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 (IC50 = 24 ng) and RsKI (IC50 = 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.

中文翻译：

---

从木豆野生近缘木豆 (Schumach.) Meikle 种子中纯化和表征 Bowman-Birk 和 Kunitz 同种抑制剂

Rhynchosia sublobata 是木豆的野生近缘种，具有防御性蛋白酶/蛋白酶抑制剂 (PI)。使用 2-D 凝胶电泳和 Edman 降解方法通过柱色谱法对从种子中分离的胰蛋白酶特异性 PI (RsPI) 进行表征，鉴定了亚叶红薯具有 Bowman-Birk 同种抑制剂 (RsBBI) 和 Kunitz 同种抑制剂 (RsKI)。基于 RsBBI 和 RsKI 在 TCA 和醋酸盐缓冲液中的不同溶解度，开发了一种快速方法来将它们从 RsPI 中分离出来。通过 MALDI-ISD 对 RsBBI 和 RsKI 进行 N 端测序，确定了亚叶蔷薇中存在 Bowman Birk 和 Kunitz 型同种抑制剂。RsBBI (9216 Da) 和 RsKI (19,412 Da) 分别通过使用抗 BBI 抗体的蛋白质印迹和 MALDI-TOF 肽质量指纹分析显示出自关联模式。RsBBI 和 RsKI 的生化、生物物理和杀虫特性差异很大。RsBBI 通过非竞争模式抑制胰蛋白酶 (Ki = 128.5 ± 4.5 nM) 和胰凝乳蛋白酶 (Ki = 807.8 ± 23.7 nM) 的活性，而 RsKI (Ki = 172.0 ± 9.2 nM) 单独抑制胰蛋白酶的活性。RsBBI 的胰蛋白酶抑制剂 (TI) 和胰凝乳蛋白酶抑制剂 (CI) 活性在高达 100 °C 时保持稳定。但是，RsBBI 在用 3 mM DTT 还原时完全失去了其 TI 和 CI 活性。相反，RsKI 在 100 °C 加热时失去其 TI 活性，并在 3 mM DTT 存在下保留其 60% 以上的 TI 活性。RsBBI 和 RsKI 的 CD 光谱研究显示它们的二级结构元素按以下顺序排列：无规卷曲 > β-折叠/β-转角 > α-螺旋。然而，RsKI 显示可逆变性中点 (Tm) 为 75 °C。更多，