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Biomedical subjects

B Mattiasson

Publications and source records attributed to B Mattiasson.

At least 127 records · Page 7Linked to original sources
PubMed1990

Interesterification of phosphatidylcholine with lipases in organic media.

Lipases were investigated with respect to their ability to catalyse the incorporation of fatty acids into phosphatidylcholine (PC) by interesterification reactions. The enzymes were dried onto solid support materials and the conversions were carried out in water-saturated toluene. Three lipases (two fungal and one plant enzyme) had the desired activity; immobilized lipase from Mucor miehei (Lipozyme) was the most active enzyme. The Lipozyme-catalysed interesterification was selective for the sn-1 position of PC and during 48 h of reaction around 50% of the fatty acids in this position were replaced with heptadecanoic acid, a fatty acid which was practically absent in the original phospholipid. Due to adsorption on the support material and the competing hydrolysis reaction the total amount of PC in the reaction solution decreased to about 40% of the original amount. Higher interesterification rates were obtained with free fatty acids as acyl donors than with fatty acid esters.

Chromatography, Gas↗
PubMed1990

A dual streaming potential device used as an affinity sensor for monitoring hybridoma cell cultivations.

A dual streaming potential device was used for determining the content of monoclonal antibodies in cultivation medium for hybridoma cells. Samples of culture medium were analyzed as discrete pulses, as a continuous flow of constant concentration as well as with fluctuating concentrations. Tests were done with two subclasses of IgG as well as with IgM. Finally, the analytical device was applied to the registration of production of monoclonal antibodies in a cultivation.

Animals↗
PubMed1990

Enzymatic peptide synthesis in organic media: a comparative study of water-miscible and water-immiscible solvent systems.

Peptide synthesis was carried out in a variety of organic solvents with low contents of water. The enzyme was deposited on the support material, celite, from an aqueous buffer solution. After evaporation of the water the biocatalyst was suspended in the reaction mixtures. The chymotrypsin-catalyzed reaction between Z-Phe-OMe and Leu-NH2 was used as a model reaction. Under the conditions used ([Z-Phe-OMe]0 less than or equal to 40 mM, [Leu-NH2]0/([Z-Phe-OMe]0 = 1.5) the reaction was first order with respect to Z-Phe-OMe. Tris buffer, pH 7.8, was the best buffer to use in the preparation of the biocatalyst. In water-miscible solvents the reaction rate increased with increasing water content, but the final yield of peptide decreased due to the competing hydrolysis of Z-Phe-OMe. Among the water-miscible solvents, acetonitrile was the most suitable, giving 91% yield with 4% (by vol.) water. In water-immiscible solvents the reaction rate and the product distribution were little affected by water additions in the range between 0% and 2% (vol. %) in excess of water saturation. The reaction rates correlated well with the log P values of the solvent. The highest yield (93%) was obtained in ethyl acetate; in this solvent the reaction was also fast. Under most reaction conditions used the reaction product was stable; secondary hydrolysis of the peptide formed was normally negligible. The method presented is a combination of kinetically controlled peptide synthesis (giving high reaction rates) and thermodynamically controlled peptide synthesis (giving stable reaction products).

Chromatography, High Pressure Liquid↗
PubMed1989

Flow-injection binding assays: a way to increase the speed in binding analyses.

A flow-injection analysis system was equipped with a small column containing immobilized concanavalin A. Pulses containing glucosides or glycoproteins were passed over the column, the lectin bound the carbohydrates. By using horseradish peroxidase as a labeled carbohydrate and letting it compete with other glucosides or mannosides a competitive binding assay for the latter was set up. When the enzyme activity had been evaluated, the column was rinsed and reconditioned, allowing a new assay to be run. To speed up the assay, substrates for the enzyme marker, peroxidase, were present in the perfusing buffer. A computerized evaluation of the absorbance peak allowed the time of the assay cycle to be reduced to 70 s. The sensitivity of this binding assay was fully comparable with those reported for other systems using the same reactants.

Antibodies↗
PubMed1988

The influence of water on protease-catalyzed peptide synthesis in acetonitrile/water mixtures.

Protease-catalyzed peptide synthesis in acetonitrile/water mixtures, containing 0-90% water, was investigated. alpha-Chymotrypsin, as well as thermolysin, were deposited on solid supports, prior to exposure to the reaction media. Peptide syntheses were performed using both a kinetically controlled process (chymotrypsin) and an equilibrium-controlled synthesis (thermolysin). The activity of chymotrypsin decreased at low water contents. However, at low water contents (1-10%) hydrolytic side reactions were suppressed and high yields of dipeptides were obtained. Optimal water content for the thermolysin-catalyzed reaction was 4-8%. The dipeptides produced were fully soluble in the reaction mixtures. High operational stability for alpha-chymotrypsin was obtained during 216 h of reaction.

Acetonitriles↗
PubMed1988

Affinity chromatography on a hydrophobic matrix using a heterobifunctional ligand.

Affinity adsorption chromatographic purification using heterobifunctional ligands and a hydrophobic support is described. Soybean trypsin inhibitor was modified with a detergent, with the result that hydrophobic residues were attached to the inhibitor. On passage through an octyl-Sepharose column the inhibitor was bound to the support. The modified inhibitor was still able to bind trypsin either in free solution or after the inhibitor was bound by the support. This procedure forms the basis for a robust affinity purification technique that can be made very general.

Chemical Phenomena↗
PubMed1988

On the importance of the support material for bioorganic synthesis. Influence of water partition between solvent, enzyme and solid support in water-poor reaction media.

alpha-Chymotrypsin was adsorbed on solid support materials and the catalytic activity of the preparations in organic solvents was studied. The activity was highly dependent on the nature of the support material and on the amount of water present in the reaction mixture. There appears to be competition for the water in the system between the enzyme, the support material and the solvent. The support materials were characterized by measuring their ability to absorb water from water-saturated diisopropyl ether. For the quotient: (amount of water on the support)/(amount of water in the solvent) in the model system the term aquaphilicity was proposed. The activity of adsorbed chymotrypsin in diisopropyl ether decreased with increasing aquaphilicity of the support material. The same trend was observed when the activity of horse liver alcohol dehydrogenase adsorbed on different supports was measured in diisopropyl ether.

Acetonitriles↗
PubMed1987

Activity and stability of horse-liver alcohol dehydrogenase in sodium dioctylsulfosuccinate/cyclohexane reverse micelles.

Horse liver alcohol dehydrogenase (EC 1.1.1.1) solubilized in sodium dioctylsulfosuccinate (AOT)/cyclohexane reverse micelles was used for the oxidation of ethanol and reduction of cyclohexanone in a coupled substrate/coenzyme recycling system. The activity of the enzyme was studied as a function of pH and water content. The enzyme was optimally active in microemulsions prepared with buffer of pH around 8. An increase in enzymatic activity was observed as a function of increasing water content. The Km values for the substrates were calculated based on the total reaction volume. The apparent Km for ethanol in reverse micelles was about eight times lower as compared to that in buffer solution, whereas the Km for cyclohexanone was almost unaltered. Storage and operational stability were investigated. It was found that the specific activity of the alcohol dehydrogenase operating in reverse micellar solution was good for at least two weeks. The steroid eticholan-3 beta-ol-17-one was also used as a substrate. In this case the reaction rate was approximately five times higher in a reverse micellar solution than in buffer.

Alcohol Dehydrogenase↗
PubMed1986

Streaming potential--a general affinity sensor.

Electrokinetic phenomena, such as streaming potential or streaming current, have so far been used for studies of unspecific adsorption of ionic compounds on various materials. This paper shows that streaming potential measurements can also be used for studies of biospecific interactions. Several different interactions were studied, e.g. lectin-carbohydrate, IgG-Protein A. Regardless of the type of interaction an increasing change in streaming potential was obtained with increasing concentration of the interacting molecule. The authors also observed a correlation between streaming potential and affinity constants for carbohydrate-induced desorption of Concanavalin A from partially hydrolyzed Sephadex G50. In conclusion, it is shown that streaming potential measurements can be used to study molecular interactions, and to determine concentrations and relative binding constants of interacting molecules.

Antigen-Antibody Complex↗
PubMed1985

A sensitive method to introduce membrane-bound proteins into recipient cells based on affinity enrichment of lipid vesicles to the recipient cell prior to fusion.

A sensitive analytical procedure for studying membrane-bound structures has been developed. Membrane glycoproteins inserted into liposomes were transferred to recipient cells by use of a lectin, concanavalin A, bound to the cells as a bridge to generate proximity between the recipient cell and the glycoprotein-containing liposome, prior to exposure to the fusing agent, poly(ethylene glycol). Partially purified histocompatibility antigen from rats was introduced into the membrane of human lymphocytes. After treating the cells with poly(ethylene glycol) under fusion conditions, some of the antigen present in the preparation could not be eluted with alpha-methyl mannoside and EDTA, indicating that incorporation in the cell membrane had taken place. This antigen remained exposed on the lymphocyte surface for approximately 1 h as demonstrated by sensitivity of the lymphocytes to the lytic effect of an antiserum to the histocompatibility antigen in the presence of complement. Some of the lectin molecules seemed to be internalized in the cells but no induction of cell mitosis was observed. The described method gives an opportunity to work with small amounts of membrane proteins inserted into liposomes, introducing them into recipient cells for analysis of their biological activities.

Animals↗
PubMed1984

Lectin-mediated binding of liposome-inserted membrane proteins to red blood cells. A method to detect binding of antibodies to purified rat histocompatibility antigen or binding of insulin to the insulin receptor.

Partially purified membrane proteins such as the rat RT-1 histocompatibility antigen or the insulin receptor of porcine liver were inserted into liposomes. These liposomes then bound efficiently to Con A-coated red blood cells. After attachment of the liposomes to the cells, cell-liposome conjugates could be separated from free liposomes by centrifugation. Binding of FITC-labeled specific antibodies or insulin to membrane proteins inserted into the liposomes could then be analyzed with a cell flow cytofluorometer. The amount of RT-1 antigen that became associated with the cells depended on the composition and concentration of phospholipids during liposome formation. No association of RT-1 antigen to the cells was obtained in the presence of 50 mM alpha-methyl mannoside. The technique allows detection of micrograms amounts of the histocompatibility antigen associated with the red blood cells. It was possible to detect binding of insulin to cells to which approximately 9 ng (3 X 10(-14) mol) insulin receptor/10(6) cells had been attached. This amount of membrane protein was close to the detection limit of the method.

Animals↗
PubMed1984

Combination of solid-phase second antibody and internal sample attenuator counting techniques. Radioimmunoassay of thyroid-stimulating hormone.

A method is described for the quantitation of free and bound antigen in a new radioimmunoassay which does not require centrifugation or decantation steps. The method involves coupling a second antibody to agarose spheres which also contain entrapped crystals of bismuth oxide (Bi2O3) in order to attenuate the gamma-radiation from the bound fraction of the 125I-labelled antigen. The system is designed so that the samples may be measured in a gamma counter 15 min after the spheres have been added. This new method was compared to a conventional radioimmunoassay for the analysis of the thyroid-stimulating hormone (TSH). Data were obtained with the modified radioimmunoassay which showed both high sensitivity and precision and also had a good correlation (r = 0.951) with data obtained using a conventional method.

Animals↗
PubMed1984

Fractionation of glycoproteins according to lectin affinity and molecular size using a high-performance liquid chromatography system with sequentially coupled columns.

The lectin phytohaemagglutinin was coupled to porous silica (10 micron) and used as adsorbent in a high-performance liquid affinity column sequentially coupled to a TSK-G 3000SW gel permeation column. This system was used for high-speed separation/analysis of human serum glycoproteins according to their lectin affinity and molecular size. Serum samples could be resolved in at least six different peaks representing glycoproteins exhibiting different molecular weights but with a carbohydrate content compatible with the specificity of phytohaemagglutinin.

Chromatography, Affinity↗