Absence Of Buffer Research Articles

Germinability of coat-lacking spores of [formula omitted][formula omitted

Upon treatment with acid, the germinability of both intact and coat-lacking spores of Bacillus megaterium ATCC 19213 exhibited similar features. Namely, when the spores previously germinated by alanine in the presence of phosphate buffer were converted to H-spores by treatment with nitric acid, germination proceeded at a very low speed in a same germination medium. When H-spores converted to Ca-spores by treatment with calcium acetate and subsequently germinated, germination proceeded at a speed higher than that of native spores and occurred even in the absence of buffer. These results suggest that the site of exchangeable cations concerned with germinability must not exist in the coat.

Correlation of photochemical cycle, H + release and uptake, and electric events in bacteriorhodopsin

The kinetics of 3 photoinduced responses of bacteriorhodopsin have been compared: spectral changes, pH shifts in a suspension of open purple membrane sheets and electric potential generation by the sheets incorporated into a lipid-impregnated collodion film. In the presence of a pH-buffer, the H + release by bacteriorhodops in was shown to correlate with the formation of the M412 intermediate and the microsecond phase of the potential generation. The H +/M412 ratio is equal to 0.7±0.1 if the ionic strength of the solution is high. In the absence of the buffer, the H + release proved to be much slower than spectral and electric responses. The kinetics of H + uptake by bacteriorhodopsin is close to M412 decay and to the electrogeneous millisecond phase in both the presence and absence of the pH buffer. The bacteriorhodopsin-induced proton release phase accounts for about 20%, and the uptake phase for about 80% of the overall potential. This is compatible with the model assuming that the proton start-out point - possibly, the protonated Schiff base connecting lysine 216 with retinal - is closer to the outer rather than the inner (cytoplasmic) surface of the bacterial membrane.

Oxygen-18 isotope effect in carbon-13 nuclear magnetic resonance spectroscopy. 8. Oxygen exchange of 2,4,6-trimethylpyrylium cation

The rate of the oxygen-exchange reaction in an aqueous solution of a heterocyclic oxygen compound, 2,4,6-trimethylpyrylium perchlorate, was quantitated. The /sup 18/O isotope effect in /sup 13/C NMR spectroscopy affords a direct, continuous, and relatively simple analysis of the reaction and was used to obtain data on the incorporation of /sup 18/O from solvent H/sub 2//sup 18/O into the pyrylium salt. Due to the low solubility of this salt in aqueous solutions, 2,4,6-trimethyl(2-/sup 13/C)pyrylium perchlorate and 2,4,6-trimethyl(2,6-/sup 13/C/sub 2/)pyrylium perchlorate were synthesized to facilitate the acquisition of the data on an NTC-200 spectrometer operating at 50.31 MHz. Upon incorporation of /sup 18/O into the pyrylium salt, the /sup 13/C NMR signal of the directly bonded carbon atom is shifted upfield 0.038 ppm. A /sup 13/C isotope effect is also detected in the doubly /sup 13/C-enriched pyrylium salt where the /sup 13/C NMR signal for /sup 13/C-O-/sup 13/C appears 0.011 ppm downfield from the /sup 13/C NMR signal for /sup 12/C-O-/sup 13/C. The rate of the oxygen-exchange reaction was studied as a function of pH (2.0-4.8), buffer concentration, added electrolyte, and temperature. A positive salt effect is observed. The oxygen-exchange reaction is subject to specific hydroxide ion catalysis and to generalmore » base catalysis by buffers such as acetate. The /sup 18/O-exchange reaction is compared to the other reactions of the pyrylium salt including hydrolysis and /sup 2/H exchange. The apparent energy of activation for the oxygen-exchange reaction at pH 3.1 in the absence of buffer and electrolyte is approximately 25 kcal mol/sup -1/ while at pH 4.7 in 50 mM sodium acetate, ..mu.. = 0.2 M with NaCl, it is 10 kcal mol/sup -1/. 36 references, 2 figures, 1 table.« less

Isolation and Characterization of Inner Membrane-Associated and Matrix NAD-Specific Isocitrate Dehydrogenase in Potato Mitochondria

The isotherm for isocitrate oxidation by potato (Solanum tuberosum L. var. Russet Burbank) mitochondria in the presence of exogenous NAD is characterized by a hyperbolic phase at isocitrate concentrations below 3 millimolar, and a sigmoid, or positively cooperative phase from approximately 3 to 30 millimolar. The two forms of isocitrate dehydrogenase were separated and characterized following the sonication of mitochondria in 15% glycerol in the absence of buffer, followed by fractionation in a density step gradient to yield inner membrane and matrix components. The membrane-associated isocitrate dehydrogenase was found to have a Hill, or cooperativity, number of 1, while the Hill number of the matrix enzyme was 2.5. Upon digitonin extraction the cooperativity number of the membrane enzyme rose to 3.5. The isocitrate K(m) for the membrane enzyme was calculated to be approximately 5.9 x 10(-4) molar, while the S(0.5) for the matrix was 6.9 x 10(-4) molar. The NAD K(m) for both enzymes was 150 micromolar. Whereas the membrane enzyme proved indifferent to adenine nucleotides, the matrix enzyme was arguably inhibited by AMP and ADP, and inhibited some 25% by 5 millimolar ATP. Both enzymes were negatively responsive to the mole fraction of NADH, the membrane enzyme being 50% inhibited at a mole fraction of 0.26, and the matrix enzyme by a mole fraction of 0.32. The suggestion is offered that the enzymes in question constitute two forms of a single enzyme, one peripherally associated with the inner membrane, and one soluble in the matrix. It is proposed that a degree of regulation may be achieved by the apportionment of the enzyme between the bound and free forms.

ChemInform Abstract: REACTIONS OF METHYLMERCURY(II), MERCURY(II), AND ZINC(II) IONS WITH 2‐METHYL‐2‐(2‐PYRIDYL)THIAZOLIDINE, INCLUDING STOPPED‐FLOW RAPID‐SCANNING SPECTROPHOTOMETRIC STUDIES

AbstractDie sich zwischen der Titelverbindung (I) u:nd Methyl‐Hg‐chlorid‐ oder ‐acetat, Hg‐perchlorat, Zn‐perchlorat oder ‐acetat bildenden Gleichgewichte werden 1H‐ und 13C‐NMR‐ sowie UV‐spektroskopisch untersucht.

35] Carbonic anhydrase: Oxygen-18 exchange catalyzed by an enzyme with rate-contributing Proton-transfer steps

This chapter reviews the application of an 18 O exchange method to study the catalysis of the hydration of CO 2 by carbonic anhydrase, which, with a maximal turnover number near 10 6 sec –1 , is one of the fastest known enzymic reactions. The catalysis is so rapid that proton transfer steps between the enzyme and its environment, which are not ordinarily rate limiting for slower enzymic reactions, become rate-limiting and more amenable to investigation with carbonic anhydrase. The 18 O exchange method is chosen to investigate this problem because it is an equilibrium method that may be used in the absence of buffer or in the presence of excess buffer concentration, and buffers are sources of protons for transfer to the enzyme. With this method, it is possible to elucidate the role of proton transfer in the pathway of the catalytic hydration of CO 2 . A further result is that the fate of oxygen in the catalysis can be followed; hence, this method reveals the rate of release from the enzyme of water bearing substrate oxygen, when the substrate is 18 O-labeled bicarbonate.

Slow Transients in the Activity of the NAD Malic Enzyme from Crassula

The NAD malic enzyme from Crassula argentea shows a slow reaction transient in the form of a lag before reaching a steady-state rate in assays. This lag, which has a half-time or tau ranging from seconds to many minutes under various conditions, poses problems in the interpretation of kinetic data with this enzyme. The NAD malic enzyme from Kalanchoë daigremontiana has a similar lag.The lag is greatest in freshly prepared enzyme and diminishes with storage at -70 degrees C, but the activity of the enzyme also diminishes with storage.The lag is inversely proportional to the concentration of enzyme, both in the assay and in storage prior to assay. The lag is also inversely proportional to the concentration of malate used in the assay, which poses particular problems because the lag with low malate concentrations may be so long that activity begins to be lost before the steady-state rate is reached.Various buffer ions produce different lags, but the lag with all buffers is longer than in the absence of buffer. The effectors CoA and SO(4) (2-) in the assay substantially decrease the lag. The lag is shorter with Mn(2+) as the required divalent cation than when Mg(2+) is used.The response of enzyme activity to pH shows that the intrinsic activity is greater with magnesium than with manganese, although the rate actually attained is lower with Mg(2+) because the pK values for the response to pH are closer together when that cation is used. The enzyme has a higher optimum pH and a broader response to pH when Mn(2+) is used. The change in lag with pH follows the general pattern of activity with longer lags at intermediate pH values.Preincubation of the enzyme with various reaction components and effectors reduces the lag, with NADH being the most effective. The presence of NADH in the assay is much more effective, but none of the treatments tried will completely eliminate the lag of freshly prepared enzyme.

Reactions of methylmercury(II), mercury(II), and zinc(II) ions with 2-methyl-2-(2-pyridyl)thiazolidine, including stopped-flow rapid-scanning spectrophotometric studies

Equilibrium studies of the reactions of 2-methyl-2-(2-pyridyl)thiazolidine (A) with [HgMeX](X = Cl, MeCO2), HgII and ZnII ions have been investigated by 1H and 13C n.m.r. and by u.v. spectrophotometry, and kinetic studies of the reactions with HgII and ZnII ions by stopped-flow rapid-scanning u.v. spectrophotometry. The n.m.r. results show that opening of the thiazolidine ring occurs with the loss of a proton, to give thiolate-bound Schiff-base chelates. In the absence of buffer, the reaction with [HgMeCl] also gives a second species in which the thiazolidine ring is not opened, and addition of acetate buffer causes this minor species to deprotonate and convert to the ring-opened product. Kinetic studies using HgII reveal a single, very rapid reaction, close to the limits of stopped-flow detection, and approximately first order in [HgII]. Two distinct kinetic stages are apparent in the reaction with zinc(II) perchlorate in methanol solution, the first stage attributed to metal binding and the pH-dependent second stage to the opening of the thiazolidine ring. Using zinc(II) acetate the first stage is too rapid for stopped-flow detection, but the second stage is kinetically simpler because of the buffering effect of the acetate ions.

PH oscillations in transport processt

Oscillations in pH have been observed when a substrate passes through a synthetic membrane containing enzymes which possess the autocatalytic character in pH. In this paper, we present a mathematical model and computations to show the role of buffer on the oscillation. The theory shows that the oscillation strongly depends on the buffer concentration, in agreement with the experimental observation. In addition, we have found that depending on the nature of a buffer, a buffer can either enhance or suppress the oscillation. We have also shown that in the absence of buffer, oscillations in the papain-BAEE system which corresponds to those observed by Naparstek et al. arise only if the diffusion constant of OH − is at least 25 times larger than that measured by the experiment. An explanation for this discrepancy is given in this paper.

Action Pattern and Substrate Specificity of alpha‐Amylase K, a Novel Amylase from a Strain of Bacillus subtilis

AbstractThe action pattern of α‐amylase K on amylopectin, glycogen and a number of starches is reported. It was found that the predominant component of the hydrolysis mixture was maltohexaose under most conditions with varying amounts of maltoheptaose, maltopentaose and maltotriose being present depending on the time of hydrolysis. Michaelis constants and maximum rates of reaction were determined for a number of substrates which indicated that the enzyme was most active against amylopectin. Conditions for large scale industrial use of the enzyme, including high substrate concentration and the absence of buffer from the hydrolysis mixture are discussed. A thin layer chromatographic technique for the rapid separation and identification of the hydrolysis products (up to maltodecaose) is reported.

Effects of Trace Elements on Phosphatase Activity in Soils

AbstractStudies to evaluate the effects on the activity of acid and alkaline phosphatases of 20 trace elements showed that all trace elements inhibit the activity of phosphatases in soils. Results showed that the relative effectiveness of the trace elements in inhibition of phosphatase activity depends on the soil. When the trace elements were compared by using 25 µmole/g soil, the average inhibition of acid phosphatase in three soils showed that Hg(II), As (V), W(VI), and Mo(VI) were the most effective (average inhibition >50%) and that Ba(II), Co(II), and As(III) were the least effective (average inhibition <10%) inhibitors. Other elements that inhibited acid phosphatase activity in soils were: Cu(I), Ag(I), Cd(II), Cu(II), Zn(II), Mn(II), Sn(II), Ni(II), Pb(II), Fe(II), Cr(III), Fe(III), B(III), Al(III), V(IV), and Se(IV); their degrees of effectiveness varied in the three soils used.The most effective inhibitors of alkaline phosphatase activity in soils were Ag(I), Cd(II), V(IV), and As(V). All the trace elements that inhibited acid phosphatase activity also inhibited alkaline phosphatase activity. The order of effectiveness of the trace elements in inhibition of acid phosphatase activity was different from that of inhibition of alkaline phosphatase activity. Generally, the inhibitory effect of trace elements was less in the presence of 2.5 µmole/g soil than in the presence of 25 µmole/g soil. In addition to the effect of trace elements, phosphate ion inhibited acid and alkaline phosphatase activities in soils. Related anions such as NO2‐, NO3‐, Cl‐, and SO42‐ were not inhibitory in the presence and absence of buffer.

The binding of bromophenol blue to aspartate aminotransferase

Several experiments are presented that suggest that the sulfonphthalein dye, bromophenol blue, binds at, or near to, the active site of pig heart extramitochondrial aspartate aminotransferase (EC 2.6.1.1.). The binding is characterized at pH 8.0 by a bathochromic shift in the dye's visible spectrum from 590 to 599 nm. Changes in absorption at 612 nm are used to calculate dissociation constants for the enzyme-dye complex. These constants are a linear function of buffer concentration, and the dissociation constant in the absence of buffer is calculated to be 0.6 × 10 −6 m. Acetate and glutarate are shown to be competitive inhibitors of the dye's binding. However, similar experiments and kinetic data indicate that the substrates, erythro-β-hydroxy- l-aspartate and l-aspartate, displace the dye in an uncompetitive fashion. Direct spectroscopic evidence proves the existence of a ternary complex of bromophenol blue, aspartate aminotransferase, and erythro-β-hydroxy- l-aspartate. Rapid reaction kinetic experiments further show that the dye may be displaced by erythro-β-hydroxy- l-aspartate through formation of this ternary complex. Addition of 1.9 m sucrose to a solution of the aspartate aminotransferase-bromophenol blue complex causes a 2-nm bathochromic shift in the bound dye's visible spectrum; the free dye has a 5-nm bathochromic shift under the same set of conditions. It is concluded that the dye molecule binds through multiple interactions at the protein surface. Such multiple interaction loci can explain the differences observed in the way that substrates and inhibitors displace the dye molecule from the enzyme.

A study of the histidine residues of human carbonic anhydrase C using 270 MHz proton magnetic resonance.

Abstract Nine resonances in the 270 MHz proton magnetic resonance spectrum of human carbonic anhydrase B have been identified with imidazole C (2) protons of histidine residues, six of which are observed to titrate with p K a values in the range 4.7 to 7.4. The behaviour of the nine resonances has been studied in the presence of the inhibitors, iodide, cyanide, acetate, hexacyanochromate, and imidazole. Measurements have also been made of the enzyme in its apo, cobalt, and mono-alkylated forms. Used in conjunction with the crystal structure, these results have enabled the tentative assignment of all nine resonances to particular histidine residues in the amino-acid sequence. Three of the active-site histidines at positions 64, 67, and 200 have low p K a values and cannot be directly linked to the activity of the enzyme. However, the resonances assigned to the three metal-liganding histidines do exhibit changes on anion binding and with pH, which parallel changes in the esterase activity. These results are consistent with the model of an ionizable water molecule bound to the zinc ion. Linewidth measurements of the resonances of the histidine residues on the enzyme surface are used to estimate pseudo-first-order rate constants of the order of 4 × 10 3 s −1 for D + exchange between imidazole N and solvent in the absence of buffer. These rates are observed to increase in the presence of small amounts of the buffers Tris and imidazole.

Comparison of methods of assaying urease activity in soils

Analyses of 16 soils selected to obtain a wide range in properties showed that the results obtained by a buffer method of assaying urease activity were markedly higher than those obtained by a non-buffer method. Both methods gave reproducible results, but the non-buffer method was the more precise, and studies reported showed that it provides a much better index than the buffer method of the ability of soils to hydrolyze urea under natural conditions. The buffer method detects urease activity that does not occur when soils are treated with urea in the absence of buffer. This is a disadvantage if the purpose of the assay is to obtain an index of the ability of soils to hydrolyze fertilizer urea, but is an advantage if the purpose is to detect urease in soils or soil fractions. Use of toluene to inhibit microbial activity in assay of urease activity by the non-buffer method described did not significantly affect the results obtained by this method, but it is decreased the precision of the method.

A study of the histidine residues of human carbonic anhydrase B using 270 MHz proton magnetic resonance

Nine resonances in the 270 MHz proton magnetic resonance spectrum of human carbonic anhydrase B have been identified with imidazole C (2) protons of histidine residues, six of which are observed to titrate with p K a values in the range 4.7 to 7.4. The behaviour of the nine resonances has been studied in the presence of the inhibitors, iodide, cyanide, acetate, hexacyanochromate, and imidazole. Measurements have also been made of the enzyme in its apo, cobalt, and mono-alkylated forms. Used in conjunction with the crystal structure, these results have enabled the tentative assignment of all nine resonances to particular histidine residues in the amino-acid sequence. Three of the active-site histidines at positions 64, 67, and 200 have low p K a values and cannot be directly linked to the activity of the enzyme. However, the resonances assigned to the three metal-liganding histidines do exhibit changes on anion binding and with pH, which parallel changes in the esterase activity. These results are consistent with the model of an ionizable water molecule bound to the zinc ion. Linewidth measurements of the resonances of the histidine residues on the enzyme surface are used to estimate pseudo-first-order rate constants of the order of 4 × 10 3 s −1 for D + exchange between imidazole N and solvent in the absence of buffer. These rates are observed to increase in the presence of small amounts of the buffers Tris and imidazole.

The effect of alkaline earth ions on the co-operativity of the oxygenation of Helix pomatia haemocyanin

1. 1. The most pronounced sigmoidal oxygenation curve is obtained for Helix pomatia haemocyanin, in the absence of buffer, at pH 7·9 in the presence of 6 mM Ca 2+ or 10 mM Sr 2+. Mg 2+ and Ba 2+, on the contrary, have no effect on the co-operativity of the oxygenation. 2. 2. The influence of alkaline earth ions on the stabilization of H. pomatia haemocyanin at pH 7·9 follows a different order: Ca 2+>Mg 2+>Sr 2+ = Ba 2+, suggesting two types of binding sites. The type with the higher affinity could be responsible for the stabilization, the type with the lower affinity for the co-operativity of the oxygen binding. 3. 3. An interpretation is suggested for the co-operative oxygen binding of H. pomatia haemocyanin by postulating a deoxy and an oxy structure in a relaxed and in a tense state. In the relaxed state the transition between the two structures is easy and the oxygen affinity high, in the tense state, stabilized at pH 7·9 by Ca 2+ or Sr 2+, the transition is difficult and the oxygen affinity low.

The urease-catalyzed hydrolyses of some substituted ureas and esters of carbamic acid.

By use of a new technique for the assay of products resulting from hydrolyses catalyzed by urease, the following substituted ureas and esters of carbamic acid have been found to be substrates for urease: methyl-, ethyl-, formyl-, and allylurea, and methyl and ethyl carbamates. The rate–pH profiles have been determined for urea and these substrates, under conditions of enzyme saturation and in the absence of buffer. Variations of rate with substrate concentration have been studied at the optimum pH in each case; approximate Michaelis–Menten kinetics was observed in all cases, but with substrate inhibition for the substituted ureas, not for the carbamates. All of the reactions were inhibited by the NH4+ ions formed.

Reversal mechanisms for the photohydrates of cytosine and its derivatives

1. Dehydration rate constants of the photohydrates of cytosine, cytidine and cytidine monophosphate are presented as a function of pH with a view to elucidating the reaction mechanisms. 2. Photohydrates were produced by ultraviolet irradiation at a high exposure rate at 0° and pH 8–9 in the absence of buffer. These conditions lead to a high degree of reversibility after nearly complete photolysis. 3. Buffers with p K's between 3 and 7 were found to accelerate dehydration of cytosine and cytidine hydrates in the pH range 2–7. 4. The ionization constants of the hydrates were determined by potentiometric titration. 5. A model involving general base catalysis is proposed to account for the observed rate-pH profiles. It agrees well with the experimental results. Rate constants for catalysis by various basic species are presented. 6. Reversal of the hydrate of cytidine monophosphate was found to be insensitive to added buffers. In this case the rate is greatly influenced by the phosphate group which acts as an effective internal catalyst. Our model accounts qualitatively for this effect.

FACTORS AFFECTING UREASE ACTIVITY IN A BLACK SPRUCE HUMUS STERILIZED BY GAMMA RADIATION

Factors affecting the activity of urease were studied in a black spruce (Picea mariana (Mill.) B S P.) humus sampled at Baie-Comeau, Quebec, and sterilized with a Co-60 source. The enzyme was almost completely inactivated by 4–5,000 kr (about four times the sterilization dose). For a given dose, the measured activity was somewhat lower when a lower radiation flux was used with a longer exposure time. A substrate concentration of 50–100 mg/g urea-N was required for maximum rate of hydrolysis. Even at a much lower concentration (3.5 mg/g), hydrolysis was linear during the first 12 hr of reaction at 20 °C. Urease activity was approximately proportional to temperature in the range 10–40 °C. Activity showed a slight decrease as water content was increased from 60 to 140% of maximum holding capacity. In humus buffered at different pH's, the activity was at a maximum at about pH 7.0. In the absence of buffer, however, activity was higher than when buffered at either the initial or the final pH value of the unbuffered system. Activity did not change as buffer concentration in the samples was increased from 0.25 to 1.00 M.

Comparative Radiolability of Amino Acids of Proteins and Free Amino Acids

Despite considerable work in the field of radiation chemistry of amino acids and proteins, the mechanisms of radiation damage to free amino acids and amino acids of proteins are not well understood, and more knowledge of the generalities of comparative radiolabilities of free amino acids and amino acids of proteins is needed. Drake et al. (1) determined the radiolability of amino acids of insulin in pH 3.0 solution and found that cystine, tyrosine, phenylalanine, proline, and histidine were the most radiolabile. Recently, we determined the radiolability of amino acids of cytochrome c, hemoglobin, and ovalbumin at pH 7 (2, 3). Although there is some variation among these proteins, the rank in terms of radiolability of amino acids is methionine, phenylalanine, histidine, cystine, serine, and threonine. However, whether this radiolability of amino acids is also true for other proteins and free amino acids was not ascertained. Our knowledge of the comparative radiolability of free amino acids in solutions is limited to results reported in a few studies. Dale et al. (4) compared the yield of ammonia from various irradiated amino acids with glycine as the reference. However, the extent of deamination of amino acids by radiation did not reveal a definite pattern of radiolability of amino acids. Barron (5) did not obtain ammonia from radiation of dilute solutions of glycine, and similar discrepancies have been observed by others (6, 7). Critical appraisal of observations, by different workers, of the effect of radiation on amino acids in solution is complicated because of several variables. Concentration, solubility and pH of amino acid solutions, presence or absence of buffer, oxygen tension of the medium, and quality and quantity of radiation dose employed often modify the effect of radiation. The purpose of this research was to determine the comparative radiolability of amino acids of proteins and free amino acids.