Enzyme spacer effect

With Manfred FrOhler, Walter Dasch, Werner Knittl, Hans Lansky, and Lothar Mayring. Influence of Spacers between Matrix and Protein on the Activity and Stability of Enzymes and Effect of Oligomerisation . Proceedings of lUPAC 26th Internat. Sympos. on Macromolecules, Mainz (September 1979), Vol. Ill, pp. 1563-1566. 

The immobilized enzyme should undergo strong deformation, especially in the lower surface concentration region without spacer (Figure 8A), whereas the immobilized GOD molecule widi spacer (Figure 8B) must be protected from the structure deformation even in the lower si ace concentration region owing to the spacer effect... 

Jervis used porous silica coated with chemisorbed polyacrylhydrazide for immobilization of adenosine monophosphate (AMP) [117]. After periodate oxidation of its ribose residue the ligand was coupled to the carrier and used for isolation of lactate dehydrogenase from rabbit muscle. The specific capacity was 2 mg of protein/g adsorbent with a ligand content of 10 pmol/g, whereas recovery of enzymatic activity after elution was 85%. Hipwell et al. [118] found that for effective binding of lactate dehydrogenases on AMP-o-aminoalkyl-Sepharose the spacer arm length required at least 4 methylene links. Apparently, a macromolecule of polyacrylhydrazide acts itself like an extended spacer arm and thus allow AMP to bind the enzyme. 

Example 1 Attachment of an enzyme to a quartz surface is usually effected via a spacer chain.1119]... 

Although affinity chromatography has not been used directly as an analytical method, it may be modified in the future to produce a viable technique. Leucovorin has been used as an effective spacer in obtaining active samples of dihydrofolate reductase.79 If the enzyme could be immobilized without losing its activity, perhaps it could be used to separate folates. 

Some of these sites can effect function in zinc proteins as well as stabilizing structure. The hydrolase class of zinc enzymes are good examples of this action. In this case, one or more amino acid residues within the active site may be provided by the amino acid spacers between zinc ligands (Figure 12). The side chain of these amino acids may be involved in substrate binding, bond cleavage or modulating the chemical enviromnent of the active site. In addition, other active-site residues are often provided by... 

Labelling with enzymes drastically alters the properties of the labelled compounds, because of the large sizes of the enzjune labels, and the effect is more pronounced for small haptens than for larger compounds (e.g., peptides and proteins). A practice often used is to optimize the length of the spacer arm between the structure containing the antigenic determinants and the enzyme label, so that hindering effects are avoided [97,98]. 

Thus, the active functions of the carrier material, which is usuaUy a naturally occurring or synthetically prepared fxjlymer, can be of almost any nature. Introducticm of the spacer molecules (functionalisation) is also the phase determining the activity of the immobilised enzyme preparation the more spacer molecules per unit area, the menzyme molecules can be attached. Of course, steric hindrance exerts a limit on this number. After the carrier has been functionalised, excess reagent is removed by hltration and washing and the enzyme can be attached to the support The immobilised enzyme thus obtained is usually stored in an aqueous medium in order to avoid dehydration, which may lead to irreversible deactivation of d e enzyme. Just before use, die beads containing die enzyme are collected by filtration, wash, and added to die aqueous solution of substrate. Once die desired conversion has been effected, the beads are removed by filtration, washed, and either stored or reused direcdy afterwards. 

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