Have you tried adding reducing agents like sodium sulphite, cysteine, glutathione, mercaptoethanol or dithiothreitol (0.1–10 mM)? It should enhance the effect of either protease you choose on your compact protein stabilised by disulphide bridges.

For low pH, pepsin is the obvious choice. at neutral pH I would try elastase.

Pepsins purified from salmon, tuna or other fish are less temperature dependent than porcine pepsin (See: Preparation and properties of crystalline salmon pepsin. by Earl R. Norris and Daniel W. Elam. J. Biol. Chem. 1940, 134:443-454. Preparation, properties, and crystallization of tuna pepsin. by Earl R. Norris and James C. Mathies. J. Biol. Chem. 1953, 204:673-680.). Increasing the salt concentration, denaturing or reducing the disulphide bonds prior to digestion would also help.

you can use pepsin. it can cleave peptide bond in GdnHcl solution pH 0.9 and incubate it for 24 hours at room temp. or at lower temp.

Fungal protease XIII. Do a search for deuterium exchange mass spec papers. This method always uses these proteases and there is a new one that was published.

I use pepsin and FXIII at pH 2.7 and 4C and get reproducible cleavage when performing deuterium exchange experiments.

Pepsin, proteases XIII and XVIII, Nepenthesines I and II.

Thank you all for the great suggestions!

I should have mentioned earlier, but I am looking for a protease already food grade or at least non GM as my target proteins are in a food matrix. As you can imagine this complicates things significantly as the enzyme I am looking for ideally could just be added to a food preparation without the need for treatments aimed to favour the enzymatic acitvity (eg no reducing agent, special buffers and so on...)

I have tried pepsin before but only the porcine one with little results but I haven't tried the Salmon one suggested by Russel, or others that came up in this discussion.

thanks again!

Since you are working with a food matrix, you could try doing hydrolysis in the presence of high or low pH and heat.

I have tried protein degradation at higher temperatures in presence of aspartil proteases and it worked, but I am after a method that does not involve heat or changing the pH

OK. You could try ultrasonication or a glass bead mill to break up some of the solid matter before adding the proteinase.

On a separate note, what dry white Australian wine would you recommend?

Hi Steingrimur and thanks for your input...I will give it a go.

For the wine suggestion I would it depends what you prefere in style, but in general Hunter valley Semillon are pretty good, Clare valley Rieslings, Chardonnay from Western Australia, and Sauvingon Blanc from the Adelaide Hills !

I don't know, sorry.