Electrochemical consideration on the optimum pH of bilirubin oxidase

Steady-state current-potential curves were obtained for the direct electron transfer (DET) of bilirubin oxidase (BOD) at a highly oriented pyrolytic graphite electrode, and the theoretical analysis based on nonlinear regression enabled us to determine the formal redox potential (E degrees') of BOD in a wide pH range of 2.0 to 8.5. Cyclic voltammetric measurements were also performed for substrates, including p-phenylenediamine (PPD), o-aminophenol (OAP), and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and their E degrees ' values or the anodic peak potentials (for OAP) were determined at various pH values. The difference in the redox potentials between BOD and substrates (DeltaE degrees') showed a maximum at pH 6.5 to 8.0, pH 6.5 to 8.0, and pH 3.5 to 4.5 for PPD, OAP, and ABTS, respectively. These pH ranges should be thermodynamically most favorable for the electron transfer between BOD and the respective substrates. In practice, the pH ranges showing a maximum DeltaE degrees' corresponded well with the optimum pH values for the O(2) reduction activity of BOD: pH 6.5 to 7.5, pH 8.0 to 8.5, and pH 4.0 for PPD, OAP, and ABTS, respectively. Thus, it was suggested that DeltaE degrees ' should be one of the primary factors determining the activity of BOD with the substrates.     

The optimum pH of renal adenosine triphosphatase in rats: influence of vanadate, noradrenaline and potassium

In the presence of vanadate, the optimum pH of renal (Na+, K+)-ATPase in rats is reduced and lies in the range of intracellular pH. This explains the difference in optimum pH observed with ATP extracted from equine muscle. Removal of vanadate from such ATP (with noradrenaline) raises the optimum to the accepted range obtained with synthetic ATP. Changes in the sensitivity of the enzyme to potassium concentration contribute to the alterations in optimum pH. The optimum pH of Mg-ATPase is unaffected by vanadate. Since vanadate may be an intracellular regulator of (Na+, K+)-ATPase changes of optimum pH in relation to intracellular pH could well contribute to the regulation of sodium pump activity.     

Effects of buffers and pH on rabbit red blood cell hexokinase

The kinetic properties of rabbit red blood cell hexokinase in different buffer systems have been studied. At pH 8.0 the reaction velocity (v) is about 30% higher in glycylglycine compared to Tris, Tea, Hepes or ammonium acetate buffers. The enzyme stability, heat-dependence and spectral properties of the enzyme are also affected by the buffer utilized. None of the following kinetic properties of red blood cell hexokinase varies with pH in the range 6.8-8.5: Km of glucose; Km of ATP and Ki of glucose 6-phosphate.     

The effect of buffers and chelators on the reaction of luminol with Fenton's reagent near neutral pH

The chemiluminescence of the system luminol +Fe²⁺ + H₂O₂ was measured in aqueous buffer at pH 7.2. In veronal (5,5-diethybarbiturate) buffer, the luminescence is strongly quenched by ethanol and mannitol, but only weakly by t-butanol, benzoate and superoxide dismutase (SOD); complexing Fe²⁺ with 1,10-phenanthroline or 2,2′-dipyridyl causes a decrease of light production that can be partially obviated by the simultaneous addition of SOD. In phosphate buffer, the luminescence is higher than in veronal and it is efficiently quenched by all four OH · quenchers and by SOD. In Tris buffer, no light production is observed as long as the Fe²⁺ is not complexed. When Fe²⁺ is complexed by pyrophosphate or phytate, there is a strong chemiluminescence in all three buffers, which is quenched by all four OH · quenchers and by SOD. When Fe²⁺ is complexed by EDTA or DTPA, very little luminescence is observed. The luminol analogue phthalhydrazide, which was suggested by Merényi and Lind as a reliable OH · detector, can replace luminol only in phosphate buffer, and thus turns out to be very specific indeed for free OH ·.     

The structure of aconitase

The crystal structure of the 80,000 Da Fe-S enzyme aconitase has been solved and refined at 2.1 A resolution. The protein contains four domains; the first three from the N-terminus are closely associated around the [3Fe-4S] cluster with all three cysteine ligands to the cluster being provided by the third domain. Association of the larger C-terminal domain with the first three domains creates an extensive cleft leading to the Fe-S cluster. Residues from all four domains contribute to the active site region, which is defined by the Fe-S cluster and a bound SO4(2-) ion. This region of the structure contains 4 Arg, 3 His, 3 Ser, 2 Asp, 1 Glu, 3 Asn, and 1 Gln residues, as well as several bound water molecules. Three of these side chains reside on a three-turn 3(10) helix in the first domain. The SO4(2-) ion is bound 9.3 A from the center of the [3Fe-4S] cluster by the side chains of 2 Arg and 1 Gln residues. Each of 3 His side chains in the putative active site is paired with Asp or Glu side chains.     

The influence of pH on arsenazo III.

None of the methods already reported for elimination of pectins from rRNA extracts allowed the complete removal of methylated polysaccharides from methyl-labeled cytoplasmic 17 and 26 S rRNA preparations of sycamore (Acer pseudoplatanus L.) cells. An improved procedure for purifying large amounts of higher plant cytoplasmic rRNA labeled on the methyl groups was investigated. Bulk cellular RNA from sycamore cells incubated for 24 to 36 h with methyl-labeled methionine was extracted at 4°C by the phenol-extraction procedure. Most of the pectic compounds (that accounted for about 30% of the total label of RNA extracts) was selectively precipitated, before the 66% ethanol precipitation of nucleic acid, by bringing the deproteinized aqueous layer to 10% ethanol ?0.15 m sodium acetate. Cytoplasmic rRNA, 17 and 26 S, were isolated by repeated sucrose gradient sedimentations and further chromatographed on a methylated albumin kieselgurh (MAK) column. The old-fashioned MAK chromatography proved to be very useful for elimination of residual pectins, since these compounds eluted in the void volume of the column. This purification procedure gave in a reproducible way cytoplasmic 17 and 26 rRNA virtually free of any labeled DNA, mRNA, plastid rRNA, and pectic compounds.     

Reaction of phenylglyoxal with arginine. The effect of buffers and pH.

Phenylglyoxal reacts much more rapidly with N²-acetylarginine than with either N²-acetyllysine or N-acetylcysteine. The rate of the reaction of phenylglyoxal with either N-acetylarginine or arginine increases with increasing pH from 7.5 to 11.5. The model reaction with arginine is much faster in bicarbonate, diethylamine, or triethylamine buffer than in N-ethylmorpholine, borate, phosphate, or Tris buffer. This activation by various buffers should be taken into consideration when glyoxal derivatives are used to modify arginyl residues.     

The influence of pH and temperature on the properties of myosin

1. The rate of denaturation of myosin solutions at temperatures between 32 degrees and 45 degrees and at pH values between 5.3 and 6.2 has been studied, by using adenosine-triphosphatase activity and solubility in m-potassium chloride at pH6.1 as criteria. 2. Myosin, when heated, loses its adenosine-triphosphatase activity before it becomes insoluble. 3. The loss of adenosine-triphosphatase activity and solubility are both first-order and pH-dependent reactions. Myosin, however, becomes insoluble only when heated within a narrow range of pH values. 4. The thermodynamic functions found for the two processes of denaturation are compared and discussed. 5. The possibility is discussed that, in muscle undergoing rigor, conditions may obtain that would denature myosin.     

The influence of pH on the absorption spectrum of arsenazo III

The absorption spectrum of arsenazo III in media containing K⁺, Mg²⁺ and Ca²⁺ is sharply influenced by pH in the range of 7.5–5.0. The effect of pH is particularly pronounced in the wavelength range 532–602 nm due to the large pH dependence of the dissociation constant of Mg-arsenazo III complex. Therefore absorption changes at these wavelengths during muscle contraction cannot be used as reliable indicators of free ionized Ca²⁺ concentration in the cell. The effect of pH is less pronounced, but still noticeable at the wavelength pairs 575–650 or 660–685 nm.Multiple layers of muscle cells grown on polystyrene coils permit measurement of absorption changes of arsenazo III, introduced into the cells, by equilibration with 0.5 nM arsenazo III under routine culture conditions. The absorbance changes recorded at 660–685 nm are probably related to changes in intracellular free Ca²⁺ concentration.     

The influence of pH on the enzymatic decarboxylation of oxalacetic acid

The pH-activity curve of the oxalacetic carboxylase of Cucurbita pepo seeds which catalyzed the decarboxylation of oxalacetic acid was examined for the possible factors whose variation with pH might be responsible for the shape of the curve between pH 3.5–5.0.

1.

1. It was shown that the shape of the curve remains essentially the same regardless of whether the reactions were conducted in buffers of constant ionic strength or of constant molarity. However, ionic strength had a definite effect on the over-all rate of the enzymatic reaction.     

The influence of pH on the convertogenic activity of plant phenolics

The genotoxicity of plant phenolics, including pyrogallol, gallic acid, resorcinol and catechin, and a water extract and tannin fraction of betel nut (Areca catechu) was examined at pH levels ranging from 5 to 10. Strain D7 of Saccharomyces cerevisiae was used since the cells can withstand a wide range of pH levels without any loss of viability. At alkaline pH ranges, the examined phenolics and betel nut extracts induced mitotic conversion, whereas they lacked this capacity at acid pH levels. This phenomenon may be due to the rapid autoxidation of phenolics under alkaline conditions, which leads to the generation of H2O2 and free radicals. The results indicate that plant phenolics may pose a genotoxic hazard during chewing of lime-containing betel quid and tobacco which causes the salivary pH to rise above 8.