Preventing proteolysis by denaturation

If proteins of interest are not required in an active form, as in the case of total cell protein analysis by SDS-PAGE, the most direct means of preventing proteinase activity is to use strongly denaturing conditions either during or immediately following cell disruption. Protein extraction can be carried out in the presence of urea, SDS or guanidinium hydrochloride. However, proteinases are often more resistant to denaturation than other proteins, and mild denaturation conditions may accelerate proteolysis by exposing sites on target proteins without inactivating the proteinases. Proteinase inhibitors should be included whenever mild denaturation conditions are used, for example if samples are mixed

Table 5 The avoidance of proteolysis during sample preparation

Factor Comment pH and buffers Use a buffer with a pH either above or below the optimum for proteinase activity. It is almost certain that there is no range of pH at which all proteolytic enzymes could be considered to be inactive, but above neutral pH the non-specific and highly active enzymes of the lysosomes or vacuoles will be minimally active. If, as in yeasts, the cellular pH is normally lower than this, a high concentration of buffer should be used to make certain that the required pH is obtained when the cell contents are released. A neutral pH favours interactions between proteinases and any endogenous inhibitors that could provide some degree of protection. Some stabilizing effects have been noted with particular buffers, for example phosphate (41)

Low temperature Proteinase activity is minimized by working throughout at low temperature. Extended procedures should be carried out in the cold and samples stored frozen

Time The shorter the preparation time, the less opportunity for proteolysis to occur. Lengthy procedures such as overnight dialysis should not be undertaken without the inclusion of proteinase inhibitors in the dialysis buffer. In some systems, for example yeasts and some filamentous fungi, prolonged incubation of extracts results in proteinase activation because endogenous inhibitors are degraded

Stabilizing agents Protein stabilizing reagents can provide protection against proteolysis.

The inclusion of glycerol (15-35% v/v) or DMSO (10% v/v) may be useful during preparation and storage. Reducing agents such as DTT (0.1-1 mm) or mercaptoethanol (1-10 itim) ensure that free thiol groups are not oxidized but will activate cysteine proteinases. The reagents could affect the activity of the protein of interest and reducing agents will diminish the effectiveness of some proteinase inhibitors

Exogenous protein Proteins such as BSA (1-5 g/l) may provide protection by offering an alternative substrate to endogenous proteinases. Additional steps in protein preparation may have to be included in order to remove this exogenous protein later

Effectors Low molecular weight effectors such as substrates, substrate analogues and cofactors can help to maintain a protein in a stable conformation and prevent proteolytic attack. Not all substrates or cofactors will necessarily be effective. Each substrate and cofactor should be tested as part of general screening process for protective agents. Any protective effect has to be balanced against the cost of using expensive chemicals in a large-scale preparation

Activators omitted

Potential proteinase activators such as divalent cations (these can

activate and/or stabilize the enzyme) should be excluded from the

extraction buffer

Denaturation

See Section 4.3

Proteinase inhibitors See Section 4.4

with reducing agent and SDS but not boiled. If the samples are heated as part of the denaturation process (this should ensure the inactivation of most proteinases) this should be carried out immediately after addition of denaturing agents. Incubation at 100 °C for 2-3 min is sufficient for the preparation of samples for electrophoretic analysis. To ensure rapid temperature equilibration when samples are placed in a boiling-water bath, sample volumes should be as small as possible; slow warming will encourage proteolysis. Some proteinases can survive fairly harsh denaturation conditions and their activity will be restored when dénaturants are removed. Denaturation may not, therefore, provide a permanent solution. Even heating samples with SDS and mercaptoethanol has not always eliminated proteolytic problems associated with SDS-PAGE analysis. Other methods of sample preparation or alternative electrophoretic procedures should be investigated in these circumstances.

Two examples of the analysis of plant proteins illustrate some relevant points. The proteins of petunia anthers are highly susceptible to proteolysis, and high-Mr proteins are degraded within a few minutes of homogenization in buffered solutions (30). Even the presence of 1% SDS in the homogenization buffer failed to prevent a complete loss of these proteins in SDS-PAGE analysis. The inclusion of proteinase inhibitors (PMSF, benzamidine) did not eradicate the problem. An alternative extraction method involving denaturation with TCA (trichloroacetic acid) was devised (Protocol 7).

Protocol 1

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  • Semhar
    Does proteolytic enzymes denature?
    11 months ago

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