Isoflurane's Effect on Protein Conformation as a Proposed Mechanism for Preconditioning

Hindawi Publishing Corporation Biochemistry Research International Volume 2011, Article ID 739712, 8 pages doi:10.1155/2011/739712 Research Article Isoflurane’s Effect on Protein Conformation as a Proposed Mechanism for Preconditioning Michelle R. Baker,1 Sean K. Benton,1 Christopher S. Theisen,2 Chad A. McClintick,1 Eugene E. Fibuch,1 and Norbert W. Seidler2 1 Department of Anesthesiology, University of Missouri-Kansas City School of Medicine, 4401 Wornall Road, Kansas City, MO 64108, USA 2 Department of Biochemistry, Kansas City University of Medicine and Biosciences, 1750 Independence Avenue, Kansas City, MO 64106, USA Correspondence should be addressed to Norbert W. Seidler, Received 3 June 2011; Accepted 13 July 2011 Academic Editor: Trevor Creamer Copyright © 2011 Michelle R. Baker et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Persistent alteration of protein conformation due to interaction with isoflurane may be a novel molecular aspect of preconditioning. We preincubated human serum albumin with isoflurane, dialyzed to release agent, and assessed protein conformation. Susceptibility to chemical modification by methylglyoxal and nitrophenylacetate was also examined. Isoflurane had a persistent effect on protein conformation. An increase in the susceptibility of surface residues to chemical modification attended this change in conformation. Modification of isoflurane-treated HSA included intra- and intersubunit cross-linking that may be a consequence of anesthetic-induced changes in multimeric subpopulations. This irreversible effect of isoflurane may represent a mechanism for preconditioning. 1. Introduction The prevailing view of inhaled anesthetics is that they exhibit their desired effects on consciousness and response to pain by binding to target neuronal proteins [1]. The specific conformational changes that occur on binding of inhaled anesthetics remain an area of substantial research interest. Crystallographic and biochemical data reveal that binding sites exhibit considerable heterogeneity [2, 3]. Nevertheless, certain commonalities may exist [4]. The binding of inhaled anesthetics appear to affect interfacial regions in proteins that may be at sites interfacing two domains or subunits [1, 5]. These sites are considered to be less hydrated than substrate binding sites [6, 7]. Occupancy of these cavities by inhaled anesthetics may play a role in limiting conformational exploration of proteins. We think that upon release of anesthetic agents from these sites, a persistent alteration in conformation may occur that contributes to preconditioning. The susceptibility of proteins to chemical modification by carbohydrate and lipid fragmentation products increases following exposure to inhaled anesthetics [8, 9]. The current study further explores the consequence of isoflurane binding using serum albumin as a surrogate protein in order to elucidate the effects of inhaled anesthetics on protein conformation. Human serum albumin (HSA) has several well-characterized anesthetic-binding sites [2, 3]. We think that the anesthetic-induced increase in susceptibility to chemical modification, such as glycation, may ultimately lead to cell signals associated with preconditioning. Interestingly, inhaled anesthetics upregulate heat shock proteins [10], which are involved in repairing protein misfolds. The unfolded protein response is thought to play a part in conferring cellular preconditioning [11]. We previously observed that protein unfolding increases the susceptibility to glycation [12], suggesting that susceptibility to chemical modification is dependent upon the conformational integrity of the protein. 2 Biochemistry Research International In the present study we tested whether glycation and acetylation of HSA is affected by isoflurane. Methylglyoxal (MG), a reactive di-carbonyl, is a glycating agent elevated in diabetics [13], and p-nitrophenylacetate (NPA) is a potent synthetic acetylating reagent that reacts with lysine residues that are common targets of monocarbonyl glycating agents. Albumin exhibits an esterase-like activity [14, 15], which recently has been redefined as chemical modification via acetylation of multiple sites by NPA that occurs in a biphasic manner with specific residues being sequentially modified [16]. We also looked at the recovery of protein conformation following extensive dilution of isoflurane-treated HSA. In addition to describing the appearance of a persistent isoflurane-directed misfold, we discuss the role of isoflurane on HSA oligomerization and MG-induced crosslinking. Our results suggest a novel mechanism of preconditioning, which is a curious attribute of isoflurane [17, 18]. 2. Materials and Methods 2.1. Chemicals and Reagents. Unless otherwise stated, HSA (Sigma-Aldrich; A-8763, fraction V) solutions (150 μM) were prepared in a 20 mM sodium phosphate (pH 7.4) buffer. Stock bottles of MG (Sigma-Aldrich; M-0252) and isoflurane (Hospira Laboratories, Lake Forest, IL; lot #35526-DK) were used within three months after opening; MG (40% in water) was kept at 4◦ C and isoflurane was purged with nitrogen gas following use. p-Nitrophenylacetate (NPA) was purchased from Sigma (N8130) and freshly prepared (37.5% acetone) for each experiment. 8-Anilino1-naphthalene sulphonic acid (ANS) was from Sigma. Water was deionized using Millipore’s Milli-Q system to 18.2 MΩ/cm. All other chemicals were of reagent grade or better. 2.2. Exposure of HSA to Isoflurane. Unless otherwise indicated, HSA (950 μL of 150 μM) was treated with isoflurane (500 μL) at room temperature by gently mixing in airtight vials (Supelco) for 30 min prior to brief centrifugation and removal of upper aqueous phase containing HSA. Isoflurane is immiscible in water with a preference for the gas phase. The aqueous isoflurane concentrations were therefore determined using the ideal gas law and the partition coefficient for isoflurane in water/gas as described in [19]. Briefly, the partial pressure of isoflurane (ppI) in the air compartment of the vial (gVi ) was calculated using the following equation: ppI = gVi × 760 mmHg. (1) The ideal gas law, PV = nRT, where P is ppI, V is volume of the headspace (hsV ), T is temperature in kelvins, and R is the gas constant (62.363 L · mmHg · ◦ K−1 · mol−1 ), was used to determine the number of moles of isoflurane in the gas compartment (n(g)):   n g =    ppI · hsV . RT (2) The water/gas partition coefficient (λw) for isoflurane was used to calculate the amount of moles of isoflurane in the water compartment (n(w)):   (n(w)) λw =    . n g (3) The concentration of isoflurane in the water compartment (C(w)) was obtained by dividing the number of moles of isoflurane (n(w)) by the volume in liters of the water compartment (lVw ) in the vial and a (...truncated)