Proteolytic inhibitors

In some instances it may be necessary to demonstrate that all traces of specific contaminants have been removed prior to final product filling. This would be true, for example, of many proteolytic inhibitors added during the initial stages of downstream processing to prevent proteolysis by endogenous proteases. Some such inhibitors may be inherently toxic, and many could (inappropriately) inhibit endogenous proteases of the recipient patient. 

Polymers may also inhibit proteolytic enzymes or be used to augment the activity of proteolytic inhibitors (Table 11.6). The administration of small molecule protease inhibitors in conjunction with the peptide, however, has been examined with some success (Fujii et al. 1985) however, the ability of these molecules to protect the protein has been hampered by the fact that they tend to cause systemic side effects (Yagi et al. 1980 McCaffrey and Jamieson 1993 Plumpton et al. 1994). Many protease inhibitors have been specific for the proteases in the stomach and intestine, but some of these factors are not specific and some control over absorption of the... 

In the absence of any specific information about the nature of such activities, it is often best to mix a cocktail containing several or all of the available proteolytic inhibitors. These include such compounds as 1,10-phenanthroline, soybean trypsin inhibitor, leupeptin, benzamidine, antipain, aprotinin, phenyl-methanesulfanyl fluoride, and diisopropylfluorophosphate. As a precaution against any enzyme destruction, such a cocktail is best added to the buffer in which the cellular lysis will be carried out (Fig. 5.6). 

Figure 5.6 Overview of steps involved in the preparation of a cell-free lysate. The cells are resuspended in a buffered solution at a specified cell density. To this suspension is added a cocktail containing several proteolytic inhibitors. The cells in the suspension are lysed (here by homogenization). Finally the lysate is subjected to a very low speed centrifugation such as 5000g for 10 minutes to remove unbroken cells.

Benzamidine hydroehloride hydrate [206752-36-5] M 156.6 (anhydrous), m 86-88°, pK ° 11.6 (see free base above). Recrystallise it from dilute HCl (crystals contain XH2O) or EtOH/few drops HCl dry it in a vacuum. It is a proteolytic inhibitor [Jeffcoate White J Clin Endocrinol Metab 38 155 1974, Beilstein 9 IV 898.]... 

Unlike uteroferrin, which produces a single band of expected mobility for its molecular size by SDS-FAGE, bovine spleen add phosphatase yields two bands, even when isolated in the continuous presence of proteolytic inhibitors However, the extensive sequence homology between the spleen and uterine enzyme, and the fact that isolation of the spleen enzyme is accomplished by prolonged add extraction of spleen homogenates, make it likely that the purple add phosphatase of spleen is also a single-chain protein ... 

Whereas proteolytic activities in mitochondria, nuclei, and the microsomal fraction are relatively high, very little proteolytic capacity is found in the cell cytosol. In part this may reflect technical difficulties, because the highly efficient proteolytic inhibitors found in serum, especially a2-macroglobulin and aj-antitrypsin, would occur in the cytosol fraction of an homogenized tissue. Nevertheless, separation of cytosol proteins from serum proteolytic inhibitors by Sephadex chromatography does not give rise to any fractions with substantial proteolytic capacity (Bohley et al., 1971). 

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