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PubMed1975

Conformation and spin state in methemoglobin.

The properties of human methemoglobin have been investigated under a wide variety of conditions to determine its conformation and to test for evidence of the T state conformation which has been proposed by Perutz to exist in the presence of high spin ligands and inositol hexaphosphate (IHP). Subunit dissociation was measured as a criterion for the T state since marked differences in the tetramer-dimer equilibrium exist for oxyhemoglobin (R state) and deoxyhemoglobin (T state). In the absence of IHP, complexes of methemoglobin with both high spin ligands (water, fluoride) or low spin ligands (azide, cyanide) show extensive dissociation in 2,2-bis(hydroxymethyl)-2,2',2"-nitriloethanol buffers, pH 6, 0.1 M NaCl, with values of the tetramer-dimer dissociation constant (K4,2) near 10-5 M. The addition of IHP lowers K4,2 to a value near 10-5 M for all forms of methemoglobin. Combination of IHP with methemoglobin promotes a conformational change, but the change is apparently independence of spin state. The conformation acquired in the presence of IHP is not identical with the T state (K4,2 similar to 10-12 M) and can also occur with hemoglobin in the ferrous form, as revealed by a substantial reduction in K4,2 for CO-hemoglobin upon addition of IHP. Subunit dissociation has also been measured using the haptoglobin reaction, since haptoglobin binds only to hemoglobin dimers. The haptoglobin experiments give results that are qualitatively in agreement with the conclusions reached by ultracentrifuge measurements. Similar results are also obtained by estimating the degree of dissociation on the basis of the material which aggregates following mixing with dithionite. The effect of IHP on azide-binding kinetics with methemoglobin has also been examined. Changes in reactivity is observed upon addition of IHP, but the principal effect is observed upon addition of IHP, but the principal effect is an enhancement of the rate of reaction of the beta chains. Changes in the reactivity of the beta93 sulfhydryl group of methemoglobin also accompany addition of IHP, but in a manner which is largely independent of the spin state of the iron. Similar changes are again found with CO-hemoglobin upon addition of IHP. The rate of binding of bromthymol blue also shows some changes upon addition of IHP, but the changes are more pronounced for deoxyhemoglobin than for methemoglobin. Since the results obtained did not appear to indicate a significant role for spin state in the changes observed, additional studies were undertaken using EPR spectroscopy.

Azides↗
PubMed1975

Effect of phospholipase A on the structure and functions of membrane vesicles from Mycobacterium phlei.

The phospholipid composition of the electron transport particles and coupling factor-depleted electron transport particles of Mycobacterium phlei are the same, but they differ in contents. The accessibility of partially purified phospholipase A to these membrane phospholipids was found to be different. Treatment of membranes of Mycobacterium phlei with phospholipase A impairs the rate of oxidation as well as phosphorylation. The inhibition of phosphorylation can be reversed by washing the membranes with defatted bovine serum albumin. The reconstitution of membrane-bound coupling factor-latent ATPase activity to phospholipase A-treated depleted electron transport particles and their capacity to couple phosphorylation to oxidation of substrates remained unaffected after phospholipase A treatment. However, the pH gradient as measured by bromthymol blue was not restored after reconstitution of phospholipase A-treated depleted electron transport particles with membrane-bound coupling factor-latent ATPase. These findings show that the phosphorylation coupled to the oxidation of substrates can take place without a pronounced pH gradient in these membrane vesicles. The dye 1-anilino-8-naphthalene sulfonic acid (ANS) exhibited low levels of energized and nonenergized fluorescence in phospholipase A-treated membranes. This decrease in the level of ANS fluorescence in phospholipase A-treated membranes was found to be directly related to the amount of phospholipids cleaved. The decrease in the energy-dependent ANS response in phospholipase A-treated electron transport particles, as compared with untreated electron transport particles, was shown to be a result of a change in the apparent K-d of the dye-membrane complex, and of a decrease in the number of irreversible or slowly reversible binding sites, with no change in the relative quantum efficiency of the dye. The decrease in ANS fluorescence in phospholipase A-treated particles appears to be due to a decrease in the hydrophobicity of the membranes.

Anilino Naphthalenesulfonates↗
PubMed1985

Determination of chloroquine and its metabolites in urine: a field method based on ion-pair extraction.

A new straightforward photometric method for the assay of the antimalarial drug chloroquine and its metabolites in urine is described. The method involves an ion-pair extraction procedure with dichloromethane using the acid-base indicator bromthymol blue as counter-ion. The ion pair formed with chloroquine in the organic phase is yellow, and absorbance is measured at lambda = 410 nm using a filter photometer. The absorbance is a linear function of concentration up to 400 mumol/l (120 mg/l) chloroquine. The method is suitable for the determination of chloroquine and its metabolites in urine down to a limiting concentration of about 10 mumol/l (3 mg/l). Additionally, the method is suitable for semiquantitative visual estimation of the concentration of chloroquine in urine. A single dose of 5 mg/kg chloroquine base could be determined in urine from two volunteers for at least 8 days after administration of the drug. The results obtained for the analysis of chloroquine and its metabolites with the colorimetric method described here correlate well with those obtained using high performance liquid chromatography.

Chloroquine↗
PubMed1984

[Changes in the quantitative composition of cytochromes and the functional activity of heart mitochondria during ischemia].

Differential cytochrome spectra and their fourth degree derivatives were recorded at 77 degrees K temperature. During myocardial ischemia (2-h autolysis), only cytochrome c content was found to be decreased in isolated mitochondria. According to these data mitochondrial state 3 respiration with succinate decreased only in a medium without cytochrome c. Before ADP addition mitochondrial respiration increased but in a medium with cytochrome c. This was followed by an increase in the respiration rate minimized by bromthymole blue, an inhibitor of dicarboxylate transport. It is inferred that these alterations seen in ischemia are linked with increased permeability of mitochondrial membranes: external for cytochrome c, and internal for inorganic ions and low-molecular compounds.

Animals↗
PubMed1984

[Improved method for isolating Yersinia enterocolitica].

Investigations were carried out to restore and isolate Yersinia enterocolitica from both pure and mixed cultures as well as from sewage waters through enriching and alkaline treatment, employing the method of Aulisio and coll. It was found that the peptone used for the purpose (containing sorbitol, 10.0, yeast extract, 5.0, sodium taurocholate, 2.0, bacto-peptone, 2.0, bacto-tryptone, 2.0, bromthymol blue, 0.007, and 1/15 phosphate buffer, 1000 cm3 at pH = 7) made it possible for Y. enterocolitica to replicate at 4 degrees C in 8 to 10 days and inhibited the development of many other organisms. Enriching the medium at 4 degrees C with the alkaline treatment that followed enabled the isolation of Y. enterocolitica from mixed cultures and sewage waters from milk-processing enterprises in a very high percent of the cases as against enriching at 22 degrees C. Such enriching broth could be used not only for the enriching of Y. enterocolitica at 4 degrees C, but also for the differentiation of pure cultures in culturing at the same temperature.

Bacteriological Techniques↗
PubMed1978

Liposomal membrane. I. Chemical damage of liposomal membranes with functional detergent.

The interaction and reaction between liposomal membrane and a functional detergent, N-hexadecyl-N-(imidazol-4-yl)methyl-N,N-dimethylammonium chloride hydroperchlorate (Im-I), have been investigated in conjunction with the leakage of bromothymol blue encapsulated as a marker in the bilayers of liposomes. Im-I carries an imidazole moiety and was expected to behave as a simple lipase model. The reaction with Im-I significantly enhanced the leakage of bromothymol blue encapsulated in the egg lecithin and dipalmitoyl phosphatidylcholine liposomes. During the course of reaction with Im-I, the formation of acyl-imidazole intermediate was clearly identified, which was certainly connected with the bromothymol blue release. From various kinetic results on bromothymol blue release and acyl-imidazole formation, it has been suggested that the bromothymol blue release from liposomal bilayer may be caused by the local and instantaneous decomposition of lipids when Im-I penetrates into the bilayer. However, it has also been demonstrated that the immediate reconstruction of liposomes retains the barrier function to protect against the further release of bromothymol blue.

Biological Transport↗
PubMed2006

A colorimetric assay for measuring phospholipase A2 degradation of phosphatidylcholine at physiological pH.

Phospholipase A(2) is an important enzyme in various pathologies. Although fluorescent substrate assays for it have been recently developed, there is a need for an assay with inexpensive commercially available substrates, useful when samples interfered with fluorescent assays, that is nonisotopic, continuous, conducted at physiological pH, and in a 96 well format. A reaction using bromothymol blue was developed that meets all these requirements.

Animals↗
PubMed2003

Integration of polymer waveguides for optical detection in microfabricated chemical analysis systems.

Multimode polymer waveguides and fiber-to-waveguide couplers have been integrated with microfluidic channels by use of a single-mask-step procedure, which ensured self-alignment between the optics and the fluidics and allowed a fabrication and packaging time of only one day. Three fabrication procedures for obtaining hermetically sealed channels were investigated, and the spectrally resolved propagation loss (400-900 nm) of the integrated waveguides was determined for all three procedures. Two chemical absorbance cells with optical path lengths of 100 and 1000 microm were furthermore fabricated and characterized in terms of coupling loss, sensitivity, and limit of detection for measurements of the dye bromothymol blue.

Bromthymol Blue↗