Properties of Bromphenol Blue as an Electron Donor for Higher Plant NADH: Nitrate Reductase

Bromphenol blue, which was reduced with dithionite, was found to support nitrate reduction catalyzed by squash NADH:nitrate reductase at a rate about 5 times greater than NADH with freshly prepared enzyme and 10 times or more with enzyme having been frozen and thawed. Kinetic analysis of bromphenol blue as a substrate for squash nitrate reductase yielded apparent K_m values of 60 micromolar for bromphenol blue at 10 millimolar nitrate and 500 micromolar for nitrate at 0.2 millimolar bromphenol blue. With the same preparation of enzyme the apparent K_m values were 9 micromolar for NADH at 10 millimolar nitrate and 50 micromolar nitrate at 0.1 millimolar NADH. Bromphenol blue was found to be a noncompetitive inhibitor versus NADH with a K_i of 0.3 millimolar. When squash NADH:nitrate reductase activity was inactivated with p-hydroxymercuribenzoate or denatured by heating at 40°C, the bromphenol blue nitrate reductase activity was not lost. These results were taken to indicate that bromphenol blue and NADH donated electrons to nitrate reductase at different sites. When monoclonal antibodies prepared against corn and squash nitrate reductases were used to inhibit the nitrate reductase activities supported by NADH, bromphenol blue, and methyl viologen, differential inhibition was found which tended to indicate that the three electron donors were interacting with the enzyme at different sites. One monoclonal antibody prepared against squash nitrate reductase inhibited all three activities of both corn and squash nitrate reductase. It appears this antibody may bind to a highly conserved antigenic site in the nitrate binding region of the enzyme.     

Seeing gel wells well

Indicator dyes have been incorporated in the stacking gel monomer used in acrylamide gels to render readily visible and delineate the sample wells. Some dyes, such as bromphenol blue, migrate during the electrophoresis, whereas others, such as the ortho-unsubstituted phenol red, become chemically bound to the polymerized gel. The method can be used for agarose gels.     

用染色法鉴别蛋白质晶体

本文报导了蛋白质晶体能被汞溴酚蓝、氨基黑10B和考马斯亮蓝R250或G250染色,而无机盐晶体不被染色.结晶学家可用此法鉴别蛋白质晶体.     

New artificial electron donors for in vitro assay of nitrate reductase isolated from cultured tobacco cells and other organisms

The capacity of bromphenol blue and its analogs to act as electron donors for measurement of in vitro nitrate reductase activity from tobacco cells (Nicotiana tabacum var Techné SP 25 strain) was determined. Competitive inhibition was demonstrated to occur between NADH, the natural electron donor, and bromphenol blue, the artificial electron donor, suggesting that both donors bind to a similar active site on the enzyme. NADH-dependent or bromphenol blue-dependent nitrate reductase activity was carried out by a similar molecular weight protein exhibiting similar antigenic sites. Following ammonium sulfate precipitation, sucrose density gradient and two chromatographic steps, nitrate reductase activity from tobacco cells was purified near homogeneity using bromphenol blue as an electron donor in the absence of measurable NADH-dependent activity. The enzyme is composed of two identical subunits of 83 kilodaltons < ω < 94 kilodaltons.     

Dichromatism of bromphenol blue, with an improvement in the mercuric bromphenol blue technic for protein.

Staining of protein in sections using the mercuric bromphenol blue technic is improved by staining with 1% HgCl2 and 0.05% bromphenol blue in 2% aqueous acetic acid for 15 min at room temperature. Rinse slides 20 min in 2 changes of 0.5% aqueous acetic acid. Blot and give 2 fast changes in absolute ethanol with agitation before transferring to xylene. Transfer slide to 0.5% n-butylamine in xylene for a few seconds until the section is blue, then, after 2 changes of xylene, mount in DPX. Spectrophotometric analysis of this blue dye at different concentrations and with or without heparin showed that the reddish hues are due to dichromatism and not metachromasia.     

CHANGES IN NUCLEAR HISTONES DURING FERTILIZATION, AND EARLY EMBRYONIC DEVELOPMENT IN THE PULMONATE SNAIL, Helix aspersa

Calf thymus histories comprising two fractions, one rich in lysine, the other having roughly equal amounts of lysine and arginine, Loligo testes histones rich in arginine, and salmine, are compared with respect to their amino acid compositions, and their staining properties when the proteins are fixed on filter paper. The three types of basic proteins; somatic, arginine-rich spermatid histones, and protamine can be distinguished on the following basis. Somatic and testicular histones stain with fast green or bromphenol blue under the same conditions used for specific staining of histones in tissue preparations. The former histones lose most or all of their stainability after deamination or acetylation. Staining of the arginine-rich testicular histones remains relatively unaffected by this treatment. Protamines do not stain with fast green after treatment with hot trichloracetic acid, but are stained by bromphenol blue or eosin after treatment with picric acid. These methods provide a means for the characterization of nuclear basic proteins in situ. Their application to the early developmental stages of Helix aspersa show the following: After fertilization the protamine of the sperm is lost, and is replaced by faintly basic histones which differ from adult histones in their inability to bind fast green, and from protamines, by both their inability to bind eosin, and their weakly positive reaction with bromphenol blue. These "cleavage" histones are found in the male and female pronuclei, the early polar body chromosomes, and the nuclei of the cleaving egg and morula stages. During gastrulation, the histone complement reverts to a type as yet indistinguishable from that of adult somatic cells.     

Dichromatism of Bromphenol Blue, with an Improvement in the Mercuric Bromphenol Blue Technic for Protein

Staining of protein in sections using the mercuric bromphenol blue technic is improved by staining with 1% HgCl₂ and 0.05% bromphenol blue in 2% aqueoua acetic acid for 15 min at room temperature. Rinse slides 20 miu in 2 changes of 0.5% aqueous acetic acid. Blot and give 2 fast changes in absolute ethanol with agitation before transferring to xylene. Transfer slide to 0.5% n-butylamine in xylene for a few seconds until the section is blue, then, after 2 changes of xylene, mount in DPX. Spectrophotometric analysis of this blue dye at different concentrations and with or without heparin showed that the reddish hues are due to dichromatism and not metachromasia.     

Sorption of sulfophthaleinic dyes by the brain synaptosomes of rats deprived of parodoxical sleep

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.     

Histology,histochemistry, and emptying mechanism of the venom glands of some elapid snakes

The venom glands of several species of elapid snakes are described. The main venom gland consists of many tubules which usually contain large amounts of secretion product. The accessory gland surrounds the entire venom duct and is usually composed of uniform mucous epithelium. The epithelium lining the tubules of the accessory gland of Naja naja is composed of two distinct types of cells. Histochemical tests indicate that the main venom gland reacts with mercury bromphenol blue and PAS but not with alcian blue. The accessory gland reacts with PAS and alcian blue, and not with mercury bromphenol blue. Treatment of sections with sialidase demonstrates the presence of a sialomucin in the accessory gland. Stimulation of the muscles associated with the venom gland offers an indication of the venom expulsion mechanism of Bungarus caeruleus. A comparison of the venom apparatus of elapid and viperid snakes emphasizes marked differences in the internal anatomy of the venom glands, muscles associated with the gland, and arrangement of glandular components. The morphological differences and dissimilar venom expulsion mechanisms support the recent view of the polyphyletic origin of venomous snakes.     

Salmonella Testing of Pooled Pre-Enrichment Broth Cultures for Screening Multiple Food Samples

A method has been described for testing multiple food samples for Salmonella without loss in sensitivity. The method pools multiple pre-enrichment broth cultures into single enrichment broths. The subsequent stages of the Salmonella analysis are not altered. The method was found applicable to several dry food materials including nonfat dry milk, dried egg albumin, cocoa, cottonseed flour, wheat flour, and shredded coconut. As many as 25 pre-enrichment broth cultures were pooled without apparent loss in the sensitivity of Salmonella detection as compared to individual sample analysis. The procedure offers a simple, yet effective, way to increase sample capacity in the Salmonella testing of foods, particularly where a large proportion of samples ordinarily is negative. It also permits small portions of pre-enrichment broth cultures to be retained for subsequent individual analysis if positive tests are found. Salmonella testing of pooled pre-enrichment broths provides increased consumer protection for a given amount of analytical effort as compared to individual sample analysis.     

Fine structure of endosperm protein bodies inSetaria lutescens (Gramineae)

Summary Endosperm protein bodies are membrane bound. Internally, each body shows a pattern of dark and light concentric layers. A median dense core may also be present. These bodies stain for protein with mercuric bromphenol blue, but not for acid phosphatase.Based on a portion of a dissertation submitted to the Graduate College of Iowa State University in partial fulfillment of the requirements for the Ph. D. degree.     

HPLC-MS/MS determination of a hardly soluble drug in human urine through drug-albumin binding assisted dissolution

ABT-263 is under development for treatment of cancer. In order to support clinical trials, an analytical method for ABT-263 quantification in human urine became necessary. Due to the extremely poor solubility of ABT-263 in aqueous and most common organic solvents, a critical step was to dissolve the drug into urine matrix. Although other potential approaches could be used, addition of powder albumin was found to be the most advantageous. Albumin powder does not significantly alter urine sample volume (相似文献   

The Differentiation of Live From Dead Sperms in Fowl Semen

A combined stain solution is made by dissolving 0.1 gm bromphenol blue and 0.2 gm nigrosin in 100 ml of a M/15 buffer solution of KH₂PO₄ and Na₂HPO₄ adjusted to pH 7.5. This staining solution was used to prepare stained fowl semen smears. Such smears give stable differentiation of live from dead sperms. The dead sperms are stained with a dark violet color while the live ones are not stained.     

Bichromatophilia of rat liver proteins during azo dye carcinogenesis

Summary In the livers of rats fed the azo dye 4-dimethylamino-azobenzene, nucleic acids in connective tissue trabeculae, preneoplastic foci and hepatomas were found to stain intensely with toluidine blue. With the indicator dye bromphenol blue, proteins were observed to stain similarly in these hyperbasophilic tissues but differently from those in surrounding parenchyma.These observations indicate that preneoplastic regions and tumors do not differ from surrounding tissue only by the increased basophilia due to cytoplasmic ribonucleic acid, but also in protein staining. Thus, the change in RNA responsible for hyperbasophila is paralleled by alterations in protein histochemistry. It is suspected that the differential staining of proteins might correspond either to variations in the acidic-basic protein ratios or to the presence of unusual proteins synthesized by such an altered RNA.The tissue similarities in nucleic acid as well as in protein staining observed in the proliferating connective tissue elements and cells undergoing the neoplastic transformation remain an obscure phenomenon.This work was supported by a grant from The Quebec Medical Research Council.     

Direct liquid chromatography method for retinol, alpha- and gamma-tocopherols in rat plasma

An HPLC method for Vitamins A and E in rat plasma has been developed. The main goals of the method are the small amount of sample, 50 microl, and the direct extraction of analytes in one step with acetone, which is a solvent compatible with the reverse-phase mobile phases. Recoveries, as compared with classical and more tedious methods, were near 100%. The method employs a Supelco Discovery C18 column and methanol/water (95:5, v/v) as mobile phase. After being developed, the method was validated following ICH guidelines, with UV, fluorescence and electrochemical detectors. It proved to be selective, lineal, accurate and precise. This method greatly simplifies sample treatment and that is a critical point when working with a large number of samples.     

Unique modification of human heart glycerol 3-phosphate dehydrogenase by blue agarose

The major form of glycerol phosphate dehydrogenase in human heart (GPDH-1) is a minor form (less than 15%) in brain and other tissues and is extremely labile. After GPDH-1 was eluted from an agarose column to which Cibacron blue F3GA had been covalently linked, (a) it was no longer labile (t 1/2 at 40 degrees C changed from 1.6 min to greater than 180 min); (b) it could now be stained for activity on native gels following electro-phoresis; and (c) it now migrated with the bromphenol blue dye front. The results suggest that this stabilized form of GPDH-1 is due to the covalent binding of charged ligands from the column and that this technique may be useful for studying the molecular structure and/or the active site of GPHD-1 and possibly of other enzymes which bind to blue agarose.     

Effect of capping agent on the particle size of CdSe nanoparticles

CdSe nanoparticles were synthesized by green route and chemical route methods. In the green route method the samples were capped by starch and in the chemical route method the samples were capped by mercaptoacetic acid (MAA). The samples were characterized by powder X‐ ray diffraction (XRD) and transmission electron microscopy (TEM). Both the samples showed zinc blend structure. The optical absorption spectra and Fourier transform infrared (FTIR) spectra were also studied. A blue shift was seen in the absorption spectra as compared with the bulk as well as the sample capped by starch. TEM images showed agglomeration for the starch‐capped sample as compared with the MAA‐capped sample. The particle size for the sample capped by MAA was found to be less as compared with the starch‐capped sample. A blue shift in the photoluminescence (PL) spectra was also recorded for the samples prepared by the chemical route as compared with the sample prepared by the green route as well as the bulk. The PL peak shifted towards the red side and increase in the peak intensity occurred with the change in the excitation wavelength. Change in PL intensity was observed with different pH at 685 nm. Copyright © 2016 John Wiley & Sons, Ltd.     

Electrophoretic extraction-concentration of ribonucleic acid from agarose-acrylamide composite gels

A simple technique for the electrophoretic extraction of RNA, under constant monitoring by bromphenol blue line, in a concentrated form from agarose-acrylamide composite gels run according to A. C. Peacock and C. W. Dingman, (Biochemistry 7, 668–674, (1968)) has been developed. Typically, the extraction time from 5 gel slices/tube containing 56 μg of RNA was 3.5 h, and the maximum concentration of the recovered RNA was 0.37 mg/ml with a total recovery of more than 90%. The desired scale can be adjusted by manipulating the number of tubes or number of slices.     

A specific stain for the detection of nonheme iron proteins in polyacrylamide gels.

Nonheme iron proteins can be visualized as blue bands in native polyacrylamide gels using a staining method that is both simple and rapid. The reaction of potassium ferricyanide with protein-bound iron atoms to form royal blue complexes occurs almost instantaneously and is sensitive enough to detect 1 microgram of analytical-grade ferritin and 2 micrograms of purified ferredoxin from cyanobacteria. No special treatment of reagents or apparatus was necessary. On comparison, this stain was found to be more specific than the Ferene S stain, not detecting bovine serum albumin even when present as a hundredfold excess over ferritin. The method was found to be effective for isoelectric focusing gels as well.     

A radioimmunoassay for deoxycorticosterone in human peripheral plasma using sephadex LH-20 chromatography

An accurate, precise, sensitive and relatively simple radioimmunoassay method for the measurement of deoxyocorticosterone (DOC) in human plasma is described. A hexane pre-extraction Is carried out when the sample contains a high progesterone level. A Sephadex LH-20 column (45 × 0.9 cm, in the system dichloromethane:methanol, 98:2) provides adequate purification before radioimmunoassay. However, it has been observed that progesterone inteference is likely in plasma of pregnant females. A specific antibody was generated against DOC-3-oxime coupled to bovine albumin. The Intra and inter assay precision yielded a coefficient of variation of 12.4% for five samples and 27% for 17 samples. The accuracy was checked by measuring recovery of DOC added to pre-extracted plasma (98.5 ± 12.4 (S.D.)%). The sensitivity (10 pg) and blank values (1.5 ng/100 ml) are satisfactory. The normal plasma DOC level obtained (6.4 ± 4.4 ng/100 ml, n = 14) is in agreement with previously reported values.