Purification and characterization of a lectin from Trigonella foenum-graecum (fenugreek) seeds and its porphyrin binding studies

Abstract

Lectin (TfgL) was purified from the seeds of Trigonella foenum-graecum (Fenugreek) belonging to fabaceae family by ammonium sulphate precipitation, ion exchange and followed by size exclusion chromatography. SDS–PAGE analysis revealed that TfgL molecular weight is approximately 27 kDa. 2D-PAGE reveals the existence of two isolectins (pI values of 6.3 and 6.7) with acidic nature and charge heterogeneity. The MALDI-TOF–MS and peptide mass fingerprinting investigation of TfgL showed sequence similarity with a lectin. The hemagglutinating activity of TfgL was stable in broad range of temperature 37–90 °C and at varied pHs 3, 7.6 and 10. Far-UV circular dichroism measurements showed that TfgL is mostly composed of α-helix (84.5%), β-sheet (6.5%), β-turns (5%) and unordered structures (4%). TfgL showed conformational stability in wide range of temperatures (20‒90 °C) and pHs (3, 7.6 and 10) but lost its secondary structure in the presence of 6 M Gdn.HCl. Quenching titrations were carried out with acrylamide and iodide quenchers in order to investigate the exposure and accessibility of the protein tryptophan residues. Maximum quenching observed with acrylamide compared to iodide revealed that the Trp residues of TfgL are buried in the protein core, which is hydrophobic in nature. TfgL showed binding affinity towards porphyrin, the association constant (K_a), for MnTSPP and MnTMPyP was calculated to be 1.2 × 10⁶ M^‒1 and 3.45 × 10⁶ M^‒1, respectively.