Background

In higher plants, small proteinaceous protease inhibitors are wound-induced molecules produced as a part of its defense system against insect attack (Graham et al., 1981; Villanueva et al., 1998). Among the studied inhibitors, only two are specific for MCP, i.e., the potato carboxypeptidase inhibitor (PCI) and its close homolog found in tomato plants (TCI). Over the last few decades, the presence of MCP inhibitors in Solanaceae has been extensively reported, revealing potato (Solanum tuberosum) as one of the most important sources of MCP inhibitors (Hass et al., 1979; Obregón et al., 2012; Lufrano et al., 2015). In humans, MCP action is exquisitely regulated and dysregulation of its function might lead to disease or even to cell death (Arolas et al., 2007). In fact, MCP have been associated with human pathologies such as acute pancreatitis (Appelros et al., 1998), diabetes (Cool et al., 1997), several types of cancer (Ross et al., 2009; Sun et al., 2016; Abdelmagid et al., 2008; Tsakiris et al., 2008), fibrinolysis (Valnickova et al., 2007), inflammation (Deiteren et al., 2009) or neurodegeneration (Rogowski et al., 2010). In this context, there is an interest in the discovery of new MCP inhibitors, and thus we focus our studies in potatoes that are native from the Andean region of South America. In this region, thousands of different potato varieties coexist, constituting a natural reservoir for the discovery of novel MCP inhibitors (Figure 1).


Figure 1. Andean potatoes and potato extract CPA inhibitory activity. A. Picture displaying the large number of potato varieties found within the Andean region. Currently in this region coexist thousands of Andean varieties of Solanum tuberosum (Machida-Hirano, 2015; Clausen et al., 2010). B. Effects of potato extracts on bCPA activity. The activity of bovine CPA (bCPA) was measured using the substrate N-(4-methoxyphenylazoformyl)-Phe-OH determining the decrease in absorbance at 340 nm in function of the time. Due to its high content in MCP inhibitors, the addition of potato extract to the reaction decreases the rate of substrate hydrolysis in a dose-response manner.

Here, we describe a simple protocol to determine the specific and dose-response carboxypeptidase A inhibitory activity present in Andean potatoes and in other biological extracts using microplates. The major advantage of this protocol over other available approaches (Yanes et al., 2007) is that the use of microplates allows multiple enzymatic measurements to be done in a single experiment, therefore reducing the time and costs.