Some special features of glyoxyl supports to immobilize proteins

Abstract

This paper described that glyoxyl activated supports are only able to quantitatively immobilize proteins at alkaline pH values and using highly activated supports (otherwise, immobilization is negligible). Furthermore, the immobilization rate depends on the surface density of glyoxyl groups and not on the total concentration of groups in the suspension. Moreover, the temperature presented a dramatic effect on enzyme immobilization rate and different enzymes become immobilized at very different rates. Finally, it was not possible to detect immobilization of mono aminated compounds at neither alkaline nor neutral pH values. In fact, these supports may be used to purify enzymes due to these great differences in immobilization rate (e.g., glutamate racemase). These special features are not shared with other supports usually utilized for covalent immobilization of enzymes (e.g., glutaraldehyde, cyanogen bromide supports) that are able to immobilize proteins by just one very stable bond and suggesting an unique mechanism for glyoxyl agarose supports. In fact, all these results suggested that each individual amino-glyoxyl bond is very weak. Therefore, the first immobilization of proteins on supports activated with glyoxyl groups, in absence of reducing reagents, needs the simultaneous establishment of, at least, two attachments between the protein and the support. This mechanism promotes that proteins become immobilized by the area/s where the highest lysine residues density is located and explain the high enzyme stabilization usually achieved by using this immobilization technique.