Physico-Chemical and Antifungal Properties of a Trypsin Inhibitor from the Roots of Pseudostellaria heterophylla

• Published in: Molecules (Basel, Switzerland) Vol. 23; no. 9; p. 2388
• Main Authors: Cai, Xixi, Xie, Xiaoli, Fu, Nanyan, Wang, Shaoyun
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 18.09.2018; MDPI
• Subjects: Aluminum; Amino Acid Sequence; Ammonium; Ammonium sulfate; antifungal; Antifungal agents; Antifungal Agents - chemistry; Antifungal Agents - isolation & purification; Antifungal Agents - pharmacology; Antimicrobial agents; Blood coagulation; Carcinogenesis; Carcinogens; Caryophyllaceae - chemistry; Cell membranes; Chromatography; Chromatography, Gel; Circular Dichroism; Conformation; Dichroism; Disruption; Edman degradation; Enzyme Stability - drug effects; Enzymes; Ferric ions; Fluorescence; Fluorescence spectroscopy; Food; Fungi - drug effects; Fungicides; Fusarium oxysporum; Gel chromatography; Gel filtration; Herbal medicine; Hydrogen-Ion Concentration; Inactivation; Inhibitors; Ion exchange; Ion-exchange chromatography; Iron; Kinetics; Manganese ions; Metal ions; Microbial Sensitivity Tests; Microorganisms; Molecular Weight; Organic chemistry; Peptidase; Peptides; Phytochemicals - chemistry; Phytochemicals - isolation & purification; Phytochemicals - pharmacology; Plant Extracts - chemistry; Plant Extracts - isolation & purification; Plant Extracts - pharmacology; Plant Roots - chemistry; Platelet aggregation; Proteins; Proteolysis; Pseudostellaria; Pseudostellaria heterophylla; Regulators; Roots; Seeds; Spectrum analysis; Temperature; Trypsin; trypsin inhibitor; Trypsin inhibitors; Trypsin Inhibitors - chemistry; Trypsin Inhibitors - isolation & purification; Trypsin Inhibitors - pharmacology; Tryptophan; Tyrosine; China; antifungal; Pseudostellaria heterophylla; trypsin inhibitor; fluorescence spectroscopy; circular dichroism
• ISSN: 1420-3049; 1420-3049
• DOI: 10.3390/molecules23092388

Plant peptidase inhibitors play essential roles in the defense systems of plants. A trypsin inhibitor (PHTI) with a molecular mass of 20.5 kDa was isolated from the fresh roots of the medicinal herb, . The purification process involved ammonium sulfate precipitation, gel filtration chromatography on Sephadex G50, and ion-exchange chromatography on DEAE 650M. The PHTI contained 3.7% α-helix, 42.1% β-sheets, 21.2% β-turns, and 33% disordered structures, which showed similarity with several Kunitz-type trypsin inhibitors. Inhibition kinetic studies indicated that PHTI was a competitive inhibitor, with a K value of 3.01 × 10 M, indicating a high affinity to trypsin. The PHTI exhibited considerable stability over a broad range of pH (2⁻10) and temperatures (20⁻70 °C); however, metal ions, including Fe , Ba , Mn , and Al , could inactivate PHTI to different degrees. Results of fluorescence spectroscopy and circular dichroism showed that Fe could bind to TI with an association constant of 2.75 × 10⁵ M to form a 1:1 complex, inducing conformation changes and inactivation of PHTI. In addition, PHTI could inhibit the growth of the phytopathogens, and , through disruption of the cell membrane integrity. The present study extended research on proteins and makes PHTI an exploitable candidate as an antifungal protein for further investigation.