I want to perform chemical cleavage of a proteoglycan heparan sulfate chains with borohydride. I have found protocols but they always use as a starting material a purified fraction of the proteoglycan. In my case, I am using total larval drosophila extracts to latter do a WB with an antibody against the core of the proteoglycan. When I apply the protocoI, I get protein degradation even if I add protein inhibitors. Is there a way around this problem?
Did you use reductive cleavage of O-glycosidic linkages (beta-elimination)? (NaOH/KBH4) ? Peptide bonds are destroyed during this reaction, even when using diluted alkali and low temperature. If you omit the reducing agent and work at low temperature i.e. 4°C you reduce the protein degradation but you can have a "peeling" reaction of the polysaccharide. Enzymatic degradation of the polysaccaharide using protease free preparations is one idea to completely preserve the protein backbone.
Dear Yannis, yes, I did use beta-elimination (1M NaBH4 0.5M NaOH) and I did perform the reaction on ice. The first time I did it overnight without protease inhibitors and got a smear of degradation on my WB. I tried a second time on ice for only 3h in the presence of protease inhibitors and had the same problem. I am actually not even sure that the protease inhibitors are active in these alkili conditions (I am not a biochemist). How would you get a protease free preparations starting from animal extracts? (immunoprecipitation of the proteoglycan is not an alternative as I have very little antibody)
Even if you use protease inhibitors, your problem is that the chemical treatment destroys your protein. My question is if you can use an heparinase to hydrolyse the polysaccharide backbone, in which case you should use an protease-free enzyme.
My second option was indeed to use heparinase (protease free). I will still have to face the problem of proteases in my sample prep but it's worth a try. Thank you for your answers!
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