Proteolytic enzymes, unstable

The specific cleavage of collagen by mammalian collagenases produces two fragments that are unstable at body temperature and begin to uncoil into individual chains. These are then susceptible to attack by other proteolytic enzymes. 

Analyte stability is a great source of concern, and it often varies with matrix type as each matrix has a distinct combination of binding proteins, lipids, and proteolytic enzymes that may act on the drug. Analyte stability can be determined in experiments meant to mimic the conditions encountered by study samples such as room temperature or refrigerator incubations and multiple freeze thaw cycles. Analyte stability determined for one matrix may not be extrapolated to another matrix as an analyte found to be stable in monkey or human serum could be highly unstable in rat or mouse serum. Where an analyte stability issue is anticipated, it is recommended that the study samples are collected in the presence of a protease inhibitor cocktail and kept on ice... 

The study of the co-enzyme has established the fact that this substance is in reality very unstable. Its activity dinoinishes perceptibly in the presence of 2.5 per cent K2CQ3 at 35°, and is completely destroyed if maintained at 130 for 4 hours. The co-enzyme is indifferent to the action of the proteolytic enzymes, but is destroyed, on the contrary, vmder the influence of lipase, due to the setting free of phosphoric acid. As noted, this substance resembles in certain ways, particularly injts chemical nature, the anti-protease referred to above, but differs from the latter in its sensitiveness to alkalis and its lower resistance to heat. Anti-protease, in fact, not only is not capable of activating zymase, but it is not even destroyed by K2COJ, nor by heating 4 hours at 130 . 

In a review of their work, Caplan and Naparstek pointed out that a simpler system might have been an enzyme-free membrane separating the alkahne BAEE solution from a small chamber containing the papain [56]. The chamber could be treated as homogeneous, and quasi-steady-state ordinary differential equations could account for transport of substrate, acid, and base across the membrane as well as for the enzyme-catalyzed reaction. By fixing the external concentrations of BAEE and H+ (and hence OH since the dissociation product is constant), it was shown that conditions exist under which diffusional and reaction fluxes that balance each other are unstable, and the system is directed to a hmit cycle. This simplified membrane-chamber system was further investigated theoretically by Ohmori et al [59], who identified regions of parameter space that are predictive of pH oscillations for compartmentalized papain and other proteolytic... 

For ribonuclease, removal by pepsin proteolysis of only a tetrapeptide sequence at the C-terminus (Asp-Ala-Ser-Val) lead to an inactive enzyme and unstable structure (Anfinsen 1956 Taniuchi, 1970). When this shortened enzyme, the so-called des(121-124) ribonuclease or pepsin inactivated ribonuclease (PIR), is reduced, it cannot reoxidize to 3deld the native pairing of disulfide bonds (Taniuchi, 1970). Removal of six C-terminal residues to form RNase 1-118 also yields a structureless and inactive enzyme (Lin, 1970 Andria and Taniuchi, 1978). The importance of the C-terminal end to the folding of these two proteins was also emphasized by the data reported in Chapter 9. The information contained in the C-terminal sequence of the two nucleases appears to be crucial for their refolding. It was proposed that the polypeptide chain of nuclease and ribonuclease cannot achieve the native structure, during biosynthesis, until the termination of the polypeptide chain. For RNase even the N-terminal end seems to be important for folding. After removal by proteolytic action of subtilisin of the first 20 amino adds (Richards, 1958), the RNase S protein is unstable and cannot refold correctly when disulfide bridges are reduced. 

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