Journal of Colloid and Interface Science 296 (2006) 324–331 www.elsevier.com/locate/jcis A fluorimetric and circular dichroism study of hemoglobin—Effect of pH and anionic amphiphiles Swati De ∗ , Agnishwar Girigoswami Department of Chemistry, University of Kalyani, Kalyani 741235, India Received 3 June 2005; accepted 22 August 2005 Available online 11 October 2005 Abstract In this work, bovine hemoglobin (Hb) has been studied mainly by the fluorescence method. pH has been found to exert a profound effect on Hb structure. This has been confirmed by fluorescence and circular dichroism (CD) studies. The pH-induced change in quaternary structure of Hb indirectly affects its secondary structure. This in turn affects ligand binding to Hb at various pH. The binding of two amphiphiles, a bile salt and a surfactant, have been investigated. The pH-induced structural modification of Hb has been confirmed by studies with the well-known denaturant urea and the polarity probe ANS, which has been used as an extrinsic fluorophore.  2005 Published by Elsevier Inc. 1. Introduction Hemoglobin (Hb) is a physiologically important globular protein [1–4]. It has a molecular weight of 64.5 kDa. The Hb molecule contains four globin chins, of which two are α-chains and two are β -chains [3–6]. Each of the four globin chains con- tain the prosthetic group, i.e., heme. In the center of each heme group is an Fe 2+ . The heme group is held in position by interac- tions with the histidine side chain of the globins. The tetramer is assembled from two symmetrical (αβ ) dimers. Each (αβ ) dimer contains 3 tryptophan (Trp) residues, adding to a total of six Trp residues in the tetramer. The three Trp residues in each dimer are named as α-14 Trp, β -15 Trp, and β -37 Trp, depending on their location. Fluorescence of intact heme proteins is of- ten difficult to detect due to quenching of Trp fluorescence by the neighboring heme groups [7,8]. However, Hirsch et al. [5– 8] and other workers [9–11] have overcome this problem by using dilute protein solutions (i.e., <5 μM) and front-face opti- cal alignment. Fluorescence quenching of external labels upon binding to heme proteins can be used to ascertain the nature of interaction between the two [12]. Alpert et al. [13] showed that the intrinsic fluorescence of Hb primarily originates from the * Corresponding author. Fax: +91 3325828282. E-mail address: swati_de1@rediffmail.com (S. De). β -37 Trp and is sensitive to the R–T transition. The R form is the oxy/ligand bound form while the T form is the deoxy form [1–4]. The R and T forms show significant changes in relative fluorescence intensity. When oxygen binds to Hb, the iron atom moves into the plane of the porphyrin ring and consequently the histidine residue bound to the fifth coordination site of Fe 2+ moves. This histidine is part of the globin α-helix which also moves thus leading to a structural change. Previous workers have reported significant changes in Trp fluorescence intensity and lifetime of Hb due to the R–T transition [14–17]. The pri- mary emitting fluorophore of Hb, i.e., β -37 Trp, lies in the protein interior in a hydrophobic environment. The residence site of β -37 Trp is also the major site of quaternary change during the R–T transition. Thus Trp fluorescence emission re- ports faithfully on the allosteric R–T transition and dissociation state [5]. Against this backdrop of literature, this work was performed to study the effect of pH on Hb fluorescence and on the R–T transition. The effect of binding of anionic amphiphiles to Hb was also investigated. Studies were also carried out with urea, the well-known protein denaturant. 2. Experimental Hemoglobin from bovine blood, Fluka, was used without further purification. The concentration of Hb used was low, 0021-9797/$ – see front matter  2005 Published by Elsevier Inc. doi:10.1016/j.jcis.2005.08.047