Relative Abundance and Inhibitory Distribution of Protease Inhibitors in Potato Juice from cv. Elkana L. Pouvreau, † H. Gruppen, †,‡ S. R. Piersma, †,§ L. A. M. van den Broek, †,‡ G. A. van Koningsveld, † and A. G. J. Voragen* ,†,‡ Centre for Protein Technology, TNO-WU, P.O. Box 8129, 6700 EV Wageningen, The Netherlands, Laboratory of Food Chemistry, Department of Agrotechnology and Food Sciences, Wageningen University, Wageningen, The Netherlands, and Department of Protein and Meat Technology, TNO Nutrition and Food Research, Zeist, The Netherlands Protease inhibitors from potato juice of cv. Elkana were purified and quantified. The protease inhibitors represent ca. 50% of the total soluble proteins in potato juice. The protease inhibitors were classified into seven different families: potato inhibitor I (PI-1), potato inhibitor II (PI-2), potato cysteine protease inhibitor (PCPI), potato aspartate protease inhibitor (PAPI), potato Kunitz- type protease inhibitor (PKPI), potato carboxypeptidase inhibitor (PCI), and “other serine protease inhibitors”. The most abundant families were the PI-2 and PCPI families, representing 22 and 12% of all proteins in potato juice, respectively. Potato protease inhibitors show a broad spectrum of enzyme inhibition. All the families (except PCI) inhibited trypsin and/or chymotrypsin. PI-2 isoforms exhibit 82 and 50% of the total trypsin and chymotrypsin inhibiting activity, respectively. A strong variation within the latter activities was shown within one family and between protease inhibitor families. Keywords: Potato; Solanum tuberosum; protease inhibitor; inhibiting activity; trypsin; chymotrypsin INTRODUCTION Although protease inhibitors have been long consid- ered only as antinutritional factors, they have regained interest in recent years because of their possible anti- carcinogenic (1) and positive dietary effects (2). Potato tuber protease inhibitors have been reported to act as anticarcinogenic agents by interfering in tumor-cell proliferation (3), H 2 O 2 formation (4), and processes resulting from solar-UV irradiation (5). In addition, by intervening with cholecystokinin (2), one of the protease inhibitors can act as a satiety agent. Potato tubers (Solanum tuberosum) contain approxi- mately 1.5% protein on a fresh weight basis (6). It has been reported that protease inhibitors represent about 30% of the total tuber protein (7). In contrast to patatin, the major potato tuber protein (8), the protease inhibi- tors are a more heterogeneous group of proteins. They differ with respect to molecular mass, amino acid sequence, and inhibitory activity. The most studied protease inhibitors from potato tuber are protease inhibitor I (PI-1), protease inhibitor II (PI-2), and potato carboxypeptidase inhibitor (PCI). PI-1 is a pentameric serine protease inhibitor composed of five 7-8-kDa isoinhibitor protomers and inhibits chymotrypsin (and with lower affinity also trypsin) (9, 10). PI-2 is a dimeric serine protease inhibitor composed of two 10.2-kDa subunits (11). A disulfide bridge links the subunits, and the protein behaves like a single domain protein (12). The 4.3-kDa PCI is the smallest inhibitor present in potato tuber, is a single subunit peptide (13) and is remarkably thermo-stable (14, 15). In addition to PI-1, PI-2, and PCI, other protease inhibitors have been identified in potato tubers as well. The reported protease inhibitors include Kunitz-family inhibitors (16), cysteine protease inhibitors (17), and cathepsin D inhibitors (18). In industrial processes, potato proteins are recovered as a byproduct of potato starch production (19). This is done by an acidic heat-treatment of the so-called potato juice and results in irreversibly precipitated proteins which have lost all functionality (19). To use specific protease inhibitor fractions of the potato juice in indus- trial, food, or pharmaceutical applications, the proteins in this group should be identified and quantified. To our knowledge no reports have appeared which describe the relative abundance and activity of the different protease inhibitors in potato tuber. Therefore, in this report we describe a general fractionation method to obtain the most important protease inhibitors from potato tubers, in particular of cv. Elkana. The choice of Elkana cultivar was determined by the economical importance of this cultivar in the potato starch industry in The Nether- lands. Subsequently, the inhibitors are identified on the basis of their subunit molecular mass, isoelectric pH, and their activity against various proteases. The amount of protein relative to the total protein content has been determined for each protease inhibitor fraction and an overview of the most abundant protease inhibitor fami- lies will be presented. MATERIALS AND METHODS Materials. Porcine pancreas trypsin (T-0134, lot 100H0658), bovine chymotrypsin (C-4129, lot 58H7001), papaya latex papain (P-9886, lot 66H7130), bovine pancreas carboxypepti- * Corresponding author. Phone number: +31 (0) 317 483209. Fax number: +31 (0) 317 484893. E-mail: office@ chem.fdsci.wag-ur.nl. † Centre for Protein Technology, TNO-WU. ‡ Wageningen University. § TNO Nutrition and Food Research. 2864 J. Agric. Food Chem. 2001, 49, 2864-2874 10.1021/jf010126v CCC: $20.00 © 2001 American Chemical Society Published on Web 05/25/2001