie probably between the values 1.7 x 10^–15 and 3.2 x 10^–15. From solubility measurements the value of this same constant is shown to lie probably between 2.0 and 2.2 x 10^–15, and the isoelectric point of sulfanilic acid is thus at a cH of 0.056 or a pH of 1.25. From conductivity ratios the acid ionization constant of sulfanilic acid is shown to be 7.05 x 10^–4 at room temperature (21°C.). Calculations are made, from data published in preceding papers, of the ionization constants of glycine, K_a being 2.3 x 10^–10, and K_b being 2.2 x 10^–12.     

CONCENTRATION AND PURIFICATION OF BACTERIOPHAGE

1. A method for isolating a nucleoprotein from lysed staphylococci culture is described. 2. It is homogeneous in the ultracentrifuge and has a sedimentation constant of 650 x 10^–13 cm. dyne^–1 sec.^–1, corresponding to a molecular weight of about 300,000,000. 3. The diffusion coefficient varies from about 0.001 cm.²/day in solutions containing more than 0.1 mg. protein/ml. to 0.02 in solutions containing less than 0.001 mg. protein/ml. The rate of sedimentation also decreases as the concentration decreases. It is suggested, therefore, that this protein exists in various sized molecules of from 500,000–300,000,000 molecular weight, the proportion of small molecules increasing as the concentration decreases. 4. This protein is very unstable and is denatured by acidity greater than pH 5.0, by temperature over 50°C. for 5 minutes. It is digested by chymo-trypsin but not by trypsin. 5. The loss in activity by heat, acid, and chymo-trypsin digestion is roughly proportional to the amount of denatured protein formed under these conditions. 6. The rate of diffusion of the protein is the same as that of the active agent. 7. The rate of sedimentation of the protein is the same as that of the active agent. 8. The loss in activity when susceptible living or dead bacteria are added to a solution of the protein is proportional to the loss in protein from the solution. Non-susceptible bacteria remove neither protein nor activity. 9. The relative ultraviolet light absorption, as determined directly, agrees with that calculated from Gates'' inactivation experiments in the range of 2500–3000 Å. u. but is somewhat greater in the range of 2000–2500 Å. u. 10. Solubility determinations showed that most of the preparations contained at least two proteins, one being probably the denatured form of the other. Two preparations were obtained, however, which had about twice the specific activity of the earlier ones and which gave a solubility curve approximating that of a pure substance. 11. It is suggested that the formation of phage may be more simply explained by analogy with the autocatalytic formation of pepsin and trypsin than by analogy with the far more complicated system of living organisms.     

THE DIFFUSION COEFFICIENT OF CRYSTALLINE TRYPSIN

The diffusion coefficient of crystalline trypsin in 0.5 saturated magnesium sulfate at 5°C. is 0.020 ±0.001 cm.² per day, corresponding to a molecular radius of 2.6 x 10^–7 cm. The rate of diffusion of the proteolytic activity is the same as that of the protein nitrogen, indicating that these two properties are held together in chemical combination and not in the form of an adsorption complex.     

THE EFFECT OF DETERGENTS ON THE CHLOROPHYLL-PROTEIN COMPOUND OF SPINACH AS STUDIED IN THE ULTRACENTRIFUGE

1. The chlorophyll-protein compound of the spinach leaf has been studied in the air-driven ultracentrifuge using the Svedberg light-absorption method, and a direct-reading refractive index method. 2. When the untreated extracts are centrifuged at low speeds, the green protein sediments with a purely random spread of particle sizes confirming the fact that the protein is not in true solution. 3. In the presence of digitonin, bile salts, and sodium desoxycholate, the extracts are clarified. These detergents split the chlorophyll from the protein and the protein itself shows a sedimentation constant of 13.5 x 10^–13 equivalent to a molecular weight of at least 265,000 as calculated from Stokes'' law. This probably represents the minimum size of the protein in native form. 4. Sodium dodecyl sulfate, a detergent which also clarifies the leaf extracts, shows a different behavior. The prosthetic group remains attached to the protein but the protein is split into smaller units. In 0.25 per cent SDS, S ₂₀ is 2.6 x 10^–13 over a pH range of 5 to 9, although at the acid pH chlorophyll is converted to phaeophytin. In 2.5 per cent SDS, S ₂₀ is 1.7 x 10^–13 suggesting a further splitting of the protein. 5. No differences in behavior were found for the various chloroplast pigments.     

A Proline-Hinge Alters the Characteristics of the Amphipathic α-helical AMPs

HP (2–20) is a 19-aa, amphipathic, α-helical peptide with antimicrobial properties that was derived from the N-terminus of Helicobacter pylori ribosomal protein L1. We previously showed that increasing the net hydrophobicity of HP (2–20) by substituting Trp for Gln¹⁷ and Asp¹⁹ (Anal 3) increased the peptide''s antimicrobial activity. In hydrophobic medium, Anal 3 forms an amphipathic structure consisting of an N-terminal random coil region (residues 2–5) and an extended helical region (residues 6–20). To investigate the structure-activity relationship of Anal 3, we substituted Pro for Glu⁹ (Anal 3-Pro) and then examined the new peptide''s three-dimensional structure, antimicrobial activity and mechanism of action. Anal 3-Pro had an α-helical structure in the presence of trifluoroethanol (TFE) and sodium dodecyl sulfate (SDS). NMR spectroscopic analysis of Anal 3-Pro''s tertiary structure in SDS micelles confirmed that the kink potential introduced by Pro¹⁰ was responsible for the helix distortion. We also found that Anal 3-Pro exhibited about 4 times greater antimicrobial activity than Anal 3. Fluorescence activated flow cytometry and confocal fluorescence microscopy showed that incorporating a Pro-hinge into Anal 3 markedly reduced its membrane permeability so that it accumulated in the cytoplasm without remaining in the cell membrane. To investigate the translocation mechanism, we assessed its ability to release of FITC-dextran. The result showed Anal 3-Pro created a pore <1.8 nm in diameter, which is similar to buforin II. Notably, scanning electron microscopic observation of Candida albicans revealed that Anal 3-Pro and buforin II exert similar effects on cell membranes, whereas magainin 2 exerts a different, more damaging, effect. In addition, Anal 3-Pro assumed a helix-hinge-helix structure in the presence of biological membranes and formed micropores in both bacterial and fungal membranes, through which it entered the cytoplasm and tightly bound to DNA. These results indicate that the bending region of Anal 3- Pro peptide is prerequisite for effective antibiotic activity and may facilitate easy penetration of the lipid bilayers of the cell membrane.     

STUDIES ON THE PURIFICATION OF BACTERIOPHAGE

A simple method of concentrating and purifying bacteriophage has been described. The procedure consisted essentially in collecting the active agent on a reinforced collodion membrane of a porosity that would just retain all the active agent and permit extraneous material to pass through. Advantage was taken of the fact that B. coli will proliferate and regenerate bacteriophage in a completely diffusible synthetic medium with ammonia as the only source of nitrogen, which permitted the purification of the bacteriophage by copious washing. The material thus obtained was concentrated by suction and after thorough washing possessed all the activity of the original filtrate. It was labile, losing its activity in a few days on standing, and was quickly and completely inactivated upon drying. This material contained approximately 15 per cent of nitrogen and with 2 or 3 mg. samples of inactive dry residue it was possible to obtain positive protein color tests. The concentrated and purified bacteriophage has about 10^–14 mg. of nitrogen, or 6 x 10^–17 gm. of protein per unit of lytic activity. Assuming that each unit of activity represents a molecule, the calculated maximum average molecular weight would be approximately 36,000,000, and on the assumption of a spherical shape of particles and a density of 1.3, the calculated radius would be about 22 millimicra. By measurement of the diffusion rate, the average radius of particle of the fraction of the purified bacteriophage which diffuses most readily through a porous plate was found to be of the order of magnitude of 9 millimicra, or of a calculated molecular weight of 2,250,000. Furthermore, when this purified bacteriophage was fractionated by forcing it through a thin collodion membrane, which permits the passage of only the smaller particles, it was possible to demonstrate in the ultrafiltrate active particles of about 2 millimicra in radius, and of a calculated molecular weight of 25,000. It was of interest to apply this method of purification to a staphylococcus bacteriophage. Since this organism does not readily grow in synthetic medium, a diffusate of yeast extract medium was employed. The better of two preparations contained about 10^–12 mg. of nitrogen per unit of lytic activity. Although this is about one hundred times the amount of nitrogen found in an active unit of B. coli bacteriophage, nevertheless, the diffusion rate experiments gave results which paralleled those obtained with the coliphage. The diffusible particles of the crude staphylococcus bacteriophage had a radius of about 7 millimicra, and a calculated molecular weight of about 1,000,000, while the particles of the same phage which appeared in the ultrafiltrate through a thin collodion membrane had a radius of about 2.4 millimicra and a calculated molecular weight of about 45,000. It appears, therefore, that the active principle is distributed as particles of widely different sizes. However, since the smaller particles have all the properties of bacteriophage, the larger particles probably do not represent free molecules, but either are aggregates, or more likely, inactive colloids to which the active agent is adsorbed. The protein isolated, which bears the phage activity, is capable of stimulating the production of antilytic antibodies on parenteral injection into rabbits or guinea pigs. It retains its specific antigenicity when inactivated by formalin, but not when inactivated by drying.     

ELECTRIFICATION OF WATER AND OSMOTIC PRESSURE

1. Amphoteric electrolytes form salts with both acids and alkalies. It is shown for two amphoteric electrolytes, Al(OH)₃ and gelatin, that in the presence of an acid salt water diffuses through a collodion membrane into a solution of these substances as if its particles were negatively charged, while water diffuses into solutions of these electrolytes, when they exist as monovalent or bivalent metal salts, as if the particles of water were positively charged. The turning point for the sign of the electrification of water seems to be near or to coincide with the isoelectric point of these two ampholytes which is a hydrogen ion concentration of about 2 x 10^–5 N for gelatin and about 10^–7 for Al(OH)₃. 2. In conformity with the rules given in a preceding paper the apparently positively charged water diffuses with less rapidity through a collodion membrane into a solution of Ca and Ba gelatinate than into a solution of Li, Na, K, or NH₄ gelatinate of the same concentration of gelatin and of hydrogen ions. Apparently negatively charged water diffuses also with less rapidity through a collodion membrane into a solution of gelatin sulfate than into a solution of gelatin chloride or nitrate of the same concentration of gelatin and of hydrogen ions. 3. If we define osmotic pressure as that additional pressure upon the solution required to cause as many molecules of water to diffuse from solution to the pure water as diffuse simultaneously in the opposite direction through the membrane, it follows that the osmotic pressure cannot depend only on the concentration of the solute but must depend also on the electrostatic effects of the ions present and that the influence of ions on the osmotic pressure must be the same as that on the initial velocity of diffusion. This assumption was put to a test in experiments with gelatin salts for which a collodion membrane is strictly semipermeable and the tests confirmed the expectation.     

PERMEABILITY OF ERYTHROCYTES TO RADIOACTIVE POTASSIUM

The diffusion coefficients for the exchange of potassium across the membrane of erythrocytes of humans, rats, and rabbits have been determined by the use of artificially radioactive potassium, both into and out of the erythrocytes both in vitro and in vivo. The diffusion coefficients found in minutes^–1 were 0.2 to 0.25 x 10^–3 for human, 0.32 to 0.665 x 10^–3 for rabbits, and 1.0 x 10^–3 for rat erythrocytes. Rabbit erythrocytes appear to be more permeable in vivo. Reasons are advanced to explain the failure of earlier workers to demonstrate appreciable exchange of potassium in erythrocytes.     

CONTRIBUTION TO THE THEORY OF PERMEABILITY OF MEMBRANES FOR ELECTROLYTES

From experiments on such membranes as apple skin, parchment paper membrane, and a membrane of completely dry collodion, results have been obtained which could be interpreted by the assumption that these membranes are less permeable for anions than for cations. In parchment paper there is only a relative diminution of the mobility of the anions, in the apple skin and in the dry collodion membrane there is practically no permeability for anions at all. The theory is developed which explains how the decrease or complete lack of mobility of anions influences the electromotive effects of the membrane and the diffusibility of electrolytes across a membrane. The results of the theory are compared with the experimental results. In membranes impermeable for anions the permeability for cations gives the same order of cations as for the mobilities in a free aqueous solution. But the differences of the mobilities are enormously magnified, e.g. the mobilities of H^• and Li^•, which are in the proportion of about 1:10 in aqueous solution, are in proportion of about 1:900 in the collodion membrane. The general cause for the retardation of ionic mobility within the membrane may be supposed to be the increased friction of the water envelope dragged along by the ion in the capillary canals of the membrane. The difference of the effect on the cations and on the anions may be attributed to the electric charge of the walls of the canals.     

ELECTRON BOMBARDMENT OF BIOLOGICAL MATERIALS : II. THE RATE OF DEATH OF FUNGUS SPORES BOMBARDED IN VACUUM WITH CATHODE RAY BEAMS FROM 4 KV.–15 KV.

A study has been undertaken of the rate of inactivation of spores of the ascomycete fungus Aspergillus niger when bombarded in vacuum, with homogeneous beams of cathode rays of energies from 4 to 15 electron kv. and current densities of 1 x 10^–7 to 3 x 10^–6 amperes per square cm. These velocities and densities are in the range of those of showers of secondary electrons produced in biological materials irradiated with moderately soft x-rays, and so may be made to serve as quantitative indicators of the mechanics of x-ray action. Four qualitative effects are described.     

THE RATE OF ESCAPE OF HEMOGLOBIN FROM THE HEMOLYZED RED CORPUSCLE

A theoretical treatment is given of the rate of escape of hemoglobin from the hemolyzed red corpuscle. For complete permeability of the surface, as may perhaps be produced by strong lysins, the time taken for the hemoglobin to decrease to 10 per cent of its original concentration is calculated to be 0.16 seconds (for the human cell). For dilute saponin, giving complete lysis of human cells in 3 minutes, Ponder found a time of escape of 4 seconds, from which the permeability of the membrane to the pigment is calculated to be µ_H = 5 x 10^–5 cm./sec.