Rolf Erik Axel Verner Axen

Preparation of cyanogen bromide-activated agarose gels

Abstract By performing the cyanogen bromide activation of hydroxylic gels (e.g., agarose) in alkaline phosphate solutions of very high buffer capacity, the pH control that has hitherto been necessary can be omitted. Strongly, moderately and weakly activated gels can easily be prepared in a simple and reproducible manner.

Radioimmunosorbent assay for proteins. Chemical couplings of antibodies to insoluble dextran

Abstract The use of immunosorbents for radioimmunoassays of proteins and polypeptides has considerably simplified these methods. The immunosorbents were prepared by chemical coupling of antibodies to derivatives of insoluble dextran (Sephadex). Four coupling methods were compared: three cyanogen halide methods and one isothiocyanato-method. It was concluded that immunosorbents prepared by CNBr-activation of Sephadex are stable for at least three months, have a high antigen binding capacity, have a very small tendency for nonspecific adsorption, and are easiest to prepare in comparison with the other immunosorbents investigated. This method is recommended for the preparation of immunosorbents to be used in the radioimmunosorbent assay system.

[3] Immobilization of enzymes to agar, agarose, and sephadex support

Publisher Summary This chapter discusses the immobilization of enzymes to agar, agarose, and Sephadex supports. From a purely economic point of view, cellulose and starch are perhaps the most attractive starting materials for immobilization. Their chemistry is well understood. In spite of these facts, they have certain disadvantages that make them less suitable for granular immobilized enzymes, the most serious of which are an improper macroporous structure and a susceptibility to microbial disintegration. Dextran in cross-linked form, Sephadex, under certain conditions is superior to cellulose and starch. Enzymes fixed to Sephadex exhibit higher relative activity than the corresponding cellulose-bound enzymes. Presumably, the microenvironment within Sephadex gels is less disruptive for the exertion of catalytic action than in fibrous cellulose with its microcrystalline regions. Starch is more easily attacked by microorganisms than is Sephadex. Agarose has been found to be very well suited as a matrix for the production of biospecific adsorbents, although less extensively purified agar may be a better choice for economic reasons in technical applications.

A theoretical model describing steady-state catalysis by enzymes immobilized in spherical gel particles. Experimental study of α-chymotrypsin-sepharose

Abstract Analytical expressions describing steady-state kinetics are given for systems with enzymes enclosed or covalently bound in spherical gel particles. In experiments using immobilized CT ∗ the rate of product formation was found to be proportional to the square root of the enzyme content at low substrate concentration, and proportional to the enzyme content at high substrate content. These findings are in agreement with the results predicted from the theoretical model. The determination of the turnover number and Michaelis-Menten constant for immobilized enzymes is outlined. Some possible consequences for the description of enzyme kinetics in vivo are discussed.

Covalent fixation of pepsin to agarose derivatives

In recent years the covalent attachment of enzymes to water-insoluble carriers has become fairly routine [ 1,2] . Proteolytic enzymes have received particular attention. Pepsin, however, has presented some difficulty since it is rapidly and irreversibly denatured at the alkaline pH values where most fixation procedures proceed at optimal rate. The only polymer-linked pepsin preparations that have been reported [3-5] are characterized by low catalytic activity or by rather summary washing after the fixation to remove noncovalent bound enzyme molecules. We shall report here the preparation of catalytically active, water-insoluble pepsin conjugates. The enzyme has been attached to amino derivatives of SepharoseR by reaction with an isocyanide and an aldehyde [6]. The insolubilized pepsin is able to digest haemoglobin and bovine serum albumin.

[28] Characterization of immobilized enzymes by chemical methods

Publisher Summary This chapter presents the methods for determining the protein contents of resulting conjugates and ways of assessing the proportion of active enzyme are described. The properties of the final enzyme-carrier conjugate will depend, among other factors, on the number of chemically reactive structures on the carrier. Whereas the total amount of protein bound increases with increasing concentration of reactive groups, the specific activity of the enzyme after immobilization tends to go down with increasing protein contents in the conjugates. Thus, it may be of no great use to maximize the amount of enzyme bound to a gel, as the total activity may increase only fractionally. In other instances—for example, when trying to immobilize subunits of polymeric enzymes, s the concentration of reactive groups in the gel should be low, in order not to bind the polymeric protein by more than one covalent link. In addition, the number of links by which a protein is bound can influence its physical-chemical or enzymic properties—for example, its resistance against denaturation. Methods have been described to determine the concentration of various groups in polymers: carboxyl groups, amino groups, diazo groups, oxirane groups, halogenoacetyl groups, carbonyl groups, sulfhydryl groups, dye-triazine groups, and anhydride groups.