RESEARCH PAPER Biophysical aspects of lysozyme adduct with monocrotophos Sreenivasa Rao Amaraneni & Sudhir Kumar & Samudrala Gourinath Received: 12 February 2014 /Revised: 20 May 2014 /Accepted: 5 June 2014 /Published online: 27 June 2014 # Springer-Verlag Berlin Heidelberg 2014 Abstract The present study on in vitro formation and char- acterization of lysozyme adduct with monocrotophos (MP) evaluates the potential of lysozyme to be used as a sensitive biomarker to monitor exposure levels to the commonly used organophosphorus pesticide monocrotophos. Crystallization of lysozyme protein adduct with monocrotophos was also undertaken to understand the adduct formation mechanism at a molecular level. The binding of organophosphorus pesti- cides to lysozyme is one of the key steps in their mutagenicity. The formation and structural characterization of lysozyme adduct with monocrotophos was done using MALDI- TOFMS, fluorescence, UV/Vis spectroscopy, circular dichro- ism, and X-ray diffraction studies. We report the crystal struc- ture of lysozyme adduct with monocrotophos at 1.9 Å. It crystallized in the P4 3 space group with two monomers in one asymmetric unit having one molecule of monocrotophos bound to each protein chain. The results proved that the fluorescence quenching of lysozyme by monocrotophos is due to binding of monocrotophos with a tryptophan residue of lysozyme. Monocrotophos interacts most strongly with the Trp-108 and Asp-52 of lysozyme. The interactions of the monocrotophos molecule with the lysozyme suggest the formation of a stable adduct. In addition, the alteration of lysozyme secondary structure in the presence of monocrotophos was confirmed by circular dichroism and fluorescence inhibition of lysozyme by increasing monocrotophos and UV/Vis spectrophotometry. The forma- tion of lysozyme adduct with monocrotophos was confirmed by MALDI-TOFMS. Keywords Protein adducts . X-ray diffraction . Organophosphorus pesticide . Monocrotophos . Lysozyme . Crystal structure Introduction Pesticides are widely used in agriculture to prevent the loss in production caused by pests and pest-borne diseases. Indiscriminate use of pesticides may have serious effects on human health and the environment [1–3]. Pesticide contami- nation in water, food, air, and fish may have various effects on human health as they enter humans through the food chain [4]. Proteins in the human body tend to bind to oranophosphorus toxicants and form adducts which cause toxicity. Proteins are considered as potential biomarkers of organophosphorus pes- ticide contamination. Organophosphorus (OP) toxicants are known to cause acute toxicity due to inhibition of acetylcho- line esterase (AChE) [1, 5–7]. Pesticides which cause genotoxic cancer are a structur- ally diverse group. The corresponding molecular mecha- nisms of pesticide poisoning and cancer by formation of DNA and protein adducts are poorly understood [8–14]. Many neurotoxicants (organochlorines, herbicides, organ- ophosphate pesticides) react with DNA and protein resulting in the formation of the adducts which lead to disruption of the structure and function of DNA and S. R. Amaraneni (*) Department of Chemistry, Alliance College of Engineering and Design, Alliance University, Bangalore 562106, India e-mail: sramaraneni@yahoo.com S. Kumar Department of Zoology and Biotechnology, HNB Garhwal University, Srinagar, Uttarakhand 246174, India S. Gourinath (*) School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India e-mail: sgourinath@mail.jnu.ac.in Anal Bioanal Chem (2014) 406:5477–5485 DOI 10.1007/s00216-014-7953-y