Purification and Characterization of a 43 kD Hepatoprotective Protein from the Herb Cajanus indicus L. Kasturi Sarkar, 1 Ayantika Ghosh, 1 Michael Kinter, 2 Barsanjit Mazumder, 3 and Parames C. Sil 1,4 Cajanus indicus L, a herb, is popularly known for its hepatoprotective activity. Aqueous extract of the leaves of this plant contains hepatoprotective and hepatostimulatory molecule(s). Present study was aimed to isolate, purify and characterize the active principle(s) responsible for that activity. A hepatoprotective protein molecule has been purified to homogeneity (approximately 300 fold). Homogeneous preparation of the protein was achieved by homogenization, (NH 4 ) 2 SO 4 precipitation, ion-exchange chromatography, gel filtration and high performance liquid chromatography. The protein purified is composed of a single polypeptide chain having an apparent molecular mass of 43 kD as determined by SDS-PAGE and gel filtration through sephadex G-75 column. The isoelectric point of the protein determined was 4.8. Loss of biological activity after heat and protease treatment confirmed that the active molecule is a protein. Peptide fragments of the protein generated by trypsin cleavage were subjected to MALDI-TOF as well as LC-MS analyses and among the various fragments, four were very prominent and used for the determination of the amino acid sequence of the hepatoprotective protein. While one of the peptide fragment revealed strong sequence homology with plastocyanin, another fragment showed some similarity with a tomato protein present in the NCBI non-redundant database. The third peptide, on the other hand, is unique as it did not show any sequence homology with any known protein in the database. The protein showed maximum hepatoprotective activity when administered at a dose of 2 mg/kg body weight for five days after CCl 4 administration. Histopathological studies also supported the hepatoprotective nature of the protein. Along with its curative property, the protein also possesses preventive role against a number of toxin induced hepatic damages. KEY WORDS: Amino acid sequence; Cajanus indicus; hepatotoxicity; hepatoprotective protein; purifi- cation & characterization. Kasturi Sarkar and Ayantika Ghosh contributed equally in the study 1 Department of Chemistry, Bose Institute, 93/1, A.P.C. Road, Calcutta, 700009, West Bengal, India. 2 Department of Cell Biology, The Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH, 44195, USA. 3 Department of Biology, Geology and Environmental Science, Cleveland State University, 2121 Euclid Avenue, Cleveland, OH, 44115, USA. 4 To whom correspondence should be addressed. E-mail: parames@ bosemain.boseinst.ac.in/parames_95@yahoo.co.in Abbreviations: BSA, bovine serum albumin; PH, phytohaemag- glutinin; Fip, fungal immunomodulatory proteins; (NH 4 ) 2 SO 4 , ammonium sulphate; SDS-PAGE, sodium dodecyl sulphate poly- acrylamide gel electrophoresis; KD, kilodalton; CCl 4 , carbon tet- rachloride; HPLC, high performance liquid chromatography; mRNA, messenger ribonucleic acid; DEAE, diethyl amino ethyl; SGPT, serum glutamate pyruvate transaminase; DNPH, dinitro phenyl hydrazine; LC-MS, Liquid Chromatography Mass Spec- trometry; MALDI-TOF, Matrix Assisted Laser Desorption/Ioni- zation-Time of Flight. 411 1572-3887/06/0900-0411/0 Ó 2006 Springer Science+Business Media, LLC The Protein Journal, Vol. 25, No. 6, September 2006 (Ó 2006) DOI: 10.1007/s10930-006-9030-7 Published Online: November 8, 2006