Covalent binding method

Km reflects the affinity between the enzyme and the substrate, and the Km of immobilized enzyme changes little or much, depending on the interaction between immobilized enzyme and carrier. When enzyme is immobilized using carrier binding, due to the electrostatic interaction between the immobilized enzyme and the carrier. Km of the immobilized enzyme decreases. Maximum reaction rate may differ in terms of fixed methods. The maximum reaction rate of the invertase, immobilized by porous glass using covalent binding method is the same as the free enzyme while the maximum reaction rate of the invertase embedded by... 

The use of electroactive polymers as a DNA immobilization platform or as reporters of DNA hybridization was described in the literature. The immobilization of DNA using a polymer film is very simple, and the adsorption or covalent binding method can be applied simultaneously. The characterization of a biofunctional electroactive polymer, poly(5-hydroxy-1,4-naphthoquinone) (juglone)-co-5-hydroxy-3-thioacetic acid-1,4-naphthoquinone, used for direct... 

To develop a continuous process, the immobilisation of aminoacylase of Aspergillus oryzae by a variety of methods was studied, for example ionic binding to DEAE-Sephadex, covalent binding to iodo-acetyl cellulose and entrapment in polyacrylamide gel. Ionic binding to DEAE-Sephadex was chosen because the method of preparation was easy, activity was high and stable, and regeneration was possible. 

Covalent binding of peptides to polymer surfaces is now a standard method to improve their biocompatibility. The primary amino acid sequence of a peptide can be chosen to mimic the putative... 

Note that in some cases one may follow the time course of covalent E-A formation by equilibrium binding methods (e.g., LC/MS, HPLC, NMR, radioligand incorporation, or spectroscopic methods) rather than by activity measurements. In these cases substrate should also be able to protect the enzyme from inactivation according to Equation (8.7). Likewise a reversible competitive inhibitor should protect the enzyme from covalent modification by a mechanism-based inactivator. In this case the terms. S and Ku in Equation (8.7) would be replaced by [7r] and K respectively, where these terms refer to the concentration and dissociation constant for the reversible inhibitor. 

This process involves the suspension of the biocatalyst in a monomer solution which is polymerized, and the enzymes are entrapped within the polymer lattice during the crosslinking process. This method differs from the covalent binding that the enzyme itself does not bind to the gel matrix. Due to the size of the biomolecule it will not diffuse out of the polymer network but small substrate or product molecules can transfer across or within it to ensure the continuous transformation. For sensing purposes, the polymer matrix can be formed directly on the surface of the fiber, or polymerized onto a transparent support (for instance, glass) that is then coupled to the fiber. The most popular matrices include polyacrylamide (Figure 5), silicone rubber, poly(vinyl alcohol), starch and polyurethane. 

In addition to the covalent binding, some methods derived from bioaffinity chromatography can be used for non covalent attachment of antibodies to a surface by the inactive Fc portion. The advantage is that antigen binding sites stay undamaged and accessible for the analytes due to the orientation of antibody with the active Fab portions towards the tested medium. 

An alternative approach used to investigate the covalent binding of B[a]P to mouse skin has been to release the hydrocarbon-DNA adducts from the isolated DNA by acid hydrolysis (60.61). Since, in the case of BPDE-DNA adducts, they are acid labile and the tetraols produced are not only more fluorescent than the adducts themselves but also more easily extracted from the large excess of unmodified bases, this provides a convenient approach. The method does, however, require the certainty that hydrolysis of the adducts will occur and that the products will be stable or, if degradation occurs, that they can still be recognized. 

This procarcinogen undergoes metabolic conversion to benzo[a]pyrene diol epoxides, BPDEs (5,28-31), which have been the focus of structural and conformational studies by theoretical and experimental methods. These chemically reactive BPDEs are involved in covalent binding to DNA (13-22). 

The crosslinking of polymers in a binary membrane complex via covalent binding is a traditional method for improving the impact resistance of materials. Generally, high molecular crosslinkers have been successful (e.g., dextran dialdehyde... 

Immunochemical methods are valuable because of their sensitivity and specificity. The sensitivity depends on the method used to determine an end point. One of the reaction components may be tagged with radioactivity, or tagged by covalent binding of an enzyme capable of being detected, or by covalent binding of a totally unrelated species (i.e., fluorescein). 

Methods of indicator immobilization in sol-gels include physical and chemical (covalent binding) doping by incorporation of an indicator or reagent during formation of the sol-gel glass. 

Immobilization method Covalent binding Adsorption Ion pair formation Entrapment or ship-in-a-bottle ... 

There have been many reports in which the immobilization method was covalent binding. In fact, many pH indicators used in above reports own at least one active amino or carboxyl group so that they can be covalently bound relatively easily to a solid substrate [165,166], Kostov et al. had discussed the immobilizing process of Congo red, neutral red and phenol... 

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