Physicochemical characterization of cross-linked human serum albumin dimer and its synthetic heme hybrid as an oxygen carrier Teruyuki Komatsu a , Yukiko Oguro a , Yuji Teramura a , Shinji Takeoka a , Junpei Okai b , Makoto Anraku b , Masaki Otagiri b , Eishun Tsuchida a, * a Advanced Research Institute for Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan b Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto 862-0973, Japan Received 13 May 2004; received in revised form 9 August 2004; accepted 10 August 2004 Available online 11 September 2004 Abstract The recombinant human serum albumin (rHSA) dimer, which was cross-linked by a thiol group of Cys-34 with 1,6- bis(maleimido)hexane, has been physicochemically characterized. Reduction of the inert mixed-disulfide of Cys-34 beforehand improved the efficiency of the cross-linking reaction. The purified dimer showed a double mass and absorption coefficient, but unaltered molar ellipticity, isoelectric point (pI : 4.8) and denaturing temperature (65 8C). The concentration dependence of the colloid osmotic pressure (COP) demonstrated that the 8.5 g dL 1 dimer solution has the same COP with the physiological 5 g dL 1 rHSA. The antigenic epitopes of the albumin units are preserved after bridging the Cys-34, and the circulation lifetime of the 125 I-labeled variant in rat was 18 h. A total of 16 molecules of the tetrakis{(1-methylcyclohexanamido)phenyl}porphinatoiron(II) derivative (FecycP) is incorporated into the hydrophobic cavities of the HSA dimer, giving an albumin–heme hybrid in dimeric form. It can reversibly bind and release O 2 under physiological conditions (37 8C, pH 7.3) like hemoglobin or myoglobin. Magnetic circular dichroism (CD) revealed the formation of an O 2 –adduct complex and laser flash photolysis experiments showed the three-component kinetics of the O 2 -recombination reaction. The O 2 -binding affinity and the O 2 -association and -dissociation rate constants of this synthetic hemoprotein have also been evaluated. D 2004 Elsevier B.V. All rights reserved. Keywords: Human serum albumin dimer; Cross-linking; Colloid osmotic pressure; Synthetic heme; Albumin–heme dimer; Oxygen carrier 1. Introduction Human serum albumin (HSA) is the most abundant plasma protein and contains 35 cysteines, of which 17 couples form intramolecular disulfide bonds to fold a single polypeptide as a unique heart-shape structure [1–4]. Only the first thiol residue in the chain, namely Cys-34, does not participate in the S–S bonding and functions as a binding site for the SH-involving ligands (cysteine, glutathione, and captopril), as well as for the various metal ions and nitric oxide [1,5]. Interestingly, two albumin molecules can associate to produce a dimer through an intermolecular disulfide bridge of Cys-34; approximately 5% of HSA is actually in a dimeric form in our bloodstream [6]. Hughes [7] initially prepared the HSA dimer by the addition of bifunctional HgCl 2 , which causes Cys-34 to connect through mercury. Subsequent oxidation of this mercury dimer by treatment with iodine gave a disulfide-linked HSA [8]. It can also be directly prepared by oxidation of HSA with ferricyanide [9]. However, electron spin resonance measurements of HSA and the latest crystal structural analysis of the recombinant HSA (rHSA) revealed that Cys- 34 locates in a hydrophobic crevice at a depth of 9.5 2 from the surface [2–4,10]. This implies that the intermolecular Cys-34 disulfide bridging might lead to flattening of the pocket. We have linked two rHSA molecules with a flexible bola-shape spacer, 1,6-bis(maleimido)hexane (BMH), which is long (16 2) enough to connect the Cys-34 residues, to produce a new type of rHSA dimer (Fig. 1) [11]. 0304-4165/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2004.08.010 * Corresponding author. Tel.: +81 3 5286 3120; fax: +81 3 3205 4740. E-mail address: eishun@waseda.jp (E. Tsuchida). Biochimica et Biophysica Acta 1675 (2004) 21 – 31 http://www.elsevier.com/locate/bba