Exploring albumin-sulfonephthalein dyes interactions by a chemometric-assisted and multi-technique equilibria analysis in solution.

Albumin is undoubtedly the most studied protein thanks to its widespread diffusion and biochemistry; despite its binding ability towards different dyes, provoking dye's colour change, has been exploited for decades for quantification purposes, the joint effect of working pH, ionic strength, and dye's pKa still remains only sporadically discussed. In the present study, the interaction of Bovine Serum Albumin (BSA) with five dyes belonging to the sulfonephthalein group, Bromophenol Blue (BPB, pKa = 3.75), Bromocresol Green (BCG, pKa = 4.42), Chlorophenol Red (CPR, pKa = 5.74), Bromocresol Purple (BCP, pKa = 6.05) and Bromothymol Blue (BTB, pKa = 6.72), is investigated at four working pH values (3.5, 6.0, 7.5 and 9.0) and two ionic strength conditions by UV-Vis spectroscopy. Principal Component Analysis is then applied to rationalize dye behavior upon BSA addition at each pH value and to summarize the protein effect on dyes' spectral features, identifying three general behaviors. The most relevant systems are then submitted to further characterization involving a solution equilibria study aimed at determining conditional binding constants for the selected DSA-dye adducts and fluorescence, CD, and 1H NMR spectroscopy to evaluate the binding effect on the species involved.

Ligand binding to natural and modified human serum albumin.

This paper reports evaluation of ligand binding constants for unmodified or biotinylated HSA (HSAB) for two well-known HSA binding ligands, naproxen and bromocresol green. Results demonstrate differential scanning calorimetry (DSC) is a reliable quantitative method for straight-forward and rapid evaluation of ligand binding constants for HSA and modified derivatives. DSC measured the thermodynamic stability of free and ligand-bound HSA and HSAB at pH = 6.0, 7.4 and 8.0. DSC analysis provided a quantitative gauge of responses of HSA and HSAB thermodynamic stability to ligand binding. The influence of different levels of biotinylation of HSAB on ligand binding, and how ligand binding varied as a function of pH for these molecules was also examined. In the three pH environments, biotinylation increased stability of HSAB alone compared to free HSA at pH 7.4. Stabilities of free protein and ligand-bound complexes varied with pH in the order, pH = 6.0>7.4>8.0. Our analytical approach provided very accurate estimates for known binding constants of these ligands for HSA. Results revealed, for both ligands, extent of biotinylation of HSAB affected binding, reducing binding constants from three to 100-fold. DSC analysis was able to delineate inter-relationships between molecular structure and thermodynamic stability of HSA and HSAB bound by ligands; and their variations with pH.

Aqueous extract of broccoli mediated synthesis of CaO nanoparticles and its application in the photocatalytic degradation of bromocrescol green.

CaO nanoparticles have been prepared using CaCl2 and aqueous extract of broccoli as a precursor and reducing agent, respectively. Different volumes of the aqueous broccoli extract were utilised to obtain Ca(OH)2 and subsequent calcination gave CaO nanoparticles. The synthesised CaO was confirmed by powder X-ray diffraction (XRD). The morphology was studied using transmittance electron microscopy (TEM), and the surface composition of Ca(OH)2 was explored using Fourier transform infrared spectroscopy. The major functional groups present in the capping material responsible for the reduction of the metal salt and the surface passivation of Ca(OH)2 were identified. The XRD pattern revealed cubic phase for all the CaO nanoparticles, and the crystallite size was estimated using Scherrer's equation showed a variation which is dependent on the volume of the extract used. TEM analysis showed different shapes, while the selected area electron diffraction (SAED) results confirmed the crystallinity of the nanoparticles. Thermogravimetric analysis of Ca(OH)2 showed the decomposition product to be CaO. Sample C3, which has the smallest particle size, was used as a catalyst for the degradation of bromocresol green via photo irradiation with ultraviolet light and the result revealed a degradation efficiency of 60.1%.

A simple medium modification for isolation, growth and enumeration of Acidithiobacillus thiooxidans (syn. Thiobacillus thiooxidans) from water samples.

High concentrations of H(2)S in groundwater are commonly removed using Biological Trickling Filter (BTF) that contains high numbers of biofilm immobilized sulfur oxidizing bacteria (mainly Thiobacillus thiooxidans). BTF performance requires continuous monitoring of these bacteria at several sampling points. The Most Probable Number (MPN) technique is at the moment the method of choice to enumerate viable T. thiooxidan cells under the above conditions. However, this method is extremely time-consuming (7-10days) and not always suitable for environmental monitoring. In the present study, Thiobacillus agar recommended for isolation and cultivation of Thiobacillus species by Spread plate method was modified by addition of bromocresol green (BCG) in order obtain a clear-cut resolution of the growing colonies resulting in similar or higher numbers compared to other methods. Visual emergence of bacterial colonies on the 3rd and 4th days, from the initial plating, was associated with sulfuric acid production, resulting in an unambiguous color change from blue to yellow, around each colony. This study revealed that BCG modified Thiobacillus agar is substantially time saving and much easier to infer compared to MPN technique.

Triphenylmethane dyes, an alternative for mediated electronic transfer systems in glucose oxidase biofuel cells.

The bioelectrochemical behavior of three triphenylmethane (TPM) dyes commonly used as pH indicators, and their application in mediated electron transfer systems for glucose oxidase bioanodes in biofuel cells was investigated. Bromophenol Blue, Bromothymol Blue, Bromocresol Green were compared bioelectrochemically against two widely used mediators, benzoquinone and ferrocene carboxy aldehyde. Biochemical studies were performed in terms of enzymatic oxidation, enzyme affinity, catalytic efficiency and co-factor regeneration. The different features of the TPM dyes as mediators are determined by the characteristics in the oxidation/reduction processes studied electrochemically. The reversibility of the oxidation/reduction processes was also established through the dependence of the voltammetric peaks with the sweep rates. All three dyes showed good performances compared to the FA and BQ when evaluated in a half enzymatic fuel cell. Potentiodynamic and power response experiments showed maxima power densities of 32.8 µW cm(-2) for ferrocene carboxy aldehyde followed by similar values obtained for TPM dyes around 30 µW cm(-2) using glucose and mediator concentrations of 10 mmol L(-1) and 1.0 mmol L(-1), respectively. Since no mediator consumption was observed during the bioelectrochemical process, and also good redox re-cycled processes were achieved, the use of triphenylmethane dyes is considered to be promising compared to other mediated systems used with glucose oxidase bioanodes and/or biofuel cells.

Applications of a microplate reader in yeast physiology research.

Microplate readers have been useful assistants of researchers for several decades. This work is focused on the applications of a simple absorbance microplate reader in yeast physiology research, and its advantages and limitations in comparison with alternative methods are discussed. The two main procedures involved are measuring growth curves and monitoring the pH changes of medium using two different pH indicators. We suggest mathematical formulas for converting absorbance data into pH values. With a microplate reader as many as 96 samples can be simultaneously analyzed, while medium consumption is minimized to 100 microL per sample. The results can be observed in 24-48 h (for growth curves) or in 1-3 h (for pH changes) with minimal hands-on time required.

Effects of minimally invasive puncture and drainage of intracranial hematoma on the blood-brain barrier in patients with cerebral hemorrhage.

The effects of minimally invasive surgery on the blood-brain barrier (BBB) of 30 patients with cerebral hemorrhage were investigated. Difference of the BBB index and serum MBP concentration were assessed in 15 cases of conservative treatment group and 15 cases of minimally invasive surgery group. The BBB index in minimally invasive surgery group was significantly lower than in conservative treatment group (P<0.05), and the BBB index in the two treatment groups was significantly higher than in control group (P<0.01). Serum MBP concentration in minimally invasive surgery group was significantly lower than in conservative treatment group (P<0.05), and that in the two treatment groups was significantly higher than in control group (P<0.01). It was suggested the permeability of BBB in patients with cerebral hemorrhage was increased, and BBB index and serum MBP concentration in patients with cerebral hemorrhage were increased. Minimally invasive surgery can reduce the lesion of cytotoxicity to BBB and cerebral edema.

Interaction of bromocresol green with different serum albumins studied by fluorescence quenching.

The binding of bromocresol green to bovine serum albumin at micromolar concentrations leads to quenching of protein fluorescence. This property has been used here to study interaction of bromocresol green with bovine serum albumin as a function of pH and ionic strength. The transformation of fluorescence quench data obtained with bromocresol green into Scatchard plots yielded an association constant of 3.06 x 10(7) 1 M-1 and a binding capacity of about 1.0. The affinity of bromocresol green for bovine serum albumin remains virtually unchanged between pH 4.0 and 8.0 but decreases by about 7 fold with increase in ionic strength from 0.01 to 1.0. Six other serum albumins obtained from cat, dog, human, pig and sheep have also been studied for bromocresol green binding. Although all the albumins studied bind bromocresol green, they show considerable differences in their affinities towards the dye. It appears that despite a great degree of overall similarity in their structure and conformation, serum albumins from different species differ in their ligand binding properties.

Bromcresol green assay is nonspecific for rat plasma albumin.

Bromcresol green (BCG) assay has been used in microvascular studies to determine albumin in rat plasma. The purpose of this study was to demonstrate that BCG overestimates rat plasma albumin partly because BCG binds to transferrin, a beta-globulin. The light absorbance of a transferrin-BCG reagent solution is shown to increase with time; appreciable binding occurs within a few seconds. Pure transferrin produced BCG assay results (P less than 0.001) that could be expressed as pseudoalbumin concentrations. Albumin and transferrin solutions of equal concentrations were mixed in equal parts; a plot of albumin concentration determined by BCG vs. actual albumin concentration had a slope greater than the expected value of 1.0 (P less than 0.001). Plasma samples were obtained from six rats and assayed for albumin. Electrophoresis yielded a plasma albumin of 2.97 +/- 0.11 g/dl, whereas BCG assay yielded a significantly (P less than 0.01) greater value of 3.58 +/- 0.07 g/dl. We conclude that BCG assay estimates of albumin-to-globulin ratio (A/G), in which G is determined from the difference between total protein and albumin, are especially subject to error.

Effects of estrone on the biliary excretion of xenobiotics.

The effect on the biliary excretion of exogenous organic anions and the mechanism of action of estrone has been studied. Female Wistar rats weighing 180-200 g were pretreated with estrone (2.5 mg/kg ip daily for 4 days). This produced a significant decrease in the biliary flow and biliary excretion of bromcresol green and amaranth. Administration of taurocholate did not abolish the depressing effect of estrone; however, phenobarbital given in combination with estrone decreased or prevented the effects of estrone on the biliary excretion rate of bromcresol green and amaranth. These results indicate that changes in the bile salt independent fraction might be responsible for the effects of estrone on the biliary flow and biliary excretion of xenobiotics.

Biliary excretion of organic anions in diabetic rats.

Little definitive data are available concerning the effects of insulin deficiency on the hepatic uptake and biliary excretion of endogenous or xenobiotic substances. To expand our understanding of this area, male Sprague-Dawley rats were pretreated with streptozotocin (45 mg/kg i.v.) to induce uncontrolled diabetes. Four to five weeks later, diabetic rats exhibited elevations in serum glucose (640 +/- 13 mg/dl), biliary glucose (307 +/- 35 mg/dl), urine output (166 +/- 11 ml/24 hr), basal bile flow rate (73 +/- 2 microliter/min/kg), liver weight/body weight ratio and bile acid pool size. Polyphagia and generalized muscle atrophy were also evident. Plasma disappearance and biliary excretion of several organic anions were studied after i.v. administration. There were no differences between control and diabetic rats in the plasma elimination and biliary excretion of eosin, phenol-3,6-dibromphthalein disulfonate and sulfobromophthalein. Although hepatic uptake was unchanged, the biliary excretion of amaranth was decreased 30% in diabetic rats. There were no differences in bile flow rate in control or diabetic rats after administration of these four anions. In contrast, administration of indocyanine green, bromcresol green and rose bengal did not depress bile flow in diabetic rats as was observed in control rats. In addition, the rate of maximal biliary excretion was increased by 390, 240 and 151% for rose bengal, indocyanine green and bromcresol green, respectively. Plasma clearance of rose bengal was 65% higher in diabetic rats. Total body clearance and steady-state volume of distribution values for all other anions were not different after induction of diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)

Underestimation of albumin content by bromocresol green, induced by drug displacers and uremia.

Phenylbutazone and clofibric acid, two drugs strongly bound to human albumin, produce low readings of albumin content in serum when the bromocresol green immediate reaction is used. This abnormality is observed at drug concentrations within the range obtained during therapeutic use, and tends to be more marked in diluted samples of serum. Abnormally low values of albumin content are also obtained when the bromocresol green method is used in uremic sera, and the disparity seems related to the degree of carbamylation of these samples. The reported interferences are great enough in some cases as to suggest that the use of the immediate reaction between bromocresol green and serum should not be considered a valid measure of albumin content when these factors cannot be totally excluded.

[Sorption of sulfophthaleinic dyes by the brain synaptosomes of rats deprived of parodoxical sleep]. / Sorbtsiia sul'foftaleinovykh krasitelei sinaptosomami golovnogo mozga krys, lishennykh paradoksal'noi fazy sna.

The influence of paradoxical sleep deprivation on sorption of bromphenol blue, bromcresol green and bromthymol blue by rat's brain synaptosomes was studied. Effect of sleep disturbance (increase in the number of dye bindings) was shown to augment with the increase in hydrophobicity of the sulfophtaleinic dye.

A study of the interaction between bromocresol green dye and bovine, ovine and equine serum globulins.

In human medicine it has been shown that the bromocresol green (BCG) dye-binding method for the determination of serum albumin is not entirely specific, the dye reacting also with certain human serum globulins. This causes over-estimation of albumin when reaction times are prolonged beyond 30 seconds. In the present study, serum albumin values obtained from three animal species by the "immediate", i.e. less than 30 seconds, BCG reaction were compared with those by the 10-minute BCG reaction. Albumin-depleted sera were prepared using an affinity chromatography technique and their reactions and those of purified gamma globulin preparations with the dye were studied. In cattle, sheep and horses, serum albumin values obtained by the 10-minute reaction were higher than those obtained by the "immediate" BCG reaction, the differences being statistically significant. Purified gamma globulin did not react with the BCG dye after 10 minutes, but other globulins did. There were differences between the species in the magnitude of the reaction of their globulins with BCG dye.

[Selective, time dependent accumulation of the triphenylmethane dyes bromphenol blue, bromoresol green and iodophenol blue in mouse tumors]. / Uber eine selektive, zeitabhängige Anreicherung der Triphenylmethanfarbstoffe Bromphenolblau, Bromkresolgrün und Jodphenolblau in Mäusetumoren.

1. A selective late dye concentration dependent on the time is described for 3 triphenylmethane dyes namely bromphenol blue, bromcresol green and iodophenol blue after intravenous application in high dosage in malignant inoculated tumors and experimental tumor metastases in the mouse. 2. The possible mechanisms of this dye concentration phenomenon in the tumor tissue as well as some chances of the eventual therapeutic and tumor diagnostic utilization were discussed.

Inverse dependence of binding constants upon albumin concentration. Results for L-tryptophan and three anionic dyes.

The interactions of methyl orange, bromocresol green, 2-(4'-hydroxybenzeneneazo)benzoic acid (HABA) and L-tryptophan with human albumin at pH 7.4 were investigated by equilibrium dialysis at 37 degrees C. Binding characteristics of each of the three dyes were studied by two approaches: (1) variation of total ligand concentration with a single albumin concentration and (2) variation of albumin concentration with a single total ligand concentration. Both approaches gave typical Scatchard plots with negative slope for methyl orange and bromocresol green, with good agreement between the two sets of data for each dye. In contrast, approach (2) gave Scatchard plots with a positive slope for HABA and L-tryptophan, indicating a decrease in the number of binding sites (n) and/or association constant (k) as the albumin concentration increased. This inverse dependence of nk upon albumin concentration for 2-(4'-hydroxybenzeneazo)benzoic acid was mainly due to changes in n which were still observed in the presence of inhibitory chloride ions at pH 5.75. Reasons for this type of binding behaviour are discussed together with general implications for binding studies. The results show 2-(4'-hydroxybenzeneazo)benzoic acid to be a useful ligand for investigation of this problem.

[Primary selection method for microorganism producers of organic acids]. / Metod pervichnogo otbora mikroorganizmov-produtsentov organicheskikh kislot.

A method is suggested for primary selection of microorganisms producing organic acids. The methods is based on the use of diagnostic media containing the indicator bromocresol green. When yeast fungi of various taxonomic groups were grown in indicator media, 49 yeast cultures producing organic acids were found. Among these, 38 cultures accumulated from 5.0 to 30.0 mg% of citric acid when grown in a medium containing n-paraffins.

Effects of barbiturates on the hepatic cytochrome P-450 dependent enzyme system and biliary excretion of exogenous organic anions in rats.

Effect of pretreatment (150 mumol/kg i.p. twice daily for 5 days) wtih six barbiturates (barbital, butobarbital, pentobarbital, amobarbital, phenobarbital and thiopental) on the hepatic microsomal drug methabolizing enzyme system and biliary excretion of bromcresol green and bromsulphthalein-glutathione conjugate has been investigated in rats. All six barburates produced microsomal induction. Phenobarbital, thiopental, barbital and butobarbital increased liver weight, biliary flow and biliary excretion of bromcresol green and bromsulphthalein-glutathione conjugate, however, pentobarbital and amobarbital were ineffective in these parameters. Our results seem to indicate that the enhanced biliary excretion of exogenous anions produced by barbiturates is independent of microsomal enzyme induction.