Proteolytic immobilization

The proteolytic enzymes, trypsin, chymotrypsin, and chymoral [8076-22-0] in combination, have been used for the treatment of post-operative hand trauma, athletic injuries, and sciatica (214—216). Trypsin has also been used successfully in treating hyaline membrane disease of newborn babies, a condition usually fatal without treatment (217). Immobilized preparations of trypsin are useful in treating acute radiation cystitis following pelvic x-irradiation therapy (218). 

Purification entails use of an immunoaffinity column containing immobilized murine antifactor VII antibody. It is initially produced as an unactivated, single-chain 406 amino acid polypeptide, which is subsequently proteolytically converted into the two-chain active factor Vila complex. After sterilization by filtration, the final product is aseptically filled into its final product containers, and freeze-dried. 

Purification entails use of an immunoaffinity column containing immobilized murine antifactor VII antibody. It is initially produced as an unactivated, single chain 406 amino acid polypeptide, which is subsequently proteolytically converted into the two-chain active factor Vila complex. After sterilization by filtration, the final product is aseptically filled into its final product containers and freeze-dried. The excipients present in the product include sodium chloride, calcium chloride, polysorbate 80, mannitol and glycylglycine. When freeze-dried in the presence of these stabilizing substances and stored under refrigerated conditions, the product displays a shelf-life of at least 2 years. It has proved effective in the treatment of serious bleeding events in patients displaying anti-factor VIII or IX antibodies. 

The increased platelet adhesion onto HCP surfaces is mainly due to the high surfacial concentration of fibrinogen associated with immobilized heparin. Therefore one of the solutions to the problem could have been provided by simultaneous immobilization of heparin and proteolytic enzymes which could hydrolyze the adsorbed fibrinogen. The immobilized enzyme could also provide the lysis of the fibrin clot, in case it was formed, for instance in stagnation regions of the complicated devices. 

At present, the binary water-soluble preparation of heparin and proteolytic enzymes is being applied for the treatment of thromboses. For instance, injection into the bloodstream of heparin-plasmin complex or a heparin-plasmin-streptokinase preparation leads to the total dissolution of the thrombus, while if introduced separately, heparin and streptokinase do not display the lytic action at all, and plasmin, alone or together with streptokinase, dissolves the thrombus only partially 132>. The treatment of acute thrombophlebitis with trypsin resulted in a full dissolution of the thrombus and in an increase of antithrombin III in the blood 133). Administration of trypsin together with heparin has an effect similar in efficiency to the action of the heparin-plasmin complex 134>. The use of a mix of heparin and urokinase for improving tbrom-boresistance of polymeric materials was also described 13S). These substances were immobilized by preliminary coating of the surface of a polymer with a graphite layer and subsequent adsorption of heparin and the enzyme. 

More extensive studies were carried out on polymeric materials modified with a hydrogel containing covalently immobilized heparin and trypsin 136,137>. Table 18 compiles the results of in vitro tests of the thus-modified low-density polyethylene. Obviously, co-immobilization of heparin and proteolytic enzyme onto polyethylene results in an increase of the blood clotting time and decreases the number on adhered platelets as compared to immobilization of heparin alone. 

Svec, F. (2006) Less common applications of monoliths I. Microscale protein mapping with proteolytic enzymes immobilized on monolithic supports. Electrophoresis, 27, 947. 

Routinely, common chemical and enzymatic techniques are used to obtain protein fragments. Unfortunately, when enzymatic digestion techniques and nanograms quantities of proteins are used, the method become ineffective due to dilution and reduced enzymatic activity. An alternative approach to overcome this problem is the use of proteolytic enzymes immobilized to a solid support and a small-bore reactor column. Using trypsin immobilized to agarose, tryptic digests of less than 100 ng of protein can be reproducible obtained (49). 

In the specific case of affinity chromatography with immobilized protein A and protein G, very popularly used in the purification of antibodies, it has been shown that there is some level of ligand leakage due proteolytic action. Released peptides can still be associated with the purified antibody and are very difficult to evidence and to separate. Special process steps addressing this specific point are indicated elsewhere. 

Thus, rather than trial-and-error development of functionality, it should be possible to design functionality based on the principles of protein structure and function and the specificities of the enzymes used for modification. Use of immobilized exo- and endopeptidases in such technology could be especially attractive for the reasons listed in Table I, particularly since problems associated with autolysis would be eliminated and the extent of proteolytic reactions could be controlled with some precision. 

Endopeptidases. Our expanding understanding of the relationship between structure and functionality of food proteins presents the opportunity for designing functionality into proteins by selective, specific proteolytic modification. Control of reaction and prevention of autolysis offered by immobilization are essential to establish the conditions for a highly selective modification. Hydrolysis at specific positions in the primary structure of proteins could be coupled with resynthesis of peptide bonds by selection of conditions, for example, as in the plastein reaction. By careful choice of enzymes and conditions according to the characteristics of the substrate proteins, it may be possible to design new structures from known food proteins. 

Another approach for studying a cell s capacity to degrade a specific substrate lies midway between zymography and the immobilized component method described above. Here, the cells are seeded on the bottom of a dish, which is then overlaid with soft agar containing a substrate for proteolytic activity. After incubation under appropriate conditions, lithic plaques appear within the agar, indicative of enzymatic activity. The assay is described below to evidence urokinase-plasminogen activator (uPA) activity. 

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