There are no proteinase inhibitors that will inhibit 100% of all serine/cysteine/aspartic/metalloproteinases. These inhibitors are giving you a false sense of security because you only need miniscule amount of proteinase to ruin a prep.

Proteinase contamination. You don't need a lot of proteinases in a prep to do major damage. Never store your protein at 4oC. Always aliquote it and store at -80oC.

When I worked with kinases, we did everything as cold as possible (as in, we purified them on ice in a 4C cold room). Everything was also stored at -80. Kinases are notoriously sensitive to proteases as well as to freeze-thaw cycles, because their 3D structure factors into their activity. So, I'd say aliquot and store at -80.

Is it possible that extensive freeze thawing may lead to loss in the activity?

I forgot to mention that suitable protease inhibitors have been used in my preparations in appropriate concentrations;in spite of that,i am observing the degradation pattern

There are no proteinase inhibitors that will inhibit 100% of all serine/cysteine/aspartic/metalloproteinases. These inhibitors are giving you a false sense of security because you only need miniscule amount of proteinase to ruin a prep.

If by extensive freeze-thaw you mean multiple times (or even if you mean temperatures greater than 4 degrees C for the thaw), rest assured that can cause extensive degradation. Besides the protease problems, simple freeze-thaw can cause dehydration/elimination reactions or even hydrolysis from the solution being stratified as it thaws. There are other mechanisms possible along with the consideration of molecular instability and degradation because of the energy applied to the molecule at higher temperatures-even 4 degrees C.

As others might have suggested, I recommend making aliquots of your kinase (each enough for 1-2 experiments) and freezing at -80 degrees C (if not -80, at least -20 degrees C for the short term). We never use an aliquot more than 3-4 times and that is pushing it (because of cost considerations). I prefer to not use it more than twice after the aliquot is made.

To follow up on the aliquot-comments, it is also best to make aliquots in volumes of no less then 30µl. Enzymes in smaller volumes in the -80°C tend to lose activity more quickly than the larger ones. The exact causes for this is not really known, but I would suspect the hydrophobicity of plastic tubes may have something to do with it.

Another consideration to take into account would be your storage buffer. Do you have stabilising agents (glycerol, PEG, BSA) in your samples? If your proteins are in low concentration, think about putting in BSA, unless BSA would completely foul future experiments. Glycerol is needed to prevent water crystalisation during freezing from denaturing your proteins. PEG works as a crowding agent to help stabilization, as does PVA.

Many thaw-freeze cycles are not good for maintaining native protein enzymes. Physically during freezing and thawing ice crystals may form and shear in structures of proteins (just like the nano knives), of course with additional reasons mentioned above such as protease contamination during your manipulation (your hands, water used etc) that can degrade your proteins even at low temperature of 4oC. Your results of SDS-PAGE may answer that possible reasons though you may need native-PAGE to confirm surely. In addition, denaturation of protein and its missed refolding may also involve to cause activity reduction or losing. Aliquote and store at -80oC is the best as many above advices. You can also add DTT (reductive reagent) to protein sample to avoid protein oxidation (fat, protein fouling at low temperature most likely caused by oxidation chain reaction). If you do not have -80oC, just quick frozen by liquid N2 and at least keep protein enzymes at -20oC. Be careful with CO2 dried ices when used coz it could make your sample becomes more acidic (CO2 + H2O -----> H2CO3 (weak acid)) as well.

You didn't indicate whether your kinase sample was prepared and handled under sterile conditions or if the sample contains microbial inhibitors of any kind. If there are any bacteria or mycoplasma in your prep, that is the reason that it has been degraded. Also, as others have indicated, enzymes of any kind are very labile and must be aliquoted and stored frozen, preferably at -80C to maintain activity.

Could be many factors. You may have an auto cleavage effect.

You could have a very low level impurity protease in your sample.

Typically, proteins store best at -20 or lower & when the protein concentration is re;actively high and in the presence of other noninteracting proteins. such as BSA. Store in small aliquots so that you do not have one vial that you take out numerous times.