Showing papers on "Reagent published in 1978"

Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation

Abstract: Large unilamellar and oligolamellar vesicles are formed when an aqueous buffer is introduced into a mixture of phospholipid and organic solvent and the organic solvent is subsequently removed by evaporation under reduced pressure. These vesicles can be made from various lipids or mixtures of lipids and have aqueous volume to lipid ratios that are 30 times higher than sonicated preparations and 4 times higher than multilamellar vesicles. Most importantly, a substantial fraction of the aqueous phase (up to 62% at low salt concentrations) is entrapped within the vesicles, encapsulating even large macromolecular assemblies with high efficiency. Thus, this relatively simple technique has unique advantages for encapsulating valuable water-soluble materials such as drugs, proteins, nucleic acids, and other biochemical reagents. The preparation and properties of the vesicles are described in detail.

Protein thiolation and reversible protein-protein conjugation. N-Succinimidyl 3-(2-pyridyldithio)propionate, a new heterobifunctional reagent

Abstract: A heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio)propionate, was synthesized. Its N-hydroxysuccinimide ester group reacts with amino groups and the 2-pyridyl disulphide structure reacts with aliphatic thiols. A new thiolation procedure for proteins is based on this reagent. The procedure involves two steps. First, 2-pyridyl disulphide structures are introduced into the protein by the reaction of some of its amino groups with the N-hydroxysuccinimide ester sie of the reagent. The protein-bound 2-pyridyl disulphide structures are then reduced with dithiothreitol. This reaction can be carried out without concomitant reduction of native disulphide bonds. The technique has been used for the introduction of thiol groups de novo into ribonuclease, gamma-globulin, alpha-amylase and horseradish peroxidase. N-Succinimidyl 3-(2-pyridyldithio)propionate can also be used for the preparation of protein-protein conjugates. This application is based on the fact that protein-2-pyridyl disulphide derivatives (formed from the reaction of non-thiol proteins with the reagent) react with thiol-containing proteins (with native thiols or thiolated by, for example, the method described above) via thiol-disulphide exchange to form disulphide-linked protein-protein conjugates. This conjugation technique has been used for the preparation of an alpha-amylase-urease, a ribonuclease-albumin and a peroxidase-rabbit anti-(human transferrin) antibody conjugate. The disulphide bridges between the protein molecules can easily be split by reduction or by thiol-disulphide exchange. Thus conjugation is reversible. This has been demonstrated by scission of the ribonuclease-albumin and the alpha-amylase-urease conjugate into their components with dithiothreitol. N-Succinimidyl 3-(2-pyridyldithio)propionate has been prepared in crystalline form, in which state (if protected against humidity) it is stable on storage at room temperature (23 degrees C).

Multilayer film elements for clinical analysis: applications to representative chemical determinations.

Abstract: Using the general concept of a dry multilayer analytical element, we can change chemical procedures and configurations to assay several blood components. In the assay of serum urea nitrogen, urease in the reagent layer catalyzes the hydrolysis of urea. A semipermeable membrane excludes aqueous base, but allows ammonia to diffuse to an underlying indicator layer. For the amylase determination, the enzyme hydrolyzes a dyed-starch substrate coated on top of the spreading layer; this produces small fragments, which diffuse to a registration layer. The increase of absorbance at 540 nm is correlated with amylase activity. Bilirubin complexes with a cationic polymer at the interface between the spreading and reagent layers. The direct reading at 460 nm allows determination of total bilirubin in the range 1 to 500 mg/liter. Tirglycerides are hydrolyzed in the spreading layer, and the resulting soluble glycerol readily diffuses into the reagent layer, where it is phosphorylated and subsequently oxidized by glycerophosphate oxidase to yield dihydroxyacetone phosphate and hydrogen peroxide. Peroxidase catalyzes production of a color commensurate with the hydrogen peroxide produced.

Methodology and measurement of adenylate energy charge ratios in environmental samples

Abstract: A method for measuring ATP, ADP and AMP levels in environmental samples was devised, and applied to seawater and bacterial cell extracts. This procedure is specifically designed for measuring the extremely low concentrations of total adenine nucleotides ([AT]=[ATP]+[ADP]+[AMP]) that are apt to occur in most natural ecosystems (i.e., ≤10 ng AT ml-1 of sample extract). Although the current assay methodology can be used with purified firefly luciferase reagents, it has been suitably modified to accept crude luciferase preparations as well. ATP, ADP and AMP levels have been measured, and the corresponding energy charge (EC) ratios determined for seawater samples collected off the Southern California coast. The EC ratios ranged from 0.50 to 0.89, with peak values corresponding to the subsurface maxima in ATP and chlorophyll a concentrations, and the minimum values corresponding to the deepest water sampled (1500 m). The measurement of adenylate energy charge ratios in environmental samples can be a useful indicator of mean community metabolic activity and potential for cell growth.

Element for analysis of liquids

Abstract: A multilayer analytical element for the analysis of liquids, particularly small samples of biological liquids, having at least two layers including a reagent layer and a registration layer. The reagent layer contains an interactive composition including a nondiffusible material having a preformed detectable moiety, such composition being interactive in the presence of liquid containing an analyte of choice to provide a diffusible product comprising the preformed detectable moiety. The registration layer receives the diffusible product released from the reagent layer. The layers present in the analytical element are composed such that the detectable moiety released from the reagent layer or that remaining unreleased in the reagent layer can be selectively detected in the element. In one embodiment, there is disclosed an integral element which can include a support, preferably radiation-transmissive, on which a registration layer and a reagent layer are carried. Optionally, a spreading or analyte metering layer can be provided in the element adjacent the reagent layer to facilitate delivery of a uniform concentration of analyte to the reagent layer. In a preferred embodiment of this integral element, a radiation-blocking layer, permeable to the diffusible product, can be provided in the element intervening the reagent layer and the registration layer. The radiation-blocking layer, such as an opaque reflecting layer, can enhance the detection of the preformed detectable moiety in the registration layer or in the reagent layer by reflection densitometry or other appropriate radiometric technique. The multilayer element of the invention is particularly useful, for example, in the assay of amylase as well as a variety of other analytes.

Effect of reagent orientation and rotation upon product state distribution in the reaction Sr+HF (v=1,J) →SrF(v′, J′) +H

Abstract: The Letters to the Editor section is subdivided into four categories entitled Communications, Notes, Comments and Errata. The textual material of each Letter is limited to 1200 words minus the following: (a) 200 words for a square figure one-column wide. Larger figures are scaled in proportion to their area. (b) 50 words for each displaved equation; (c) 7 words for each line of table including headings and horizontal rulings. Proof will be sent to authors. See the issue of 1 JulV 1978 for a fuller description of Letters to the Editor.

New Silicon-Phosphorus Reagents in Organic Synthesis. Carbonyl and Conjugate Addition Reactions of Silicon Phosphite Esters and Related Systems

Abstract: The 1,2- and 1,4-addition reactions of organosilicon tervalent phosphorus esters, XlPOSiR3(X = OMe, NMe2, Ph), with saturated and a,@-unsaturated aldehydes and ketones have been studied. These addition reactions have been compared with the complementary reactions of alkyl phosphorus esters, X2POCH3, and R3SiCI with carbonyl substrates. With a,@-un- saturated aldehydes, a judicious choice of reagent and conditions leads to the regioselective 1,2- or 1,4-addition mode. Some of the mechanistic details of these addition reactions have been elucidated.

Methods for the detection and determination of ligands

Abstract: A method for determining the presence of a ligand in a liquid medium which utilizes enzyme labeled avidin and biotin labeled reagent in a specific binding process wherein the ligand to be detected is contacted with an insoluble phase containing specific binding substance for the ligand. Various protocols may be used to assay the resulting enzyme activity which is related to the amount of ligand in the liquid medium.

p-NN'-phenylenebismaleimide, a specific cross-linking agent for F-actin.

Abstract: Covalent cross-links can be inserted between the subunits of F-actin by using p-NN′-phenylenebismaleimide. Cross-linking reaches its maximum value when one molecule of reagent has reacted with each actin subunit. p-NN′-Phenylenebismaleimide reacts initially with a cysteine residue on one subunit, the slower cross-linking reaction involving a lysine residue on a neighbouring subunit. Hydrolysis of the actin-bound reagent limits the extent of cross-linking. Quantitative analysis of the amounts of cross-linked oligomers seen on polyacrylamide gels containing sodium dodecyl sulphate suggests that neither the binding of the reagent to actin nor the formation of cross-links introduces strain into the structure. The cross-links do not join together different F-actin filaments, and evidence is presented that suggests that the cross-links join subunits of the same long-pitched helix.

Extraction of heavy metal ions sorbed on clays

Abstract: The ability of seventeen different chemical solutions to displace heavy metal ions (Pb, Zn, Cu, Cd), pre-adsorbed on clay (kaolinite, illite and montmorillonite) at either pH 5 or 7, has been examined and the relative efficiency of each extractant ascertained. Of the reagents used, only EDTA (0.001 M, pH 7) quantitatively released all four ions from the three clays; oxalic acid (0.1 M, pH 3.3), totally displaced at least three ions from each clay. Other reagents, for example ammonium oxalate (0.1 M), ammonium nitrate (0.01 M), nitric acid (0.1 M) and sodium citrate (0.01 M) effectively displaced one or more heavy metal ions from individual clays. Near quantitative displacement by an excess of Na (0.1 M) or Ca (0.05 M) ions was observed only on montmorillonite. Pre-adsorption at pH 7 was accompanied by precipitation of excess metal ion, and the extraction efficiency in these systems was determined by the ability of the reagent to both dissolve hydrous oxide species and displace sorbed metal ions. The implications of the results with respect to the nature of the adsorption process and relevance to environmental systems have been considered.

Method of making printed reagent test devices

Abstract: A test device for determining the presence of a constituent in a sample, and a method for making it are disclosed. The test device comprises reactants (e.g. reagents, enzymes, etc.) incorporated with a carrier matrix such that when the device is wetted with a test sample, the reactants and the constituent react to produce a detectable response. The reactants are positioned separately from each other on the matrix in substantially, discrete, non-contacting areas. Hence, reactants are maintained substantially separate from each other until the test device is wetted with the sample.

The response of cell walls of Bacillus subtilis to metals and to electron-microscopic stains

Abstract: Purified cell walls of Bacillus subtilis were subjected to solutions of 40 independent metals and the metal uptake, the electron-scattering power of thin sections, and the type of staining response evaluated. This was repeated for six typical electron-microscopic stains (uranyl acetate, uranyl magnesium acetate, osmium tetroxide, Os-meth, osmium-dimethylethylenediamine, and ruthenium red) and one new staining reagent (a potassium platinum chloride - dimethylsulfoxide complex) whose specificity is for amine functions. The reaction of select metals can be specific in terms of both uptake and staining response. Of the metals studied most transition elements had a high affinity for the wall fabric and some (i.e., Sc III, most lanthanides, UIV, ZrIV,HfIV, Fe III, Pd II, Ru III, and In III) may be suitable as contrasting agents for electron microscopy. Furthermore, when the thickness of metal-reacted walls was compared to freeze-each and ultracryotomy data, statistical-dimensional differences were commonly seen, which indicates that wall ultrastructure can be profoundly affected by the type of metal and (or) staining reagent.

Purification and Properties of 5′‐Nucleotidase from Lymphocyte Plasma Membranes

Abstract: 5′-Nucleotidase is purified from lymphocyte plasma membranes by teo affinity chromatog-raphies. The first one, on Lens culinaris lectin-Sepharose 4B yields a fraction of twelve lectin-binding alyvoproteins (lectin-receptor fractin). The second one on 5′-AMP-Sepharose 4B leads to pure enzyme. This enzyme is a glycoprotein with a molecular weigtht of 30000; it gives a single band in polyacrylamide/dodecylsulfte electrophoresis and displays a very high specific activity (2500–3000 μmol Pi h−1 mg−1). Some propertes of purified 5′-nucleotidase are similar to those of membrance-bound enzyme: substrate speciticity, temperature dependence, effects of ions and SH-blocking reagents. Others are completely differnt for the teo systems and these differences result form an interaction between the enzyme molecule and other Lens culinarid lectin binding proteins.