## 3 Results and Discussion
### Purification and Characterization of \(\alpha\)AI-1  
For this study, \"purified \(\alpha\)AI-1 protein\" of common bean varieties were successfully purified by FPLC technique. The enzyme inhibitor activities of the \"purified \(\alpha\)AI-1 protein\" of the four bean varieties were measured and then calculated, which were between 91 and 94% in the final preparations. As we expected, it was much higher than that of the \"\(\alpha\)AI-1 extract\" (38-53%). The effect of heat treatment on \(\alpha\)AI-1 activity was also monitored in the case of Pinto bean cooked flour, where the \(\alpha\)AI-1 activity was lost. The presence and distribution of bean proteins were monitored by SDS-PAGE (not included). \"\(\alpha\)AI-text\" of common beans had several bands between the molecular weight of 14 and 31 kDa (not only and exclusively \(\alpha\)AI). However, in the case of the \"purified \(\alpha\)AI protein\" sample, we could detect two bands: one band--corresponding to the active \(\alpha\)AI-1--was observed at around 14-15 kDa and another band--corresponding to the unprocessed pre-protein (precursor) of \(\alpha\)AI-1--was found at around 30-31 kDa (see in Section 3.2). LC-MS data have already proved the presence of \(\alpha\)- and \(\alpha\)-chains in these bands, which explained the detectability of the two protein bands [8]. [PERSON] et al. [29] found similar observations for common beans. [PERSON] et al. (2018) purified \(\alpha\)AIs from Red, Pinto, White, and Cowpea beans by ethanol fractionation and they detected similar molecular weights at 14 and 18 kDa and two fractions around 27 to 32 kDa [13]. In contrast to the _Phaseolus vulgaris_ varieties, \(\alpha\)AI from _Mucuna pruriens_ showed 25.6 kDa molecular weight [14]. In the case of the \"purified \(\alpha\)AI-1 protein\" of four common bean varieties, Pinto bean \"\(\alpha\)AI-1 extract\", Pinto bean meal, and Pinto bean cooked meal samples, we made LOC separation analyses in order to obtain a more sensitive resolution (Table 1, Figure 1). The advantages of using LOC electrophoresis are it is automated, fast (usually 15-30 min), can test small volumes (up to 1 \(\upmu\)L), and ensures low-concentration detection (sensitivity). For the analyses of the \"purified \(\alpha\)AI-1 protein\" (and the in vivo stomach and intestinal samples, see Section 3.2), the LOC electrophoresis technique was particularly advantageous, as only a few of these samples were available. In general, all samples contained protein peaks with \(\mathrm{t_{R}}\) values between 24.91 and 30.12 s. We assumed that the major peaks that probably corresponded to active \(\alpha\)AI-1s were observed between 24.91 and 29. 23 and the peak found between 30.01 and 30.12 s corresponded to the pre-protein. As the purification of \(\alpha\)AI-1 was based on the collection of those protein fractions that had \(\alpha\)AI-1 activity, the preparations contained only traces of nonactive pre-protein (2.99-6.84%) in the total protein content. According to the total protein LOC separation of Pinto bean meal, 12 out of 25 peaks were observed, while, in the case of Pinto bean cooked meal, 11 out of 23 peaks were detected in the \(\mathrm{t_{R}}\) area of \(\alpha\)AI-1 (see in Section 3.2). The t (%) values of Pinto bean meal and Pinto bean cooked meal were increased (16.01% and 18.46%, respectively) compared to the t (%) values of Pinto bean \"purified \(\alpha\)AI-1 protein\". This was due to the fact that the t (%) values of bean meals included the corresponding pre-protein t values too.