Assay of 24,25-dihydroxyvitamin D by competitive protein binding

A competitive protein binding radioassay for 24,25-dihydroxyvitamin D in human serum has been developed, which is relatively simple and rapid. Acetonitrile is used for sample extraction and protein precipitation. column chromatography is then performed in a Sep-pak cartridge. High pressure liquid chromatography follows. The dried eluate is assayed using rat serum as the source of binding protein. Since 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 are equipotent in their competitive displacement of tritiated 25-hydroxyvitamin D3 from at serum, 25-hydroxyvitamin D3 can be used as the assay standard.     

A dye binding method for measurement of total protein in microalgae

Protein is a large component of the standing biomass of algae. The total protein content of algae is difficult to measure because of the problems encountered in extracting all of the protein from the cells. Here we modified an existing protein assay to measure total protein in microalgae cells that involves little or no extraction of protein from the cells. Aliquots of fresh or pretreated cells were spotted onto filter paper strips. After drying, the strips were stained in a 0.1% (w/v) solution of the protein stain Coomassie Brilliant Blue R-250 for 16 to 24 h and then destained. The stained protein spots were cut out from the paper, and dye was eluted in 1% (w/v) sodium dodecyl sulfate (SDS). Absorbance at 600 nm was directly proportional to protein concentration. Cells that were recalcitrant to taking up the dye could be either heated at 80°C for 10 min in 1% SDS or briefly sonicated for 3 min to facilitate penetration of the dye into the cells. Total protein measured in Chlorella vulgaris using this method compared closely with that measured using the total N method. Total protein concentrations were measured successfully in 12 algal species using this dye binding method.     

Assay of cyclic AMP by an isotope dilution test with binding protein

The radioisotope dilution test for cyclic AMP with binding protein from beef muscle can be described mathematically with the assumption that one molecule of binding protein binds one molecule of cyclic AMP. Formulas for the dose response under saturation and nonsaturation conditions are given. Quasilinear plots are calculated from them even under nonsaturation conditions. Good agreement exists between calculated and measured curves.     

The structure and protein binding of amyloid-specific dye reagents

The self-assembling tendency and protein complexation capability of dyes related to Congo red and also some dyes of different structure were compared to explain the mechanism of Congo red binding and the reason for its specific affinity for beta-structure. Complexation with proteins was measured directly and expressed as the number of dye molecules bound to heat-aggregated IgG and to two light chains with different structural stability. Binding of dyes to rabbit antibodies was measured indirectly as the enhancement effect of the dye on immune complex formation. Self-assembling was tested using dynamic light scattering to measure the size of the supramolecular assemblies. In general the results show that the supramolecular form of a dye is the main factor determining its complexation capability. Dyes that in their compact supramolecular organization are ribbon-shaped may adhere to polypeptides of beta-conformation due to the architectural compatibility in this unique structural form. The optimal fit in complexation seems to depend on two contradictory factors involving, on the one hand, the compactness of the non-covalently stabilized supramolecular ligand, and the dynamic character producing its plasticity on the other. As a result, the highest protein binding capability is shown by dyes with a moderate self-assembling tendency, while those arranging into either very rigid or very unstable supramolecular entities are less able to bind.     

Stoichiometry of metachromatic dye binding by deoxyribonucleic acid

Summary DNA is a weak chromotrope and induces less hypsochromic and hypochromic metachromasia in basic dyes. DNA induced metachromasia is also more susceptible to the presence of salts. In the presence of excess polyanion as well as salts, the spectral shift to a shorter wave-length, which is observed when DNA and dye are present in equivalent amounts, does not appear. The compounds of DNA with dye like methylene blue and acridine orange are not stoichiometric generally; DNA and dye form 11 compound only when forced by the presence of excess of dye.C.S.I.R. Junior Research Fellow.     

Human adenosine deaminase binding protein. Assay, purification, and properties

In many human tissues adenosine deaminase exists as a complex composed of two proteins; one protein has adenosine deaminase activity while the other represents a binding protein with no other known binding activity. A rapid, quantitative assay for human adenosine deaminase binding protein has been developed utilizing 125I-labeled calf adenosine deaminase. In addition this binding protein has been purified 1,690-fold from human kidney using adenosine deaminase affinity chromatography and appears to be homogenous by sedimentation equilibrium, sodium dodecyl sulfate, and native polyacrylamide gel electrophoresis. This highly purified binding protein exists as a dimer of native molecular weight 190,000, complexes with calf adenosine deaminase in a ratio of 1:2, respectively, and contains carbohydrate which reacts specifically with phytohemagglutinin and ricin lectins. A second form of this adenosine deaminase binding protein may exist, resulting from degradation of its carbohydrate moiety.     

Increased sensitivity of chitosan determination by a dye binding method

Chitosan is a topic of current research in pharmaceutics, medicine, biotechnology, and beyond. This note describes an improved quantification of chitosan using the dye Cibacron Brilliant Red 3B-A. The method is sensitive and of a good reproducibility and linearity in the range of 10-80 microg/mL.     

Quantitation of dye binding by cell monolayers in a microtiter system

Summary A semi-automated system has been developed for the quantitation of dye binding to cultured eukaryotic cells. It is based on staining precisely controlled numbers of cells seeded into microtiter trays. Cell-bound stain is then released using an appropriate solvent and quantitatedin situ by measuring absorbance in a single beam ELISA reader with an interactive microcomputer link. In order to illustrate potential applications of this approach, the time course of dye—monolayer association and influence of cell number and stain concentration on staining has been examined for four dyes, Crystal Violet, Naphthol Yellow S, Ethyl Green and Pyronin Y. In addition, the effect of sequential and simultaneous staining was examined for Ethyl Green and Pyronin Y. The results provide evidence for the overall reliability of this approach as well as revealing several interesting features in the individual procedures examined. The combination of microtiter technology and computer link make the system particularly well suited to the efficient investigation of the permutations involved in optimizing conditions for a given staining procedure, as well as analysis of the thermodynamics of dye substrate interaction. Overall, the approach is viewed as an intermediate between artificial gel systems and microdensitometry.     

Cationic dye binding by hyaluronate fragments: dependence on hyaluronate chain length

Sodium hyaluronate, digested with bovine testicular hyaluronidase, yielded a mixture of oligosaccharides with identical repeating disaccharide structures and differing molecular weights. The oligosaccharides were separated into a ladder-like series of bands by electrophoresis on a 10% polyacrylamide gel matrix. Coelectrophoresis of purified oligosaccharides has established that adjacent bands differ in chain length by one disaccharide unit. This procedure formed the basis for a rapid screening method in which the binding of cationic dyes by hyaluronate oligosaccharides may be assayed. As a function of chain length, the oligosaccharides showed a marked change in dye binding. Species containing less than seven repeating disaccharide units are not detected by any dye tested, even at very high sample loads. Larger oligosaccharides show an increase in dye binding. The chain length at which constant maximal dye binding is reached depends on the dye structure and solvent conditions, varying from approximately 12 to 30 disaccharide units. The hyaluronate fragments of sufficient chain length to duplicate polymer behavior should be useful models for the study of hyaluronate structure and interactions in solution.     

Partial characterization of nuclear binding sites for retinol delivered by cellular retinol binding protein

Retinol (vitamin A alcohol), which plays an important role in the differentiation of epithelia, can be transferred to chromatin in vitro. Rat liver chromatin can accept retinol in a specific and saturable manner only when the retinol is presented as a complex with cellular retinol-binding protein (CRBP). A partial characterization of the nuclear components responsible for accepting retinol is reported here. A preparation of solubilized chromatin isolated from liver nuclei was able to accept retinol from its complex with CRBP as described previously for nuclei and chromatin. The binding of retinol to chromatin was noncovalent. However, chromatin prepared from nuclei which were incubated with DNase I or micrococcal nuclease did not accept retinol specifically. Chromatin in the form of mono and dinucleosomes also did not accept retinol. However, treatment of nuclei with RNase did not affect the specific binding of retinol. Furthermore, it has been found that retinol was not transferred to purified double or single stranded DNA. These results are interpreted to indicate that the transfer of retinol to specific nuclear binding sites requires a higher order of chromatin structure than that occurring in nucleosome preparations.     

Evidence for ATP binding and double-stranded DNA binding by Escherichia coli RecF protein.

RecF protein is one of the important proteins involved in DNA recombination and repair. RecF protein has been shown to bind single-stranded DNA (ssDNA) in the absence of ATP (T. J. Griffin IV and R. D. Kolodner, J. Bacteriol. 172:6291-6299, 1990; M. V. V. S. Madiraju and A. J. Clark, Nucleic Acids Res. 19:6295-6300, 1991). In the present study, using 8-azido-ATP, a photo-affinity analog of ATP, we show that RecF protein binds ATP and that the binding is specific in the presence of DNA. 8-Azido-ATP photo-cross-linking is stimulated in the presence of DNA (both ssDNA and double-stranded DNA [dsDNA]), suggesting that DNA enhances the affinity of RecF protein for ATP. These data suggest that RecF protein possesses independent ATP- and DNA-binding sites. Further, we find that stable RecF protein-dsDNA complexes are obtained in the presence of ATP or ATP-gamma-S [adenosine-5'-O-(3-thio-triphosphate)]. No other nucleoside triphosphates served as necessary cofactors for dsDNA binding, indicating that RecF is an ATP-dependent dsDNA-binding protein. Since a mutation in a putative phosphate-binding motif of RecF protein results in a recF mutant phenotype (S. J. Sandler, B. Chackerian, J. T. Li, and A. J. Clark, Nucleic Acids Res. 20:839-845, 1992), we suggest on the basis of our data that the interactions of RecF protein with ATP, with dsDNA, or with both are physiologically important for understanding RecF protein function in vivo.     

New insights into the binding interaction of food protein ovalbumin with malachite green dye by hybrid spectroscopic and molecular docking analysis

Communicated by Ramaswamy H. Sarma     

Phosphorylation of CCAAT-enhancer binding protein by protein kinase C attenuates site-selective DNA binding.

Four DNA-recombinant proteins, corresponding to the DNA-binding domain of CCAAT/enhancer binding protein (C/EBP), were phosphorylated in vitro by protein kinase C (PKC). High-performance liquid chromatography-peptide mapping of 32P-labeled C/EBP indicated the presence of three major 32P-labeled peptides: S299 (P)RDK, AKKS277 (P)VDK, and GAAGLPGPGGS248 (P)LK. Phosphorylation of C/EBP by PKC or M-kinase resulted in an attenuation of binding to a 32P-labeled CCAAT oligodeoxynucleotide. Three other truncated forms of C/EBP, C/EBP87, C/EBP87S-C, and C/EBP60, were studied to define the sites of phosphorylation affecting DNA binding. Phosphorylation of the C/EBP87, containing sites Ser299 and Ser277, and C/EBP60, containing only site Ser299, by PKC also resulted in attenuation of DNA binding. In contrast, phosphorylation of C/EBP87S-C, which retained Ser277 but had a Cys in place of Ser299, had no effect on DNA binding. Ser299 could not be phosphorylated by PKC if the protein is already bound to specific DNA. Phosphorylation of intact C/EBP from liver nuclear extract by PKC or M-kinase occurred at Ser299 and Ser277 and at an additional site, as demonstrated by immunoprecipitation and peptide mapping.     

Application of Remazol dye for isolation of protein subunits by preparative SDS-polyacrylamide gel electrophoresis

A practical and simple method for separation and collection of polypeptides in small volumes after polyacrylamide gel electrophoresis is described. Identification of the bands is achieved by prestaining a portion of the protein to be electrophoresed, thus making spectrophotometric monitoring unnecessary. Isolation of protein subunits having quite similar molecular weights has been achieved, and the application of the system to obtaining milligram quantities of globulin subunits from French bean seeds is presented.     

NifI inhibits nitrogenase by competing with Fe protein for binding to the MoFe protein

Reduction of substrate by nitrogenase requires direct electron transfer from the Fe protein to the MoFe protein. Inhibition of nitrogenase activity in Methanococcus maripaludis occurs when the regulatory protein NifI_1,2 binds the MoFe protein. This inhibition is relieved by 2-oxoglutarate. Here we present evidence that NifI_1,2 binding prevents association of the two nitrogenase components. Increasing amounts of Fe protein competed with NifI_1,2, decreasing its inhibitory effect. NifI_1,2 prevented the co-purification of MoFe protein with a mutant form of the Fe protein that forms a stable complex with the MoFe protein, and NifI_1,2 was unable to bind to an -stabilized Fe protein:MoFe protein complex. NifI_1,2 inhibited ATP- and MoFe protein-dependent oxidation of the Fe protein, and 2OG relieved this inhibition. These results support a model where NifI_1,2 competes with the Fe protein for binding to MoFe protein and prevents electron transfer.     

Specificity of the synergistic anion for iron binding by ferric binding protein from Neisseria gonorrhoeae

Ferric binding protein in Neisseria gonorrhoeae (nFbpA) transports iron from outer membrane receptors for host proteins across the periplasm to a permease in an alternative pathway to the use of siderophores in some pathogenic bacteria. Phosphate and nitrilotriacetate, both at pH 8, and vanadate at pH 9 are shown to be synergistic in promoting ferric binding to nFbpA, in contrast to carbonate and sulfate. Interestingly, only phosphate produces the fully closed conformation of nFbpA as defined by native electrophoresis. The role of phosphate was probed by constructing three mutants: Q58E, Q58R, and G140H. The anion and iron binding properties of the Q58E mutant are similar to the wild-type protein, implying that one phosphate oxygen is a hydrogen bond donor and may in part define the specificity of nFbpA for phosphate over sulfate. Phosphate is a weakly synergistic anion in the Q58R and G140H mutants, and these mutants do not form completely closed structures. Ferric binding was investigated by both isothermal titration and differential scanning calorimetry. The apparent affinity of nFbpA for iron in a solution of 30 mM citrate is 1 order of magnitude larger in the presence (K(app)= 1.7 x 10(5) M(-1)) of phosphate than in its absence (K(app) = 1.6 x 10(4) M(-1)) at pH 7. Similar results were obtained at pH 8. This increase in affinity with phosphate as well as the formation of closed structure allows nFbpA to compete for free ferric ions in solution and suggests that ferric binding to nFbpA is regulated by the synergistic phosphate anion at sites of iron uptake.     

Prion protein fate governed by metal binding

The conversion of the normal cellular prion protein to an abnormal isoform is considered to be causal to the prion diseases or transmissible spongiform encephalopathies. The prion protein is a copper binding protein but under some conditions may bind other metals. In particular, the binding of manganese has been suggested to convert the prion protein (PrP) to a protease resistant isoform. Therefore, the differences in the way the protein binds copper and manganese might be revealing in terms of the mechanism of conversion of the protein or its normal cellular activity. We report the use of near-infrared spectroscopy for studies on aqueous solutions of prion protein binding Cu or Mn. These alloforms of the protein were analyzed by spectral data acquisition and multivariate analysis. Our results indicate that PrP binds both Mn and Cu differently. Analyses of Cu binding suggest that the PrP-Cu complex protected Cu from the water increasing protein stability. PrP-Mn does not protect Mn from water interactions. A real-time study of the protein alloforms showed that PrP-Cu remains stable in solution, but that PrP-Mn underwent highly different changes that led to fibril formation.     

Analysis of dye binding by and membrane potential in spores of Bacillus species

Aims:  To determine roles of coats in staining Bacillus subtilis spores, and whether spores have membrane potential.
Methods and Results:  Staining by four dyes and autofluorescence of B. subtilis spores that lack some ( cotE , gerE ) or most ( cotE gerE) coat protein was measured. Wild-type, cotE and gerE spores autofluorescenced and bound dyes, but cotE gerE spores did not autofluorescence and were stained only by two dyes. A membrane potential-sensitive dye DiOC₆(3) bound to dormant Bacillus megaterium and B. subtilis spores. While this binding was abolished by the protonophore FCCP, DiOC₆(3) bound to heat-killed spores, but not to dormant B. subtilis cotE gerE spores. However, DiOC₆(3) bound well to all germinated spores.
Conclusions:  The autofluorescence of dormant B. subtilis spores and the binding of some dyes are due to the coat. There is no membrane potential in dormant Bacillus spores, although membrane potential is generated when spores germinate.
Significance and Impact of the Study:  The elimination of the autofluorescence of B. subtilis spores may allow assessment of the location of low abundance spore proteins using fluorescent reporter technology. The dormant spore's lack of membrane potential may allow tests of spore viability by assessing membrane potential in germinating spores.