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.     

PURIFIED DIPHTHERIA ANTITOXIN IN THE ULTRACENTRIFUGE AND IN THE ELECTROPHORESIS APPARATUS

Ultracentrifugation studies of diphtheria antitoxin showed that: 1. Purified antitoxin of high activity obtained from horse plasma without enzymatic treatment has exactly the same sedimentation constant as the globulin fraction obtained in a similar way from normal horse plasma s ₂₀ ^water = 6.9 x 10^–13. 2. Purified antitoxin obtained with trypsin digestion of the toxin-antitoxin complex has a sedimentation constant of s ₂₀ ^water = 5.5 ± 0.1 x 10^–13, a diffusion constant of D ₂₀ ^water = 5.7₆ x 10^–7, and a molecular weight of about 90,000. Electrophoresis experiments demonstrated that: 1. The trypsin-purified antitoxin has an isoelectric point not far from pH 7.0. 2. The reversible spreading noticed at about pH 7.3 cannot be attributed to heterogeneous preparation. 3. The large increase in the γ-globulin fraction occurring during immunization consists either of antitoxin of various degrees of activity or of some inert protein in addition to the antitoxin.     

STUDIES IN THE PHYSICAL CHEMISTRY OF THE PROTEINS : II. THE RELATION BETWEEN THE SOLUBILITY OF CASEIN AND ITS CAPACITY TO COMBINE WITH BASE. THE SOLUBILITY OF CASEIN IN SYSTEMS CONTAINING THE PROTEIN AND SODIUM HYDROXIDE.

1. The solubility in water of purified, uncombined casein has previously been reported to be 0.11 gm. in 1 liter at 25°C. This solubility represents the sum of the concentrations of the casein molecule and of the soluble ions into which it dissociates. 2. The solubility of casein has now been studied in systems containing the protein and varying amounts of sodium hydroxide. It was found that casein forms a well defined soluble disodium compound, and that solubility was completely determined by (a) the solubility of the casein molecule, and (b) the concentration of the disodium casein compound. 3. In our experiments each mol of sodium hydroxide combined with approximately 2,100 gm. of casein. 4. The equivalent combining weight of casein for this base is just half the minimal molecular weight as calculated from the sulfur and phosphorus content, and one-sixth the minimal molecular weight calculated from the tryptophane content of casein. 5. From the study of systems containing the protein and very small amounts of sodium hydroxide it was possible to determine the solubility of the casein molecule, and also the degree to which it dissociated as a divalent acid and combined with base. 6. Solubility in such systems increased in direct proportion to the amount of sodium hydroxide they contained. 7. The concentration of the soluble casein compound varied inversely as the square of the hydrogen ion concentration, directly as the solubility of the casein molecule, Su, and as the constants Ka₁ and Ka₂ defining its acid dissociation. 8. The product of the solubility of the casein molecule and its acid dissociation constants yields the solubility product constant, Su·Ka₁·Ka₂ = 2.2 x 10^–12 gm. casein per liter at 25°C. 9. The solubility of the casein molecule has been estimated from this constant, and also from the relation between the solubility of the casein and the sodium hydroxide concentration, to be approximately 0.09 gm. per liter at 25°C. 10. The product of the acid dissociation constants, Ka₁ and Ka₂, must therefore be 24 x 10^–12N. 11. It is believed that these constants completely characterize the solubility of casein in systems containing the protein and small amounts of sodium hydroxide.     

THE ELECTRICAL CONDUCTIVITY OF SODIUM AND POTASSIUM GUAIACOLATES IN GUAIACOL

The data of the author and Uhlig, and new data, on the conductivity of sodium and of potassium guaiacolates in guaiacol at 25° have been computed with an improved conductance equation which is valid to somewhat higher concentrations than the equations formerly used. The new constants are, Λ₀ = 9.0, K = 2.8 x 10^–5 for sodium guaiacolate and Λ₀ = 9.5, K = 3.4 x 10^–5 for potassium guaiacolate.     

THE ACTIVATION OF STARFISH EGGS BY ACIDS : II. THE ACTION OF SUBSTITUTED BENZOIC ACIDS AND OF BENZOIC AND SALICYLIC ACIDS AS INFLUENCED BY THEIR SALTS.

1. Comparison of the rates of activation of unfertilized starfish eggs in pure solutions of a variety of parthenogenetically effective organic acids (fatty acids, carbonic acid, benzoic and salicylic acids, chloro- and nitrobenzoic acids) shows that solutions which activate the eggs at the same rate, although widely different in molecular concentration, tend to be closely similar in C_H. The dissociation constants of these acids range from 3.2 x 10^–7 to 1.32 x 10^–3. 2. In the case of each of the fourteen acids showing parthenogenetic action the rate of activation (within the favorable range of concentration) proved nearly proportional to the concentration of acid. The estimated C_H of solutions exhibiting an optimum action with exposures of 10 minutes (at 20°) lay typically between 1.1 x 10^–4 M and 2.1 x 10^–4 M (pH = 3.7–3.96), and in most cases between 1.6 x 10^–4 M and 2.1 x 10^–4 M (pH = 3.7–3.8). Formic acid (C_H = 4.2 x 10^–4 M) and o-chlorobenzoic acid (C_H = 3.5 x 10^–4 M) are exceptions; o-nitrobenzoic acid is ineffective, apparently because of slow penetration. 3. Activation is not dependent on the penetration of H ions into the egg from without, as is shown by the effects following the addition of its Na salt to the solution of the activating acid (acetic, benzoic, salicylic). The rate of activation is increased by such addition, to a degree indicating that the parthenogenetically effective component of the external solution is the undissociated free acid. Apparently the undissociated molecules alone penetrate the egg freely. It is assumed that, having penetrated, they dissociate in the interior of the egg, furnishing there the H ions which effect activation. 4. Attention is drawn to certain parallels between the physiological conditions controlling activation in the starfish egg and in the vertebrate respiratory center.     

Photodegradation of Moxifloxacin in Aqueous and Organic Solvents: A Kinetic Study

The kinetics of photodegradation of moxifloxacin (MF) in aqueous solution (pH 2.0–12.0), and organic solvents has been studied. MF photodegradation is a specific acid-base catalyzed reaction and follows first-order kinetics. The apparent first-order rate constants (k_obs) for the photodegradation of MF range from 0.69 × 10⁻⁴ (pH 7.5) to 19.50 × 10⁻⁴ min⁻¹ (pH 12.0), and in organic solvents from 1.24 × 10⁻⁴ (1-butanol) to 2.04 × 10⁻⁴ min⁻¹ (acetonitrile). The second-order rate constant (k₂) for the [H⁺]-catalyzed and [OH⁻]-catalyzed reactions are 6.61 × 10⁻² and 19.20 × 10⁻² M⁻¹ min⁻¹, respectively. This indicates that the specific base-catalyzed reaction is about three-fold faster than that of the specific acid-catalyzed reaction probably as a result of the rapid cleavage of diazabicyclononane side chain in the molecule. The k_obs-pH profile for the degradation reactions is a V-shaped curve indicating specific acid-base catalysis. The minimum rate of photodegradation at pH 7–8 is due to the presence of zwitterionic species. There is a linear relation between k_obs and the dielectric constant and an inverse relation between k_obs and the viscosity of the solvent. Some photodegraded products of MF have been identified and pathways proposed for their formation in acid and alkaline solutions.KEY WORDS: acid-base catalysis, kinetics, moxifloxacin, photodegradation, rate–pH profile, solvent effect     

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.     

Heat-induced formation of reactive oxygen species and 8-oxoguanine, a biomarker of damage to DNA

Heat-induced formation of 8-oxoguanine was demonstrated in DNA solutions in 10^–3 M phosphate buffer, pH 6.8, by enzyme-linked immunosorbent assays using monoclonal antibodies against 8-oxoguanine. A radiation-chemical yield of 3.7 × 10^–2 µmol J^–1 for 8-oxoguanine production in DNA upon γ-irradiation was used as an adequate standard for quantitation of 8-oxoguanine in whole DNA. The initial yield of heat-induced 8-oxoguanine exhibits first order kinetics. The rate constants for 8-oxoguanine formation were determined at elevated temperatures; the activation energy was found to be 27 ± 2 kcal/mol. Extrapolation to 37°C gave a value of k₃₇ = 4.7 × 10^–10 s^–1. Heat-induced 8-oxoguanine formation and depurination of guanine and adenine show similarities of the processes, which implies that heat-mediated generation of reactive oxygen species (ROS) should occur. Heat-induced production of H₂O₂ in phosphate buffer was shown. The sequence of reactions of thermally mediated ROS formation have been established: activation of dissolved oxygen to the singlet state, generation of superoxide radicals and their dismutation to H₂O₂. Gas saturation (O₂, N₂ and Ar), D₂O, scavengers of ¹O₂, O₂^–• and OH^• radicals and metal chelators influenced heat-induced 8-oxoguanine formation as they affected thermal ROS generation. These findings imply that heat acts via ROS attack leading to oxidative damage to DNA.     

STIMULATION BY MINERAL AND FATTY ACIDS IN THE BARNACLE BALANUS BALANOIDES

1. Stimulation in the rock barnacle Balanus balanoides by hydrochloric, sulfuric, and nitric acids, and by the first seven members of the normal aliphatic acid series has been studied. The hydrogen ion concentrations of the solutions tested varied from 3.2 x 10^–8 to 5.889 x 10^–6. The criterion of response was percentage closure in groups of individuals, recorded at 1 minute intervals until maximum closure occurred. 2. The intensity of stimulation by these acids is proportional to the effects of two forces, one related to the change in the (H⁺), and the other to the field of force around the anion of the acid added to the environment. 3. A preliminary interpretation of the results led to the development of the following expression which fits approximately the data obtained at the end of 4 minutes: Per cent closure = 100 – 100e ^{–0.1z+(0.003125)2–0.1z+(0.003125)2n(z–0.4)} where z is the (H⁺) x 10⁷ and n is the number of carbon atoms (if present) in the anion of the acid. This equation assumes that the anions of the mineral acids enter into the reaction stoichiometrically, and emphasizes the difference in the fields of force around the anion of the fatty acids, a difference which is correlated with the length of the carbon chain. 4. A further analysis of the data revealed the presence of three or more receptor groups which appeared to be differentially affected by forces originating from the anions of the acids. 5. The order of stimulating efficiency for the mineral acids was found to be: HCl>H₂SO₄>HNO₃. 6. The order of stimulating efficiency for the fatty acids was found to be: heptylic>caproic>valeric>butyric = acetic>propionic = formic.     

STUDIES ON CELL METABOLISM AND CELL DIVISION : III. OXYGEN CONSUMPTION AND CELL DIVISION OF FERTILIZED SEA URCHIN EGGS IN THE PRESENCE OF RESPIRATORY INHIBITORS

1. The effects of a number of respiratory inhibiting agents on the cell division of fertilized eggs of Arbacia punctulata have been determined. For eggs initially exposed to the reagents at 30 minutes after fertilization at 20°C., the levels of oxygen consumption prevailing in the minimum concentrations of reagents which produced complete cleavage block were (as percentages of the control): In 0.4 per cent O₂-99.6 per cent N₂, 32; in 0.7 per cent O₂-99.3 per cent CO, 32; in 1.6 x 10^–4 M potassium cyanide, 34; in 1 x 10^–3 M phenylurethane, 70; in 4 x 10^–3 M 5-isoamyl-5-ethyl barbituric acid, 20; in 3 x 10^–4 M iodoacetic acid, 53. 2. The carbon monoxide inhibition of oxygen consumption and cell division was reversed by light. The percentage inhibition of oxygen consumption by carbon monoxide in the dark is described by the usual mass action equation with K, the inhibition constant, equal to approximately 60, as compared to values of 5 to 10 for yeast and muscle. In 20 per cent O₂-80 per cent CO in the dark there was a slight stimulation of oxygen consumption, averaging 20 per cent. 3. Spectroscopic examination of fertilized and unfertilized Arbacia eggs reduced by hydrosulfite revealed no cytochrome bands. The thickness and density of the egg suspension was such as to indicate that, if cytochrome is present at all, the amount in Arbacia eggs is extremely small as compared to that in other tissues having a comparable rate of oxygen consumption. 4. Three reagents poisoning copper catalyses, potassium dithio-oxalate (10^–2 M), diphenylthiocarbazone (10^–4 M), and isonitrosoacetophenone (2 x 10^–3 M) produced no inhibition of division of fertilized Arbacia eggs. 5. These results indicate that the respiratory processes required to support division in the Arbacia egg may perhaps differ in certain essential steps from the principal respiratory processes in yeast and muscle.     

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.     

Variations in the FRA10AC1 Fragile Site and 15q21 Are Associated with Cerebrospinal Fluid Aβ1-42 Level

Proteolytic fragments of amyloid and post-translational modification of tau species in Cerebrospinal fluid (CSF) as well as cerebral amyloid deposition are important biomarkers for Alzheimer’s Disease. We conducted genome-wide association study to identify genetic factors influencing CSF biomarker level, cerebral amyloid deposition, and disease progression. The genome-wide association study was performed via a meta-analysis of two non-overlapping discovery sample sets to identify genetic variants other than APOE ε4 predictive of the CSF biomarker level (Aβ_1–42, t-Tau, p-Tau_181P, t-Tau:Aβ_1–42 ratio, and p-Tau_181P:Aβ_1–42 ratio) in patients enrolled in the Alzheimer’s Disease Neuroimaging Initiative (ADNI) study. Loci passing a genome-wide significance threshold of P < 5 x 10⁻⁸ were followed-up for replication in an independent sample set. We also performed joint meta-analysis of both discovery sample sets together with the replication sample set. In the discovery phase, we identified variants in FRA10AC1 associated with CSF Aβ_1–42 level passing the genome-wide significance threshold (directly genotyped SNV rs10509663 P _FE = 1.1 x 10⁻⁹, imputed SNV rs116953792 P _FE = 3.5 x 10⁻¹⁰), rs116953792 (P _one-sided = 0.04) achieved replication. This association became stronger in the joint meta-analysis (directly genotyped SNV rs10509663 P _FE = 1.7 x 10⁻⁹, imputed SNV rs116953792 P _FE = 7.6 x 10⁻¹¹). Additionally, we identified locus 15q21 (imputed SNV rs1503351 P _FE = 4.0 x 10⁻⁸) associated with CSF Aβ_1–42 level. No other variants passed the genome-wide significance threshold for other CSF biomarkers in either the discovery sample sets or joint analysis. Gene set enrichment analyses suggested that targeted genes mediated by miR-33, miR-146, and miR-193 were enriched in various GWAS analyses. This finding is particularly important because CSF biomarkers confer disease susceptibility and may be predictive of the likelihood of disease progression in Alzheimer’s Disease.     

AREA AND VISUAL THRESHOLD

1. The variation of threshold with field area was measured in fields homogeneous in rod-cone composition. At 15° above the fovea, an increase in field diameter from 1° to 5° reduces the threshold sevenfold, at 25° above the fovea tenfold. 2. These changes are shown to follow qualitatively from simple statistical properties of the retinal mosaic. Analytic treatment leads to the expression, (A – n_t)^k I = C, in which A = area, n_t = constant threshold number of elements, I = threshold intensity, and k and C are constants. This equation describes the available data accurately, and is the general form of previous empirical area-threshold formulae.     

Solvent Effect on the Photolysis of Riboflavin

The kinetics of photolysis of riboflavin (RF) in water (pH 7.0) and in organic solvents (acetonitrile, methanol, ethanol, 1-propanol, 1-butanol, ethyl acetate) has been studied using a multicomponent spectrometric method for the assay of RF and its major photoproducts, formylmethylflavin and lumichrome. The apparent first-order rate constants (k_obs) for the reaction range from 3.19 (ethyl acetate) to 4.61 × 10⁻³ min⁻¹ (water). The values of k_obs have been found to be a linear function of solvent dielectric constant implying the participation of a dipolar intermediate along the reaction pathway. The degradation of this intermediate is promoted by the polarity of the medium. This indicates a greater stabilization of the excited-triplet states of RF with an increase in solvent polarity to facilitate its reduction. The rate constants for the reaction show a linear relation with the solvent acceptor number indicating the degree of solute–solvent interaction in different solvents. It would depend on the electron-donating capacity of RF molecule in organic solvents. The values of k_obs are inversely proportional to the viscosity of the medium as a result of diffusion-controlled processes.KEY WORDS: dielectric constant, kinetics, photolysis, riboflavin, solvent effect, viscosity     

Efflux and Influx of Erythrocyte Water

Rabbit erythrocytes were washed in buffered NaCl solutions isotonic with rabbit serum (Δ^t -0.558°C.) and suspended in buffered NaCl solutions of tonicity equidistant from intracellular tonicity (Δ^t = -0.558°C. ± 0.112°C.) of varying pH and incubated at varying temperatures. After incubation, the freezing point depression (Δ^t) was measured on the supernatant. Change in the Δ^t measured change in the water content of the extracellular solutions—water being withdrawn by erythrocytes (W_I) from the hypotonic solutions and added (W_E) to the hypertonic solutions. W_E was always less than W_I and was inversely proportional to the pH in the range 6.5–8.0. W_E was significantly increased by lowering the temperature of the cell suspension to 4°C. W_I was increased by raising or lowering the pH or raising the temperature of the cell suspension. W_E x W_I ≠ k. W_E and W_I were affected differently by changes in pH and temperature. It was concluded that W_E and W_E were probably under different physicochemical control.     

Nitrite-Reductase and Peroxynitrite Isomerization Activities of Methanosarcina acetivorans Protoglobin

Within the globin superfamily, protoglobins (Pgb) belong phylogenetically to the same cluster of two-domain globin-coupled sensors and single-domain sensor globins. Multiple functional roles have been postulated for Methanosarcina acetivorans Pgb (Ma-Pgb), since the detoxification of reactive nitrogen and oxygen species might co-exist with enzymatic activity(ies) to facilitate the conversion of CO to methane. Here, the nitrite-reductase and peroxynitrite isomerization activities of the CysE20Ser mutant of Ma-Pgb (Ma-Pgb*) are reported and analyzed in parallel with those of related heme-proteins. Kinetics of nitrite-reductase activity of ferrous Ma-Pgb* (Ma-Pgb*-Fe(II)) is biphasic and values of the second-order rate constant for the reduction of NO₂ ^– to NO and the concomitant formation of nitrosylated Ma-Pgb*-Fe(II) (Ma-Pgb*-Fe(II)-NO) are k _app1 = 9.6±0.2 M^–1 s^–1 and k _app2 = 1.2±0.1 M^–1 s^–1 (at pH 7.4 and 20°C). The k _app1 and k _app2 values increase by about one order of magnitude for each pH unit decrease, between pH 8.3 and 6.2, indicating that the reaction requires one proton. On the other hand, kinetics of peroxynitrite isomerization catalyzed by ferric Ma-Pgb* (Ma-Pgb*-Fe(III)) is monophasic and values of the second order rate constant for peroxynitrite isomerization by Ma-Pgb*-Fe(III) and of the first order rate constant for the spontaneous conversion of peroxynitrite to nitrate are h _app = 3.8×10⁴ M^–1 s^–1 and h ₀ = 2.8×10^–1 s^–1 (at pH 7.4 and 20°C). The pH-dependence of h _on and h ₀ values reflects the acid-base equilibrium of peroxynitrite (pK _a = 6.7 and 6.9, respectively; at 20°C), indicating that HOONO is the species that reacts preferentially with the heme-Fe(III) atom. These results highlight the potential role of Pgbs in the biosynthesis and scavenging of reactive nitrogen and oxygen species.     

PHOSPHATE ION AS A PROMOTER CATALYST OF RESPIRATION

The active component of phosphate solutions, in relation to promoter action on oxidising enzymes, is the PO₄ ^'''''' ion. This is shown by the demonstration of a hyperbolic relationship between per cent production of CO₂ (of Elodea) and pPO₄, the measure of the phosphate ion potential. This is consistent with the rate of respiration as affected by changing pPO₄ through change of total phosphate concentration while pH is kept constant. The equation for this relationship is (CO₂ – a) (pPO₄ – b)ⁿ = K where a, b, n, and K are constants and n = 1. The same relationship to phosphate ion concentration, expressed by the equation (Activity of enzyme) (pPO₄)ⁿ = K, where n and K are constants and n varies from 1 to 6 under different conditions, appears to hold for some other enzyme actions, including those of peroxidase and pancreatic lipase.     

THE APPARENT DISTORTION OF BRIEF RECTANGULAR ELECTRICAL STIMULI IN NERVE

If it is assumed that the kinetics of the process of excitation in nerve is given by dp/dt = KI – kp, I being the actual exciting component of the current, p the state of excitation, and K and k constants, it is necessary to postulate that on application of a rectangular stimulus of voltage, V, the current, I, undergoes a transient exponential variation, usually a decrease, in order that the integral of the differential equation (above) may fit the strength-duration data in V and t. This hypothesis is substantiated by data by Sakamoto on single fibers of the sciatic nerve of the frog. The time constant of the postulated current transient is of the order of 10^–4 sec. for single fibers and of the order of 10^–5 sec. or less in the sciatic nerve trunk. The latter value is about the same as that found by Cole in the same tissue by purely physical measurements. Some criticisms by Rushton (1934) are discussed.     

Reduction of ferricytochrome c by tyrosyltyrosylphenylalanine

Cytochrome c (cyt c) was reduced by a tyrosine-containing peptide, tyrosyltyrosylphenylalanine (TyrTyrPhe), at pH 6.0–8.0, while tyrosinol or tyrosyltyrosine (TyrTyr) could not reduce cyt c effectively under the same condition. Cyt c was reduced at high peptide concentration, whereas the reaction did not occur effectively at low concentration. The reaction rate varied with time owing to a decrease in the TyrTyrPhe concentration and the production of tyrosine derivatives during the reaction. The initial rate constants were 2.4×10^–4 and 8.1×10^–4 s^–1 at pH 7.0 and 8.0, respectively, for the reaction with 1.0 mM TyrTyrPhe in 10 mM phosphate buffer at 15°C. The reciprocal initial rate constant (1/k_int) increased linearly against the reciprocal peptide concentration and against the linear proton concentration, whereas logk_int decreased linearly against the root of the ionic strength. These results show that deprotonated (TyrTyrPhe)^–, presumably deprotonated at a tyrosine site, reduces cyt c by formation of an electrostatic complex. No significant difference in the reaction rate was observed between the reaction under nitrogen and oxygen atmospheres. From the matrix-assisted laser desorption ionization time-of-flight mass spectra of the reaction products, formation of a quinone and other tyrosine derivatives of the peptide was supported. These products should have been produced from a tyrosyl radical. We interpret the results that a cyt c_ox/(TyrTyrPhe)^–cyt c_red/(TyrTyrPhe) equilibrium is formed, which is usually shifted to the left. This equilibrium may shift to the right by reaction of the produced tyrosyl radical with the tyrosine sites of unreacted TyrTyrPhe peptides.     

Papain-catalysed hydrolysis of some hippuric esters. A new mechanism for papain-catalysed hydrolyses

1. The Michaelis–Menten parameters for the papain-catalysed hydrolysis of a number of alkyl, aryl and alkyl-thiol esters of hippuric acid have been determined. 2. For all the aryl esters and most of the alkyl esters studied, the catalytic constant, k₀, is 2–3sec.⁻¹ and most probably represents deacylation of the common intermediate, hippuryl-papain. 3. Two alkyl esters and hippurylamide, however, have catalytic rate constants, k₀, less than 2–3sec.⁻¹. It is possible to interpret all the available kinetic data in terms of a three-step mechanism in which an enzyme–substrate complex is first formed, followed by acylation of the enzyme through an essential thiol group, followed by deacylation of the acyl-enzyme. 4. The logarithm of the ratio of the Michaelis–Menten parameters, which reflect the acylation rate constant, for four aryl esters of hippuric acid studied give a linear Hammett plot against the substituent constant, σ. Arguments are presented that indicate acid as well as nucleophilic catalysis in the acylation process and that the most likely proton donor is an imidazolium ion. 5. It is suggested that this imidazolium ion is part of the same histidine residue that has been tentatively implicated in the deacylation process (Lowe & Williams, 1965b). 6. A new mechanism is proposed for the papain-catalysed hydrolysis of N-acyl-α-amino acid derivatives.