Enzyme Leakage and Multipoint Attachment of Agarose‐Bound Enzyme Preparations

Abstract

The solvolytic detachment of leucine aminopeptidase from Sepharose-enzyme conjugates with multiple and single anchoring bonds has been studied under a variety of conditions by radiochemical and enzymological methods. The release of the single-point-fixed conjugate could be described by a leakage function, derived previously, yielding the first-order rate constant of the cleavage of the enzyme-matrix bond. The nucleophile hydroxylamine increased the detachment rate considerably. The release of the immobilized enzyme was incomplcte in all experiments even after prolonged times. The enzyme leakage from multipoint-attached conjugates was still high enough to prohibit a long-term use of such preparations in routine work at room temperature.Although the first covalent attachments of biologically active proteins to insoluble carriers date back to the early fifties [1,2], the widespread use of artificially immobilized enzymes had to await the advent of a simple and straightforward method resulting in a high efficiency of binding and the preservation of most of the activity. The introduction of the cyanogen bromide activation of insoluble polysaccharides for the coupling with ligand molecules [3] filled this gap.Recent studies have, however, drawn attention to the fact that this most widely used method suffers from a serious drawback : low-molecular-weight ligands are slowly released from the polymeric support in aqueous surroundings above pH 5 [4-61. This detachment may beashighas 1 %ofthetotalamountofimmobilized ligand per hour, as has been shown with Sepharosebound ~-phenylalanine-4-nitroanilide at pH 7.8 and 25 "C [7]. The chemical mechanism of this relatively facile solvolytic detachment has been elucidated in considerable detail [5], though the surmised anchimeric assistance of neighbouring groups of the carrier gel needs further studies.In the presence of strong nucleophiles the detachment rate is increased considerably so that even multiAbbreviations. Thiol-Sepharose, glutathionine-derivative of Sepharose; Sepharose-aminopeptidase, leucine amino-peptidase covalently bound to Sepharose by a multitude of bonds (CNBr method); Sepharose-S-S-aminopeptidase. leucine amino-peptidase attached to thiol-Sepharose by a disulfide bond. On the other hand it has been demonstrated that derivatives of high stability can be prepared by coupling the multivalent mediators, polylysine, polyornithine or polyvinylamine, to Sepharose [ll].It seems that the possibility of a release of BrCNimmobilized enzymes around the neutral point in the absence of strong nucleophiles has so far been considered negligible and not studied systematically.Some attempts have been made to describe mathematically the leakage of low-molecular-weight ligands from affinity gels [12,13]. However, with immobilized enzymes, where quite a number of heterogeneous bonds between carrier and macromolecular ligand can be anticipated, the assumptions on which the derivations of leakage functions are based will hold only in exceptional cases.So far, the multiplicity and ...

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