http://informahealthcare.com/ipi ISSN: 0892-3973 (print), 1532-2513 (electronic) Immunopharmacol Immunotoxicol, 2014; 36(2): 138–144 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/08923973.2014.884136 RESEARCH ARTICLE Leucoagglutinating phytohemagglutinin: purification, characterization, proteolytic digestion and assessment for allergenicity potential in BALB/c mice Sandeep Kumar 1,2 , Akanksha Sharma 1,3 , Mukul Das 1,3 , S. K. Jain 2 , and Premendra D. Dwivedi 1,3 1 Food Toxicology Division, CSIR – Indian Institute of Toxicology Research, Lucknow, Uttar Pradesh, India, 2 Department of Biotechnology, Faculty of Science, Jamia Hamdard, Hamdard Nagar, New Delhi, India and 3 Academy of Scientific & Innovative Research (AcSIR), New Delhi, India Abstract Red kidney bean (Phaseolus vulgaris) is consumed worldwide as a vegetarian protein source. But, at the same time the allergenicity potential of red kidney bean is a matter of concern. This study is aimed towards purification, characterization, thermal stability, proteolytic digestion and allergenicity assessment of one of the clinically relevant allergens of red kidney bean. The purification of red kidney bean allergic protein was carried out with the help of column chromatography, IgE immunoblotting and reverse phase high- pressure liquid chromatography (RP-HPLC). The purified protein was characterized by peptide mass finger printing (PMF) and studied for its thermal stability, and proteolytic resistance using simulated gastric fluid (SGF) assay. The allergenicity potential of the purified protein was studied in BALB/c mice. The purified protein was identified as leucoagglutinating phytohemagglutinin (PHA-L) with molecular weight 29.5 kDa. The PHA-L showed resistance to heat as well as proteolytic enzyme. Higher levels of total IgE, specific IgE, and histamine were observed in PHA-L treated BALB/c mice when compared to control. Overall, PHA-L possesses characteristics of allergens and may play a potential role in the red kidney bean induced allergy. Keywords Allergic mediators, food allergy, IgE, legumes, peptide mass fingerprinting History Received 11 November 2013 Revised 19 December 2013 Accepted 13 January 2014 Published online 19 February 2014 Introduction Leguminous foods play crucial role in food induced allergic reactions throughout the globe. Legume allergy is well evident in developed as well as developing countries. The prevalence of legume allergy is proportional to the consump- tion scenario within the territory as higher consumption of legume induces higher probability of allergy. This is quite evident by the example of peanut, soybean, and nut allergy which have been reported in US, UK and India, respectively 1–3 . The allergenicity potential of several legumes including peanut, soybean, lentil, chickpea, red gram, green gram, red kidney bean and green bean have been well studied 4 . Along with allergenic assessment of these legumes the allergens are well identified and characterized, including Ara h1-Ara h7 of peanut, Gly mBD of soybean, Vig r2-Vig5 of green gram, Caj c1-Caj c6 of red gram and Pha v3 of green bean 4 . The allergenic proteins have been reported to be purified by bacterial expressions, column chromatography, fast protein liquid chromatography (FPLC), reverse phase high-pressure liquid chromatography (HPLC) and the several other techniques. The characterization of purified proteins can be performed by matrix-assisted laser desorption ionization- time of flight (MALDI-TOF), peptide mass fingerprinting (PMF), liquid chromatography tandem mass spectrometry (LC-MS/MS), and other proteomics techniques. Most of the allergenic proteins of legumes have ability to resist against temperature and proteolytic enzymes but exceptions are also there 5,6 . The allergenic proteins mainly cause hypersensitivity reactions via immunoglobulin E (IgE) and Th2 cytokines 7 . Red kidney bean (Phaseolus vulgaris) is a member of Fabaceae family that is produced and consumed throughout the world 8 . The allergenicity of red kidney bean proteins has been reported in the allergic patients, murine model and passively sensitized rat basophilic leukemia (RBL-2H3) cells 9–11 . Further, the role of phytohemagglutinins (PHAs) in the red kidney bean-induced allergic reactions have also been studied that revealed the pivotal role of PHAs 12 . But, still very few allergenic proteins from red kidney bean have been purified so far. Allergens are responsible for the induction of food allergic reaction, so it is an important aspect to assess allergenicity Address for correspondence: Dr. Premendra D. Dwivedi, Principal Scientist, Food Toxicology Division, CSIR – Indian Institute of Toxicology Research (CSIR-IITR), P.O. Box No. 80, Mahatma Gandhi Marg, Lucknow – 226 001, Uttar Pradesh, India. Tel: +91 522 2620107, 2620106, 2616191. Fax: +91 522 2628227. E-mail: pddwivedi@ yahoo.com