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Biomedical subjects

W H Scouten

Publications and source records attributed to W H Scouten.

At least 19 recordsLinked to original sources
PubMed1992

Evaluation of the mode of binding of immunoglobulin to activated agarose.

Determining the orientation of the immobilization of proteins to solid-phase matrices is of critical importance in the development of systems that employ immobilized proteins. Among these are enzyme-linked immunoassays, immobilized enzymes and affinity chromatography matrices. To determine the orientation of immunoglobulin G (IgG) on activated agaroses, we coupled the immunoglobulin covalently to various activated matrices. The IgG was then cleaved with papain and the liberated fragments collected and analyzed using high-performance liquid chromatography. Only Fab fragments could be detected regardless of the activation method used. This implies that IgG binds to these matrices predominantly via the Fc domain. In order to develop a quantitative method of measuring the Fab and Fc fragments, we compared the binding of IgG and its papain cleavage fragments to S-Zephyr columns and Mono-S columns. Comparison between these columns showed that IgG is bound more tightly to the S-Zephyr column and, in contrast, its retention on Q-Zephyr is less than on a comparable Mono-Q column. The resolution of IgG and its fragments was better in all cases on S-Zephyr than on Mono-S under the conditions employed.

Buffers↗
PubMed1992

Reversible immobilization of antibodies on magnetic beads.

A streptavidin-biotin system was utilized to prepare an antibody-polyadenylic acid conjugate which was subsequently attached to commercially available magnetic beads, Dynabeads oligo(dT)25. Biotinylated polyadenylic acid was combined with streptavidin and the resulting polyadenylic acid-streptavidin was conjugated with an antibody-biotin derivative. The immobilized antibody-polyadenylic acid conjugate was separated from the reaction mixture by hybridization with complementary oligonucleotide immobilized on the surface of Dynabeads oligo(dT)25. The immobilized antibody-polyadenylic acid can be released from the carrier, utilizing low-ionic-strength buffers. The system is intended to be utilized in cell sorting, using immobilized antibodies against cell surface antigens. Dissociation of antibody-containing conjugate from magnetic beads is essential for the isolation of viable cells via positive cell sorting.

Adenosine Monophosphate↗
PubMed1991

Affinity chromatography for protein isolation.

Thousands of reports concerning protein purification have appeared in the past year, and over 150 of these involved, at least in part, the affinity chromatography process. Immobilized membrane affinity chromatography, temperature-programmed elution, and centrifugal affinity chromatography are among the most significant new techniques amid the myriads of applications in this mature field.

Animals↗
PubMed1991

S-Zephyr, a new high performance ion exchange chromatography column matrix.

S-Zephyr, a new column material for high performance cation exchange chromatography of proteins, is compared with Mono-S. The comparison is based on a retentivity study, a model separation of an artificial protein mixture, a sample load capacity experiment and the development of the separation performance at a column overload situation. S-Zephyr is found to be a good matrix for cation exchange chromatography, for analytical separations as well as for small and large scale protein purification applications.

Anion Exchange Resins↗
PubMed1991

Immobilization of perfluoroalkylated enzymes in a biologically active state onto Perflex support.

Perflex has been introduced by E. I. du Pont de Nemours and Co., Inc., as a new fluorocarbon-based technology for protein immobilization. Due to the hydrophobic character of the support, however, significant loss of enzymatic activity may occur upon immobilization of certain enzymes, which appears to be due to a large conformational change of the protein ("inversion"). Pretreatment of the Perflex support with a neutral fluorosurfactant lessened the surface hydrophobicity, thus decreasing the hydrophobic interaction between the support and the protein. Modification of enzymes with a high number of fluorocarbon residues, which forms a hydrophobic "envelope" around the protein, also appears to prevent enzyme inactivation upon immobilization on Perflex support. Moreover, preactivation of the support with either perfluorooctylpropylisocyanate or reactive poly(fluoroalkyl) sugar reagents greatly improves the enzyme particle activity by increasing the amount of immobilized enzyme. Fluorosurfactant treatment of the support activated with perfluorooctylpropylisocyanate improves the retention of activity for sensitive enzymes such as alpha-chymotrypsin and increases the wetability and ease of handling of the Perflex particles.

Adsorption↗
PubMed1990

Centrifugal affinity chromatography.

A new technique termed centrifugal affinity chromatography (CAC) is presented in this paper. CAC combines a high flow-rate, created by centrifugal force, with the specificity of affinity chromatography. This technique has been used for the purification of human immunoglobulin G. Furthermore this technique has been used to remove human albumin from serum and the effect of centrifugal force, ionic strength and pH has been studied. A test for determining the percentage of glycosylated hemoglobin in hemolysates has also been developed. This test, employing centrifugal chromatography, is more than three times faster than commonly used gravity flow methods.

Centrifugation↗
PubMed1990

Dye-ligand centrifugal affinity chromatography.

A fast method for the screening of a large number of immobilized dyes for the purification or binding of proteins called dye-ligand centrifugal affinity chromatography, is described. The ease and speed of this method is demonstrated by screening 65 immobilized dyes for the binding of purified goat IgG. Two immobilized dyes (Drimarene Blue K-R and Drimarene Rubine R/K-5BL) with a high affinity for goat IgG were found to bind specifically the Fc-fragment of the IgG.

Animals↗
PubMed1988

Centrifugal column chromatography for small sample purification.

We evaluated several gel filtration materials for suitability in spun column chromatography a rapid desalting method. The materials were evaluated for resistance to column cracking and to radial shrinkage for sample recovery and for salt separation. A series of graphs can be derived from these evaluations which relates the gel heights to sample volumes for maximum recovery and salt separation while maintaining low sample dilution.

Centrifugation↗
PubMed1986

Sulfonyl chloride activation of hydroxylic materials.

We have discovered that chromophoric sulfonyl chlorides and affinity ligand-containing sulfonyl chlorides prepared based upon fluorinated carbon skeletons are excellent activating agents for agarose. The primary basis for development of such reagents has been pentafluorobenzenesulfonyl chloride and tresyl chloride, both of which are excellent activating agents. Activation using the sulfonyl chlorides whose synthesis is described here has yielded matrices that are very reactive to nucleophilic displacement by amines and thiols and, thus, should be excellent agents for the immobilization of affinity ligands, enzymes, cells, etc. The resulting material is based on covalent coupling and, thus, is more stable than affinity ligands or enzymes immobilized by other methods.

Chemical Phenomena↗
PubMed1978

Fluorescence energy-transfer studies on the pyruvate dehydrogenase complex isolated from Azotobacter vinelandii.

Fluorescence energy transfer has been employed to estimate the minimum distance between each of the active sites of the 4 component enzymes of the pyruvate dehydrogenase multienzyme complex from Azotobacter vinelandii. No energy transfer was seen between thiochrome diphosphate, bound to the pyruvate decarboxylase active site, and the FAD of the lipoamide dehydrogenase active site. Likewise, several fluorescent sulfhydryl labels, which were specifically bound to the lipoyl moiety of lipoyl transacetylase, showed no energy transfer to either the flavin or thiochrome diphosphate. These observations suggest that all the active centers of the complex are quite far apart (greater than or equal to 40 nm), at least during some stages of catalysis. These results do not preclude the possibility that the distances change during catalysis. Several of the fluorescent probes used possessed multiple fluorescent lifetimes, as shown by determination of lifetime averages by both phase and modulation measurements on a phase fluorimeter. These lifetimes are shown to result from multiple factors, not necessarily related to multiple protein conformations.

Acetylcysteine↗
PubMed1977

Lipoamide dehyrogenase immobilized on porous glass.

Lipoamide dehydrogenase (NADH:lipoamide oxidoreductase, EC 1.6.4.3) isolate from pig heart and Escherichia coli was covalently coupled by both diazonium and amide bonds to controlled pore glass beads (96% silica). When the enzyme was immobilized in the presence of NAD+, the enzyme no longer exhibited its normal requirement for NAD+ for full activity. If the immobilized enzyme was then treated with NADase, the requirement for NAD+ was restored. Enzyme immobilized in the absence of NAD+ exhibited normal NAD+ dependence both prior to an after NADase treatment. These results are discussed in terms of co-immobilization of NAD+ at or near the allosteric site of the enzyme.

Allosteric Site↗
PubMed1976

Photooxidation of methionine with immobilized methylene blue as photooxidizer.

Methylene blue immobilized on porous glass beads was used to catalyze the photooxidation of methionine alone and the methionine residues of lysozyme. A solution of 2 mM methionine in 50% acetic acid was oxidized to methionine sulfoxide in the presence of immobilized methylene blue after 6 h of photooxidation at 37 degrees C. Selective photooxidation of the methionyl residues in lysozyme was achieved after 26 h of reaction in 84% acetic acid at 4 degrees C. The specific activity of lysozyme exposed to light in the presence of methylene blue decreased by 94%, while that of a lysozyme solution in the presence of methylene blue not exposed to light decreased by 21%. The lysozyme solution exposed to light but not containing the methylene blue beads lost 33% of its specific activity after the same period of photooxidation. It was shown that the decrease in enzyme activity was not caused by adsorption of the enzyme onto the beads.

Hydrogen-Ion Concentration↗