Cross-linked human serum albumin dimer incorporating sixteen (tetraphenylporphinato)iron(II) derivatives: Synthesis, characterization, and O-2-binding property

Recombinant human serum albumin (rHSA) was dimerized by bis(maleimido)hexan e through the free thiol at Cys34. The molecular mass of the dimer [(rHSA)( 2)] was determined by native PAGE electrophoresis and MALDI-TOFMS spectrome try. As expected, the colloid osmotic pressure was only half that of the mo nomeric rHSA solution. Incorporation of (2-[8-{N-(2-methylimidazolyl)}-octa noyloxymethyl]-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 dioxyg en under physiological conditions tin aqueous media, pH 7.3, 37 degrees C) like hemoglobin and myoglobin. A maximum of 16 hemes (FePs) were incorporat ed into the (rHSA)(2) structure. On the basis of the isoelectric focusing m easurement, the surface charge distributions of the (rHSA)(2) and (rHSA-FeP )(2) are identical to that of rHSA. The O-2-binding affinity (P-1/2: 30 Tor r at 37 degrees C) and O-2-association and -dissociation rate constants of (rHSA-FeP)(2) (k(on), 2.4 x 10(7) M-1 s(-1); k(off), 4.7 x 10(2) s(-1)) sat isfy the requirements for a synthetic O-2 carrier as a red cell substitute.