Occurrence and physico-chemical properties of protease inhibitors from potato tuber (Solanum tuberosum)

Laurice Pouvreau Occurrence and physico-chemical properties of protease inhibitors from potato tuber (Solanum tuberosum) Ph.D. thesis Wageningen University, Wageningen, The Netherlands, 2004 Protease inhibitors represent approximately 50% of the total amount of proteins in potato juice (cv. Elkana). Potato protease inhibitors have a broad spectrum of inhibitory activity, showing inhibition against serine proteases, cysteine proteases, aspartate proteases and metallo-proteases. They can be classified in 7 different groups based on the class of enzymes they inhibit and additionally on their molecular mass, their protein architecture and their isoelectric pH. Using an antibody assay it was shown that approximately 70% of the protease inhibitors in potato presumably belongs to the Kunitztype inhibitors. The most abundant group of protease inhibitors, denoted Potato Serine Protease Inhibitor (PSPI), is a Kunitz-type serine protease inhibitor, and represents approximately 44% of the total amount of protease inhibitors in potato. PSPI isoforms represent 80% and 50% of the total trypsin and chymotrypsin inhibiting activity, respectively. Based on its structural characteristics, PSPI was classified as a β-II protein; i.e. a protein in which most of the residues are involved in irregular and short β-sheets. PSPI shows no changes in secondary and tertiary structural characteristics in the pH range 3.0 to 7.5, at ambient temperature. Also, the heat induced unfolding behaviour does not change within this pH range. Unfolding studies showed that the thermal as well as the guanidinium-induced unfolding of PSPI occurs via a non-two state mechanism in which at least two parts of the protein unfold more or less independently. Aggregation, upon heating, seems to occur via a specific mechanism in which the end product is a tetrameric form of PSPI. Isoforms of the Potato Cysteine Protease Inhibitor (PCPI) group showed very similar structural characteristics as those of PSPI, and were, therefore, also classified as β-II proteins. At pH 4.0, isoforms of PCPI unfold at approximately 67°C. Similar to PSPI, PCPI isoforms (especially for PCPI 8.3) do not unfold via a two state mechanism and at least one intermediate is present. Upon heating, PCPI aggregates are formed, but the size of the aggregates (>100kDa) differs from those of PSPI. Recombinant Potato Inhibitor I (PI-1) showed very similar structural characteristics to those of PI-1 purified from potato, as well as a similar transition temperature (88°C). Using recombinant PI-1, it was suggested that PI-1 is a hexameric protein rather than a pentamer, as previously described in literature. Differential scanning calorimetry analysis showed that PI-1 unfolds having a dimer instead of a monomer as a cooperative unit. SYMBOLS AND ABBREVIATIONS PI(s) Protease inhibitor(s) PI-1 Potato Inhibitor I PI-2 Potato Inhibitor II PSPI Potato Serine Protease Inhibitor PCPI Potato Cysteine Protease Inhibitor PAPI Potato Aspartate Protease Inhibitor PKPI Potato Kunitz-type Protease Inhibitor OSPI Other Serine Protease Inhibitor PCI Potato Carboxypeptidase A Inhibitor NID Novel Inhibitor of Cathepsin D PDI Potato Cathepsin D Inhibitor BPTI Bovine Pancreatic Trypsin Inhibitor BBI Bowman-Birk Inhibitor SSI Streptomyces Subtilisin Inhibitor STI Soybean Trypsin Inhibitor