Studies of the Distribution of Proteins Bound to CNBr‐Activated Sepharose 6B at the Electron‐Microscopic Level

Lasch, Jürgen; Iwig, M; Koelsch, R; David, Heinz; Marx, I

doi:10.1111/j.1432-1033.1975.tb20988.x

Cited by 43 publications

(6 citation statements)

References 14 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Early efforts of protein imaging analysis used laborious microtome sectioning of the solid supports [60,61]. Layers thus prepared were then analyzed microscopically.…”

Section: Direct Visualization Of Protein Distribution In Solid-suppormentioning

confidence: 99%

Advanced characterization of immobilized enzymes as heterogeneous biocatalysts

2016

View full text Add to dashboard Cite

“…Early efforts of protein imaging analysis used laborious microtome sectioning of the solid supports [60,61]. Layers thus prepared were then analyzed microscopically.…”

Section: Direct Visualization Of Protein Distribution In Solid-suppormentioning

confidence: 99%

Advanced characterization of immobilized enzymes as heterogeneous biocatalysts

2016

View full text Add to dashboard Cite

“…Recent reports [5][6][7] have presented conflicting evidence concerning the distribution of immobilised ligands throughout the beaded agarose matrix. Therefore we cannot eliminate the explanation that a percentage of the immobilised ligand is substituted on the surface of the bead; the extent of this substitution having a direct bearing on the enzyme-ligand interaction.…”

Section: Resultsmentioning

confidence: 99%

“…Recently the microscopic structure of these polysaccharide matrices has been investigated both with respect to their ability to withstand various activation procedures, in particular cyanogen bromide [4], and also the distribution of the immobilised ligand throughout the matrix following coupling [5,6], which in itself may be a function of the CNBr activation [7]. In a previous communication [4] we reported that beaded cross-linked dextrans were susceptible to structural damage during CNBr activation, whilst beaded agarose was not obviously affected provided that vigorous stirring was avoided.…”

Section: Introductionmentioning

confidence: 99%

The potential of Ultrogel®, an agarose‐polyacrylamide copolymer, as a matrix for affinity chromatography

1976

View full text Add to dashboard Cite

“…Substitution of these variables in equations ( 1 1)- (13) results in the following dimensionless equations:…”

Section: Model Formulationmentioning

confidence: 99%

Distribution of heparinase covalently immobilized to agarose: Experimental and theoretical studies

Bernstein

Yang

Langer

1987

Biotech & Bioengineering

View full text Add to dashboard Cite

An immobilized enzyme reactor has been developed to remove heparin, the anticoagulant that is required in all extracorporeal devices for patients undergoing open-heart surgery or kidney dialysis. The device uses the enzyme heparinase (EC 4.2.2.7), which is covalently linked to agarose with cyanogen bromide. A critical parameter in the development of a model for the degradation of heparin catalyzed by immobilized heparinase is the radial concentration profile of the enzyme within the agarose matrix. Experimental determinations of bound enzyme con centrations have been conducted previously for several enzyme systems using radioactive or fluorescent labels. For the development of the heparinase reactor it is necessary to use catalytically but not electrophoretically pure enzyme, and thus it is not possible to use the labeling techniques. To obtain information about the bound enzyme distribution, an experimental study of the intrinsic binding kinetics of heparinase to cyanogen bromide-activated agarose was conducted. The binding reaction was studied as a function of both the concentration of heparinase and the gel-reactive group. At conditions of functional group excess, the binding kinetics were pseudo first order in heparinase concentration with a rate constant equal to 0.12 C(c[triple chemical bond]n) (h(-1)), where C(c[triple chemical bond]n) is the gel-reactive group concentration. The reactive group concentration remained constant within the 2-4-h experiments. Competitive binding between heparinase and the protein contaminants was unimportant. A model was formulated for the immobilization procedure based on the diffusion of heparinase within the porous network and the binding kinetics as determined above. The model predicted the immobilization of heparinase to be kinetically controlled and the enzyme to distribute uniformly within the agarose matrix. These experimental techniques could be applied to predict the immobilized enzyme distribution for different enzyme systems that are not electrophoretically pure.

show abstract

Studies of the Distribution of Proteins Bound to CNBr‐Activated Sepharose 6B at the Electron‐Microscopic Level

Cited by 43 publications

References 14 publications

Advanced characterization of immobilized enzymes as heterogeneous biocatalysts

Advanced characterization of immobilized enzymes as heterogeneous biocatalysts

The potential of Ultrogel®, an agarose‐polyacrylamide copolymer, as a matrix for affinity chromatography

Distribution of heparinase covalently immobilized to agarose: Experimental and theoretical studies

Contact Info

Product

Resources

About