Bubble extraction and affinity chromatography techniques for human trypsin inhibitors enrichment and purification

Bubble extraction combined with affinity chromatography can be considered as fully reliable techniques to concentrate and separate proteins and enzymes, and could be applied to the treatment of large samples to purify analytes from diluted solutions, on a preparative scale. In order to assess the possibility of employment of these techniques for the preconcentration of proteins in biological fluids, they have been applied to isolate and purify Kazal-type and Kunitz-type trypsin inhibitors which are usually present in human urine in very low concentration and of renowned biomedical interest, especially for their anti-metastatic activity. A three-step procedure, including direct RP-HPLC monitoring of the inhibitors at intermediate and final steps, is described here. Recoveries obtained are in the range 70-90%, and the highly purified inhibitors display a fully retained trypsin-inhibiting activity.