COUNTERACTION OF THE INHIBITING EFFECTS OF VARIOUS SUBSTANCES ON NITELLA

When living cells of Nitella are first exposed to (1) phosphate buffer mixture, or (2) phosphoric acid, or (3) hydrochloric acid, or (4) sodium chloride, or (5) sodium borate, and are then placed in a solution of brilliant cresyl blue made up with a borate buffer mixture at pH 7.85, the rate of penetration of the dye into the vacuole is decreased as compared with the rate in the case of cells transferred directly from tap water to the same dye solution. When cells exposed to any one of these solutions are placed in the dye solution made up with phosphate buffer solution at pH 7.85, the rate of penetration of dye into the vacuole is the same as the rate in the case of cells transferred from the tap water to the same dye solution. It is probable that this removal of the inhibiting effect is due primarily to the presence of certain concentration of sodium and potassium ions in the phosphate buffer solution. If a sufficient concentration of sodium ions is added to the dye made up with a borate buffer mixture the inhibiting effect is removed just as it is in the case of the dye made up with the phosphate buffer mixture. The inhibiting effect of some of these substances is found to be removed by the dye containing a sufficient concentration of bivalent cations, or by washing the cells with salts of bivalent cations. The inhibiting effect and its removal are discussed from a theoretical standpoint.     

THE FLOCCULATION MAXIMUM (pH) OF FIBRINOGEN AND SOME OTHER BLOOD-CLOTTING REAGENTS. (RELATIVE TURBIDIMETRY WITH THE EVELYN PHOTOELECTRIC COLORIMETER)

By means of a novel adaptation of the Evelyn photoelectric colorimeter to the measurement of relative turbidities, the question of the flocculation maximum (F.M.) in acetate buffer solutions of varying pH and salt content has been studied on (a) an exceptionally stable prothrombin-free fibrinogen and its solutions after incipient thermal denaturation and incomplete tryptic proteolysis, (b) plasma, similarly treated, (c) prothrombin, thrombin, and (brain) thromboplastin solutions. All the fibrinogens show a remarkable uniformity of the precipitation pattern, viz. F.M. =4.7 (±0.2) pH in salt-containing buffer solutions and pH = 5.3 (±0.2) in salt-poor buffer (N/100 acetate). The latter approximates the isoelectric point (5.4) obtained by cataphoresis (14). There is no evidence that denaturation or digestion can produce any "second maximum." The data support the view that fibrin formation (under the specific influence of thrombin) is intrinsically unrelated to denaturation and digestion phenomena, although all three can proceed simultaneously in crude materials. A criticism is offered, therefore, of Wöhlisch''s blood clotting theory. Further applications of the photoelectric colorimeter to coagulation problems are suggested, including kinetic study of fibrin formation and the assay of fibrinogen, with a possible sensitivity of 7.5 mg. protein in 100 cc. solution.     

Separation and sensitive determination of i-urobilin and 1-stercobilin by high-performance liquid chromatography with fluorimetric detection

i-Urobilin and 1-stercobilin were separated by high-performance liquid chromatography on a reversed-phase octadecylsilane-bonded column and detected fluorimetrically through formation of phosphor with zinc ions in the eluent. The separation and the intensity of the fluorescence response were affected by concentrations of zinc acetate and sodium borate buffer, pH and methanol content in the eluent. The optimal eluent used consisted of 0.1% zinc acetate in 75 mM boric acid buffer (pH 6.0)—methanol (25:75). The detection limit was 0.2 μg/l for both i-urobilin and 1-stercobilin (signal-to-noise ratio 2), which makes the method 250–2500 times more sensitive than conventional methods.     

Solution properties of polygalacturonic acid

1. The specimen of polygalacturonic acid used in these studies was shown to contain very little neutral sugar, methyl ester groups or ash, and only residues of galacturonic acid. Its electrophoretic homogeneity was examined in pyridine–acetic acid buffer at pH6·5 and in borate buffer at pH9·2. The distribution of effective particle weights was shown to be fairly narrow. 2. The pH-titration curve of the polymer gave a pK value of 3·7. 3. The interaction of the polymer with Ruthenium Red was studied and titration curves were obtained for the spectral shifts associated with the formation of a complex. 4. Optical-rotatory-dispersion studies showed that the Drude constant, λ_c, was dependent on pH. 5. Polygalacturonic acid was shown to display non-Newtonian properties in solution and to have an anomalously high relative specific viscosity at low concentrations. 6. Studies were made of the pH-dependence of the sedimentation coefficient of the polymer. 7. These results are discussed in terms of the structure of the molecule and their relevance to the properties of pectic substances.     

Borate buffer inhibition of peroxidatic intermediate formation in the deutero-ferriheme-hydrogen peroxide system

The rate of formation of peroxidatically active reaction intermediate(s) via oxidation of the iron(III)-porphyrin complex, deuteroferriheme, with hydrogen peroxide decreases with increasing borate content of mixed borate-carbonate buffer solutions. Studies at pH = 9.25 in 0.035 M borate buffer and 0.035 M carbonate buffer suggest borate to function as an uncompetitive inhibitor. A comparison of slopes and intercepts of double reciprocal plots for inhibited and uninhbited reactions allows calculation of selected parameters for the deuteroferriheme-H₂O₂ reaction at pH = 9.25 in terms of a typical enzymatic stoichiometric mechanism for heme activity. This includes the Michaelis constant (K_m = 8.1 × 10^?5 M) and the first-order rate constant for conversion of heme-substrate complex to intermediate(s) (k₃ = 7.4 sec^?1). A tentative mechanistic model involving reversible interaction of borate inhibitor with heme-substrate complex is considered, and pseudo-first-order rate constants calculated on the basis of this scheme are in reasonable agreement with those obtained experimentally. It is suggested that comparable inhibitory action may be responsible for some previously reported cases of decreased catalase enzyme activity in borate buffer solutions     

Electrophoretic and other studies on haem pigments from Rhodopseudomonas palustris: Cytochrome 552 and cytochromoid c

1. Cytochrome 552 and cytochromoid c were extracted from Rhodopseudomonas palustris cells, purified and obtained in crystalline form. 2. Extinction ratios and amino acid compositions of the two pigments are reported. 3. When subjected to starch-gel electrophoresis in borate buffer, pH8·8, each pigment migrated towards the cathode; oxidized cytochromoid c migrated more rapidly than its reduced form. 4. By a determination of electrophoretic mobilities in buffers of I0·1 by using the moving-boundary method, the isoelectric point of cytochrome 552 was found to be at pH10·6 and that of cytochromoid c at pH9·7. 5. As obtained, cytochrome 552 was non-autoxidizable; cytochromoid c was autoxidizable but became considerably less so on alkaline treatment. 6. Discussion of the results includes a consideration of the isoelectric points of the pigments in terms of their amino acid composition.     

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.     

The interaction of borate and sulfite with pyridine nucleotides.

The kinetics and equilibria of the borate interaction at ribose with NAD+ and NMN+ have been measured using as a chromophoric probe the perturbation effect borate has on the addition of sulfite to the 4 position of the nicotinamide ring. NAD+ and NMN+ have more favorable borate association constants than do their corresponding sulfite addition complexes. The rate of interaction of the ribose moiety with borate at low borate buffer concentration is dependent on the concentration of both borate and boric acid. At high borate concentration the rate becomes independent of borate concentration, indicating the existence of a two-step process for the interaction of NAD-sulfite with borate with a change of rate-determining step from the interaction of the ribose hydroxyl group with borate at low borate to an elimination of sulfite at high borate concentration. A linear free energy relationship with a slope of 0.94 describes an increased reactivity of the nucleotide for sulfite as the affinity of the nucleotide for sulfite increases.     

ISOELECTRIC POINTS FOR THE MYCELIUM OF FUNGI

1. Mycelium of Rhizopus nigricans when stained with certain acid and basic dyes and washed with buffer mixtures of 0.1 M phosphoric acid and sodium hydroxide responded much like an amphoteric colloid with an isoelectric point near pH 5.0. 2. When grown on potato dextrose agar the reaction of which was varied with phosphoric acid the extent of colony growth of Rhizopus nigricans plotted against the initial Sörensen value of the agar produced a double maximum curve with the minimum between the two maxima at initial pH 5.2. 3. When grown in potato dextrose broth the reaction of which was varied with phosphoric acid the dry matter produced by Rhizopus nigricans plotted against the Sörensen value of the broth produced a double maximum curve with the minimum between the two maxima at initial pH 5.2 or average pH 4.9. 4. Mycelium of Rhizopus nigricans placed in buffer mixtures of 0.01 M phosphoric acid and sodium hydroxide of pH 4.1 to 6.3, changed the reaction in most cases toward greater alkalinity. 5. Mycelium of Fusarium lycopersici stained with certain acid and basic dyes and washed with buffer mixtures of 0.1 M phosphoric acid and sodium hydroxide responded much like an amphoteric colloid with an isoelectric point near pH 5.5.     

THE R?LE OF THE ACTIVITY COEFFICIENT OF THE HYDROGEN ION IN THE HYDROLYSIS OF GELATIN

1. The hydrolysis of gelatin at a constant hydrogen ion concentration follows the course of a monomolecular reaction for about one-third of the reaction. 2. If the hydrogen ion concentration is not kept constant the amount of hydrolysis in certain ranges of acidity is proportional to the square root of the time (Schütz''s rule). 3. The velocity of hydrolysis in strongly acid solution (pH less than 2.0) is directly proportional to the hydrogen ion concentration as determined by the hydrogen electrode i.e., the "activity;" it is not proportional to the hydrogen ion concentration as determined by the conductivity ratio. 4. The addition of neutral salts increases the velocity of hydrolysis and the hydrogen ion concentration (as determined by the hydrogen electrode) to approximately the same extent. 5. The velocity in strongly alkaline solutions (pH greater than 10) is directly proportional to the hydroxyl ion concentration. 6. Between pH 2.0 and pH 10.0 the rate of hydrolysis is approximately constant and very much greater than would be calculated from the hydrogen and hydroxyl ion concentration. This may be roughly accounted for by the assumption that the uncombined gelatin hydrolyzes much more rapidly than the gelatin salt.     

THE PENETRATION OF BASIC DYE INTO NITELLA AND VALONIA IN THE PRESENCE OF CERTAIN ACIDS,BUFFER MIXTURES,AND SALTS

When living cells of Nitella are exposed to an acetate buffer solution until the pH value of the sap is decreased and subsequently placed in a solution of brilliant cresyl blue, the rate of penetration of dye into the vacuole is found to decrease in the majority of cases, and increase in other cases, as compared with the control cells which are transferred to the dye solution directly from tap water. This decrease in the rate is not due to the lowering of the pH value of the solution just outside the cell wall, as a result of diffusion of acetic acid from the cell when cells are removed from the buffer solution and placed in the dye solution, because the relative amount of decrease (as compared with the control) is the same whether the external solution is stirred or not. Such a decrease in the rate may be brought about without a change in the pH value of the sap if the cells are placed in the dye solution after exposure to a phosphate buffer solution in which the pH value of the sap remains normal. The rate of penetration of dye is then found to decrease. The extent of this decrease is the greater the lower the pH value of the solution. It is found that hydrochloric acid and boric acid have no effect while phosphoric acid has an inhibiting effect at pH 4.8 on stirring. Experiments with neutral salt solutions indicate that a direct effect on the cell (decreasing penetration) is due to monovalent base cations, while there is no such effect directly on the dye. It is assumed that the effect of the phosphate and acetate buffer solutions on the cell, decreasing the rate of penetration, is due (1) to the penetration of these acids into the protoplasm as undissociated molecules, which dissociate upon entrance and lower the pH value of the protoplasm or to their action on the surface of the protoplasm, (2) to the effect of base cations on the protoplasm (either at the surface or in the interior), and (3) possibly to the effect of certain anions. In this case the action of the buffer solution is not due to its hydrogen ions. In the case of living cells of Valonia under the same experimental conditions as Nitella it is found that the rate of penetration of dye decreases when the pH value of the sap increases in presence of NH₃, and also when the pH value of the sap is decreased in the presence of acetic acid. Such a decrease may be brought about even when the cells are previously exposed to sea water containing HCl, in which the pH value of the sap remains normal.     

THE KINETICS OF THE REACTION WHICH TAKES PLACE BETWEEN IODOACETIC ACID AND GLYCINE

1. The kinetics of the reaction which takes place between glycine and iodoacetic acid was studied by means of the polarographic method. 2. On the basis of kinetic equations, evidence was obtained that (a) The reaction proceeds in two steps in which the hydrogens of the amino group are consecutively replaced by the acetyl radicals, the velocity constants being in the ratio 2:1. (b) Only the anionic form of glycine is able to react since the velocity constants at any pH are proportional to the concentration of glycine anion. (c) The reaction is of the ionic type, showing a positive salt catalysis, which, according to Brönsted''s hypothesis, involves the primary and the secondary salt effects. 3. The fact that only the glycine anion is able to react was explained as being due to the existence of an unbonded pair of electrons on the nitrogen in the NH₂ group. The NH₃ ⁺ group, however, in which these electrons are shared by H⁺, must, therefore, be inactive.     

THE KINETICS OF TRYPSIN DIGESTION : V. SCHUTZ'S RULE.

The rate of digestion of concentrated casein solutions by low concentrations of trypsin at 0° has been followed. Under these conditions the enzyme is inhibited by the product of the reaction and under certain conditions this effect should lead to Schütz''s rule, i.e. the amount of hydrolysis should be proportional to the square root of the product of the time into the enzyme concentration. This is the result obtained. Both Schütz''s rule and Arrhenius'' equation fail to hold accurately owing to the incorrect relation assumed to hold between the rate of hydrolysis and the substrate concentration.     

FORMATION OF TRYPSIN FROM TRYPSINOGEN BY AN ENZYME PRODUCED BY A MOLD OF THE GENUS PENICILLIUM

1. A powerful kinase which changes trypsinogen to trypsin was found to be present in the synthetic liquid culture medium of a mold of the genus Penicillium. 2. The concentration of kinase in the medium is increased gradually during the growth of the mold organism and continues to increase for some time even after the mold has ceased growing. 3. Mold kinase transforms trypsinogen to trypsin only in an acid medium. It differs thus from enterokinase and trypsin which activate trypsinogen best in a slightly alkaline medium. 4. The action of the mold kinase in the process of transformation of trypsinogen is that of a typical enzyme. The process follows the course of a catalytic unimolecular reaction, the rate of formation of a definite amount of trypsin being proportional to the concentration of kinase added. The ultimate amount of trypsin formed, however, is independent of the concentration of kinase used. 5. The formation of trypsin from trypsinogen by mold kinase is not accompanied by any measurable loss of protein. 6. The temperature coefficient of formation of trypsin from trypsinogen by mold kinase varies from Q _5–15 = 1.70 to Q _25–30 = 1.25 with a corresponding variation in the value of µ from 8100 to 4250. 7. Trypsin formed from trypsinogen by means of mold kinase is identical in crystalline form with the crystalline trypsin obtained by spontaneous autocatalytic activation of trypsinogen at pH 8.0. The two products have within the experimental error the same solubility and specific activity. A solution saturated with the crystals of either one of the trypsin preparations does not show any increase in protein concentration or activity when crystals of the other trypsin preparation are added. 8. The Penicillium mold kinase has a slight activating effect on chymo-trypsinogen the rate being only 1–2 per cent of that of trypsinogen. The activation, as in the case of trypsinogen, takes place only in an acid medium. 9. Mold kinase is rapidly destroyed when brought to pH 6.5 or higher, and also when heated to 70°C. In the temperature range of 50–60°C. the inactivation of kinase follows a unimolecular course with a temperature coefficient of Q ₁₀ = 12.1 and µ = 53,500. The molecular weight of mold kinase, as determined by diffusion, is 40,000.     

THE SOLUBILITY OF TYROSINE IN ACID AND IN ALKALI

Measurements have been made of the solubility at 25°C. of tyrosine in hydrochloric acid and in sodium hydroxide solutions varying from 0.001 to 0.05 M, and also in distilled water. The pH of the saturated solutions was measured with the hydrogen electrode. The following values for the ionization constants of tyrosine have been obtained from the measurements: k_b = 1.57 x 10^–12, k_a1 = 7.8 x 10^–10, k_a2 = 8.5 x 10^–11. The changes in solubility with pH can be satisfactorily explained by the use of these ionization constants.     

Oxidation of a methionine residue in subtilisin-type proteinases by the hydrogen peroxide/borate system — an active site-directed reaction

Subtilisin-type proteinases (thermitase, subtilisin Carlsberg, alkaline proteinase ZIMET 10911, proteinase K) are partially inactivated by hydrogen peroxide in the alkaline pH range only in the presence of boric acid or phenylboronic acid. A model is presented to describe the inactivation mechanism. Both boric acid and perboric acid existing in equilibrium in the presence of hydrogen peroxide bind competitively at the active site of the enzyme. The inactivation, which is known to be caused by sulfoxide formation from the methionine residue in the active site (Stauffer C.E. and Etson D. (1969) J. Biol. Chem. 244, 5333–5338), is due to the enzyme-bound perboric acid species. The dissociation constants for the boric acid-thermitase and perboric acid-thermitase complexes are 36 ± 7 and 4 ± 1 mM, respectively. The first-order rate constant of inactivation is k = 0.63 ± 0.14 min⁻¹. The same mechanism of inactivation holds true for phenylboronic acid in alkaline hydrogen peroxide solutions.     

THE EFFECT OF ACETATE BUFFER MIXTURES,ACETIC ACID,AND SODIUM ACETATE,ON THE PROTOPLASM,AS INFLUENCING THE RATE OF PENETRATION OF CRESYL BLUE INTO THE VACUOLE OF NITELLA

When living cells of Nitella are exposed to a solution of sodium acetate and are then placed in a solution of brilliant cresyl blue made up with a borate buffer mixture at pH 7.85, a decrease in the rate of penetration of dye is found, without any change in the pH value of the sap. It is assumed that this inhibiting effect is caused by the action of sodium on the protoplasm. This effect is not manifest if the dye solution is made up with phosphate buffer mixture at pH 7.85. It is assumed that this is due to the presence of a greater concentration of base cations in the phosphate buffer mixture. In the case of cells previously exposed to solutions of acetic acid the rate of penetration of dye decreases with the lowering of the pH value of the sap. This inhibiting effect is assumed to be due chiefly to the action of acetic acid on the protoplasm, provided the pH value of the external acetic acid is not so low as to involve an inhibiting effect on the protoplasm by hydrogen ions as well. It is assumed that the acetic acid either has a specific effect on the protoplasm or enters as undissociated molecules and by subsequent dissociation lowers the pH value of the protoplasm. With acetate buffer mixture the inhibiting effect is due to the action of sodium and acetic acid on the protoplasm. The inhibiting effect of acetic acid and acetate buffer mixture is manifested whether the dye solution is made up with borate or phosphate buffer mixture at pH 7.85. It is assumed that acetic acid in the vacuole serves as a reservoir so that during the experiment the inhibiting effect still persists.     

Fluorometric characterization of dityrosine: complex formation with boric acid and borate ion

Borate/boric acid solutions have distinctive effects on the absorption and fluorescence emission spectra of dityrosine. In the presence of excess borate/boric acid, the fluorescence emission maximum of the singly ionized dityrosine chromophore shifts from 407 nm (quantum yield = 0.80) to 374 nm (quantum yield = 0.14). Fluorescence measurements performed as a function of pH and concentration are consistent with a 1:1 complex which may dissociate to either boric acid and singly ionized dityrosine (K1 = 17 mM) or to monoborate ion and unionized dityrosine (K2 = 0.10 mM). As a consequence of the pKa values characteristic of dityrosine and boric acid, complex formation is maximal near pH 8. 2,2'-Dihydroxy-biphenyl shows similar interactions. The fluorescence of dityrosyl calmodulin (0 Ca2+) also responds to the addition of boric acid, giving K1 = 42 mM and K2 = 2 mM. Singly ionized dityrosine produced through dissociation occurring in the excited state does not interact with boric acid.     

TEMPERATURE CHARACTERISTIC FOR THE ANAEROBIC PRODUCTION OF CO2 BY GERMINATING SEEDS OF LUPINUS ALBUS

The rate of anaerobic production of CO₂ by germinating seeds of Lupinus albus was studied as a function of temperature between 7.5° and 18°C. The mean value for the temperature characteristic was found to be 21,500± calories, which is slightly lower than that for the same process under aerobic conditions (23,500± calories). The values for the individual µ''s in the two cases overlap considerably. The possible identity of the processes underlying the production of CO₂ aerobically and anaerobically is discussed.     

STIMULATION OF FUNDULUS BY OXALIC AND MALONIC ACIDS AND BREATHING RHYTHM AS FUNCTIONS OF TEMPERATURE

1. Chemical stimulation as a function of temperature was studied by using oxalic acid in fresh and salt water and malonic acid in salt water as stimulating agents on Fundulus. According to the Arrhenius equation the following µ values were obtained for the various acid solutions between 0 and 29°C.: for 0.002N oxalic in fresh water—15,800; 33,000; for 0.0008N oxalic in fresh water—15,800; 33,000; 48,000; for 0.002N oxalic in salt water—19,400; 24,100; 56,500; for 0.004N and 0.002N malonic in salt water—20,600; 65,000. At a critical temperature there is a sharp transition from one thermal increment to another. 2. The chemical processes controlling stimulation do not change with concentration, for different normalities of a single acid yield the same µ values. Distinctly different temperature characteristics were obtained for stimulation by oxalic in salt and fresh water. Likewise stimulation by oxalic and malonic in salt water yielded very different increments. This temperature study indicates that the controlling chemical reactions determining rate of response are different for the same acid in two different environments, or for two dibasic acids in the same environment. Other work indicates, however, that the fundamental stimulation system is the same for all the adds in both environments. Chemical rather than physical processes limit the rate of response since all the values are above 15,000. Stimulation depends upon a series of interrelated chemical reactions, each with its own temperature characteristic. Under varying conditions (e.g. change of temperature, environment, or acid) different chemical reactions may become the slowest or controlling process which determines the rate of response. 3. The variation of response, as measured by the probable error of the mean response time of the fish, is the same function of temperature as reaction time itself. Hence variability is not independent of reaction time and is controlled by the same catenary series of events which determine rate of response to stimulation. 4. Breathing rhythm of Fundulus as related to temperature was studied in both salt and fresh water. In salt water the temperature characteristic is 8,400 while in fresh water it is 16,400 below 9.5°C., and 11,300 above this critical temperature. These µ values are typical of those which have been reported by other workers for respiratory and oxidative biological phenomena. A change in thermal increment with an alteration in environment indicates that different chemical reactions with characteristic velocity constants are controlling the breathing rhythm in salt and fresh water.