Rhynchosia sublobata subsp. protease, (Schumach.) Meikle (Schumach.) Meikle

Mohanraj, Soundappan S., Gujjarlapudi, Mariyamma, Lokya, Vadthya, Mallikarjuna, Nalini, Dutta-Gupta, Aparna & Padmasree, Kollipara, 2019, Purification and characterization of Bowman-Birk and Kunitz isoinhibitors from the seeds of Rhynchosia sublobata (Schumach.) Meikle, a wild relative of pigeonpea, Phytochemistry 159, pp. 159-171 : 160

2.1. Purification of R. sublobata protease inhibitors “RsPI” by FPLC

The 20–60% (NH 4) 2 SO 4 fractionated CPI was resolved into two peaks in trypsin-affinity column when eluted with 0.01 N HCl ( Fig. 1A ). The peak 2 with TI activity was further loaded on a Sephadex G-50 fine gel filtration chromatography column to remove any high MW contaminants retained in the trypsin affinity fractions. The pool of peak 2 fractions with significant TI activity was referred to as R. sublobata trypsin specific protease inhibitors “RsPI” ( Fig. 1B ) and it showed two major bands when separated in Tricine SDS-PAGE ( Fig. 1C ). The lower major band apparently possessed molecular mass of ca. 13 kDa or 8 kDa when compared with commercially available protein molecular weight marker (PMWM) and soybean Bowman-Birk Inhibitor (SBBI), respectively. This discrepancy is very common in TIs, particularly among those which are known to self-associate depending on the method of molecular weight determination such as ultra-centrifugation, size exclusion chromatography, SDS-PAGE, 2-D gel electrophoresis or MALDI-TOF mass spectrometry ( Bergeron and Nielsen, 1993 and references therein; Swathi et al., 2014, 2016). However, molecular mass of lower band is assumed to be closer to 8 kDa considering its similar migration pattern with SBBI as compared to PMWM marker. In contrast, the upper band which showed molecular mass of ca. 20 kDa is well in range with both PMWM and SBBI. Further, this discrepancy in molecular mass was monitored by loading both SBBI and RsPI in all gels performed in this study.

Figures

Fig. 1. FPLC purification of ‘RsPI’ from R. sublobata seeds. Elution profiles of (A) trypsin-affinity and (B) gel filtration columns. The protein peaks active against trypsin were marked with an asterisk (*); (C) Tricine SDS-PAGE (15%) showing different fractions of purification protocol under non-reducing conditions. Lane 1 is loaded with Protein molecular weight marker (PMWM) and lanes 2–6 are loaded with 5 or 10 μg of corresponding protein sample. The gel was stained with silver nitrate. CPI – crude protease inhibitor, TAC – Trypsin affinity chromatography, GFC – Gel filtration chromatography and SBBI – Soybean BBI.