The ability of protein to form specific, stable com- plexes with other proteins is fundamental to many biolog- ical processes. Understanding the molecular basis of pro- tein–protein recognition requires knowledge of individ- ual interactions at the interface between the proteins [1- 3]. One may define two broad categories of protein–pro- tein interfaces: (i) those which resemble cross section through folded proteins in which hydrophobic residues are in the interior and hydrophilic ones at the periphery and in which productive binding is mediated largely by the former and (ii) ones in which polar and non polar residues are interspersed throughout the interface and in which both residue types make comparable contribution to complex stabilization [4]. An antibody molecule combines with its antigen through the complementarity determining region (CDR). Since the repertoire of antigen to which it combines is large, it is understandable that the amino acid residues comprising the CDR have greater functional versatility. These residues have both polar and non-polar groups in them and generally the nonpolar end is buried and the polar end protrudes out. These polar atoms have the potential of forming hydrogen bonds and additional sta- bility of the complex is provided by hydrophobic interac- tions. The interaction of the antibody with the antigen is highly specific and involves noncovalent interactions such as hydrophobic interactions and hydrogen bonding [5, 6]. With the availability of data on crystal structure of several antibody–antigen complexes [2, 7-11], it is possi- ble to quantify these interactions. Some reports suggest preponderance of hydrogen bonds while others show hydrophobic [5, 12] suggesting universal principle of interactions in the immune complexes does not exist and each complex should be characterized de novo. The nature of the interactions between antigen and active cen- ter of the antibody plays an important role both in prog- nosis of immunochemical processes in vivo and in the development of different biotechnological model sys- tems. Though there are a series of direct structural studies like site-specific mutations [13], docking thermodynam- ics [14, 15], X-ray crystallography [8-11, 16], and titra- tion calorimetry devoted to the properties of these com- plexes, such work for each antigen–antibody pair is ISSN 0006-2979, Biochemistry (Moscow), 2006, Vol. 71, Suppl. 1, pp. S31-S37. © Pleiades Publishing, Inc., 2006. Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM04-332, June 26, 2005. S31 Abbreviations: CD) circular dichroism; CDR) complementari- ty determining region; Fab) antigen binding fragment of immunoglobulin; IgG) immunoglobulin G; UV) ultraviolet. * To whom correspondence should be addressed. Hydrophobic Interactions Are the Prevalent Force in Bromelain:Fab′ Complex P. Gupta 1,2 , M. Saleemuddin 1,3 , and R. H. Khan 3 * 1 Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh 202002, Uttar Pradesh, India; fax: +91-571-272-1776; E-mail: pawan_g75@hotmail.com 2 Current address: Department of Pharmacology, Basic Science Biomedical Engineering, University of Minnesota, 6-120 Jackson hall, 321 Church Street SE, Minneapolis, MN55455, USA 3 Interdisciplinary Biotechnology Unit, Faculty of Life Sciences, Aligarh Muslim University, Aligarh 202002, Uttar Pradesh, India; fax: +91-571-272-1776; E-mail: rizwanhkhan1@yahoo.com; msaleemuddin@rediffmail.com Received November 3, 2004 Revision received March 30, 2005 Abstract—Antibromelain polyclonal antibodies against stem bromelain were raised in male albino rabbits and the Fab′ monomers isolated from the IgG of the immune sera as reported in our earlier communication (Gupta, P., Khan, R. H., and Saleemuddin, M. (2003) Biochim. Biophys. Acta, 1646, 131-135). Further, as evident from that communication, brome- lain:Fab′ complex has 1 : 1 stoichiometry. The stability of bromelain:Fab′ complex (1 : 1) was investigated by far and near- UV CD and fluorescence measurements. Addition of up to 1.8 M NaCl caused no significant changes in fluorescence sig- nals and near-UV CD peak pattern. However, the spectral studies together with gel filtration studies suggest dissociation of the complex beyond 5% (v/v) methanol. These results show that hydrophobic interactions play a pronounced role in the binding of Fab′ to bromelain while electrostatic interactions may be less crucial. DOI: 10.1134/S0006297906130050 Key words: bromelain, Fab′, immunocomplexes, hydrophobic interaction, electrostatic interaction, methanol, NaCl