Purification and characterization of a stable Kunitz trypsin inhibitor from Trigonella foenum-graecum (fenugreek) seeds.

Kunitz trypsin inhibitor was purified from the seeds of Trigonella foenum-graecum (TfgKTI) belonging to fabaceae family by ammonium sulphate precipitation, cation exchange, gel filtration and hydrophobic chromatography. Purity of the protein was analyzed by RP-HPLC and native-PAGE. SDS-PAGE analysis under reducing and non-reducing conditions showed that protein consists of a single polypeptide chain with molecular mass of approximately 20 kDa. Mass spectroscopy analysis revealed that the intact mass of purified inhibitor is 19,842.154 Da. One dimensional SDS gel was tryptically digested, resulting peptides were subjected to MALDI-TOF-MS analysis, and peptide mass fingerprinting (PMF) analysis of TfgKTI shows sequence similarity with Kunitz trypsin inhibitor in database search. Two dimensional electrophoresis identified presence of four isoinhibitors (pI values of 5.1, 5.4, 5.7 and 6.1). Kinetic studies showed that the protein is a competitive inhibitor and has high binding affinity with trypsin (Ki 3.01×10(-9)M) and chymotrypsin (Ki 0.52×10(-9)M). The TfgKTI retained the inhibitory activity over a broad range of pH (pH 3-10), temperature (37-100°C) and salt concentration (up to 3.5%). Far-UV circular dichroism measurements revealed that TfgKTI is predominantly composed of β-sheets (39%) and unordered structures (48%) with slight helical content (13%). TfgKTI retained over 90% trypsin inhibition upon storage at 4°C for over a period of six months.