A radioisotope method for assays of amylomaltase and D-enzyme

A method has been developed for the assay of amylomaltase based on the incorporation of a [14C]glucose moiety of uniformly 14C-labeled maltose into a maltodextrin fraction insoluble in aqueous ethanol. The presence of dextrin at a high concentration greatly enhances [14C]glucose incorporation and serves to minimize interference with the assay by contaminating enzymes that hydrolyze substrates and products in the assay mixture. Since a number of other enzymes are capable of forming glucose in the assay mixture, the 14C incorporation is a more specific method of enzyme assay than are previously reported assays based on glucose release.     

Interaction of proteins with detergents: Binding of cationic detergents with lysozyme

Binding studies of cationic detergents such as cetyl trimethylammonium bromide, Cetylpyridinium bromide and dodecyl trimethylammonium bromide with lysozyme were carried out by equilibrium dialysis, ultraviolet difference and circular dichroism techniques at 25 C. Binding isotherms at pH 5·0, 7·0 and 9·0 show cooperative binding at all concentrations of detergents and the number of available binding sites in lysozyme increases with pH. Gibbs free energy of binding calculated on the basis of Wymans’ binding potential concept increases with pH indicating increased binding strength at higher pH. The ultraviolet difference spectra of the detergent complexes with lysozyme at pH 7·0 and 9·0 in the region of 250–300 nm indicate the involvement of aromatic amino acid residues as probable binding sites and also the carboxylate groups since the binding is cooperative. The circular dichroism spectra also indicate the involvement of aromatic amino acid residues in the binding of these detergents. This is substantiated by the decrease in the intensity of the aromatic positive bands in the near ultraviolet region. The increase in the magnitude of [θ]_{222 nm} values in the far ultraviolet region with the increase in the concentration of the detergent in the complex indicates conformational changes resulting in an increase of α-helical content producing a more ordered structure of lysozyme.These binding studies show that at pH 7·0 and 9·0, hydrophobic interactions play a major role, while at pH 5·0 only electrostatic interactions play prominent role in the binding of these detergents. Paper presented at the International Symposium on Biomolecular Structure and Interactions held at the Molecular Biophysics Unit, Indian Institute of Science, Bangalore, during 17–22, December 1984.     

A rapid preparative method for isolation of neutral and acidic glycosphingolipids by radial thin-layer chromatography

An efficient method to separate neutral and acidic glycosphingolipids (GSLs) from their mixtures within a short period (45-60 min) and with low consumption of solvents (chloroform-methanol-water, 60/35/8 (v/v/v); 250-500 ml) has been developed. This method utilizes a centrifugal thin-layer chromatograph (Chromatotron) and the GSL mixtures (30-400 mg) are applied to glass plates coated with a 1-mm layer of silica gel 60 PF-254. The method (radial thin-layer chromatography) is rapid and simple and the recovery of glycosphingolipids is high (70-80%).     

A database for the life-cycle assessment of procter &; gamble laundry detergents

A Life-Cycle Inventory (LCI) and Assessment (LCA) database for laundry detergents of the Procter & Gamble Company (P&G) was constructed using SimaPro software. The input data needed to conduct a product LCI came from several different, supporting databases to cover supplier (extraction and manufacturing of raw materials), manufacturing of the detergent product, transportation, packaging, use and disposal stages. Manufacturing, packaging and transportation stages are usually representative of European conditions while the use and disposal stages are country specific and represent how consumers are using a specific product and how wastes are disposed of. The database has been constructed to allow Procter & Gamble managers to analyse detergent products from a system-wide, functional unit point of view in a consistent, transparent and reproducible manner. For demonstrative purpose, a life cycle inventory and a life cycle impact assessment of a P&G laundry detergent used in Belgium is presented. The analysis showed that more than 80% of the energy consumption occurs during the consumer use stage (mainly for heating of the water). Air and solid waste follow the same pattern, most of these being associated with die energy generation for the use stage. More than 98% of the biological oxygen demand, however, is associated with the disposal stage even after accounting for removal during treatment. Future challenges are the completion and/or updating of all detergent ingredient inventories.     

Blood group A glycolipid (Ax) with globo-series structure which is specific for blood group A1 erythrocytes: one of the chemical bases for A1 and A2 distinction

A new blood group A-active glycolipid fraction, termed Ax, showing a chromatographic mobility between Aa and Ab was found in blood group A1 erythrocytes but not in A2 erythrocytes. Ax was identified by its conversion to "globo H" by alpha-N-acetylgalactosaminidase and by 1H-NMR spectroscopy as GalNAc alpha l----3[Fuc alpha l----2]Gal beta l----3GalNAc beta l----3Gal alpha l----4Gal beta l----4Glc beta l----lCer. Globo-H (Fuc alpha l----2Gal beta l----3GalNac beta l----3Gal alpha l----4Gal beta l----4Glc beta l----lCer) was found in blood group A, and O but not in A1 erythrocytes. Thus, one of the A1-specific determinants must be an A determinant carried by globo-series structure.     

Effect of detergents on the alkaline phosphatase of Cuscuta reflexa

The non-ionic detergents, in particular Tween 20, Tween 80 and Triton X100, stimulated the alkaline phosphatase activity of Cuscuta reflexa homogenates with fructose-1,6-diphosphate and β-glycerophosphate as substrates. The order of activation was usually less than 100%, suggesting that a true latency was not involved. A differential response was found towards the two substrates, indicating the existence of two enzyme activities.     

A simple method for preparation of D-rhamnose

A rapid procedure for the preparation of D-rhamnose from bacterial lipopolysaccharide (LPS) has been developed. It involves purification of LPS from Pseudomonas syringae pv. phaseolicola by phenol extraction and hydrophobic interaction chromatography (HIC), followed by mild hydrolysis and cleavage of the O-antigen into D-fucose and D-rhamnose. The monosaccharides were separated by column chromatography, and D-rhamnose recovered after filtration over Sephadex-LH 20.     

A simple technique for eliminating interference by detergents in the Lowry method of protein determination.

The presence of nonionic and cationic detergents interfered in the Lowry method of protein estimation by causing precipitate formation. The addition of 0.5% sodium dodecylsulphate in the alkali reagent prevented this precipitation without affecting colour development, and allowed the method to be used on detergent treated membrane preparations.     

Nitroxide spin labeled analogs of the non-ionic detergent triton X-100

Two nitroxide spin labeled analogs of the non-ionic detergent, Triton X-100, have been synthesized. Electron spin resonance spectra of these compounds in solution, in egg lecithin multilayers, and in aqueous dispersion of dimyristoylphosphatidylcholine vesicles are described.     

A simple method for the separation of bile acid groups by ion-exchange chromatography

A simple ion-exchange chromatographic method for the separation of bile acid mixtures is described. The method employs Dowex 1 as anion exchanger and mixtures of aqueous ethanol and hydrochloric acid as eluants. The use of mixtures in which both the ethanol and acid concentrations are varied has permitted a clear separation of the major bile acid groups (nonconjugated, glycine conjugated, and taurine conjugated bile acids).     

Toxic effects of detergents on the alga Plagioselmis prolonga (Cryptophyta)

The effect of sodium dodecyl sulfate (SDS) and the household synthetic detergents (HSDs) Kristall and Tix (0.1, 1, and 10 mg/l) on cell motility, cell number dynamics, and the growth rate of the alga Plagioselmis prolonga (Cryptophyta) is studied. Algal cell motility proved to be the most sensitive indicator of detergent toxicity. SDS was the least toxic: 1 mg SDS/l caused a short-term loss of motility in 10% of the algal cells. The HSD Tix was the most toxic: only 70% of the cells recovered motility after a 24 h exposure to 1 mg/l. The substances tested in a concentration of 10 mg/l caused mortality of the P. prolonga population. According to their toxic effect on P. prolonga, the investigated toxicants can be arranged as follows: SDS < Kristall < Tix.     

A simple and rapid electrophoretic method for the separation of glucuronic acid and iduronic acid

A new electrophoretic method using Titan III cellulose acetate plates has been developed for the separation and quantitation of glucuronic acid and iduronic acid. This method is quite simple, and glucuronic acid and iduronic acid can be separated within 50 min. This method was applied to the analyses of uronic acids in chondroitin sulfates A and C, and dermatan sulfate.     

A simple method for calculating the productivity of polyphenol separations by polymer-based chromatography

A simple method for calculating the productivity of chromatography processes was proposed based on the iso-resolution curve concept. The model separation system was polyphenol separations by polystyrene divinylbenzene resins with the ethanol–water mixture mobile phase. The distribution coefficient K was determined as a function of ethanol concentration I by linear gradient elution experiments. The HETP-mobile phase velocity u curves were determined as a function of I. Using K and HETP, the iso-resolution curve was calculated, from which the productivity was determined as a function of I. It was found that there is an optimum I, where the highest productivity with the minimum amount of mobile phase consumption is obtained.     

A rapid and simple assay method for UDP-glucose:ceramide glucosyltransferase.

We have developed a simple rapid method for measuring UDP-glucose:ceramide glucosyltransferase; the method utilizes ceramide immobilized on the surface of silica gel and [14C]UDP-glucose as substrate. The reaction product, [14C]glucosylceramide, formed on the surface of the silica gel was easily separated from free [14C]UDP-glucose, either by centrifugation or by filtration. The reliability of this solid phase method was evaluated by using rat brain membrane fraction as an enzyme source. This enzyme had an optimal pH of 6.4-6.5 and required Mn2+, Mg2+ in the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Apparent Km values of 8.7 microM for UDP-glucose and 292 microM for ceramide were determined using the new method. Under the optimal conditions, the solid phase method yielded 2-5-times more product than did the method using micellar system. Moreover, the reaction was highly quantitative in its enzyme dose-activity relationship.