Pollutant-Induced Modulation in Conformation and β-Lactamase Activity of Human Serum Albumin

Khan RH (2012) Pollutant-Induced Modulation in Conformation and b-Lactamase Activity of Human Serum Albumin. PLoS ONE 7(6): e38372. doi:10.1371/journal.pone.0038372 Pollutant-Induced Modulation in Conformation and b- Lactamase Activity of Human Serum Albumin Ejaz Ahmad 0 Gulam Rabbani 0 Nida Zaidi 0 Basir Ahmad 0 Rizwan Hasan Khan 0 Rajagopal Subramanyam, University of Hyderabad, India 0 Interdisciplinary Biotechnology Unit, Aligarh Muslim University , Aligarh , India Structural changes in human serum albumin (HSA) induced by the pollutants 1-naphthol, 2-naphthol and 8-quinolinol were analyzed by circular dichroism, fluorescence spectroscopy and dynamic light scattering. The alteration in protein conformational stability was determined by helical content induction (from 55 to 75%) upon protein-pollutant interactions. Domain plasticity is responsible for the temperature-mediated unfolding of HSA. These findings were compared to HSAhydrolase activity. We found that though HSA is a monomeric protein, it shows heterotropic allostericity for b-lactamase activity in the presence of pollutants, which act as K- and V-type non-essential activators. Pollutants cause conformational changes and catalytic modifications of the protein (increase in b-lactamase activity from 100 to 200%). HSA-pollutant interactions mediate other protein-ligand interactions, such as HSA-nitrocefin. Therefore, this protein can exist in different conformations with different catalytic properties depending on activator binding. This is the first report to demonstrate the catalytic allostericity of HSA through a mechanistic approach. We also show a correlation with non-microbial drug resistance as HSA is capable of self-hydrolysis of b-lactam drugs, which is further potentiated by pollutants due to conformational changes in HSA. - Funding: Financial assistance to E. Ahmad, G. Rabbani and N. Zaidi in the form of a Senior Research Fellowship was supported by the Council of Scientific and Industrial Research (CSIR), New Delhi, India. R.H. Khan is an Associate Professor in A.M.U., Aligarh. No additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Rizwan Hasan Khan is a PLoS ONE Editorial Board member. This does not alter the authors adherence to all the PLoS ONE policies on sharing data and materials. Human serum albumin (HSA) is the most abundant multifunctional single chain protein in blood plasma. HSA plays important physiological and pharmacokinetical roles by binding and transporting exo- and endo-genous compounds [1,2]. It also possesses some enolase, esterase and hydrolase activities [3]. Thus, this protein contains both binding and catalytic sites [4]. This is heart-shaped and 80680630 A in size [5] and the molecular topology can be easily changed because of its flexible nature as demonstrated in physicochemical studies [6]. Transportation of solute is one of the best characterized roles of this protein which solublizes ligands and targets them to cells through binding to specific cell receptors. HSA bound to specific ligands are recognized by specific cellular receptors through the liganddependent conformations of this protein [7]. Additionally, upon ligand binding, albumin undergoes physiologically relevant structural changes as in case of HSA-oleate interaction. As a consequence of the alteration in the nature of the local environment surrounding Cys-34, the long chain of fatty acid regulates the radical-trapping antioxidant activity [8]. These ligand-dependent changes in protein conformations are specific to the type of ligands and more precisely to their capacity to accumulate in the binding pockets. The ligand-induced structural changes in HSA are suggested to mediate its role in receptormediated cellular interaction as well as solute transport in physiological conditions. We have studied the effect of pollutants on the structure and function of HSA. 1-naphthol (1N), 2-naphthol (2N) and 8quinolinol (8H) shown in Figure 1 are direct or indirect (metabolite) organic pollutants and their accumulation in body can cause cyanosis, liver damage, nephritis, circulatory collapse and even death. A detailed study on the mode of interaction between HSA and these pollutants has been already performed and reported by our group [9] and the amino acid residues to which the pollutants bind are shown in Figure 2. In the present study, the effects of pollutant binding to HSA have been analyzed by a number of techniques. UV-visible, fluorescence spectroscopy, circular dichroism and dynamic light scattering are used to investigate the structural changes in protein associated with ligand binding. Here, our study offers not only direct proof for ligand-induced conformational alterations in protein, but also a clear understanding of the nature and after effects of these changes. Materials and Methods Materials Fatty acid free human serum albumin (A1887), 1N (N2780) and 2N (185507) were from Sigma-Aldrich, USA, 8-quinolinol (8H), tris and hydrochloric acid were from Qualigens, India, whereas nitrocefin (484400) was a product of Calbiochem. Preparation of Solutions All experiments were carried out in 20 mM tris-HCl buffer, pH 7.4. Fatty acid free HSA was used exactly as it was received. The protein concentration was spectrophotometrically determined (E218%0nm = 5.3) on PerkinElmer Lambda 25. Circular Dichroism The isothermal wavelength scan and thermal denaturation studies of HSA in the absence and presence of pollutants were carried out with JASCO-J815 spectropolarimeter equipped with a Peltier-type temperature controller. The instrument was calibrated with d-10-camphorsulfonic acid. All the isothermal CD measurements were keeping at 37uC. Spectra were collected with 50 nm/ min scan speed, 0.1 nm data pitch and a response time of 2 s. Each spectrum was the average of 2 scans. For the measurement of far-UV CD spectra (190250 nm) the pathlength of cell was 0.1 cm while it was of 1 cm for near-UV CD (250300 nm) spectra. The results were expressed as MRE (mean residue ellipticity) in deg.cm2.dmol21, which is given by: where hobs is the observed ellipticity in degrees, Cp is the molar fraction and l is the length of the light path in centimeter [10]. All spectra were smoothed by the SavitzkyGolay method with 25 convolution width. The thermal denaturations were studied in the range of 2590uC with 1uC min21 temperature slope probed by far-UV CD at 222 nm and near-UV CD at 263 nm. To get the fractional populations of intermediates mediated elevated temperature, the CD values of unfolding at their respective temperatures were calculated by algebraic differentiation and the prominent peaks were considered as intermediates. Acrylamide Quenching Measurements by Steady State Fluorescence Tryptophan fluorescence is used as a probe of local environment in a protein for determ (...truncated)