Purification and characterization of Bowman-Birk and Kunitz isoinhibitors from the seeds of Rhynchosia sublobata (Schumach.) Meikle, a wild relative of pigeonpea

Abstract

Rhynchosia sublobata, a wild relative of pigeonpea, possesses defensive proteinase/protease inhibitors (PIs). Characterization of trypsin specific PIs (RsPI) separated from seeds by column chromatography using 2-D gel electrophoresis and Edman degradation method identified R. sublobata possessed both Bowman-Birk isoinhibitors (RsBBI) and Kunitz isoinhibitors (RsKI). A quick method was developed to separate RsBBI and RsKI from RsPI based on their differential solubility in TCA and acetate buffer. N-terminus sequencing of RsBBI and RsKI by MALDI-ISD ascertained the presence of Bowman Birk and Kunitz type isoinhibitors in R. sublobata. RsBBI (9216 Da) and RsKI (19,412 Da) exhibited self-association pattern as revealed by western blotting with anti-BBI antibody and MALDI-TOF peptide mass fingerprint analysis, respectively. RsBBI and RsKI varied significantly in their biochemical, biophysical and insecticidal properties. RsBBI inhibited the activity of trypsin (Ki = 128.5 ± 4.5 nM) and chymotrypsin (Ki = 807.8 ± 23.7 nM) while RsKI (Ki = 172.0 ± 9.2 nM) inhibited the activity of trypsin alone, by non-competitive mode. The trypsin inhibitor (TI) and chymotrypsin inhibitor (CI) activities of RsBBI were stable up to 100°C. But, RsBBI completely lost its TI and CI activities on reduction with 3 mM DTT. Conversely, RsKI lost its TI activity on heating at 100°C and retained > 60% of its TI activity in presence of 3 mM DTT. CD spectroscopic studies on RsBBI and RsKI showed their secondary structural elements in the following order: random coils > β-sheets/β-turns > α-helix. However, RsKI showed reversible denaturation midpoint (Tm) of 75°C. Further, the significant inhibitory activity of RsBBI (IC 50 = 24 ng) and RsKI (IC 50 = 59 ng) against trypsin-like gut proteases of Achaea janata (AjGPs) and Helicoverpa armigera (HaGPs) suggest them as potential biomolecules in the management of A. janata and H. armigera, respectively. vegetative organs during biotic and abiotic stresses (Jamal et al., 2013; Yamchi et al., 2017). They also act as pseudosubstrates of proteases and stabilize them during desiccation. The PIs are rapidly degraded during seed germination to release essential amino acids and they reappear in cotyledons to protect them from invading pests and pathogens. They also take part in programmed cell death in plants. Bowman-Birk

GASP‐1 and GASP‐2, two closely structurally related proteins with a functional duality in antitrypsin inhibition specificity: a mechanistic point of view

“…As for GASP-1 and GASP-2, we measured inhibitor/trypsin dissociation K i values of 220 and 290 nM for mGASP-1/Ku 2-2 and mGASP-2/Ku 2-1 , respectively. K i values are of the same order of magnitude as those obtained for the two recombinant mGASP proteins or several antiproteases containing a Ku domain [28,29]. Kinetic analyzes showed that the mGASP-1/Ku 2-2 chimeric protein exhibits a competitive-type inhibition mechanism similar to that described for GASP-2.…”

Response of Midgut Trypsin- and Chymotrypsin-Like Proteases of Helicoverpa armigera Larvae Upon Feeding With Peanut BBI: Biochemical and Biophysical Characterization of PnBBI

“…Besides, the self-association of BBIs not only provides stability, but also important in molecular packing as a seed storage protein (Hogg, 2003;Kumar et al, 2015). All the isoinhibitors visualized on one and two-dimensional gel electrophoresis conferred resistance against trypsin and chymotrypsin digestion (Figures 2C,D,F,G; Prasad et al, 2010a;Swathi et al, 2016;Mohanraj et al, 2019). The prominent TI activity was evidenced through wider protective zones against trypsin as compared to chymotrypsin during gelatin in-gel activity staining (Figure 2F).…”

“…The protease activity was identified by observing the formation rate of p-nitroanilide from BAPNA or GLUPHEPA (1 mM) at 410 nm using UV-visible spectrophotometer (UV-1700, Shimadzu, Japan) as described in Mohanraj et al (2019). The p-nitroanilide molar extinction coefficient (M −1 cm −1 ) is corresponding to 8800 at 410 nm.…”

“…(Oddepally et al, 2013), sombreiro (Dantzger et al, 2015) and pigeon pea cultivars (Swathi et al, 2014) as well as wild relatives (Swathi et al, 2016;Mohanraj et al, 2019) (Table 1). The most discrepancy observed in molecular mass of PnBBI determined by Tricine SDS-PAGE (∼10 kDa) as well as MALDI-TOF (6.73 kDa) analysis is not uncommon among the BBIs (Figures 1C, lane 5 and 2A; Swathi et al, 2014;Mohanraj et al, 2019). However, the observed molecular mass of PnBBI in SDS-PAGE is comparable to commercial soybean BBI.…”

Trypsin inhibitor from duck albumen: Purification and characterization

“…Moreover, TI from other sources including fish roe (Klomklao et al, 2016;Ustadi et al, 2005), chicken plasma (Rawdkuen, Benjakul, Visessanguan, & Lanier, 2005), mung bean (Klomklao, Benjakul, Kishimura, & Chaijan, 2011), and the seed of Rhynchosia sublobata (Schumach.) Meikle (Mohanraj et al, 2019) were also isolated, purified as well as characterized. Recently, protease inhibitors present in duck albumen has been proven to exhibit higher inhibitory activity than hen albumen (Quan & Benjakul, 2018a).…”