Cross-Linked Human Serum Albumin Dimer Incorporating Sixteen (Tetraphenylporphinato)iron(II) Derivatives: Synthesis, Characterization, and O2-Binding Property

Abstract

Recombinant human serum albumin (rHSA) was dimerized by bis(maleimido)hexane through the free thiol at Cys34. The molecular mass of the dimer [(rHSA)2] was determined by native PAGE electrophoresis and MALDI−TOFMS spectrometry. As expected, the colloid osmotic pressure was only half that of the monomeric rHSA solution. Incorporation of (2-[8-{N-(2-methylimidazolyl)}octanoyloxymethyl]-5,10,15,20-tetrakis(o-pivalamido)phenylporphinato)iron(II)s (FePs) into the hydrophobic cavities of the rHSA dimer provides a synthetic hemoprotein, [(rHSA-FeP)2], which can reversibly bind and release dioxygen under physiological conditions (in aqueous media, pH 7.3, 37 °C) like hemoglobin and myoglobin. A maximum of 16 hemes (FePs) were incorporated into the (rHSA)2 structure. On the basis of the isoelectric focusing measurement, the surface charge distributions of the (rHSA)2 and (rHSA-FeP)2 are identical to that of rHSA. The O2-binding affinity (P1/2: 30 Torr at 37 °C) and O2-association and -dissociation rate constants ...