Enzyme intermolecular cross-linking

Fig. 3.1 Several different methods for producing immobilized enzymes with multifunctional reagents (a) enzymes are adsorbed on the surface-active support followed by inter-molecular cross-linking, (b) functional groups are intro-duced on the support to react covalently with enzymes, and (c) enzymes are cross-linked intermolecularly.

Chemical methods that involve the formation of at least one covalent bond (attachment of enzyme to water-insoluble functionalized polymers, intermolecular cross-linking of enzyme molecules using multifunctional... 

Involvement of esters in the intermolecular cross-links accounts for the dissolution of mature collagen by hydroxylamine, hydrazine or alkali in the presence of hydrogen-bond breakers. Similarly, the participation of hexoses accounts for the dissolution of collagen by periodic acid and a hydrogen-bond breaker. Recently, it has been shown that collagen can be completely dissolved by some proteolytic enzymes (see Section II). These enzymes also cleave the intramolecular cross-links (Kiihn et al, 1963a Rubin et al., 1963). It appears therefore that either the crosslinks include a peptide chain or the chain close to the cross-links contains pepsin- and trypsin-sensitive bonds. 

Fig. 3A, B Protein repair mechanisms. Panel A demonstrates the oxidation of thiol groups, leading to the formation of an intermolecular cross-link. This modification can be repaired by two mechanisms, one involving gluathione and the other thioredoxin. Panel B demonstrates the methionine oxidation,leading to methionine sulfoxide and methionine sulfone. Methionine sulfoxide can be repaired either in its protein-bound or soluble form by two types of enzymes (1 and 11). For more detailed description see the text...

Figure 1 (A) Carrier-bound immobilized enzymes of defined size and shape. Insoluble carriers vary in iheir geometric parameters. Different shapes and types of enzyme carrier are illustrated (a) bead, (b) fiber, (c) capsule, (d) film, and (e) membrane. (B) Methods used for immobilizing enzymes onto a spherical solid support matrix 1, physical absorption 2, covalent binding 3, electrostatic binding 4, intermolecular cross-linking 5, gel entrapment 6, chelation and/or metal binding. E, enzyme M, metal.

Enzymes may be intermolecularly cross-linked by bi- or multifunctional reagents. The protein molecules may be crosstinked with each other or with another, functionally inert protein (e.g. albumin or gelatin). The biomacromolecules can also be adsorbed to a carrier or entrapped in a gel and than crosslinked. Among others, glu-taraldehyde, biisocyanate derivatives, and bisdiazobenzidine are being used as bifunctional reagents [8]. 

In the extracellular space, procollagen is further modified by enzymes which cut the C- and N-terminal portions of the molecule and make tropocollagen suitable for self-assembly into banded fibrils. The last enzymatic modification of the collagen molecule is by lysyl oxidase, which initiates a series of reactions leading to the formation of stable intermolecular cross-links. As far as lysyl oxidase is concerned, in vivo, this enzyme activity in rat skin does not seem to be significantly affected by excess of vitamin C in the diet on the contrary, lysyl oxidase was inhibited in a concentration-dependent manner in an in vitro assay in which lysyl oxidase activity of chick embryo bones was measured in the presence of increasing concentrations of ascorbic acid (Quaglino et aL, 1991). A similar reduction was also observed by Paris et al. (1984) in cultures of rabbit aortic smooth muscle cells. 

The cross-linking method relies on bifimctional reagents to form intermolecular linkages between the enzyme molecules to render them insoluble. Often albumin is added as an extender and glutaraldehyde is most commonly employed. This material can then be either formed as a free standing membrane or applied to the inner surface of the dialysis membrane... 

A bi- or multifunctional coupling agent is used to mediate between carrier and enzyme functional groups. This can also lead to intra- and intermolecular enzyme cross-linking. 

---