Enzyme desorption experiments

Because of some of the problems with bioassays and immunoassays, liquid chromatography (LC)-based techniques are increasingly applied as an alternative. While modern LC-based assays have a comparable sensitivity to immunoassays, they oftentimes are characterized by a higher selectivity [18, 19]. Muller et ah, for example, used LC/mass spectrometry with matrix-assisted laser desorption ionization in ex vivo pharmacokinetic studies in combination with enzyme inhibition experiments to investigate the complex metabolism of dynorphin Al-13, a peptide with opioid activity, up to the fifth metabolite generation [20, 21]. 

A series of covalently supported ILs with magnetite core and silica gel shell were prepared for the immobilization of C. rugosa lipase [123]. The immobihzation of lipase was realized by adsorbing the lipase onto the support in a buffer solution. The immobilization promoted the thermal resistance of Hpase dramatically. The native lipase lost 70% activity after being treated at 60 °C. After immobihzation, its activity was maintained at >90%. The supported Hpase also had better reusabihty in the esterification reaction of oleic acid and butanol. The supported hpase retained 92% of its initial activity for the second ran, while its native counterpart retained only 35%. This makes the use of expensive enzymes more practical. Moreover, desorption experiments showed that the supported Hpase could endure 200 mM NaCl, Figure 2.39, while the commercial carboxymethyl cellulose-supported hpase lost about 90% of the enzyme when the NaCl concentration was 150 mM. 

Finally, new methods of analysis have recently been developed that may allow characterization of single atoms on surfaces such as atomic force microscopy.9 In certain cases, in situ experiments can be done such as the study of electrodes, enzymes, minerals and biomolecules. It has even been shown that one atom from a tip can be selectively placed on a desired surface.10 Such processes may one day be used to prepare catalysts that may enhance selectivity. Other methods that show promise as regards detection of surface catalytic intermediates are temperature programmed desorption techniques.11 Selective poisoning of some surface intermediates with monitoring via temperature programming methods may also allow the preparation of more selective catalysts. 

The peroxidase, adsorbed onto glass beads, was added to 5 mL dioxane (containing from 5-40% v/v aqueous buffer). [In independent experiments it was determined that the enzyme remained completely adsorbed to the glass surface in dioxane concentrations of 70% and greater. At 60% dioxane, roughly three-quarters of the enzyme had desorbed from the glass (this manifests itself as completely soluble enzyme in the reaction supernatant). A similar effect was observed with acetone, while with methanol, complete retention of adsorbed peroxidase requires 80% of the organic solvent. In aqueous solutions, complete desorption of the enzyme takes place with full retention of activity.]... 

Frequently a simple contact between an electrode surface and a solution is sufficient to generate reasonable adsorptive layers without adding further reagents. This is valid in particular for carbon-containing surfaces. In some cases, however, an adsorptive bond with a carbon surface may affect the enzyme function to such a degree that it is deactivated. Sometimes even denatu-ration occurs. The majority of molecules is bound only weakly, and a part of them is later lost by progressive desorption. Electrodes with adsorptive layers preferably are used for tentative experiments. They are useful mainly for fast tests. 

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