Preparation of affinity materials for protease profiling

Proteases are proteolytic enzymes associated with some pathological conditions such as cardiovascular diseases, inflammation, cancer, osteoporosis and neurological disorders by means of their activity and expression. Therefore, proteases are potential drug targets and it is necessary to determine proteases within variable conditions to enlighten their localization, biological functions, activity and regulation mechanisms, structure-function relationship. This can be possible by profiling the proteases at dynamic conditions. For this aim, preparation of affinity materials for profiling various types of proteases under defined conditions was targeted. For this purpose egg white was chosen for preparation of affinity material on account of egg white protein protease inhibitors and high protein content. Various methods such as binding to a carrier, precipitation of egg white proteins, cross-linking with glutaraldehyde were performed in order to obtain unsoluble affinity materials. Precipitation with acetone followed by glutaraldehyde cross linking approach was chosen as the most convenient method and the resultant material named as "Egg White Protein Based Polymer (EW-PBP)". Glutaraldehyde concentration of EW-PBP was optimised as %0,5 (v/v) and protease binding efficiency was studied with different proteases. EW-PBP effectively bound trypsin (serine protease), papain (cysteine protease) and the different proteases from pancreatin which is a commercial mixture of amylase, lipase, and protease. Protease binding capacity of EW-PBP was found to be 60 mg, 70 mg and 38 mg per 1 g polymer for trypsin, papain and pancreatin respectively. 50 mM sodium phosphate buffer at pH 6,0 with 25 mM NaCl addition and 20 minutes of incubation were chosen as optimum conditions for protease binding to EW-PBP. Surface morphology of free, enyzme bound and enzyme removed polymer was investigated with Scanning Electron Microscopy (SEM) and showed protease binding changed the surface morphology of EW-PBP. Protease binding efficiency of EW-PBP was also investigated via SDS- PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) and diffusion agar plate methods. SDS-PAGE results of enzymes before introducing to the polymer and after elution from the polymer supported the protease binding efficiency of EW-PBP. Also, diffusion agar plate tests with different substrates and protease inhibitors proved the protease specifity of EW-PBP. As a conclusion, EW-PBP is able to specifically bind proteases and it is a convenient and cost-effective material for profiling and purifying of proteases. Clearly, EW-PBP will shed light on proteomic and degradomic studies as a highly specific and capacitively fractioning/enrichment material.